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Patent 1269598 Summary

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(12) Patent: (11) CA 1269598
(21) Application Number: 598511
(54) English Title: SERUM PRETREATMENT FOR TRICYCLIC ANTIDEPRESSANT DRUG ASSAYS
(54) French Title: PRETRAITEMENT DU SERUM POUR LE DOSAGE DE MEDICAMENTS ANTIDEPRESSEURS TRICYCLIQUES
Status: Deemed expired
Bibliographic Data
(52) Canadian Patent Classification (CPC):
  • 180/11
(51) International Patent Classification (IPC):
  • G01N 33/44 (2006.01)
  • G01N 30/00 (2006.01)
  • G01N 33/94 (2006.01)
(72) Inventors :
  • COLLINS, CHRISTINE GLEN (United States of America)
  • PANKAY, SUSAN CAROL (United States of America)
  • JAKLITSCH, ANNA (United States of America)
(73) Owners :
  • SYNTEX (U.S.A.) INC. (United States of America)
(71) Applicants :
(74) Agent: JOHNSON, DOUGLAS S. Q.C.
(74) Associate agent:
(45) Issued: 1990-05-29
(22) Filed Date: 1985-10-01
Availability of licence: Yes
(25) Language of filing: English

Patent Cooperation Treaty (PCT): No

(30) Application Priority Data:
Application No. Country/Territory Date
657,319 United States of America 1984-10-02

Abstracts

English Abstract



ABSTRACT OF THE DISCLOSURE

A kit is provided for purifying a serum sample to give a
tricyclic antidepressant drug sample for use in an assay for the
drug. The kit comprises a column containing alkylated silica
gel, a wash solution comprising from about 50 to 85 volume
percent of an aqueous buffered medium of pH from about 3.5 to
5.0 and from about 15 to 50 volume percent of an organic solvent
containing from 1 to 6 carbon atoms and from 1 to 5 heteroatoms
selected from the group consisting of oxygen, nitrogen, and
sulfur, and an eluent solution comprising from about 0 to 75
volume percent of an aqueous buffered medium of pH of about from
6 to 8 and from about 25 to 100 volume percent of one or more
organic solvents containing from l to 6 carbon atoms and from 1
to 5 heteroatoms selected from the group consisting of oxygen,
nitrogen, and sulfur.


Claims

Note: Claims are shown in the official language in which they were submitted.


WHAT IS CLAIMED IS:

1. A kit for purifying a serum sample to give a
tricyclic antidepressant drug sample for use in an assay
for said drug, which kit comprises -
(a) a column containing silica gel alkylated
with alkyl groups containing 1-6 carbon atoms,
(b) a wash solution comprising from about 15
to 50 volume percent of an organic solvent of from 1 to 6
carbon atoms and 1 to 5 heteroatoms selected from the
group consisting of oxygen, nitrogen, and sulfur and from
about 50 to 85 volume percent of an aqueous buffered
medium having a pH of about from 3.5 to 5.0; and
(c) an eluent solution comprising about from
25 to 100 volume percent of an organic solvent of from 1
to 6 carbon atoms and 1 to 5 heteroatoms selected from
the group consisting of oxygen, nitrogen, and sulfur, and
about from 0 to 75 volume percent of an aqueous buffered
medium having a pH of about from 6 to 8.

36

Description

Note: Descriptions are shown in the official language in which they were submitted.


~.~26~




10SERUM PRETREATMENT FOR
TRICYCLIC ANTIDEPRESSANT DRUG ASSAYS

BACKGROUND OF THE INVENTION
l. Field of the Invention.
15A number of tricyclic compounds find use in the
treatment of depression. These tricyclic antidepressants
include imipramine, desmethylimipramine (desipramine),
amitriptyline, nortriptyline 3 protriptyline, doxepin and
desmethyldoxepin (nordoxepin). In administering a
tricyclic antidepressant, it is frequently necessary to
ensure that the blood level of the antidepressant remains
within a certain narrow concentration range in order to
ensure effective dosage, while avoiding levels which rnay
be toxic or produce undesirable effects. Furthermore, it
is often necessary to detect potentially toxic levels of
tricyclic antidepressants and their metabolites.
It is therefore desirable to provide a simple and
-rapid procedure for determining or detecting the levels
of tricylic antidepressants in serum or other
physiological fluids. The procedure should provide
reproducible values and be specific for the tricyclic
compounds which are measured. Thus, the procedure must
be capable of distinguishing the tricyclic
antidepressants from other drugs and from metabolites of
the tricyclic antidepressant drug, which would otherwis~

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give an erroneous result in an assay for the detection of
tricyclic antidepressants.
Since the therapeutic range of the tricylic
antidepressant drugs is from about 50 to 300 ng/ml, it is
necessary nnt only to measure extremely small amounts of
the tricyclic antidepressant drug in serum, but also to
be able to distinguish bet~een small differences in
concentrations. Naturally occurring materials or
metabolites of the tricyclic antidepressant drug in the
serum sample may modify the observed signal so as to give
Falsely high results. It would therefore be desirable to
provide for a simple means for pretreatment of a serum
sample for a tricyclic antidepressant drug assay. The
pretreatment method should be rapid and efficient and
provide an assay sample containing the drug substantially
free of interfering substances.
2. ~rief Descr.iption of the Prior Art.
The tricyclic antidepressants are closely related
chemically to one another. Techni~ues reported for the
determination of amitriptyline in biological fluids
2~ include the use of thin layer chromatography, gas-liquid
chromatography and GLC-mass spectrometry. Gifford, et
; al., J. of Chrom., 105, 107 113 (1975); Gupta, et al.,
Clin. 3iochem., 9, 247-51 (1976); Nyberg and Martensson~
J. Chromatography, 143, 491 (1977); Watson and Stewart,
25 J. Chrom., 134, 182 (1977~; ibid. 132 155-159 (1977).
Radioimmunnoassay has been reported for amitriptyline by
Aherne, et al., Br. J. Clin. Pharmac., 3, 561 (1976),
Turner, Lancet, 180, 1316 (1977); and Aherne9 et al.,
Lancet 1214 (1977). In Aherne, et al., ibid., a
3~ synthesis for an antigen for use as an immunogen for
antibody formation is described, where nortriptyline is
substituted with aminobutylene followed by conjugation to
bovine serum albumin employing carbodiimide. In another
antigen conjugate synthesis by Kaul, et al., J. Anal._

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Tox., 1, 236 (1977), nortriptyline was conjugated to
bovine serum albumin through a succinyl group. The
resulting antibodies were found to have significant
cross-reactivity with a number of other tricyclic drugs.
U.S. Patent No. 4,275,160 describes imipramine
derivatives and poly(amino acid) conjugates. U.S.
Patents Nos. 4,223,013 and 4,3n7,245 disclose
amitriptyline conjugates to antigenic proteins and
enzymes.
N-(2-carboxyethyl) derivatives of nortriptyline and
desipramine are disclosed by Hubbard et al., J. Pharm.
Sc., 67, pp. 1571-1578 (1978) and by Hubbard et al.,
Canadian Journal of Pharmaceutical Sciences, 15,
_
pp. 89-93 (1980).
SUMMARY OF THE INVENTION
Serum samples for tricyclic antidepressant drug
assays are pretreated by passing the serum sample through
a column containing silica gel that is alkylated. After
zO application of the serum sample, the column is washed
with a wash mixture comprising from about 15 to 50 volume
percent of an organic solvent containing from 1 to 6
carbon atoms and from 1 to 5 heteroatoms selected from
the group consisting of oxygen, nitrogen, and sulfur and
from about 50 to 85 volume percent of an aqueous buffered
solution having a pH of from about 3.5 to 5Ø The drug
is eluted from the column with an eluent comprising from
~` about 25 to 100 volume percent of an organic solvent of
from 1 to 6 carbon atoms and from 1 to 5 heteroatoms
selected from the group consisting of oxygen, nitrogen,
and sulfur and from about O to 75 volume percent of an
aqueous buffered solution having a pH of about from 6 to
8. The eluted material contains the tricyclic
antidepressant drug substantially free of interfering
substances. The pretreatment method finds particular
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application in conjunction with assays employing enzyme
or fluorescent labels.

DESCRIPTI5N OF THE SPECIFIC EMBûDIMENTS
_
Blood serum or plasma samples for tricyclic
antidepressant drug assays are pretreated to provide a
sample substantially free from metabolites present in the
serum sample and in a form useful for a tricyclic
antidepressant drug assay determination. By the term
"substantially free" is meant that the tricyclic
antidepressant drug sample contains less than about from
20%, preferably less than about from 10%, of metabolites
of such drug.
The method normally employs liquid chromatography
using a column containing silica gel alkylated with alkyl
groups of from l to 12 carbon atoms, preferably l to 6
carbon atoms. The silica gel particles have a size in
the range of about 30-50 ~m, preferably about 40 ~m.
The particles are suitably silanized with methyl or ethyl
silyl groups to provide the alkylated silica gel
Z particles.
The amount of the column packing, i.e., alkylated
silica gel, employed and the dimensions of the column are
dependent on the size of the serum sample to be treated.
Generally, for a serum sample of û.5 ml, about 80 to 120
mg, preferably 90 to llO mg, of packing is used. For lOO
mg of silica gel, depending upon the manner of packing,
as well as the diameter of the column, the height of the
column can vary from about 6 to lO mm.
The column is packed by introducing the silica gel
3~ powder into an appropriate column. The column is then
condit;oned, by adding an alcohol, such as methanol, and
~ removing the alcohol by any convenient means, e.g.,
- vacuum, positive pressure, centrifugation, or the like.
After the alcohol has been removed, the column is then

3081I 24570-FF
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washed ~ith ~ater, preferably delonized water. The water
is then removed as described above, and the column is now
ready for the sample.
Prior to applying the sample to the column, the
serum sample may be subjected to other pretreatments.
Depending upon 'he nature of the sample, the sample may
be centrifu5ed, or the like.
The sample is then added to the column after all the
excess methanol and water employed in the pretreatment
and washing of the column have been removed. The sample
may then be drawn into the column by vacuum or
centrifugation~ or it may be pushed into the column by
positive pressure. The conditions for applying the
sample to the column will be generally mild, for example,
a vacuum in the range of about 10-20 inches Hg may be
used. Various conventional devices can be used, for
example, the Vac-Elut~ vacuum box (Analytichem
International).
After the sample has been applied to the column, the
column is washed with a mixture comprising a water
soluble organic solvent and an aqueous buffered medium.
The orgznic solvent generally has from 1 to 6, pre,erably
1 to 3, carbon atoms and from 1 tn 5, preferably 1 to 2,
heteroatoms selected from the group consisting of oxygen,
nitrogen and sulfur. The organic solvent may be an
alkylnitrile such as acetonitrile, propionitrile, an
alcohol such as methanol, ethanol, propanol, a ketone
such as acetone, and the like. The wash mixture
~ generally contains from about 15 to 50, preferahly 25 to
; 35, volume percent o~ the organic solvent.
The wash mixture also contains from about 50 to 85,
preferably 65 to 75, volume percent of an aqueous
buffered medium having a pH of flom about ~.5 to 5.0,
preferably from about 4.0 to 4.4. The buffered medium
may be about C.l to 1 M, preferably 0.2 to 0.4 M, in a
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metal salt of a carboxylic acid having from 2 to 4 carbon
atoms. Exemplary of such metals are alkali metals such
as sodium, potassium, and the like and exemplary of
carboxylic acids are acetic acid, propionic acid, and the
like.
The aqueous buffered medium portion of the column
wash mixture may also contain an alkyl sulfonate wherein
the alkyl group has from 5 to 7 carbon atoms such as, for
example, pentane sulfonate, hexane sulfonate, heptane
sulfonate, and the like. Conveniently, the alkyl
sulfonate is incorporated into the mixture as part of the
aqueous buffered medium. In this mode, the aqueous
buffered medium would be from about 0 to û.Olû M in alkyl
sulfonate.
The volume of the wash mixture should be sufficient
to remove substantially all of the metabolites of the
tricyclic antidepressant drug from the column. However,
the wash mixture should remove substantially none of the
drug itself. The volume of the wash solution is based
primarily on the number of theoretical plates of the
column packing. As an example, the volume of wash
mixture can be from about 0.8 to l.0 ml for about lO0 mg
of column packing. After addition of the wash solution,
the wash solution may be drawn through the column as
described above for the sample. Usually this will
involve, for an initial volume o~ l ml sample, at least
about 15 seconds and not more than about two minutes,
generally ~rom about 20 seconds to 45 seconds. Any water
remaining at the tip of the column may be removed by
blotting or other convenient means.
The tricyclic antidepressant drug is then eluted to
provide for a tricyclic an~idepressant drug sample
substantially fr~e from metabolites to be used in an
assay. To this end an elution mixture is employed
comprising one or more ~ater soluble organic solvents and
__ 35

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an aqueous buffe~ed medium having a pH of from about 6 to
8, preferably, 6.5 to 7.5, more preferably neutral pH.
The organic solvent usually comprises from about 25 to
100, preferably 70 to ~0, volume percent of the elution
mixture and the aqueous buffered medium usually comprises
from about 0 to 75, preferably 20 to 30, volume percent
of the elution mixture. Generally, the organic solvent
has the same characteristics as those described above for
the wash mixture and, conveniently, may be the same
organic solvent as that empioyed in the wash mixture~
Preferably, the elution mixture comprises at least two
organic solvents as defined above in a ratio of about
from 1:1 to 1:2. A preferred elution mixture may contain
from about 45 to 55 volume percent of an alkylnitrile as
defined above and from about ~5 to 35 volume percent of
an alcohol as de,ined above for the organic solvent.
T~ne aoueous buffered medium may comprise a phosphate
buffer such as potassium hydrogen phosphate. The
phosphate may conveniently be combined with the water
prior to combination with the other eluent components.
In this embodiment, the aqueous medium is usually about
from 0 to 0.01 M in phosphate buffer.
Elution is accomplished by adding from about 0.5 to
1.0 ml of the eluent mixture for an initial volume of
1 ml o, serum sample. Generally, the volume of eluent
mixture should be sufficient to remove substantially all
of the drug frorn the column; usually the volume
corresponds to the initial volume of the serum sample.
The eluent is drawn through the column in the same manner
described above for the wash mixture. ~he eluate is then
collected and is ready to be used in an assay since it
contains the tricyclic antidepressant ~rug substantially
free of interfering substances, i.e., those substances
which, by thei~ presence in the sample, would affect the
accur~cy of the assay.



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It is also within the scope of the present invention
to dilute the eluate prlor to conducting an assay.
Generally, about 0.5 to l ml of an aqueous buffer of pH 5
to 8, such as Tris HCl buffer is passed through the
column from which the sample was eluted. The huffer is
then combined with the eluted sample.
In assays involving labels, e.g., enzyme labels, the
components of the wash mixture and of the eiuant should
have little or no detrimental effect on the label
activity.
Tricyclic antidepressant drugs, for which samples
treatable by the method of the present invention are
assayed, are derivatives of dibenzazepine,
dibenzocycloheptadiene, and dibenzoxepin and generally
have the following formula:


~r~
(ICH2)2 (Ia)

CH3 ~
I
wherein:
~ is CH2-CH2, CH2-CH(OH), CH=CH, or CH2-0;
r is N-CH2, C=CH, or N-CH(R) wherein R is alkyl
o-f l to 3 carbon atoms, particularly CH3;
is H or CH3; and
D is hydrogen, hydroxy, or a halogen atom of atomic
number 9 to 53, preferably 7 to 35, more preferably a
chlorine atom.
Exemplary of such tricyclic antidepressant compounds
are imipramine, desmethylimipramine, amitriptyline,
protriptyline, trimipramine, chlomipramine, doxepin and
desmethyldoxepin. Also nortriptyline.
:
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Serum samples treated to give tricyclic
antidepressant drug extracts in accordance with the
present invention may be assayed for the presence of the
drug by a number of assay methodologies. The assays may
be heterogeneous or homogeneous involving labels such as
enzymes, radioisotopes, fluorescers, and the like.
The invention also includes a kit comprising, in a
packaged combination, (1) a prepacked column having
dimensions as described above and containing silica gel,
alkylated with alkyl groups containing from 1 to 12
carbon atoms, in amounts as described above, to which
column the serum sample is to be applied, (2) from 100 to
200 ml of a wash mixture as described above, and (3) from
100 to 200 ml of an eluent as described above. The wash
mixture and the eluent may be in suitable containers such
as vials made of a suitable material such as glass or
plastic. The kit may also include ancillary items such
as a device for securing the serum sample or applying the
serum sample to the column, column conditioning solutions
as described above, wash solutions such as an alcohol,
deionized water, 1~0 to 200 ml of a post elution aqueous
buffer as described above, etc., in separate containers
and so forth. The above kit may be combined with an
assay kit for performing a tricyclic antidepressant drug
assay or it may be separate therefrom.
EXAMPLE
The inventlon is further demonstrated by the
following illustrative example, which is provided by way
of illustration and not limitation.




3081I 24570-FF



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EXAMPLE
Assay for_Nortriptyli _
A 100 mg C-2 (ethyl) column from Analytichem
International was washed with approximately one ml of
methanol followed by approximately one ml of water. The
sample (500 ul) was placed on the top of the column. A
vacuum apparatus was attached to the bottom and a vacuum was
10 drawn on the column. The eluate obtained was discarded and
the column was washed with 900 ul of a solution which was 70%
0.4 M sodium acetate, 5 mM heptane sulfonate, pH 4.2, and 30
acetonitrile. A vacuum was again drawn on the column and the
eluate was discarded. Next, the column was contacted with
15 500 ul of a solution which was 50~ acetonitrile, 25%
methanol, and 25% 5 mM K2HP04, pH 7. The eluant was
collected and used in the assay procedure.
An enzyme conjugate and an antibody reagent were
prepared in accordance with the teaching of Canadian patent
20 application no.: 482064.

[The method used was as set out in Preparation A.
following.]
In carrying out the assay, a Gilford Stasar III
microsample spectrophotometer was employed with a
Thermocuvette with a flow cell. All readings were made at
340 mn. The followiny solutions were prepared as reagents
for use in the assay.
Buffer:


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0.055 M tris-HCl pH 8.1 (RT)
Enzyme Conjugate Reagent:
~uffer
0.9% NaOl
1.0% ~-lactoglobulin (BLG), pH 8.0 (RT)
Sufficient enzyme conjugate to give a maximum rate
of ~OD equal to 700-1200 in the assay medium
Assay buffer:
Buffer
0.5YO NaCl
0.01% (v/v Triton X-100, pH 8.0 (RT)
Antibody Reagent:
Buffer
0.1% BLG~
G-6-PtNa) 0.198 M,
Nicotine adenine dinucleotide (NAD) 0.12 M, pH 5.2
(RT)
Antinortriptyline optimized for assay (antibodies
prepared in sheep). All % indicated are w/v,
9/lOO ml.
. 20 The protocol employed for carrying out an assay was
as follows:
Into a diluter was drawn 15 microliters (~1~ of
the above sample. The sample was dispensed with 250
microliters of the assay buffer into a one milliliter
Croan cup followed by 15 ~1 of the antibody reagent
with 250 ~l of the assay buffer. After 50 sec.
incubation 15 ~ of the enzyme reagent and 250 ~1 of
the assay bu~fer were added. Immediately after the
~; enzyme addition, the entire sample was aspirated into the
flow cell. After 10 seconds, a first reading was taken,
followed by a second reading after a 50 second .interval.
The results are reported as the difference in absorbance
X 2.667.


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Sample Concentration
of Nortriptyline (ng~ml) ~OD

0 695*
723
761
100 807
175 8~0
250 870

* Lowest rate in assay with predetermined amount of
antibody.

EXAI~
Assay for Desmethylimipramlne
A 100 mg column (C-2 (ethyl) from Analytichem, Harbor
City, California) was washed with approximately one ml oF
methanol Followed by approximately one ml of water. The
sample (500 ul) was placed on the top of the column. A
vacuum apparatus was attached to the bottom and a vacuum was
drawn on the column. The eluate obtained was discarded and
the column was washed with 900 ul of a solution which was 70%
0.4 M sodium acetate and 5 mM heptane sulfonate, pH 4.2, 30%
acetonitrile. A vacuum was again drawn on the column and the
eluate was discarded. Next, the column was contacted with
500 ul of a solution which was 50% acetonitrile, 25%
methanol, and 25% 5 mM K2HPO4, pH 7. The eluant was
collected and used in the assay procedure.
The antibodies and the enzyme conjugate employed in this
assay for desmethylimipramine were prepared in accordance
with the teaching of U.S. Patent 4,~51,275.
[The method used was as set out in Preparation B,
following.~



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In carrying out the assay, a Gilford Stasar IIID
microsample spectrophotometer was employed with a
Thermocuvette (3017T) with a flow cell. All readings
were made at 340 nm. The following solukions were
prepared as reagents ~or use in the assay.
~uffer:
0.055 M tris-HCl pH 8.0 (RT)
Enzyme Conjugate Reagent:
Buffer
0.9% NaCl
1.0% BLG, pH 8.0 (RT)
Sufficient enzyme conjugate to give a maximum rate
of ~OD equal to 800 1200 in the assay medium
Assay buffer:
Cuffer
0.5% NaCl
0.01% (v/v) Triton X-100, pH 8.0 (RT)
Antibody Reagent:
Buffer
- 0.1% BLG,
G-6-P(Na) 0.22 M,
NAD 0.13 M, pH 5.2 (RT).
Antidesmethylimipramine optimized for assay
~antibodies were prepared in sheep)
(All % indicated are w/v, 9/lOO ml.)
The protocol employed for carrying out an assay was
as follows:
Into a diluter was drawn 15 microliters (~1) of
the above eluant. This sample was dispensed with
250 microliters of the assay buffer into a one milliliter
Croan cup followed by 15 ~1 of the antibody reagent
with 250 ~1 of the assay buffer. After 50 sec.
incubation 15 ~ of the enzyme reagent and 250 ul of
the assay buffer were added. Immediately after the
enzyme addition, the entire sample was aspirated into the
3~

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flow cell. After 10 seconds, a first reading is taken,
followed by a second reading, after a 50 second
interval. The results are reported as the difference in
absorbance x 2.667.
-- __
Sample Concentration
_f Desmethylimipramine (n~/ml) ~OD
702*
737
100 765
200 800
350 8~4
500 854
. _
* lowest rate in assay with predetermined anount of
antibody.

The assay of Example 1 was repeated except that the
serum sample was not pretreated in accordance with the
present invention. The following represents a
statistical summary of the results where the y-axis
represents the enzyme-label assay result and the x-axis
represents a reference method conducted using high
pressure liquid chromatography (HPLC).

Nortriptyline
Pretreated Sample Non-pretreated Sample

- n 14 12~
intercept 10 39 827 36
SEE 98 2~ 87
.
Two samples had rates higher than the highest
calibrator and therefore could not be accurately
quantitated.


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It is evident from the above results that the
subject method provides a sample allowing for an accurate
assay for tricyclic antidepressant drugs, particularly
involving enzyme labels. Thus, a sensitive and efficient
method is provided for treating samples for tricyclic
antidepressant ~rug assays which results in accurate
determinations of the drug.
Although the foregoing invention has been described
in some detail by way of illustration and example for
purposes of clarity of understanding, it will be obvious
that certain changes and modifications may be practiced
within the scope of the appended claims.




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PREPARATION A FOR EXAMPLE 1
,

EXAMPLE A
Prepar_tion of 3-nitrodibenzosuberone
Acetic anhydride (15 ml) was added slowly to white
fuming nitric acid (90%, 6.1 ml, 0.13 mole) at room
temperature. The résulting warm (30) solution was
cooled to 25 and was added dropwise to a solution of
; dibenzosuberone (20.8 9, 0.1 mole, from Aldrich Chemical
CoO) in 25 ml of acetic anhydride at room temperature for
a period of three hours. After addition, an aliquot was
withdrawn, and quenched in water, and partitioned in
dichloromethane; tlc showed the presence of
3-nitrodibenzosuberone, some fast moving substance, and
; starting material. The reaction mixture was then added
to 2 liters of ice water and the oily product was stirred
for half an hour. The resulting aqueous layer was
decanted and discarded, and the oily residue on the
bottom was dissolved in dichloromethane and washed with
saturated sodium bicarbonate and brine. The organic
layer was dried over MgS04 and evaporated to yield
light yellow oil which was crystallized by dissolving in
warm ether and adding hexane until cloudiness developed.
The resulting clear solution was cooled (5) overnight to
yield 6.5 9 of pale yellow solid (26~ yield) of
3-nitrodibenzosuberone. The tlc of this material showed
a major product and a small amount of impurity; the
product was used without further purification.
Anal. CalcdO for C15Hl1N03, C7 71.15; H, 4.~5; N, 5.53
Found C, 69.42; H, 4.34; N, 5.85
.


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3081I 24570-FF

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EXAMPLE B
Preparation of 3-aminodibenZosUberone
To a suspension of 3-nitrodibenzosuberone (27.2 9,
0.107 mole) from Example A in a mixture of
tetrahydrofuran (800 ml), isopropanol (800 ml) and
phosphate buffer [pH 6.5, 1.6 liters, prepared by mixing
13.6 9 KH2P04 (0.1 molar) and 27.8 ml of lN NaOH and
diluting~the resulting solution to 2 liters] was added
sodium dithionite (220 9, from East~an OrganiG Chemicals)
over a period of 5 min. The solids became soluble, and
after 15 min. tlc showed complete reaction. The
resulting clear solution was then extracted exhaustively
with ethyl acetate and the organic layer was washed with
saturated NaCl solution, and dried over Na2S04.
Evaporation of solvents gave yellow crude product which
was chromatographed on a silica gel column and eluted
with ether/hexane 1:1 to yield 8.7 9 (36% yield) pure
yellow 3-aminodibenzosuberone.

EXAMPLE C
Preparation of 3-amino=5-~3-dimethylaminopropyl~-5-
hydroxy-10~ dihydrodibenzo[b~e}cycloheptatriene
a. Preparation of N,N-dimethylpropylchloride.
~ A solution of N,N-dimethylpropyl chloride
hydrochloride (100 9, from Aldrich Chemical Co.) in about
100 ml water was made alkaline by adding 10% NaOH to pH
of about 11 - 12. The resùlting bi-layer solution was
then extracted with ether, and the ether extracts were
dried over MgS04. Then, the ether was distilled using
a simple distill:ation apparatus under 1 atm pressure, and
. the resulting liquid was distilled at 45 (60 mm
pressure) to yield 54.5 9 colorless liquid of
N,N-dimethylpropyl chloride.

.
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b. Grignard reaction~
To Mg turnings (13 9., 0.54 mole) in tetrahydrofuran
(53 ml, dried and distilled freshly over the sodium salt
of benzophenone) was added a few drops of
1,2-dibromoethane (J. T. Baker Chemical Co.) under
nitrogen. After crushing the turnings with a glass rod
and noting gas evolution, a solution of
N,N-dimethylpropyl c~loride t32.3 9, 0.27 mole) in
tetrahydrofuran (150 ml) was added dropwise. During this
addition the solution was heated sufficiently to maintain
a gentle reflux. After stirring for 1 h under reflux,
the browrl reaction mixture was cooled to room ternperature
followed by addition of 3-aminodibenzosuberone (12.4 9,
0.056 mole) from Example B in dry THF (220 ml). The
resulting brown product was allowed to stir at room
temperature for half an hour and was cautiously quenched
with saturated ammonium chloride ~200 ml). The resulting
yellow residue wa~ extracted with ethyl acetate. The
organic phase was washed with saturated NaHC03 and
~ brine, dried (Na2S04), and evaporated to yield a
;~ ~ yellow oil (17.2 9) of 3-amino-5-(3-dimethylaminopropyl)
5-hydroxy-10,11-dihydrodibenzo[b,e]cycloheptatriene.
'
EXAMPLE D
Preparation of ~-aminoamitriptyline
~5 To a solution o~ 3-amino-5-(3-dimethylaminopropyl)-
5-hydroxy-10,11-dihydrodibenzo~b,e] cycloheptatriene
(17.1 9, 0.055 mole) from Example C in dichloromethane
(300 ml) was added trifluoroacetic àcid (29.6 ml, 0.175
mole). The resulting dark brown solution was refluxed
for 18 hours; tlc of an aliquot of the reaction mixture
showed incomplete reaction. There~ore, p-toluenesulfonic
acid monohydrate (10.5 9, 0.055 mole) was added and
refluxed overnight. After 18 hours, complete reaction
was observed. The reaction product was cooled, diluted

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with ether, made alkaline with concentrated ammonia (22
ml), and then extracted with ethyl acetate. Evaporation
of solvents gave a foaming product containing two major
components and some impurities of higher Rf values. The
samples were purified using preparative HPLC (silica gel
column, NH3:MeOH:CH2C12/0.24:3:97 by volume):
Fractions were collected and analyzed using both
refractive index and analytical tlc (silica gel plate,
0.08:1:7/NH3 MeOH CH2C12). Fractions of the same
F(f values were combined and evaporated to give 6.8 9 cis-
10 3-aminoamitriptyline Rf 0.08 and 306 9 trans
3-aminoamitriptyline Rf 0.15. Total yield of the
products from 3-aminodibenzosuberone over two steps is
10.4 9 (64% yield). Anal. calcd. for cis
3-aminoamitriptyline, C20H24N2 1/2H20, C, 79.73;
15 H,8.31; N, 9.30. Found, C, 79.53; H, 7.99; N, 8.89.

EXAMPLE E
Preparation of cis -N'-(methyldithioacetyl)-3-
aminoamitriptyline
20 To a solution of cis-~3-aminoamitriptyline (2.7 9,
9.3 mmole) from ~xample D in a mixture of tetrahydrofuran
(80 ml, dried and distilled freshly from sodium
benzophenolate) and dichloromethane (20 ml, dried over
molecular sieves 3A) was added the NHS ester of
25 methyldithioacetic acid (2.9 9, 13.8 mmole). The
resulting solution was allowed to stir at room
temperature. After four days, complete reaction was
observed on ~lc. The resulting light yellow solution was
evaporated to dryness on a rotary evaporator and the
30 residue was chromatographed on a reversed phase silica
gel column [containing 300 9 silica gel 60 silanized],
and eluted with 3% MeOH/CH2C12 ~1.4 liter) and then
5% MeOH/CH2C12 (600 ml). After evaporation of
solvents, 4.3 9 light yellow foaming product, which

3081I 24570-FF
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-20-
contained the cis-N'-methyl(dithioacetyl)3-
aminoamitriptyline was obtained. The product was
dissolved in 100 ml of CH2C12, and 1 ml triethylamlne
was added. The organic layer was washed with saturated
NaCl and dried over Na2504, and evaporated to yield
3.3 9 (86% yield) foaming product oF
cis-N~~methyl(dithioacetyl)3-aminoamitriptyline.

EXAMPLE F
Preparation of cis-N(B2~ _ richloroeth~y-
carbonyl)-N-methyl(dithioacetyl)-3-aminonortriptyline
To a solution of cis-N'-(dithioacetyl)-3-
aminoamitriptyline (3 9, 7 mmole) from Example E in
dichloromethane (120 ml, dried over molecular sieves 3A)
was added trichloethyl chloroformate (9.6 ml, 7û mmoles)
dropwise at room temperature under nitrogen atmosphere
followed by triethylamine (9.7 ml, 70 mmoles) for a
period of 15 min. The slightly warm reaction mixture was
cooled using a water bath and then allowed to stand at
room temperature for 3.5 h. Complete reaction was
obtained as observed on analytical silica gel plates.
The resulting yellow solution was evaporated to dryness.
Ether (100 ml) was added; white precipitates which formed
were filtered and then washed with ether. The ether
filtrates were collected and evaporated to yield a brown
oil which was chromatographed on silica gel. Fractions
were analyzed by tlc and detected using both UV and I2
since the product showed a UV chromophore, while the
impurities had no UV absorption but showed brown spots of
higher Rf values when being developed in an I2
chamber. Fractions were combined to give (3.2 g, 77%
yield) cis-N~ trichloroethoxycarbonyl)
3-amino-N'-(methyldithioacetyl) nortriptyline as a white
foaming product. Fractions containing impurities were
either chromatographed again or discarded since the
~ .
3081I 24570-FF

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-21-
impurities interfered with the reductive cleavage of the
methyldithioacetyl derivative in the next reaction. The
sample after column chromatography showed the correct
structure.
Anal. calcd. for C25H27N203Cl352
C, 52.31; H, 4.71; N, 4.88; Cl, 18.57
S, 11.16.
Found: C, 52,41; H, 4.85; N, 4.73; Cl, 18.14;
S, 10.84.
EXAMPLE-G
Pre aration of cis-3-amino-N'-~merca~toacetYl)
P _ . .
nortript~line
All solutions used in work up of the sulfhydryl
derivative were degassed by bubbling argon through each
solution at room temperature for at least 10 min.
To a solution of cis-N~
trichloroethoxycarbonyl)-3-amino-N'-(methyldithioacetyl)
nortriptyline (500 mg, 0.87 mmole) from Example F in
glacial acetic acid (10 ml) was added activated zinc dust
(1O5 g) at room temperature under nitrogen. The zinc
dust was activated by washing well with 100 ml of 2% HCl
for 4-5 min, then filtered and zinc powder was washed
with water, ethyl alcohol, acetone and dry ether. The
powder was then dried overnight at reduced pressure at
room temperature and then used for reduction. The
reaction mixture was allowed to stir overnight at room
temperature. After 22 hours, the reaction mixture was
filtered and washed with about 40 ml of water, and the
filtrate was cooled in an ice bath. White precipitates
(Rf 0.95, I14 mg3 formed and were removed by filtration
and discarded, and the filtrate was extracted with 2 x 10
ml ether/hexane (1:1), or until the complete removal of
side product. The resulting aqueous solution was then
extracted with a total of 200 ml dichloromethane and the
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organic solution washed with brine and dried over
Na2504. Evaporation of solvents gave a clear viscous
oil of the acetate of cis 3-amino-N'-(mercaptoacetyl)
nortriptyline (104 mg, 29% yield, Rf 0.31).
The product was found to be decomposed under vacuo
at room temperature within a day. However, under acidic
conditions, e.g., the acetate salt, the product was found
to be more stable. The acetate salt of
3-amino-N'-(mercaptoacetyl) nortriptyline was stored
under nitrogen, or argon, and kept at dry ice temperature.

EXAMPLE H
Preparation of the Conjugate of Cis-3-a~ino-N~-
(mercaptoacetyl) Nortriptyline and Bromoacetylglycyl BgGa. Preparation of the NHS ester of bromoacetylglycine
To a solution of bromoacetylglycine (1 9, mp.
114-115) in 10 ml of DMF was added powdered NHS (1 9)
and EDCI (1 9, 5.2 mmole) under nitrogen at 0. The
resulting clear solution was then allowed to stir at 5
after 18 h and used directly without the isolation of the
. 20 NHS ester.
b. Conjugation of bromoacetylglycine to BgG
To a clear solution of BgG (1.5 9) in a mixture of
phosphate buffer (100 ml, pH 9, 0.05 M) and DMF (5 ml)
was added dropwise the NHS ester of bromoacetylglycine
(500 mg in 6 ml DMF9 prepared as above) at 0 for a
period of 30 min. The pH of the ~gG solùtion before the
addition of NHS solution was 8. The pH dropped to 6.3
after addition of~the NHS solution; the p~ was then
adjusted to 6.8. The resulting mixture was allowed to
stir overnight at 5. After 18 hours, the conjugate was
dialyzed against 4 x 4 liter phosphate buffer (0.0125 M,
pH 6.8) 2 x 4 liter (0.05 M, pH 6.8). The conjugate was
diluted to 15û ml and stored for further conJugation.
The concentration of this protein conjugate was
determined by UV and found to be 9.58 mg/ml.
3081I 24570-FF
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c. Conjugation of cis-3 amino-N'-(mercaptoacetyl)
nortriptyline to bromoacetylglycyl BgG
To the bromoacetylglycyl BgG solution (212 mg)
prepared as above in 30 ml of 0.1 m phosphate buffer (pH
7, pre-degassed with nitrogen) was added
cis-3-amino N~-(mercaptoacetyl) nortriptyline acetate (35
mg in 1.75 ml DMF) prepared as in Example G. The
resulting cloudy solutlon was kept under nitrogen at 5
for 70 hours. The milky solution was then dialyzed
against 2 x 4 liter NH40H-H20, pH 9, 2xl liter 8M
10 urea, 1 liter 4M urea, 1 liter 2M urea and then 5x4 liter
NH40H-H20, pH 9. The pH of the conjugate was
adjusted to 10 with 15% NH40H and centrifuged at 3K,
10 min. Supernatant was lyophilized to give a conjugate
(205 mg) of hapten number 46.

EXAMPLE I
Preparation of the Conjugate of Ois-3-amino-N'-
(mercaptoacetyl) Nortriptyline and Bromoacetylglycyl BSA
a. Preparation of conjugate of bromoacetylglycine to BSA
To a clear solution of BSA (1.5 9) in phosphate
buffer (pH 9.0, 0.05 M, 100 ml) and DMF (6 ml) was added
dropwise the NHS ester of bromacetylglycine (500 mg), in
6 ml DMF at 0 for a period of 30 min. Before the
addition of the NHS ester, the pH of the BSA solution was
about 8Ø After the addition of the NHS ester, the pH
dropped to 5-6; the pH (5.86) of the reaction mixture was
adjusted to 6.8 and stirred overnight at 5. The
resulting conjugate was then dialyzed against 3 x 4 liter
phosphate buffer (0.0125M, pH 6.8) and 2 x 4 liter
phosphate buffer (0.05 M, pH 6.~). The conjugate was
diluted to 150 ml and stored for further conjugation.
The concentration of this protein conjugate was
determined by UV and found to be 8.8 mg protein/ml
solution.

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b. Conjugation of cis-3-amino-N'-~mercaptoacetyl)
nortriptyline to bromoacetylglycyl BSA.
To the bromoacetylglycyl BSA (250 mg) prepared as above
in a mixture of phosphate buffer (41 ml. pH 7) and DMF
(8 ml), [the solutions used for conjugation were saturated
with nitrogen gas] was added cis-3-amino-N'-(mercaptoacetyl)
nortriptyline free base (65 mg, prepared as in Example G) in
3 ml of DMF. The resulting mixture was then stirred under
nitrogen at 5 for a total of 72 hours and dialyzed
exhaustively against NH40H/H20 (10 x 4 l). The conjugate
was then lyophilized to give 194 mg protein (hapten number
24).
EXAMPLE J
Preparation oF the Conjugate of trans-3-Amino-N'-
; (merca~toacetyl)nortriptyline and Bromacetylglycyl
G-6-PDH
a) Preparation of conjugate of bromoacetylglycine (8AG3 and
G-6-PDH.
The above BAG/G-6-PDH conjugate was prepared a~cording
to the procedure disclosed in U.S. Patent No. 4,220,722 at
columns 18-19. The conjugate was dialyzed against 4 l of
tris buffer without preservatives (.05% azide .005%
Thimerasol) to give 13.8 mg of conjugate in 6.1 ml.
b) Conjugation of trans-3-amino-N'-(mercaptoacetyl~-
nor~riptyline to bromoacetylglycyl G-6-PDH.
A solution of 19 mg trans-3-amino-N'-(mercaptoacetyl)-
; 30 nortriptyline from Example G in 0.5 ml of DMF containing 50
ul of glacial acetic acid was prepared. This solution (125
ul) was degassed with argon and was added to the 6.1 ml of
the dialyzed BAG/G-6-PDH from above. The resulting mixture
was stirred for 3.5 h at 4 and then centrifuged. The

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supernatant was chromatographed on a Sephadex G-50 column
and fractions containing protein were collected. The
product was 84% deactivated and 40% inhibitable.

EXAMPLE K
Preparation of the Conjugate of G-6-PDH and
5-(~-N-Methylaminopropyl)-10,11-dihydro-
10-thioacetamido-5H-dibenz~b,f]azepine
a. Preparation of the conjugate of bromoacetyl
glycine and G-6-PDH (61 mg) with 8 ml of 0.055 M tris
buffer at pH 3.0) was brought to 4 and 320 mg each of
G-6-P(Na2) salt and NADH were added and dissolved. To
this solution a 0.5 M bromoacetylglycyl NHS ester in DMF5
prepared as in Example J above, was added slowly with
stirring until the deactivation o~ the enzyme was 65%.
~5 The solution was dialyzed against tris buffer (0.055 M 9
pH 8.0, 4000 ml) for 18 h.
b. Conjugation of 5-(3-N-methylaminopropyl)-
10,11-dihydro-10-thioacetamido-5H~dibenz~b,f]azepine to
bromoacetylglycyl G-6-PDH.
The hapten material (63 mg) from Example E was
reconstituted in 1.5 ml DMF. All of the dialyzed
material from Example I was placed inside a flask and
cooled to 4~. The hapten was added dropwise until the
inhibition against anti-DMI antibodies was 45-50% (a
hapten to enzyme ratio of about 95). The G-6-PDH
conjugate was ~hen desalted at 4 over a G50 column with
tris buffer (.055 M, pH 8.0) with preservatives.

EXAMPLE L
Antibodies were produced in conventional manner in
response to the conjugate of Example H, and used in the
assay together with the enzyme conJugates prepared in
Example K.

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P~EPARATION B, FOR EXAMPLE 2

Example M
Preparation of 5-[3-N-methyl-N-(2,2,2,-trichloro-
carboethoxy)amino propyl~-~O,ll-dihydro-
5H-diben~ ~b,f] azepinone
Into a 200 ml round bottom flask was placed 8.0 9
(0.25 mol) of 5-(3-dimethylaminopropyl)-5H-dibenz [b,f]
azepin-10-one, (prepared according to the teaching of
U.S. Patent No. 4,275,160, Examples 1-5~, 45 ml of
anhydrous toluene, 14 9 (0.102 mol) of anhydrous
potassium carbonate followed 21.7 9 (0.102 mol) of
2,2,2-trichloroethyl chloroformate. The solution was
stirred vigorously and slowly brought to reflux with an
oil bath. Reflux continued for approximately 12 hours.
TLC analysis, silica gel, GF-chloroform showed some
carbamate formed after less than one hour's time
(visualization with ~V lamp and ceric sul~ate-H2504
spray). The reaction was cooled and added to a
~ 20 separatory funnel containing 200 ml water and was
; extracted several times with chloroform. The organic
phase was again extracted with two 100-ml portions of 10%
HCl, 200 ml water, dried over MgS04, filtered, and
concentrated on a rotary vaporator leaving a dark brown
oil (~15 9).
Chromatography of the oil was performed on a glass
column with dry silica gel 60-200 mesh (J. T. Baker
Chemical, Phillipsburg, N.J.) ~650 9, 5X80 cm column
dimension. The crude product, dissolved in a small
amount of dichloromethane, was placed at the top of the
column Two liters of solvent (dichloromethane) was
collected prior to collection of ~20 ml increments on
an autcmatic fraction collector. After faster moving
impurities were eluted, larger volume fractions were
~ 5 collected and TLC indicated only one spot corresponding

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to carbamate. These fractions were combined and
concentrated to yive 9.8 9 of product.
IR: CDC13 (1670 cm~l, aromatic carbonyl); (1720
cm 1 carbamate carbonyl stretch)
Pmr: 90MHz(CDC13-TMS) ~8.1 (dd,l lH, J~9Hz,
0~1.5Hz, lH aromatic); 7.13 (m, 7H aromatic);
4.64 (br.s, 2H,-OCH2CC13); 3.96 (s,2H,-CH2GO-);
3.96 (t,2H,J~6Hz,-N-CH2CH2-);
3.34 (t,2H,~Hz,-CH2CH2NCH3);
2.85 (s,3H,-NCH3); 1.89 (m,2H,CH2,CH2-CH2)
Example N
Preparation of 5-r3 N-methyl-N-(2,2,2-
trichlorocarboethoxy)amin_ propyl]-10-amino-
10,11-dihydro-5H-dibenz [b,f]azepine
Into a 100 ml pear-shaped flask equipped with a
CaC12 drying tube and a reflux condenser was placed 3.8
9 (0.00835 mol) of the product of Example M, 6.4 9
(0.0835 mol) of anhydrous ammonium acetate (dried in
dessicator under vacuum over CaC12), 50 ml of an
anhydrous methanol and 1.5 9 (0.0250 mol) of sodium
cyanotrihydridoborate. The mixture was stirred and
brought to 50~.
After 72 h, an additional 1 9 of anhydrous ammonium
acetate and 5 gm sodium cyanotrihydridoborate was added.
~5 After 100 h the reaction mixture cooled and was
poured into a separatory funnel containing 300 ml water
and was extracted with three lOO~ml portions of
chloroform. The organic phases were combined, dried with
MgS04, filtered and concentrated on a rotary vaporator.
One-half of the material was applied to twelve 20X20
cm preparative plates, 2.5 mm silica gel 60 PF-254~366
(Analtech). The plates were treated with ethyl ether 3
times. Material was then extracted from the absorbant
with lD% methanol-sO% dichloromethane and concentrated.
.
3081I 24570-FF
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This material was re-subjected to the above
chromatographic condition, but ethyl ether saturated with
ammonia gas was substituted for the ethyl ether and the
plates were treated only one time. (The major band was
collected after UV light inspection indicated the
presence of the desired material.) These two
chromatographic steps gave pure product which was
isolated in 60-70% yield.

IR: CHC13 (1720 cm 1, carbamylcarbonyl);
(3380 3350 cm 1, NH stretching modes)
Pmr: 90MHz (CDC13-TMS) ~7.07 (m,8H,aromatic);
5.64 (S,2H,0-CH2CC13);
4.48 (m~lH1-NH2-cH-cH2);
3~75 (t~2H~6Hz~N-cH2-cH2);
3.~4 (t,2H,J~6hz;
2.85 ts13H~-NcH3);
2.13 (br.s,2H,NH2)

Example 0
Preparation of N-hydroxysuccinimidylmethyl-
dithioacetic acid
Into a 50 ml round bottom flask was placed 1.2 g
(8~86 mmols) of methyl dithioacetic acid (prepared
according to P. Singh, et aI. (1979) Anal. Biochemistry
25 104, 51? 35 ml of dichloromethane, and 1.09 9 (9.54
mmols) of NHS (crystallized from ethyl acetate). The
solution was cooled in an ice bath before addition of
1.96 9 (9.54 mmols) of distilled
N,N'-dicyclohexylcarbodiimide; a mild exothermic reaction
occurred. After stirring for 4 h, the solution was
; filtered through a medium sintered glass funnel,
precipitated urea was washed with dichloromethane and
light brown filtrate concentrated on a rotary evaporation
at ambient temperature.

3081I 24570-FF
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-29-
This material was dissolved in 10 ml CH2C12 and
applied to the top of a 2.5 X 43 cm dry glass column
packed with 110 9 of silanized silica gel 60 particle
size 0~063 - 0.200 mm (70 230 mesh ASTM, E Merck).
The eluant was l/l-CH2C12/hexane. The fractions
were monitored by tlc on silanized silica gel RP-2
(E. Merck) with l/l-CH2C12/hexane as the eluant.
Fractions 22-40 were combined (dry weight 1.2 9,
approx. 60% yield). The dry material was dissolved in
CH2C12/hexane solution and cooled overnight at 0~.
More hexane was added as needed to promote
crystallization (900 9 white NHS ester, m.p. 79-81).
Microanalysis sulfur calc. 27.25%; Act. 27.24~.

Example P
Preparation of 5-(3-N-methyl N-(2t2~2-trichlorocarbn-
ethoxy)aminopropyl)-10,11-dihydro-10-methyldithio-
acetamido-5H-dibenz [b9f] azepine
A solution of 557 mg (1.22 mmols) of
5-[3-N-methyl-N-(2,2,2-trichlorocarboethoxy)amino
. 20 propyl]-10-amino-10,11-dihydro-5H-dibenz [b,f] azepine
from Example 2, 287 mg (1.22 mmols) of
N-hydroxysuccinimidyl methyl dithioacetate from Example 0
and 25 ml of anhydrous tetrahydrofuran was stirred at
ambient temperatures for 4 h.
TLC analysis was conducted on silanized silica gel
RP-2 eluant 20% hexane-80% dichloromethane; comparison
with N-hydroxysuccinimidyl methyldithioacetate,
N-hydroxysuccinimide and starting amine revealed that the
reaction was complete. (Rf value of product ~0.13.)
The reaction mixture was concentrated and dissolved
in 20% hexane-80% CH2CL2 and placed at the top of a
glass column 2.5x42 cm dry packed with 130 gm of silica
gel 60, silanized particle size 0.063-0.20 mm (70-230
mesh ASTMj RP-2. The eluant was 20~ hexane-80%

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dichloromethane and 15-20 ml fractions were collected
(approximately 96% yield).
IR: 1% in KBr (1650 cm 1 amide carbonyl);
~ 1740 cm 1 carbamate carbonyl)
Pmr: 90 MHz (CDC13-TMS) ~2.4 (s,~H,SCH3);
2.88 (br.s,3H,-NCH3) 3.38 (s,2H,-COCH2S-);
4.68 (br.s,2H,-0-CH2CC13);
5.62 (br.m, lH,-CH-NHC0-)

Example Q
Preparation of 5-(3-N-methylaminopropyl)-10,11-dihydro-
10-thioacetamido-5H-dibenz ~b,f] azepine
Into a 25 ml round bottom flask equipped with
stopper was placed 140 mg (0.243 mmol) of the product of
Example P, excess zinc dust (>70 mg), 10 ml of glacial
acetic acid. The mixture was stirred at ambient
temperatures for 24 h (a white precipitate of ZnC12
formed). Reaction progress was monitored by TLC on RP-2
silanized silia gel plates with 15% methanol-85%
dichloromethane as the eluant (the plates were observed
under UV lamp and with Ellman's reagent spray~
The reaction mixture was ~iltered to remove solids,
was washed with acetic acid-methanol, and was
concentrated on a rotary vaporator under high vacuum
without heating. The residue was taken up in
water/chloroform extracted with many portions of
chloroform dried with MgS04, filtered, and
- concentrated. This material was applied to one
preparative TLC 20x20 cm plate, 2.5 mm thickness,
silanized si}ica gel PF 254 (E. Merck). The plate was
eluted with 15% methanol-85% dichloromethane. The band
corresponding to the desired product was isolated. The
product was extracted from the above solvent with 20/80
methanol/dichlorornethane, concentrated on rotary
evaporator and under high vacuum giving a light yellow
foam, 60 mg, 70% yield.
3081I 24570-FF

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-31-
Rf value ~.62 sulfhydryl 85% CH2C12-15% methanol-
IR: CHC13 film (1650 cm~l amide carbonyl); (acetate
salt -NH~i GH3AcO , 1720 cm 1 C02_)

PMR: 90MHz (CD30D/CDC13-TM5) ~7.1 (m,8H,aromatic);
~5.54 (m,lH,-CH-NC0-); 3.87(t,2H,J~6Hz,N-CH2CH2-);
3.23 (s,-CH2SH);
3.05 (t,2H,J~6Hz,-CH2CH2-NHCH3); 2.60 (s,3H,NCH3)
2.04 (m,2H,-CH2CH2-NHCH3)

Example R
Preparation of_the Conjugate of BSA-bromoacetylglycine
with 5-(3-N-methylaminopropyl)-10~ dihydro-10-thio-
acetamido-5H-dibenz~b,f]azepine
15a. Preparation of the NHS succinimic ester of
bromoacetylglvcine
To a solution of bromoacetylglycine (1 g,
m.p. 114-115) in 10 ml of DMF was`added powdered
N-hydroxysuccinimide (1 9) and EDCI (1 9, 5.2 mmole)
. 20 under nitrogen at 0. The resulting clear solution was
then allowed to stir at 5 after 18 h and was used
directly without the isolation o~ the NHS ester.
b. Preparation of the con~ugate of
bromoacetylglycine and BSA
25To a clear solution of 8SA (1.5 9~ in phosphate
buffer (pH 9.0, 0.05 M, 100 ml) and DMF (6 ml) was added
dropwise the NHS ester of bromoacetylglycine (500 mg)
prepared in (a) above, in 6 ml DMF at 0 for a period of
30 min. Before the addition of the NHS ester, the pH of
the BSA solution was about 8Ø After the addition of
the NHS ester, the pH dropped to 5-6, the pH (5.86) of
the reaction mixture was adjusted to 6.8 and the mixture
was stirred overnignt at 5. ~The resulting conjugate was
then dialyzed against 3 x 4 liter phosphate buffer

3081I 24570-FF
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-32-
(0.0125M, pH 6.8) and 2 x 4 liter phosphate buffer (0.05
M, pH 6.8). The conjugate was diluted to 150 ml and
stored for further conjugation. The concentration of
this protein conjugate was determined by UV and found to
be 8.8 mg protein/ml solution.




c.
; and 5-(3-N-methylaminopropyI)-10,11-dihydro-
10-thioacetamido-5H-dibenz[b,f]aze_ine
Fifty ml of the BSA-bromoacetylglycine solution from
(b) above (~0.375 9) was placed in a 125 ml flask
followed by 10 ml of .4M Na2HP04-NaH2P04 buffer
pH 7.25 (pH of protein solution 7.21 by pH meter).
The solution was cooled in an ice bath at 4; then
100 mg of the mercaptoacetamido product of Example Q was
dissolved in 1 ml of DMF and was added slowly dropwise to
the stirring protein solution. An additional 2 ml of DMF
was used to rinse residual material. After the addition
was complete, the protein solution appeared considerably
turbid.
. 20 The above solution was stirred in a cold room (4)
for 3 days.
The above solution was placed in a semi-permeable
membrane (cylinder diameter 20.4 mm, M.W. cut off
6,000-8,00Q) and dialyzed against deionized water pH 9.8
with NH40H, 4 liters, 3 times for 8 h each.
The material was then chromatographed using Sephadex
G-50 medium with a bed volume of four times the volume of
the product solution. Fractions of approximately 15 ml
each were collected, UV of fractions was recorded on a
Carey 15 spectrophotometer. The appropriate fractions
were combined based on the UV data. The combined
fractio;ns were lyophilized to give 0.440 9 o~ product
with a hapten number of 24.
` 35
.
~081I 24570-FF
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Example S

with 5-(3-N-methylaminoproplyl)-10,11-
dihydro 10-thioacetamido-5H-dibenzCb ~]azepine
a. Conju~ation of bromoacetylglycine to BgG
To a clear solution of BgG (1.5 g) in a mixture of
phosphate buffer (100 ml, pH 9, 0.05 M) and DMF (5 ml)
was added dropwise the NHS ester of bromoacetylglycine
(500 mg in 6 ml DMF, prepared as in Example Ra above~ at
0 for a period of 30 min. The pH of the BgG solution
before the addition of NHS solution was 8. The pH
dropped to 6.3 after addition of the NHS solution; the pH
~as then adjusted to 6.8. The resultins mixture was
allowed to stir overnight at 5. After 18 hours, the
conjugate was dialyzed against 4 x 4 liter phosphate
15 buf~er (0.0125 M, pH 6.8) 2 x 4 liter (0.05 M, pH 6.8).
The conjugate was diluted to 150 ml and stored for
further conjugation. The concentration of this protein
conjugate was determined by UV and found to be 9.58 mg/ml.
b. Con2ugation of B~G-bromoacetylglycine and
20 5-(~-N-methylaminopropyl)-10,11-dihydro-10-thioacetamido-5H
-dibenz[b,f]azepine
Fifty ml of the BgG-bromoacetylglycine solution from
(a) above (~0.375 9) was placed in a 125 ml flask and
10 ml of 0.4M Na2HP04-NaH2P04 buffer pH 7-23 was
added. The mixture was then cooled to 4~ in an ice
bath. Next, was added 100 mg (0.281 mmol) of
5-(3-N-methylaminopropyl)-10,11-dihydro-10-thioacetamido-5H
-dibenz[b,f]azepine from Example Q dissolved in 1 ml of
DMF. The solution became turbid then very milky. An
additional 2 ml of DMF used to rinse the remaining
material into the reaction vessel.
The above solution was stirred in a cold room (4~)
~ for 3 days.
:~ ,

~.'
3081I 24570-FF

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The above solution was placed in a semi-permeable
membrane tubing (cylinder diameter 20.4 mm, M.W. cut off
6,000 8,000) and dialyzed aqainst deionized water pH 9.8
with NH40H, 4 liters, 3 times for 8 h each.
The material was then chromatographed using Sephadex
G-50 medium with a bed volume of four times the volume of
the product solution. Fractions of approximately 15 ml
each were collected, UV of fractions was recorded on
Carey 15 spectrophotometer. The appropriate fractions
were combined based on the UV data. The combined
fractions were lyophilized to give 0.440 g of product
with a hapten number of 8.
,
Example T
Preparation of the Con~jugate of G-6-PDH and
5-(3-N-methylaminopropyl)-10,11-dihydro-10-
thioacetamido-5H-dibenz[b,f]azepine
-
a. Preparation of the conjugate of brornoacetyl_
glycine and G-6-PDH
G-6-PDH (61 mg in 8 ml of G.055 M Tris buffer at pH
8.0) was brought to 4 and 32D mg each G-G-P (Na2) salt
and NADH were added and dissolved. To this solution, a
0.5 M bromoacetylglycyl NHS ester in DMF, prepared as in
Examp~ e Ra above, was a~ed unti~ tt~e e~te~ to enzyme
;: ratio was 3 :1 and the deactivation of the enzyme was
65%. ~he solution was dialysed against Tris buffer
(0.055 M, pH 8.0) 4000 ml for 18 h.

b. Conjugation of 5-(3-N-meth~aminopropyl)
10,11-dihydro-10-thioacetamido-5H-dibenz[b,f~
azepine to_bromoac_tylgIycyl G-6-PDH
The hapten material (63 mg) from Example Q was
reconstituted in 1.5 ml of DMF. All of the dialysed
material from Example Ta was placed in a side arm flask
and cooled to 4. The hapten was added dropwise until

3081I 24570-FF

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the inhibition against anti-DMI antibodies was 45-50% (a
hapten to enzyme ratio of about 95). The G-6-PDH
conjugate was then desalted at 4 over a G50 column with
1ris buffer (0.055 M, pH 8.0) with preservatives.

5Example U
Antibodies were produced in conventional manner in
response to the conjugate of Example S, and used in the
; assay together with the enzyme conjugate from Example T.




~; 2~



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3081I 24570-FF

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Representative Drawing

Sorry, the representative drawing for patent document number 1269598 was not found.

Administrative Status

For a clearer understanding of the status of the application/patent presented on this page, the site Disclaimer , as well as the definitions for Patent , Administrative Status , Maintenance Fee  and Payment History  should be consulted.

Administrative Status

Title Date
Forecasted Issue Date 1990-05-29
(22) Filed 1985-10-01
(45) Issued 1990-05-29
Deemed Expired 2001-05-29

Abandonment History

There is no abandonment history.

Payment History

Fee Type Anniversary Year Due Date Amount Paid Paid Date
Registration of a document - section 124 $0.00 1986-06-04
Application Fee $0.00 1989-05-02
Maintenance Fee - Patent - Old Act 2 1992-05-29 $100.00 1992-04-30
Maintenance Fee - Patent - Old Act 3 1993-05-31 $100.00 1993-04-05
Maintenance Fee - Patent - Old Act 4 1994-05-30 $100.00 1994-03-22
Maintenance Fee - Patent - Old Act 5 1995-05-29 $150.00 1995-04-18
Maintenance Fee - Patent - Old Act 6 1996-05-29 $150.00 1996-04-15
Maintenance Fee - Patent - Old Act 7 1997-05-29 $150.00 1997-04-14
Maintenance Fee - Patent - Old Act 8 1998-05-29 $150.00 1998-04-06
Maintenance Fee - Patent - Old Act 9 1999-05-31 $150.00 1999-05-03
Owners on Record

Note: Records showing the ownership history in alphabetical order.

Current Owners on Record
SYNTEX (U.S.A.) INC.
Past Owners on Record
COLLINS, CHRISTINE GLEN
JAKLITSCH, ANNA
PANKAY, SUSAN CAROL
Past Owners that do not appear in the "Owners on Record" listing will appear in other documentation within the application.
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Document
Description 
Date
(yyyy-mm-dd) 
Number of pages   Size of Image (KB) 
Drawings 1993-10-07 1 19
Claims 1993-10-07 1 31
Abstract 1993-10-07 1 27
Cover Page 1993-10-07 1 29
Description 1993-10-07 35 1,538
Fees 1997-04-14 1 81
Fees 1996-04-15 1 74
Fees 1995-04-18 1 94
Fees 1994-03-22 1 66
Fees 1993-04-05 1 37
Fees 1992-04-30 1 32