Phencyclidine analogs for immunoassay

Novel analogs of phencyclidine (PCP) are disclosed. The analogs are capable of reacting with anti-PCT antibodies and are useful in immunoassays. The following analogs are described: ##STR1## wherein wherein X is --OH or a reporter molecule selected from the group consisting of aminofluorescein and aminomethylfluorescein and n is an integer between 1 and 10, inclusive.

BACKGROUND AND SUMMARY OF THE INVENTION 
Phencyclidine 1-(1-phenylcyclohexyl piperidine), known as PCP was 
originally used as an anesthetic agent. PCP has become prevalent in the 
elicit drug market. It is often sold as "angel dust", "dust" or "super 
grass". The compound is a potent drug with lethal potential. and has 
become a major drug abuse problem. In 70 cases where PCP was deemed a 
factor in death, 90% of the cases had blood levels ranging from 10 to 300 
ng/mL R. Budd and Y. Liu (1982). phencyclidine Concentration in 
Postmortem Body Fluids and Tissues. J. Toxicol. Clin Toxicol. 
19(8):843-850!. PCP is extensively metabolized and about 10% is excreted 
unchanged in urine M. Wall, D. Brine, A. Jeffcoat et al. (1981). 
Phencyclidine Metabolism and Disposition in Man following 100 mcg 
Intravenous Dose. Res Comm. in Substances Abuse. 2:161-172!. The NIDA 
cut-off value requires at least 25 ng/mL before a measurement may be 
reported as positive. Thus it is desirable to have a simple and rapid 
technique for detecting the presence of PCP in body fluid such as blood 
plasma and urine. It is additionally desirable to prevent cross-reactivity 
to other drugs which may be present and have clinically similar symptoms. 
For this purpose, it is desirable to develop immunogenic conjugates of the 
phencyclidine hapten to elicit antibodies which provide the required 
degree of specificity while having high binding affinities and titer. 
A radioimmunoassay for PCP is described by Kalir et al. (1976), 
Theriogenology 6, No. 2-3, 193. This assay employed .sup.3 H-labeled 
phencyclidine derivative as .sup.3 H-4--aminophenyl phencyclidine. The use 
of radioisotope is undesirable because of the waste generated from it. PCP 
derivative having a non-oxo carbonyl functionality linked directly or 
through a linking group out of the phenyl ring of PCP is described by C-I 
Lin, and P. Singh (1981), U.S. Pat. No. 4,281,065. This compound is used 
as the derivative for an enzyme immunoassay. Another PCP derivative having 
an oxime functionality linked out of the cyclohexane ring of the PCP 
molecule is described by C-I Lin, and P. Singh (1984), U.S. Pat. No. 
4,446,065. This compound is employed as the derivative for an enzyme 
immunoassay. Still another compound having a carboxy functionality linked 
directly or through a linking group out of the phenyl ring of PCP is 
described by A. Focella (U.S. Pat. No. 4,196,185; 1980) and J. E. Heveran 
(J. Forensic Sciences, 25:79-87,1980). This compound and its derivatives 
are used in an immunoassay. Further compounds having an N-functionality 
fused to a piperidine ring of PCP are described in U.S. Pat. No. 5,221,629 
(1993), U.S. Pat. No. 5,124,457 (1992), U.S. Pat. No. 5,407,834 (1995), 
and U.S. Pat. No. 5,155,212 (1992). All of these derivatives including 
tracers and immunogens described above are made from a ring-substituted 
phencyclidines all of which posses the core structure derived from 
phencyclidine itself, the structure of which is shown below 
##STR2##

DETAILED DESCRIPTION OF THE INVENTION 
The present invention is directed toward novel analogs having structures 
similar to phencyclidine (PCP). These analogs are 
4-phenyl-4-pyrrolidinocyclohexane, thienylcyclohexylpiperidine 
(Tenocyclidine) and thienylcyclohexypyrrolidine having substituents on the 
aryl and thiophene rings as represented in formula I, II and III as shown 
below. These compounds do not possess the exact core structure of PCP as 
shown above but instead possess structures which are different in at least 
one of the cyclic components of the core. These analogs are capable of 
binding with antibodies against PCP. The substituents on these analogs can 
be linked to a variety of reporter molecules and protein carriers to 
provide useful reagents for immunoassay. When combined with the PCP 
antibodies, the compounds and the corresponding protein conjugates 
described in the present invention provide for sensitive and rapid assay 
for the detection of PCP in human urine. 
A functional group is a chemical group which can be converted by chemical 
means to another functional group for altering or enlarging the compound. 
For example, a compound which bears a carboxy functional group can be 
reacted with an amine bearing linker such as a aminoacid with the aid of a 
carbodiimide and the like, but not limited to other similar compounds, to 
form a compound of new entity bearing a peptide bond or an amide bond. 
Conversely, a compound bearing an amine can be coupled to a linker bearing 
a carboxy with the aid of carbodiimide to form compound-linker conjugate 
bearing a peptide bond. This process of linking carboxy to amine is well 
known in the art and it represents the simplest method for making a 
peptide conjugate or compound-protein conjugate (herein called 
drug-conjugate). 
Linkers are well known in the art and are commonly used to provide a spacer 
between a compound and a carrier molecule or a reporter molecule. Use of a 
linker may or may not be advantageous depending on the specific compound 
or hapten and the carrier pairs, and selection of an appropriate linking 
group is within the skill of the art. See U.S. Pat. No. 5,144,030 (column 
16, line 1) and U.S. Pat. No. 5,237,057 (column 2). Typical linker will 
vary from 1-20 carbon atoms of straight or branched chain and 0-5 
heteroatoms of N, O, and S. It is well known to those skilled in the art 
that combination of atoms or functional groups which are compatible can 
form the linking to allow covalent bonding of the compound and the carrier 
or reporter molecule. In many cases the use of linker requires more steps 
than direct coupling of a carboxy bearing compound to an amine. For 
example, compounds bearing a thiol functionality may react with a carrier 
molecule bearing thiol-reactive groups, such as maleimide groups, as 
exemplified by U.S. Pat. No. 5,237,057. This process of linking drugs to 
protein carriers involves more steps than direct approach described in 
this invention. 
A reporter molecule is a carrier molecule which bears an appropriate 
functional group ready for covalent bonding with aid of carbodiimide and 
the like. Examples of carrier molecules are thyroglobulin (BTG), bovine 
serum albumin (BSA), enzymes, and aminoacids including polyaminoacids. 
Other reporter molecules include fluorescein which bear an amine or 
carboxy function such as aminofluorescein and carboxyfluorescein 
(Molecular Probe, Eugene, Oreg.). 
In a preferred embodiment of the present invention a novel compound 
represented as formula I is useful for the preparation of the protein 
conjugate. 
##STR3## 
wherein: X is a bond, --O--, --NH-- or a linker consisting of an aminoacid 
but not limited to poly(aminoacid), aminomethylbenzoic acid where the 
amine function is attached to the oxo carbonyl, 
Y is --H, or a reporter molecule such as aminofluorescein and 
aminomethylfluorescein where the amine function is linked to the oxo 
carbonyl of X, protein carrier or enzyme where it is attached in the same 
manner to the X group. 
Alternatively, another preferred embodiment of the present invention is a 
novel compound with its substituent having formula II that is useful for 
the preparation of the protein conjugate. 
##STR4## 
wherein: X is a linker, --O--, --NH-- consisting of an aminoacid but not 
limited to poly(aminoacid), aminomethylbenzoic acid where the amine 
function is attached to the oxo carbonyl, 
Y is a --H or reporter molecule such as aminofluorescein and 
aminomethylfluorescein where the amine function is linked to the oxo 
carbonyl end of X, BSA, protein carrier or enzyme where they are attached 
in the same manner to the X group. 
Still another variation of compound having similar structure to formulas I 
and II as described above are useful. This is represented as formula III 
##STR5## 
wherein: X is a linker, --O--, --NH--consisting of an aminoacid but not 
limited to poly(aminoacid), aminomethylbenzoic acid where the amine 
function is attached to the oxo carbonyl, 
Y is --H, a reporter molecule such as aminofluorescein and 
aminomethylfluorescein where the amine function is linked to the oxo 
carbonyl end of X, BSA, protein carrier or enzyme where they are attached 
in the same manner to the X group. 
In order to prepare novel analogs having the general structure of formula 
I, cyclohexanone was used as the starting material. This compound was 
condensed with pyrrolidine in the presence of KCN and sulfuric acid to 
provide compound 1 which was further carried forward to compound 3 
according to methods described in Scheme I below. 
##STR6## 
The carboxy group of compound 3 can be linked to a variety of reporter 
molecule and aminoacids to provide useful reagents for immunoassay use. In 
a preferred embodiment, extension of compound 3 at the carboxy 
functionality with aminomethyl benzoic acid aided by EDC and NHS provided 
compound 5. This compound provided the most preferred reagent when coupled 
to BSA as described elsewhere in this invention. 
Another variation of compound of formula I having a substituent at the 
para-position of the aryl ring can be prepared according to Scheme II as 
shown below. For example, carboxy derivative 8 was activated to provide 
the N-hydroxysuccinimide derivative 9. This activated compound can be 
coupled to a variety of reporter molecules to provide a useful reagent for 
an immunoassay to detect PCP in urine and other body fluid. 
##STR7## 
Substitution of Tenocyclidine at the C-5 position of thienyl ring that 
leads to the carboxy Tenocyclidine 10, can be prepared according to method 
described by K. Sindler, Collect. Czech. Chem. Commun. 60:894-902(1995) 
Although this compound can be coupled to BSA to make useful Tenocyclidine 
conjugate, it is preferable that a linker such as aminomethylbenzoic acid 
as described in Scheme III be used and coupled at the carboxy end to 
provide compound 12. This is highly desirable as the resulting compound is 
highly chromogenic to allow ease of quantitation of the degree of drug 
substitution after coupling to proteins. Certainly other chromogenic 
molecules would be useful as well for substituting aminomethylbenzoic 
acid. This will aid in the determination of the drug incorporation on 
proteins. Scheme III illustrates the preparation of compound 12 and the 
corresponding N-hydroxysuccinimide ester 13 which can be linked to various 
reporter molecules. 
##STR8## 
Another novel derivative of Tenocyclidine substituted at the C-5 of the 
thiphene ring was obtained by direct succinylation of Tenocyclidine using 
aluminum chloride to provide compound 14 according to the preparation 
described in Scheme IV. This carboxy derivative can be linked to a variety 
of reporter molecules useful for immunoassay. 
##STR9## 
In order to prepare compounds of formula III which bears pyrrolidine in the 
molecule, compound 1 can be utilized as the starting molecule. First, 
thiophene can be treated with n-butyl lithium to provide 
2-lithiothiophene. Alternatively, 2-bromothiophene can be converted to the 
corresponding Grignard reagent. Among the two reagents, thiophene, 
2-magnesium Grignard is the preferred reagent and can be reacted with 
compound 1 described previously to afford the Tenocyclidine analog bearing 
the pyrrolidine ring. Functionalization at the C-5 position of the 
thiophene ring can be carried out analogously according to methods 
described in Scheme III and IV above. 
Other components required for the PCP immunoassay described in this 
invention are antibodies that recognize or bind to PCP itself. Immunogens 
containing PCP may elicit polyclonal antibodies to bind PCP. The PCP 
immunogen and the antisera were prepared according to the method described 
by Focella in U.S. Pat. No. 4,196,185. 
The novel compounds described in this present invention can be used in a 
variety of immunoassays for the detection of PCP and its metabolite. Such 
immunoassays may be in the form of radioimmunoassay, enzyme immunoassays 
or microparticle-based immunoassay in which agglutination of 
microparticles represents the signal detection and others. Immunoassays 
for PCP are commonly based on competitive binding between drug derivatices 
coupled to protein carrier, and free drug from a clinical sample for a 
limiting amount of antibodies. Free drug will inhibit the binding of the 
drug derivatives to the antibody. The extent to which the free drug 
inhibits the binding of the drug derivatives to the antibody is a direct 
measurement of the amount of drug present in the clinical sample. 
In a preferred embodiment of the present invention, drug derivatices are 
couples to BSA according to the coupling of carboxy to amine as described 
previously. Antibodies against PCP are bound onto microparticles by 
passive adsorption on the surface of microparticle. Both materials were 
then placed onto membrane strips as described in Example 17 and the 
calibration curve or assay run as described also in Example 17. The 
standard curve shown in FIG. 1 illustrates how novel compound 5 derived 
from the present invention, when coupled to BSA competes with free drug 
(PCP) for anti-PCP antibodies such on dyed microparticles that the 
presence of PCP results in a decrease or inhibition of the antibody-drug 
interaction and that this compound is useful as a reagent for the 
determination of PCP. FIG. 2 illustrates how drug-BSA conjugate derived 
from compound 12 competes similarly with free drug (PCP) for the anti-PCP 
antibodies on dyed microparticles such that the presence of PCP results in 
an inhibition of the antibody-drug derivative interaction and that this 
compound is useful as a reagent for the determination of PCP. 
Another preferred embodiments of immunoassays in which the compounds of the 
invention are useful is described in copending U.S. pat. application Ser. 
No. 877,189, the entire contents of which is hereby incorporated by 
reference. Such an assay is marketed by Roche Diagnostic Systems under the 
trademark OnTrak TestTstick.TM. for PCP. The assay is based on the 
principles of microparticle capture inhibition. The test relies on the 
competition between drug, which may be present in the urine being tested, 
and drug conjugate immobilized on membrane for binding to antibody-coated 
onto colored microparticles. 
When the device is immersed in the urine sample, some of the sample is 
absorbed into the sample pad. The absorbed sample travels through a 
reagent strip contained in the device by capillary action. In the reagent 
strip, the sample rehydrates and mobilizes antibody-coated blue 
microparticles. The microparticle-urine suspension continues to migrate 
through the reagent strip and comes in contact with the immobilized drug 
conjugate. In the absence of drug in the urine, the antibody-coated 
microparticles bind to the drug conjugated and a blue band is formed at 
the result window ("negative" sign). 
When drug is present in the specimen, it binds to the antibody-coated 
microparticles. If sufficient drug is present, the microparticles are 
inhibited from binding the drug conjugate and no blue band is formed at 
the result window. A positive sample causes the membrane to remain white 
("positive" sign). 
An additional antibody/antigen reaction occurs at a "TEST VALID" area. The 
"TEST VALID" blue band forms when anti-bodies, which are imbedded in the 
reagent membrane, bind to the antigen on the blue microparticles. The 
presence of the "TEST VALID" band indicates that the test has completed, 
the reagents are viable, and, the results are ready to interpret. 
EXAMPLE 1 
Preparation of 1-Pyrrolidin-1-yl-cyclohexanecarbonitrile, (Compound 1) 
To a magnetically stirred mixture of 5.0 g pyrrolidine (70.3 mmol) in 12 mL 
of ice-cold deionized water was added 5 mL of concentrated H.sub.2 
SO.sub.4. The pH of the reaction mixture was adjusted to 3.5 with 
concentrated NH.sub.4 OH. The reaction was cooled to 20.degree. C. and 6.9 
g (70.2 mmol) of cyclohexanone was added followed by a solution of 4.57 g 
(70.2 mmol) of potassium cyanide in 10 mL of water. The mixture was 
stirred at room temperature for 18 h. The reaction mixture was filtered 
and the filtrate was concentrated. This was dissolved in 200 mL of 
dichloromethane and 80 mL of brine was added. The organic layer was 
separated and the aqueous part was extracted with 2.times.100 mL of 
dichloromethane. The combined organic layer was dried with an anhydrons 
Na.sub.2 SO.sub.4 and concentrated to give 7.2 g (40.4 mmol, 58%) of 
compound 1 as colorless oil. 
EXAMPLE 2 
Preparation of 1-1-(3-Trifluoromethyl-phenyl)-cyclohexyl!-pyrrolidine, 
(Compound 2) 
A 250 mL three-necked round-bottom flask was charged with 2.12 g of 
magnesium turning and 100 mL of freshly distilled tetrahydrofuran under 
argon atmosphere. To the reaction mixture was added 4.42 g (13 mmol) of 
3-bromotrifluoromethanebenzene followed by 250 .mu.L of 1,2 dibromoethane 
and a catalytic amount of iodine crystal. The mixture was allowed to 
reflux for 4 h under argon atmosphere and cool to room temperature. A 
solution of 2 g (11.2 mmol) of compound 1 in 15 mL of freshly distilled 
tetrahydrofuran was added to the reaction mixture dropwise and this was 
allowed to stir for 18 h at room temperature. The reaction mixture was 
filtered and 60 mL of a saturated solution of ammonium chloride was added 
to the filtrate. The aqueous layer was extracted with 3.times.100 mL of 
ethyl acetate. The combined ethyl acetate layer was washed with 100 mL of 
brine, dried with anhydrous Na.sub.2 SO.sub.4 and concentrated. The 
residue was purified by column chromatography using 1:1 ethyl acetate: 
hexane to provide 1.85 g (6.29 mmol, 57%) of compound 2. 
EXAMPLE 3 
Preparation of 3-(1-Pyrrolidin-1-cyclohexyl)-benzoic acid, (Compound 3 ) 
A mixture of 1 g (3.40 mmol) of compound 2 and 2.5 mL of concentrated 
H.sub.2 SO.sub.4 was heated in an oil bath at 110.degree. C. for 2 h. The 
reaction mixture was cooled to room temperature and 5 mL of ice-water was 
added. The pH of the solution was adjusted to 7 and concentrated to 
dryness. The residue was triturated with 300 mL of 20% methanol in 
dichloromethane and the inorganic residue was filtered off. The filtrate 
was concentrated to give 902 mg (3.30 mmol, 97%) of compound 3 as 
off-white solid. 
EXAMPLE 4 
Preparation of 3-(1-Pyrrolidin-1-yl-cyclohexyl)-benzoic acid 
2,5-dioxo-pyrrolidin-1-yl ester, (Compound 4) 
To a solution of 750 mg(2.74 mmol) of compound 3 was added 790 mg (4.12 
mmol) of 1-ethyl-3(3dimethylamino-propyl)carbodiimide and 473 mg (4.10 
mmol) of N-hydroxysuccinimide. The mixture was allowed to stir at room 
temperature 18 h and 30 ml of dichloromethane was added. The organic layer 
was washed with 2.times.50 mL of water, 2.times.50 mL of saturated 
NaHCO.sub.3 and 50 mL of water. The organic layer was dried Na.sub.2 
SO.sub.4 and concentrated to give 830 mg (2.24 mmol, 82%) of compound 4 as 
white solid. 
EXAMPLE 5 
Preparation of 
4-{(3-(1-Pyrrolidin-1-yl-cyclohexyl)benzoylamino!-methyl}-benzoic acid, 
(Compound 5 ) 
To 688 mg (4.55 mmol) of aminomethylbenzoic acid was added 4.4 mL of 1N 
NaOH, 50 mL THF and 16 mL of water. The N-hydroxysuccinimidyl ester 
compound 4 (1.62 g, 4.3 mmol) was dissolved in 32 mL of freshly distilled 
THF and added dropwise to the above solution. After the addition the 
reaction mixture appeared cloudy. An additional 12 mL of water was added 
and the pH was maintained at 9-10 for 2 h. The reaction mixture was 
concentrated to remove as much THF as possible. The residual aqueous part 
was adjusted to 6.5 and extracted with chloroform (10.times.50 mL). The 
combined organic layer was dried (anh. Na2SO.sub.4) and concentrated to 
give 1.4 g(3.4 mmol, 79%) of compound 5 as white solid. 
EXAMPLE 6 
Preparation of 4-{3-(1-Pyrrolidin-1-yl-cyclohexyl) 
benzoylamino!-methyl}-benzoic acid 2,5-dioxo-pyrrolidin-1-yl ester, 
(Compound 6) 
To a mixture of 1.2 g (2.95 mmol) of compound 5 in 240 mL of 
dichloromethane was added 550 mg of N-hydroxysuccinimide and 917 mg of EDC 
hydrochloride. The reaction mixture was allowed to stir at room 
temperature for 18 h and washed with brine (2.times.125 mL), 2.times.125 
mL of saturated Na.sub.2 CO.sub.3 and water (125 mL). The organic layer 
was dried (Na.sub.2 SO.sub.4) and concentrated. The residue was purified 
by column chromatography to give 800 mg (1.58 mmol, 56%) of compound 6 as 
white solid. Likewise, compound 9 was converted to compound 15 in the same 
manner. 
EXAMPLE 7 
Preparation of 1-1-(4-Trifluoromethyl-phenyl)-cyclohexyl! pyrrolidine, 
(Compound 7) 
A 250 mL three-necked round-bottom flask was charged with 2.12 g of 
magnesium turning and 100 mL of freshly distilled tetrahydrofuran under 
argon atmosphere. To the reaction mixture was added 4.42 g (13 mmol) of 
4-bromotrifluoromethanebenzene followed by 250 .mu.L of 1,2dibromoethane 
and a catalytic amount of iodine crystal. The mixture was allowed to 
reflux for 4 h under argon atmosphere and cool to room temperature. A 
solution of 1 g (5.6 mmol) of compound 1 in 15 mL of freshly distilled 
tetrahydrofuran was added to the reaction mixture dropwise and this was 
allowed to stir for 18 h at room temperature. The reaction mixture was 
filtered and 60 mL of a saturated solution of ammonium chloride was added 
to the filtrate. The aqueous layer was extracted with 3.times.100 mL of 
ethyl acetate. The combined ethyl acetate layer was washed with 100 mL of 
brine, dried (anh. Na.sub.2 SO.sub.4) and concentrated. The residue was 
purified by column chromatography using 1:1 ethyl acetate: hexane to 
provide 910 mg (3.06 mmol, 55%) of compound 7 as colorless gum. 
EXAMPLE 8 
Preparation of 4-(1-Pyrrolidin-1-cyclohexyl)-benzoic acid, (Compound 8 ) 
A mixture of 4.3 g (14.4 mmol) of compound 7 and 10 mL of concentrated 
H.sub.2 SO.sub.4 was heated in an oil bath at 110.degree. C. for 1.5 h. 
The reaction mixture was cooled to room temperature and was added to 15 mL 
of ice-water. The pH of the solution was adjusted to 7.5 using 6N NaOH. 
During the neutralization procedure some black material started to form 
which was filtered off and the neutralization procedure continued to 
adjust the pH to 7.5. A white precipitate formed. This was left at room 
temperature 18 h and filtered to give 3.9 g(14.2 mmol, 99%) of compound 8. 
EXAMPLE 9 
Preparation of 4-(1-Pyrrolidin-1-yl-cyclohexyl)-benzoic acid 
2,5-dioxopyrrolidin-1-yl ester, Compound 9) 
To a solution of 1 g(3.65 mmol) of compound 8 in 100 mL of dichloromethane 
was added 1.8 g (9.38 mmol) of 
1-ethyl-3(3-dimethylamino-propyl)carbodiimide and 1.0 g (8.68 mmol) of 
N-hydroxysuccinimide. The mixture was allowed to stir at room temperature 
18 h. The organic layer was washed with 2.times.100 mL of water, 
2.times.100 mL of saturated NaHCO.sub.3 and 2.times.100 mL of brine. The 
organic layer was dried (anh. Na.sub.2 SO.sub.4) and concentrated. The 
crude product was purified by silica gel chromatography using ethyl 
acetate as eluent to give 1.18 g (3.18 mmol, 87%) of compound 9 as pale 
yellow amorphous solid. 
EXAMPLE 10 
Synthesis of the TCP Analog (Compound 10) 
1.76 mL (4.41 mmol) of n-butyl lithium, 2M in hexane, was added to a 
solution of 1 g (4.01 mmol) of Tenocyclidine (TCP) in 50 mL of anhydrous 
Et.sub.2 O (distilled from sodium/benzophenone) cooled to 0.degree. C. in 
an ice bath under argon. The cooling bath was removed, and the reaction 
was stirred for 45 min at ambient temperature. The reaction was then 
cooled back down to 0.degree. C., and CO.sub.2 (g) was bubbled into the 
reaction for .about.5 min. The reaction was poured into 150 mL of H.sub.2 
O forming a thick emulsion which contained an insoluble precipitate and 
slowly separated on standing. The aqueous phase was washed with 
3.times.100 mL of ET.sub.2 O, and then filtered to collect the precipitate 
which was dried under vacuum to give 459 mg (39% yield) of compound 10 as 
a white solid. A second crop of product was obtained by adjusting the pH 
of the filtered aqueous layer to 6 to 7 with 1N HC1, and the solution was 
allowed to stand at 4.degree. C. overnight. The precipitate which formed 
was filtered to give an additional 197 mg (17% yield) of compound 10. 
EXAMPLE 11 
Synthesis of the TCP Analog, (Compound 11) 
A suspension of 300 mg (1.02 mmol) of compound 10, 586 mg (5.09 mmol) of 
N-hydroxysuccinimde and 488 mg (2.55 mmol) of 
1-(3-dimethylaminopropyl)-3ethylcarbodiimide hydrochloride in 5 mL of 
anhydrous DMF and 20 mL of anhydrous CH.sub.2 Cl.sub.2 (distilled from 
CaH.sub.2) was stirred overnight at ambient temperature under argon. The 
reaction mixture was filtered through Whatman #1 filter paper to remove 
unreacted starting material, and then diluted to 100 mL with CH.sub.2 
Cl.sub.2. The reaction was washed twice with 100 mL of sat. NaHCO.sub.3 
and once with 100 mL of H.sub.2 O, dried over Na.sub.2 SO.sub.4, and the 
solvent was removed under reduced pressure. The resulting material was 
purified by chromatography on flash grade silica eluted with 1:1:1 THF 
(distilled from sodium/benzophenone)/EtOAc/hexane to give 258 mg (65% 
yield) of compound 11 as a white solid. 
EXAMPLE 12 
Synthesis of the TCP Analog, (Compound 12) 
A mixture of 400 mg (1.02 mmol) of compound 11, and 155 mg (1.02 mmol) of 
4-(aminomethyl)benzoic acid dissolved in 10 mL of THF and 2 mL of H.sub.2 
O was treated with 1N NaOH in order to adjust the pH to 8.5-9. The 
reaction was stirred for 3 h at ambient temperature with the addition of 
base as needed to maintain the pH at 8.5-9. The THF was removed under 
vacuum, and 10 mL of H.sub.2 O was added. The pH was lowered to 5.5-6 with 
1N HC1, and the precipitate which formed was collected by centrifugation. 
The solid was washed with water and dried under vacuum to give a 
recoverable yield of 96 mg (22% yield) of compound 12 as a white solid. 
EXAMPLE 13 
Synthesis of the TCP Analog, (Compound 13) 
A mixture of 82.2 mg (0.193 mmol) of compound 12, 110.9 mg (0.963 mmole) of 
N-hydroxysuccinimide and 92.3 mg (0.482 mmol) of 
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was stirred in 
5 mL of anhydrous DMF and 20 mL anhydrous CH.sub.2 Cl.sub.2 (distilled 
from CaH.sub.2) at ambient temperature under argon overnight. The reaction 
mixture was diluted to 40 mL with CH.sub.2 Cl.sub.2, washed twice with 40 
mL of sat. NaHCO.sub.3, once with 40 mL of H.sub.2 O, and once with 40 mL 
of sat. NaCl, and then dried over Na.sub.2 SO.sub.4. The solvent was 
removed under reduced pressure, and the resulting solid was purified by 
chromatography on flash grade silica eluted with 1:1:1 THF (distilled over 
sodium/benzophenone)/EtOAc/hexane to give 82 mg (81% yield) of compound 13 
as a white solid. 
EXAMPLE 14 
Synthesis of the Succinylated TCP Analog (Compound 14) (Scheme IV) 
To a solution of 200 mg (0.802 mmol) of Tenocyclidine (TCP) and 121 mg 
(1.203 mmol) of succinic anhydride in 10 mL of distilled CH.sub.2 Cl.sub.2 
cooled in an ice-water bath was added 428 mg (3.207 mmol) of aluminum(III) 
chloride and the reaction mixture was stirred overnight, with no further 
cooling. An additional 121 mg (1.203 mmol) of succinic anhydride and 107 
mg (0.802 mmol) of aluminum(III) chloride were added and the reaction was 
stirred for an additional 2 days. The reaction was quenched with 1N HC1, 
the pH adjusted to .about.6.5 to 7 with 6N NaOH, and the resulting mixture 
filtered through a sintered glass funnel to remove aluminum salts. The 
layers were separated, and the aqueous phase was extracted once with 
CH.sub.2 Cl.sub.2 and twice with EtOAc. The aqueous phase was saturated 
with sodium chloride and again extracted with EtOAc. All of the organic 
extracts were combined and evaporated. More product was recovered by 
evaporation of the aqueous phase to dryness, trituration of the resulting 
solid with ethanol, filtration, and evaporation of the ethanolic extract 
to dryness. The solids obtained from all the organic extracts were 
combined (280 mg) and purified by chromatography on flash grade silica gel 
eluted with 15% MeOH/CHCl.sub.3 to give 182 mg (65%) of the product. 
EXAMPLE 15 
Preparation of Drug-BSA Conjugate of Compound 5 
Conjugation of the PCP derivative (Compound 5) to bovine serum albumin 
(BSA) was performed as follows: To a solution of BSA (approximately 57 
mg/mL) in 50 mM potassium phosphate, pH 8, (5 mL) (cooled in an ice bath), 
dimethylsulfoxide (DMSO) was added dropwise (5 mL). After warming to 
25.degree. C., a 5 mg/mL solution of the above mentioned derivative in 
DMSO (1 ml) was added dropwise. The reaction mixture was stirred at room 
temperature for 16 hours and then transferred to dialysis tubing and 
dialyzed first against 15 volumes of 30% DMSO--potassium phosphate buffer 
(50 mM, pH 8), second against 20% DMSO - potassium phosphate buffer, and 
third against 10% DMSO--potassium phosphate buffer, and a forth for four 
times more against potassium phosphate buffer. Overall Drug: BSA ratio 
=10:1! 
EXAMPLE 16 
Absorption of Antibodies to Microparticles 
Carboxyl-modified blue polystyrene microparticles from Seradyn (0.3 micron 
particles) were first washed three times at 1% solids by centrifuiging in 
20 mM, pH 6.1 MES (2-N-morpholino!ethanesulfonic acid). The washed 
microparticles were then adjusted to 5% solids in MES and (i) sensitized 
with 2 mg/mL anti-PCP antibody for 16 hours at room temperature, (ii) 
blocked with BSA solution in MES for 1 hour at room temperature, then 
(iii) washed for three times at 1% solids in MES by centrifugation, and 
(iv) adjusted again to 5% solids. Before use, equal volumes of this latex 
and 35% w/v sucrose in MES were mixed. 
EXAMPLE 17 
Preparation of Membrane Strip 
Mylar backed large pore size nitrocellulose (10-20 micron) from Millipore 
Corp. was cut into pieces of 15 cm in length and 5 cm in width. Solution 
of PCP-BSA conjugate (about 5 mg/mL) and anti-BSA monoclonal Ab (about 2 
mg/mL), both in 50 mM potassium phosphate buffer pH 7.5, were dispensed 
using IVEK Corp. Digispense 2000.TM. system at the rate 1 .mu.l/cm onto 
nitrocellulose at a distance respectively 2 cm and 1 cm from the 15 cm 
side. Nitrocellulose segments were allowed to dry for about 30 min. at 
37.degree. C. and then were blocked with 1% w/v polyvinyl alcohol (PVA, 
m.w. 13,000-23,000) solution in 20 mM TRIS, pH 8, for 30 min. at room 
temperature. The segments were then rinsed in water and dried. 
Sample pads were prepared by cutting BioRad Laboratories gel blotter into 
0.5 cm.sup.2 pieces. The same BioRad gel blotter was used for the sink 
pads. 
The same nitrocellulose as described above in this example was used as a 
separate membrane for microparticles (top membrane). For this purpose the 
nitrocellulose, which was previously blocked in the same manner as the 
main membrane, was cut into 5 mm wide strips and latex was applied using 
the IVEK dispensing system. After drying for 30 min. at 37.degree. C., 
this membrane was placed, nitrocellulose surface down, onto the main 
membrane and laminated to the main membrane with Adhesive Research Inc. 
adhesive mylar. After this, the segment was cut into 5 mm wide strips, 
sample pad and sink pad were placed respectively at the beginning and 
terminal ends of the strips. The calibration curve was obtained by dipping 
the membrane strip in solutions containing predetermined amounts of the 
drug (PCP standards). The signal strength is determined as follows: 
2.5=dark blue, 1.5=medium blue. 0.8=light blue, and O=colorless. When the 
strip read colorless, a complete inhibition is achieved and the sample is 
indicated to contain 25 ng/mL of PCP. 
FIG. 1 illustrates that free phencyclidine (PCP) competes with the drug 
conjugate compound 5 for binding with anti-PCP antibodies such that the 
presence of PCP results in a decrease or inhibition of the 
antibody-antigen binding and that this compound is useful as a reagent for 
the determination of PCP. 
FIG. 2 illustrates that free phencyclidine (PCP) competes with the drug 
conjugate compound 12 for binding with anti-PCP antibodies such that the 
presence of PCP results in a decrease or inhibition of the 
antibody-antigen binding and that this compound is useful as a reagent for 
the determination of PCP. 
FIG. 3 illustrates that free phencyclidine (PCP) competes with the drug 
conjugate compound 15 for binding with anti-PCP antibodies such that the 
presence of PCP results in a decrease or inhibition of the 
antibody-antigen binding and that this compound is useful as a reagent for 
the determination of PCP.