# Patent

## Title
Haptens of olanzapine

## Abstract

The invention relates to compounds of Formula I, wherein R1, R2, and R3 are defined in the specification, useful for the synthesis of novel conjugates and immunogens derived from olanzapine. The invention also relates to conjugates of an olanzapine hapten and a protein.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority of the benefits of the filing of U.S. Provisional Application Ser. No. 61/691,454, filed Aug. 21, 2012. The complete disclosures of the aforementioned related U.S. patent application is/are hereby incorporated herein by reference for all purposes.


FIELD OF THE INVENTION
The invention relates to the field of immunoassays for determining the presence of olanzapine in human biological fluids.
BACKGROUND OF THE INVENTION
Schizophrenia is a chronic and debilitating psychiatric disorder affecting approximately 0.45-1% of the world's population (van Os, J.; Kapur, S. “Schizophrenia” Lancet 2009, 374, 635-645). The principal goals of treatment are to achieve sustained remission from psychotic symptoms, reduce the risk and consequences of relapse, and improve patient functioning and overall quality of life. While many patients with schizophrenia are able to achieve symptom stability with the available antipsychotic medications, poor adherence to medication is a common reason for relapse with daily administered oral medications. Several studies (Abdel-Baki, A.; Ouellet-Plamondon, C.; Malla, A. “Pharmacotherapy Challenges in Patients with First-Episode Psychosis” Journal of Affective Disorders 2012, 138, S3-S14) investigating the outcomes of non-compliance have shown that patients with schizophrenia who do not take their medication as prescribed have higher rates of relapse, hospital admission and suicide as well as increased mortality. It is estimated that 40 to 75% of patients with schizophrenia have difficulty adhering to a daily oral treatment regimen (Lieberman, J. A.; Stroup, T. S.; McEvoy, J. P.; Swartz, M. S.; Rosenheck, R. A.; Perkins, D. O.; Keefe, R. S. E.; Davis, S. M.; Davis, C. E.; Lebowitz, B. D.; Severe, J.; Hsiao, J. K. “Effectiveness of Antipyschotic Drugs in Patients with Chronic Schizophrenia” New England Journal of Medicine 2005, 353(12), 1209-1223). Therapeutic drug monitoring (TDM) is the quantification of serum or plasma concentrations of drugs, including anti-psychotic drugs, for treatment monitoring and optimization. Such monitoring permits, for example, the identification of patients that are not adhering to their medication regimen, that are not achieving therapeutic doses, that are non-responsive at therapeutic doses, that have suboptimal tolerability, that have pharmacokinetic drug-drug interactions, or that have abnormal metabolism resulting in inappropriate plasma concentrations. Considerable individual variability exists in the patient's ability to absorb, distribute, metabolize, and excrete anti-psychotic drugs. Such differences can be caused by concurrent disease, age, concomitant medication or genetic peculiarities. Different drug formulations can also influence the metabolism of anti-psychotic drugs. TDM permits dose optimization for individual patients, improving therapeutic and functional outcomes. TDM further permits a prescribing clinician to ensure compliance with prescribed dosages and achievement of effective serum concentrations.
To date, methods for determining the levels of serum or plasma concentrations of anti-psychotic drugs involve the use of liquid chromatography (LC) with UV or mass spectrometry detection, and radioimmunoassays (see, for example, Woestenborghs et al., 1990 “On the selectivity of some recently developed RIA's” in Methodological Surveys in Biochemistry and Analysis 20:241-246. Analysis of Drugs and Metabolites, Including Anti-infective Agents; Heykants et al., 1994 “The Pharmacokinetics of Risperidone in Humans: A Summary”, J Clin Psychiatry 55/5, suppl: 13-17; Huang et al., 1993 “Pharmacokinetics of the novel anti-psychotic agent risperidone and the prolactin response in healthy subjects”, Clin Pharmacol Ther 54:257-268). Radioimmunoassays detect one or both of risperidone and paliperidone. Salamone et al. in U.S. Pat. No. 8,088,594 disclose a competitive immunoassay for risperidone using antibodies that detect both risperidone and paliperidone but not pharmacologically inactive metabolites. The antibodies used in the competitive immunoassay are developed against a particular immunogen. ID Labs Inc. (London, Ontario, Canada) markets an ELISA for olanzapine, another anti-psychotic drug, which also utilizes a competitive format. The Instructions For Use indicate that the assay is designed for screening purposes and intended for forensic or research use, and is specifically not intended for therapeutic use. The Instructions recommend that all positive samples should be confirmed with gas chromatography/mass spectrometry (GC-MS), and indicate that the antibody used detects olanzapine and clozapine (see ID Labs Inc., “Instructions For Use Data Sheet IDEL-F083”, Rev. Date Aug. 8, 2011). Some of these methods, namely HPLC and GC/MS, can be expensive and labor-intensive, and are generally only performed in large or specialty labs having the appropriate equipment.
A need exists for other methods for determining the levels of anti-psychotic drugs, particularly methods that can be performed in a prescribing clinician's office (where the treatment for an individual patient can be adjusted accordingly in a much more timely manner) and in other medical settings lacking LC or GC/MS equipment or requiring rapid test results.
Olanzapine is:




SUMMARY OF THE INVENTION
The subject invention provides compounds and conjugates that permit such an improved method for determining the levels of the anti-psychotic drug olanzapine.
The invention comprises compounds of Formula I




wherein:

R1 is H,




CH2NH2, CH2NHC(O)(CH2)mCO2H, or Z—(Y)p-G;

R2 is H,




CH2NH2, CH2NHC(O)(CH2)mCO2H, or Z—(Y)p-G;

R3 is H, or W—(Y)p-G; provided that two of R1, R2, R3 must be H, and further provided that R1, R2 and R3 may not all be H simultaneously;
wherein:
Z is selected from the group consisting of:
—N(R4)—, —O—, —S—, -alkyl-, -alkoxyalkyl-, -aminoalkyl-, -thioalkyl-, -heteroalkyl-, alkylcarbonyl-,




wherein:

W is selected from the group consisting of:
—C(O)—, -alkyl-, -alkoxyalkyl-, -aminoalkyl-, -thioalkyl-, -heteroalkyl-, -alkylcarbonyl-, —N(R4)—,




R4 is H, an alkyl group, cycloalkyl group, araalkyl group or substituted or unsubstituted aryl group;
Y is an organic spacer group;
G is a functional linking group capable of binding to a carrier;
p is 0, or 1;
m is 1, 2, 3, 4, or 5;
n is 1, 2, 3, 4, or 5.
The invention comprises conjugates of compounds of the invention with immunogenic carriers such as proteins, and products produced by the process of contacting the compounds of the invention with immunogenic carriers.



BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1-3 show Competitive ELISA results generated with three different mouse fusion 11.1 hybridomas;
FIG. 4 shows the competitive immunoassay format used on a lateral flow assay device;
FIG. 5 shows a typical dose response curve generated with olanzapine antibody clone 35;
FIG. 6 shows a typical dose response curve generated with olanzapine antibody clone 61; and
FIG. 7 shows a typical dose response curve generated with olanzapine antibody 3F11.



DETAILED DESCRIPTION OF THE INVENTION
The subject invention provides compounds and conjugates that permit the determination of levels of anti-psychotic drugs. Such methods will permit clinicians to evaluate objectively at an appointment how likely it is that the worsening of a patient's symptoms may be due to lack of adherence. Alternatively, if compliant, a clinician can consider a different treatment choice. Therapeutic drug monitoring, which is enabled by such methods, is key in identifying the most effective treatment options. Moreover, clinicians believe that such TDM will help them to move into a very different relationship with their patients, i.e., to move from a hypothetical discussion on treatment non-adherence towards a more collaborative one by engaging patients to actively take ownership in optimizing their treatment regimen.
The development of the method requires first the synthesis of several immunogens, comprising a synthetic hapten linked to a protein. A hapten is a small molecule that can elicit an immune response when attached to a large carrier such as a protein. They are protein-free substances, of mostly low molecular weight, which are not capable of stimulating antibody formation alone, but which do react with antibodies. A hapten-protein conjugate is able to stimulate the production of antibodies. Specific antibody generation against small molecules is useful for immunoassay development (Pharm Res. 1992, 9(11):1375-9, Annali Dell'Istituto Superiore di Sanita. 1991, 27(1):167-74, Annali Dell'Istituto Superiore di Sanita. 1991, 27(1):149-54, Immunology Letters. 1991, 28(1):79-83).
The invention comprises compounds of Formula I




wherein:

R1 is H.




CH2NH2, CH2NHC(O)(CH2)mCO2H, or Z—(Y)p-G;

R2 is H,




CH2NH2, CH2NHC(O)(CH2)mCO2H, or Z—(Y)p-G;

R3 is H, or W—(Y)p-G; provided that two of R1, R2, R3 must be H, and further provided that R1, R2 and R3 may not all be H simultaneously;
wherein:
Z is selected from the group consisting of:
—N(R4)—, —O—, —S—, -alkyl-, -alkoxyalkyl-, -aminoalkyl-, -thioalkyl-, -heteroalkyl-, -alkylcarbonyl-,




wherein:

W is selected from the group consisting of:
—C(O)—, -alkyl-, -alkoxyalkyl-, -aminoalkyl-, -thioalkyl-, -heteroalkyl-, -alkylcarbonyl-, —N(R4)—




R4 is H, an alkyl group, cycloalkyl group, araalkyl group or substituted or unsubstituted aryl group;
Y is an organic spacer group;
G is a functional linking group capable of binding to a carrier;
p is 0, or 1;
m is 1, 2, 3, 4, or 5;
n is 1, 2, 3, 4, or 5.
Another embodiment of the invention comprises compounds of Formula I:
wherein:
R1 is H,




CH2NH2, CH2NHC(O)(CH2)mCO2H, or Z—(Y)p-G;

R2 is H,




CH2NH2, CH2NHC(O)(CH2)mCO2H, or Z—(Y)p-G; provided that either R1 or R2 must be H, and further provided that both R1 and R2 may not be H simultaneously;

R3 is H;
wherein:
Z is selected from the group consisting of:
—N(R4)—, —O—, —S—, -alkyl-, -alkoxyalkyl-, -aminoalkyl-, -thioalkyl-, -heteroalkyl-, -alkylcarbonyl-,




R4 is H, an alkyl group, cycloalkyl group, araalkyl group or substituted or unsubstituted aryl group;
Y is an organic spacer group;
G is a functional linking group capable of binding to a carrier;
p is 0, or 1;
m is 1, 2, 3, 4, or 5;
n is 1, 2, 3, 4, or 5.
Another embodiment of the invention comprises compounds of Formula I:
wherein:
R1 is H, or CH2NH—(Y)p-G;
R2 is H, or CH2NH—(Y)p-G; provided that either R1 or R2 must be H, and further provided that both R1 and R2 may not be H simultaneously;
R3 is H,
wherein:
Y is an organic spacer group;
G is a functional linking group capable of binding to a carrier;
p is 1.
Another embodiment of the invention comprises compounds of Formula I:
wherein:
R1 is H,




CH2NH2, or CH2NHC(O)(CH2)mCO2H;

R2 is H,




CH2NH2, or CH2NHC(O)(CH2)mCO2H; provided that either R1 or R2 must be H, and further provided that both R1 and R2 may not be H simultaneously;

R3 is H;
m is 1, 2, 3, 4, or 5;
n is 1, 2, 3, 4, or 5.
In another embodiment of the invention:
R1 is H,




CH2NH2, or CH2NHC(O)(CH2)mCO2H;

R2 is H,




CH2NH2, or CH2NHC(O)(CH2)mCO2H; provided that either R1 or R2 must be H, and further provided that both R1 and R2 may not be H simultaneously;

R3 is H;
m is 1, 2, 3, 4, or 5;
n is 1, 2, 3, 4, or 5.
Another embodiment of the invention is a compound of Formula I which is:







A preferred embodiment of the invention is the compound:




A preferred embodiment of the invention is the compound:




The invention further provides conjugates of the compounds of the invention with an immunogenic carrier.
Another embodiment of the invention is thus a conjugate of a compound of Formula I




wherein:

R1 is H,




CH2NH2, CH2NHC(O)(CH2)mCO2H, or Z—(Y)p-G;

R2 is H,




CH2NH2, CH2NHC(O)(CH2)mCO2H, or Z—(Y)p-G;

R3 is H, or W—(Y)p-G; provided that two of R1, R2, R3 must be H, and further provided that R1, R2 and R3 may not all be H simultaneously;
wherein:
Z is selected from the group consisting of:
—N(R4)—, —O—, —S—, -alkyl-, -alkoxyalkyl-, -aminoalkyl-, -thioalkyl-, -heteroalkyl-, -alkylcarbonyl-,




wherein:

W is selected from the group consisting of:
—C(O)—, -alkyl-, -alkoxyalkyl-, -aminoalkyl-, -thioalkyl-, -heteroalkyl-, -heteroalkyl-, -alkylcarbonyl-, —N(R4)—,




R4 is H, an alkyl group, cycloalkyl group, aralkyl group or substituted or unsubstituted aryl group;
Y is an organic spacer group;
G is a functional linking group capable of binding to a carrier;
p is 0, or 1;
m is 1, 2, 3, 4, or 5;
n is 1, 2, 3, 4, or 5; and an immunogenic carrier.
Another embodiment of the invention is a conjugate of a compound of Formula I
wherein:
R1 is H,




CH2NH2, CH2NHC(O)(CH2)mCO2H, or Z—(Y)p-G;

R2 is H,




CH2NH2, CH2NHC(O)(CH2)mCO2H, or Z—(Y)p-G; provided that either R1 or R2 must be H, and further provided that both R1 and R2 may not be H simultaneously;

R3 is H;
wherein:
Z is selected from the group consisting of:
—N(R4)—, —O—, —S—, -alkyl-, -alkoxyalkyl-, -aminoalkyl-, -thioalkyl-, -heteroalkyl-, -alkylcarbonyl-,




R4 is H, an alkyl group, cycloalkyl group, aralkyl group or substituted or unsubstituted aryl group;
Y is an organic spacer group;
G is a functional linking group capable of binding to a carrier;
p is 0 or 1;
m is 1, 2, 3, 4, or 5;
n is 1, 2, 3, 4, or 5; and an immunogenic carrier.
Another embodiment of the invention is a conjugate of a compound of Formula I
wherein:
R1 is H, or CH2NH—(Y)p-G;
R2 is H, or CH2NH—(Y)p-G; provided that either R1 or R2 must be H, and further provided that both R1 and R2 may not be H simultaneously;
R3 is H;
wherein:
Y is an organic spacer group;
G is a functional linking group capable of binding to a carrier;
p is 1; and an immunogenic carrier.
Another embodiment of the invention is a conjugate of a compound of Formula I
wherein:
R1 is H,




CH2NH2, or CH2NHC(O)(CH2)mCO2H;

R2 is H,




CH2NH2, or CH2NHC(O)(CH2)mCO2H; provided that either R1 or R2 must be H, and further provided that both R1 and R2 may not be H simultaneously;

R3 is H;
m is 1, 2, 3, 4, or 5;
n is 1, 2, 3, 4, or 5; and an immunogenic carrier.
Another embodiment of the invention is a conjugate of a compound of Formula I wherein:
R1 is H,




CH2NH2, or CH2NHC(O)(CH2)mCO2H;

R2 is H,




CH2NH2, or CH2NHC(O)(CH2)mCO2H; provided that either R1 or R2 must be H, and further provided that both R1 and R2 may not be H simultaneously;

R3 is H;
m is 1, 2, 3, 4, or 5;
n is 1, 2, 3, 4, or 5; and an immunogenic carrier.
A preferred embodiment of the invention is a conjugate of a compound selected from the group consisting of




and an immunogenic carrier.

Preferred embodiments of the invention are the above conjugates wherein the immunogenic carrier is a protein.
More preferred embodiments of the invention are the above conjugates wherein the protein is keyhole limpet hemocyanin, bovine thyroglobulin, or ovalbumin.
The invention also provides products formed from the process of contacting the above compounds with an immunogenic carrier.
Another embodiment of the invention is thus a product made by the process of contacting a compound of Formula I




wherein:

R1 is H,




CH2NH2, CH2NHC(O)(CH2)mCO2H, or Z—(Y)p-G;

R2 is H,




CH2NH2, CH2NHC(O)(CH2)mCO2H, or Z—(Y)p-G;

R3 is H, or W—(Y)p-G; provided that two of R1, R2, R3 must be H, and further provided that R1, R2 and R3 may not all be H simultaneously;
wherein:
Z is selected from the group consisting of:
—N(R4)—, —O—, —S—, -alkyl-, -alkoxyalkyl-, -aminoalkyl-, -thioalkyl-, -heteroalkyl-, -alkylcarbonyl-,




wherein:

W is selected from the group consisting of:
—C(O)—, -alkyl-, -alkoxyalkyl-, -aminoalkyl-, -thioalkyl-, -heteroalkyl-, -alkylcarbonyl-, —N(R4)—,




R4 is H, an alkyl group, cycloalkyl group, aralkyl group or substituted or unsubstituted aryl group;
Y is an organic spacer group;
G is a functional linking group capable of binding to a carrier;
p is 0, or 1;
m is 1, 2, 3, 4, or 5;
n is 1, 2, 3, 4, or 5; with an immunogenic carrier.
Another embodiment of the invention is a product made by the process of contacting a compound of Formula I
wherein:
R1 is H,




CH2NH2, CH2NHC(O)(CH2)mCO2H, or Z—(Y)p-G;

R2 is H,




CH2NH2, CH2NHC(O)(CH2)mCO2H, or Z—(Y)p-G;

R3 is H, provided that either R1 or R2 must be H, and further provided that both R1 and R2 may not be H simultaneously;
wherein:
Z is selected from the group consisting of:
—N(R4)—, —O—, —S—, -alkyl-, -alkoxyalkyl-, -aminoalkyl-, -thioalkyl-, -heteroalkyl-, -alkylcarbonyl-,




R4 is H, an alkyl group, cycloalkyl group, aralkyl group or substituted or unsubstituted aryl group;
Y is an organic spacer group;
G is a functional linking group capable of binding to a carrier;
p is 0, or 1;
m is 1, 2, 3, 4, or 5;
n is 1, 2, 3, 4, or 5; with an immunogenic carrier.
Another embodiment of the invention is a product made by the process of contacting a compound of Formula I
wherein:
R1 is H, or CH2NH—(Y)p-G;
R2 is H, or CH2NH—(Y)p-G; provided that either R1 or R2 must be H, and further provided that both R1 and R2 may not be H simultaneously;
R3 is H,
wherein:
Y is an organic spacer group;
G is a functional linking group capable of binding to a carrier;
p is 1; with an immunogenic carrier.
Another embodiment of the invention is a product made by the process of contacting a compound of Formula I
wherein:
R1 is H,




CH2NH2, or CH2NHC(O)(CH2)mCO2H;

R2 is H,




CH2NH2, or CH2NHC(O)(CH2)mCO2H; provided that either R1 or R2 must be H, and further provided that both R1 and R2 may not be H simultaneously;

R3 is H;
m is 1, 2, 3, 4, or 5;
n is 1, 2, 3, 4, or 5; with an immunogenic carrier.
Another embodiment of the invention is a product made by the process of contacting a compound of Formula I
wherein:
R1 is H,




CH2NH2, or CH2NHC(O)(CH2)mCO2H;

R2 is H,




CH2NH2, or CH2NHC(O)(CH2)mCO2H; provided that either R1 or R2 must be H, and further provided that both R1 and R2 may not be H simultaneously;

R3 is H;
m is 1, 2, 3, 4, or 5;
n is 1, 2, 3, 4, or 5; with an immunogenic carrier.
Another preferred embodiment of the invention is a product made by the process of contacting a compound which is




with an immunogenic carrier.

Another preferred embodiment of the invention is a product made by the process of contacting a compound which is




with an immunogenic carrier.

Another preferred embodiment of the invention is a product made by the process of contacting a compound which is




with an immunogenic carrier.

Another preferred embodiment of the invention is a product made by the process of contacting a compound which is




with an immunogenic carrier.

A more preferred embodiment of the invention is a product made by the process of contacting the above compounds with an immunogenic carrier wherein the immunogenic carrier is a protein and wherein the protein is keyhole limpet hemocyanin, bovine thyroglobulin, or ovalbumin.
ABBREVIATIONS
Herein and throughout the application, the following abbreviations may be used.

    AMAS N-(α-maleimidoacetoxy) succinimide ester
    BTG bovine thyroglobulin
    Bu3N tributylamine
    DCC dicyclohexylcarbodiimide
    DCM dichloromethane
    DIEA diisopropylethylamine
    DMF N,N-dimethylformamide
    DMSO dimethylsulfoxide
    EDTA ethylenediaminetetraceticacid
    KLH keyhole limpet hemocyanin
    SATA N-succinimidyl S-acetylthioacetate
    TEA triethylamine
    THF tetrahydrofuran
    TFA trifluoroacetic acid
    r.t. room temperature
    DIC diisopropylcarbodiimide
    DMAP N,N-dimethyl-4-aminopyridine
    EDC 1-ethyl-3(3-dimethylaminopropyl) carbodiimidehydrochloride
    NHS N-hydroxysuccinimide
    TFP Tetrafluorophenyl
    PNP p-nitrophenyl
    TBTU O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate
    HOBT N-Hydroxybenzotriazole
    DEPBT 3-(diethoxyphosphoryloxy)-1,2,3-benzotrazin-4(3H)-one
    BOP—Cl Bis(2-oxo-3-oxazolidinyl)phosphonic chloride
    DTT dithioerythritol


DEFINITIONS
The term “conjugate” refers to any substance formed from the joining together of separate parts. Representative conjugates in accordance with the present invention include those formed by the joining together of a small molecule, such as the compounds of Formula I, and a large molecule, such as a carrier or a polyamine polymer, particularly protein. In the conjugate the small molecule maybe joined at one or more active sites on the large molecule.
The term “hapten” refers to a partial or incomplete antigen. A hapten is a protein-free substance, which is not capable of stimulating antibody formation, but which does react with antibodies. The antibodies are formed by coupling a hapten to a high molecular weight immunogenic carrier, and then injecting this coupled product, i.e., an immunogen, into a human or animal subject.
The term “immunogen” refers to a substance capable of eliciting, producing, or generating an immune response in an organism.
An “immunogenic carrier,” as used herein, is an immunogenic substance, commonly a protein, that can join at one or more positions with haptens, thereby enabling the production of antibodies that can bind specifically with these haptens. Examples of immunogenic carrier substances include, but are not limited to, proteins, glycoproteins, complex polyamino-polysaccharides, particles, and nucleic acids that are recognized as foreign and thereby elicit an immunologic response from the host. The polyamino-polysaccharides may be prepared from polysaccharides using any of the conventional means known for this preparation.
Various protein types may be employed as immunogenic carriers, including without limitation, albumins, serum proteins, lipoproteins, etc. Illustrative proteins include bovine serum albumin, keyhole limpet hemocyanin, egg ovalbumin, bovine thyroglobulin, fraction V human serum albumin, rabbit albumin, pumpkin seed globulin, diphtheria toxoid, tetanus toxoid, botilinus toxin, succinylated proteins, and synthetic poly(aminoacids) such as polylysine.
Immunogenic carriers can also include poly amino-polysaccharides, which are a high molecular weight polymers built up by repeated condensations of monosaccharides. Examples of polysaccharides are starches, glycogen, cellulose, carbohydrate gums such as gum arabic, agar, and so forth. The polysaccharide also contains poly(amino acid) residues and/or lipid residues.
The immunogenic carrier can also be a poly(nucleic acid) either alone or conjugated to one of the above mentioned poly(amino acids) or polysaccharides.
The immunogenic carrier can also include solid particles. The particles are generally at least about 0.02 microns (μm) and not more than about 100 μm, and usually about 0.05 μm to 10 μm in diameter. The particle can be organic or inorganic, swellable or non-swellable, porous or non-porous, optimally of a density approximating water, generally from about 0.7 to 1.5 g/mL, and composed of material that can be transparent, partially transparent, or opaque. The particles can be biological materials such as cells and microorganisms, including non-limiting examples such as erythrocytes, leukocytes, lymphocytes, hybridomas, Streptococcus, Staphylococcus aureus, E. coli, and viruses. The particles can also be comprised of organic and inorganic polymers, liposomes, latex, phospholipid vesicles, or lipoproteins.
The term “derivative” refers to a chemical compound or molecule made from a parent compound by one or more chemical reactions.
The term “analogue” of a chemical compound refers to a chemical compound that contains a chain of carbon atoms and the same particular functional groups as a reference compound, but the carbon chain of the analogue is longer or shorter than that of the reference compound.
A “label,” “detector molecule,” or “reporter” is any molecule which produces, or can be induced to produce, a detectable signal. The label can be conjugated to an analyte, immunogen, antibody, or to another molecule such as a receptor or a molecule that can bind to a receptor such as a ligand, particularly a hapten. Non-limiting examples of labels include radioactive isotopes (e.g., 125I), enzymes (e.g., β-galactosidase, peroxidase), enzyme fragments, enzyme substrates, enzyme inhibitors, coenzymes, catalysts, fluorophores (e.g., rhodamine, fluorescein isothiocyanate or FITC, or Dylight 649), dyes, chemiluminescers and luminescers (e.g., dioxetanes, luciferin), or sensitizers.
As used herein, a “spacer” refers to a portion of a chemical structure which connects two or more substructures such as haptens, carriers, immunogens, labels or binding partners through a functional linking group. These spacer groups are composed of the atoms typically present and assembled in ways typically found in organic compounds and so may be referred to as “organic spacing groups”. The chemical building blocks used to assemble the spacers will be described hereinafter in this application. Among the preferred spacers are straight or branched, saturated or unsaturated carbon chains. These carbon chains may also include one or more heteroatoms within the chain, one or more heteroatoms replacing one or more hydrogens of any carbon atom in the chain, or at the termini of the chains. By “heteroatoms” is meant atoms other than carbon which are chosen from the group consisting of oxygen, nitrogen, phosphorous and sulfur, wherein the nitrogen, phosphorous and sulfur atoms may exist in any oxidation state and may have carbon or other heteroatoms bonded to them. The spacer may also include cyclic or aromatic groups as part of the chain or as a substitution on one of the atoms in the chain.
The number of atoms in the spacing group is determined by counting the atoms other than hydrogen. The number of atoms in a chain within a spacing group is determined by counting the number of atoms other than hydrogen along the shortest route between the substructures being connected. Preferred chain lengths are between 1 to 20 atoms.
A “functional linking group” refers to a reactive group that is present on a hapten and may be used to provide an available reactive site through which the hapten portion may be coupled to another moiety through formation of a covalent chemical bond to produce a conjugate of a hapten with another moiety (such as a label or carrier). The hapten may be linked in this way to a moiety such as biotin to form a competitive binding partner for the hapten.
Spacer groups may be used to link the hapten to the carrier. Spacers of different lengths allow one to attach the hapten with differing distances from the carrier for presentation to the immune system of the animal or human being immunized for optimization of the antibody formation process. Attachment to different positions in the hapten molecule allows the opportunity to present specific sites on the hapten to the immune system to influence antibody recognition. The spacer may contain hydrophilic solubilizing groups to make the hapten derivative more soluble in aqueous media. Examples of hydrophilic solubilizing groups include but are not limited to polyoxyalkyloxy groups, for example, polyethylene glycol chains, hydroxyl, carboxylate and sulfonate groups.
The term “nucleophilic group” or “nucleophile” refers to a species that donates an electron-pair to form a chemical bond in a reaction. The term “electrophilic group” or “electrophile” refers to a species that accepts an electron-pair from a nucleophile to form a chemical bond in a reaction.
The term “substituted” refers to substitution of an atom or group of atoms in place of a hydrogen atom on a carbon atom in any position on the parent molecule. Non limiting examples of substituents include halogen atoms, amino, hydroxy, carboxy, alkyl, aryl, heteroalkyl, heteroaryl, cyano, alkoxy, nitro, aldehyde and ketone groups.
The term “alkyl” refers to saturated or unsaturated linear and branched chain radicals of up to 12 carbon atoms, unless otherwise indicated, and is specifically intended to include radicals having any degree or level of saturation. Alkyl includes, but is not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, isohexyl, heptyl, octyl, 2,2,4-trimethylpentyl, nonyl, decyl, undecyl and dodecyl.
The term “cycloalkyl” refers to a saturated or partially unsaturated monocyclic or bicyclic hydrocarbon ring radical composed of from 3 to 10 carbon atoms. Alkyl substituents may optionally be present on the ring. Examples include cyclopropyl, 1,1-dimethyl cyclobutyl, 1,2,3-trimethylcyclopentyl, cyclohexyl and cyclohexenyl.
The term “heteroatom” refers to a nitrogen atom, an oxygen atom, a phosphorous atom or a sulfur atom wherein the nitrogen, phosphorous and sulfur atoms can exist in any allowed oxidation states.
The term “heteroalkyl” refers to an alkyl group that includes one or more heteroatoms within the chain, one or more heteroatoms replacing one or more hydrogens of any carbon atom in the chain, or at termini of the chains. The term “heterocyclyl” refers to a nonaromatic (i.e. saturated or partially unsaturated) ring composed of from 3 to 7 carbon atoms and at least one heteroatom selected from N, O or S. Alkyl substituents may optionally be present on the ring. Examples include tetrahydrofuryl, dihydropyranyl, piperidyl, 2,5-dimethypiperidyl, morpholinyl, piperazinyl, thiomorpholinyl, pyrrolidinyl, pyrrolinyl, pyrazolidinyl, pyrazolinyl, imidazolidinyl and imidazolinyl.
The term “hydroxyalkyl” refers to at least one hydroxyl group bonded to any carbon atom along an alkyl chain.
The term “aminoalkyl” refers to at least one primary or secondary amino group bonded to any carbon atom along an alkyl chain.
The term “alkoxyalkyl” refers to at least one alkoxy group bonded to any carbon atom along an alkyl chain.
The term “alkoxy” refers to straight or branched chain radicals of up to 12 carbon atoms, unless otherwise indicated, bonded to an oxygen atom. Examples include but are not limited to methoxy, ethoxy, propoxy, isopropoxy and butoxy.
The term “polyalkoxyalkyl” refers to long-chain alkoxy compounds and includes polyethylene glycols of discreet or monodispersed sizes.
The term “thioalkyl” refers to at least one sulfur group bonded to any carbon atom along an alkyl chain. The sulfur group may be at any oxidation state and includes sulfoxides, sulfones and sulfates.
The term “alkylcarbonyl” refers to a group that has a carbonyl group bonded to any carbon atom along an alkyl chain.
The term “carboxyalkyl” refers to at least one carboxylate group bonded to any carbon atom along an alkyl chain. The term “carboxylate group” includes carboxylic acids and alkyl, cycloalkyl, aryl or aralkyl carboxylate esters.
The term “heteroaryl” refers to 5- to 7-membered mono- or 8- to 10-membered bicyclic aromatic ring radicals, any ring of which may consist of from one to four heteroatoms selected from N, O or S where the nitrogen and sulfur atoms can exist in any allowed oxidation state. Examples include benzimidazolyl, benzothiazolyl, benzothienyl, benzoxazolyl, furyl, imidazolyl, isothiazolyl, isoxazolyl, oxazolyl, pyrazinyl, pyrazolyl, pyridyl, pyrimidinyl, pyrrolyl, quinolinyl, thiazolyl and thienyl.
The term “aryl” refers to monocyclic or bicyclic aromatic ring radicals containing from 6 to 12 carbons in the ring. Alkyl substituents may optionally be present on the ring. Examples include phenyl, biphenyl and napththalene.
The term “aralkyl” refers to a C1-6 alkyl group containing an aryl substituent. Examples include benzyl, phenylethyl or 2-naphthylmethyl.
The term “acyl” refers to the group —C(O)Ra, where Ra is hydrogen, alkyl, cycloalkyl, heteroalkyl, aryl, aralkyl, and heteroaryl. An “acylating agent” adds the —C(O)Ra group to a molecule.
The term “sulfonyl” refers to the group —S(O)2Rb, where Rb is hydrogen, alkyl, cycloalkyl, heteroalkyl, haloalkyl, aryl, aralkyl, and heteroaryl. A “sulfonylating agent” adds the —S(O)2Ra group to a molecule.
Spacers bearing reactive functional linking groups for the attachment of haptens to carrier moieties may be prepared by a wide variety of methods. The spacer may be formed using a molecule that is differentially functionalized or activated with groups at either end to allow selective sequential reaction with the hapten and the carrier, but the same reactive moiety may also be used at both ends. The groups selected for reaction with the hapten and the functional linking group to be bound to the carrier are determined by the type of functionality on the hapten and the carrier that the hapten is to be bonded with. Spacers and methods of attachment to haptens and carriers include but are not limited to those described by Brinkley, M., A., Bioconjugate Chem. 1992, 3:2-13, Hermanson, Greg T., Bioconjugate Techniques, Academic Press, London, Amsterdam, Burlington, Mass., USA, 2008 and Thermo Scientific Pierce Crosslinking Technical Handbook; available for download or hard copy request from Thermo Scientific 3747 N Meridian Rd, Rockford, Ill. USA 61101, ph 800-874-3723 or at: http://www.piercenet.com/ and references within. Many differentially activated molecules for formation of spacer groups are commercially available from vendors, for example Thermo Scientific.
For haptens bearing an amino group, modes of attachment of the spacer to the hapten include reaction of the amine on the hapten with a spacer building block bearing an acyl halide or active ester. “Active esters” are defined as esters that undergo reaction with a nucleophilic group, for example an amino group, under mild conditions to form a stable linkage. A stable linkage is defined as one that remains intact under conditions of further use, for example subsequent synthetic steps, use as an immunogen, or in a biochemical assay. A preferred example of a stable linkage is an amide bond. Active esters and methods of formation are described by Benoiton, N. L., in Houben-Weyl, Methods of Organic Chemistry, Thieme Stuttgart, New York, vol E22 section 3.2:443 and Benoiton, N. L., Chemistry of Peptide Synthesis, Taylor and Francis, N.Y., 2006. Preferred active esters include p-nitrophenyl ester (PNP), N-hydroxysuccinimide ester (NHS) and tetrafluorophenyl ester (TFP). Acyl halides may be prepared by many methods known to one skilled in the art for example, reaction of the carboxylic acid with thionyl chloride or oxalyl chloride, see: Fieser, L. F. and Fieser, M. Reagents for Organic Synthesis, John Wiley and Sons, NY, 1967 and references within. These may be converted to other active esters such as p-nitrophenyl esters (PNP) which may also be used in active bi-functional spacers as described by Wu et. al, Organic Letters, 2004, 6 (24):4407. N-hydroxysuccinimide (NHS) esters may be prepared by reaction of N,N-disuccinimidyl carbonate (CAS 74124-79-1) with the carboxylic acid of a compound in the presence of an organic base such as triethylamine or diisopropylethylamine in an aprotic solvent under anhydrous conditions as described in example 35 of WO2012012595 or by using N-hydroxysuccinimide and dicyclohexylcarbodiimide (DCC) or other dehydrating agent, under anhydrous conditions. Tetrafluorophenyl esters (TFP) may be prepared by reaction of carboxylic acids with 2,3,5,6-tetrafluorophenyltrifluoroacetate in the presence of an organic base such as triethylamine or diisopropylethylamine in an aprotic solvent under anhydrous conditions as reported by Wilbur, et. al, Bioconjugate Chem., 2004, 15(1):203. One skilled in the art will recognize that spacers shown in Table 1, among others, can be obtained using known methods and attached to amino-bearing haptens utilizing routine optimization of reaction conditions. These spacers allow attachment of the hapten to a thiol group on a carrier.







TABLE 1













 








 








 








 








 








 








 








 








 








 








 


Reasonable values for m and n 


are between 1 and 10









Direct coupling of the amine on the hapten and a carboxylic acid functionality on the spacer building block in the presence of a coupling agent may also be used as a mode of attachment. Preferred reagents are those typically used in peptide synthesis. Peptide coupling reagents include but are not limited to O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TBTU, CAS #125700-67-6), see: Pruhs, S., Org. Process. Res. Dev. 2006, 10:441; N-Hydroxybenzotriazole (HOBT, CAS #2592-95-2) with a carbodiimide dehydrating agent, for example N—N-dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), or 1-ethyl-3(3-dimethylaminopropyl)carbodiimidehydrochloride (EDC), see: König W., Geiger, R. Chem. Ber., 1970, 103 (3):788; 3-(diethoxyphosphoryloxy)-1,2,3-benzotrazin-4(3H)-one (DEPBT, CAS#165534-43-0), see: Liu, H. et. al., Chinese Chemical Letters, 2002, 13(7):601; Bis(2-oxo-3-oxazolidinyl)phosphonic chloride; BOP—Cl (CAS 68641-49-6), see: Diago-Meseguer, J et. al. Synthesis, 1980, 7:547-51 and others described in detail by Benoiton in Chemistry of Peptide Synthesis, CRC Press, Boca Raton, Fla., 2005, Chapter 2, and the technical bulletin provided by Advanced Automated Peptide Protein Technologies (aapptec), 6309 Shepardsville Rd., Louisville Ky. 40228, ph 888 692 9111; www.aapptec.com, and references within. These methods create a stable amide linkage attaching the hapten to the spacer. Examples of spacers that can be obtained using known methods and attached to amino-bearing haptens utilizing routine optimization of reaction conditions employing the methods described and cited above are shown, but not limited to those in Table 2. These spacers allow attachment of the hapten to a thiol group on a carrier.







TABLE 2










   reasonable range for n is between 1-10 


 








 















Spacers may also be constructed in a step-wise fashion by sequential attachment of appropriate chemical groups to the hapten including the step of forming the functional linking group that is capable of binding to the carrier. See illustrative examples under General Reaction Schemes.
Additionally, when the hapten has a nucleophilic group, for example a thiol group, an amino group or a hydroxyl group which will become the point of attachment of the spacer, the spacer may also be constructed by alkylation of the thiol, amine or hydroxyl group. Any alkyl group that is appropriately substituted with a moiety capable of undergoing a substitution reaction, for example, an alkyl halide, or sulfonic acid ester such as p-Toluenesulfonate, may be used to attach the spacer. Many examples of alkylation reactions are known to one skilled in the art and specific examples may be found in the general chemical literature and optimized through routine experimentation. A discussion of alkylation reactions with many references can be found in Chapter 10 of March's Advanced Organic Chemistry, Smith, M. B., and March, J., John Wiley & sons, Inc. NY, 2001. Other linkages may also be employed such as reaction of the nucleophilic moiety, for example an amine, on the hapten with an isocyanate to form a urea or reaction with an isothiocyanate to form a thiourea linkage, see: Li, Z., et. al., Phosphorus, Sulfur and Silicon and the Related Elements, 2003, 178(2):293-297. Spacers may be attached to haptens bearing hydroxyl groups via reaction with isocyanate groups to form carbamate or urethane linkages. The spacer may be differentially activated with the isocyanate functional group on one end and a functional linking group capable of reacting with the carrier, see: Annunziato, M. E., Patel, U.S., Ranade, M. and Palumbo, P. S., Bioconjugate Chem., 1993, 4:212-218.
For haptens bearing a carboxylic acid group, modes of attachment of a spacer portion to the hapten include activation of the carboxylic acid group as an acyl halide or active ester, examples of which are shown in Table 3, preparation of which are described previously, followed by reaction with an amino (—NH2—), hydrazino (—NH—NH2—), hydrazido (—C(O)—NH—NH2—) or hydroxyl group (—OH) on the spacer portion to form an amide, hydrazide, diacylhydrazine or ester linkage, or direct coupling of the carboxylic acid group with an amino group on the spacer portion or directly on the carrier with a peptide coupling reagent and/or carbodiimide dehydrating reagent, described previously, examples of which are shown in Tables 4 and 5. Procedures found in references cited previously for formation of activated esters and use of peptide coupling agents may be employed for attachment of carboxylic acid-bearing haptens to spacer building blocks and protein carriers with available amino groups utilizing routine optimization of reaction conditions.







TABLE 3










   


   Sulfo NHS and NHS 


 





   TFP 


 





   X = Cl, Br Acyl chloride 


 





   PNP
















TABLE 4










 HOBT 


 





 DEPT 


 





 BOP-Cl 


 





 TBTU
















TABLE 5










 diisopropylcarbodiimide (DIC) 


 





 Dicyclohexylcarbodiimide (DCC) 


 





 1-ethyl- 3(3-dimethylaminopropyl) carbodiimide•HCl (EDC)









Other electrophilic groups may be present on the hapten to attach the spacer, for example, a sulfonyl halide




or electrophilic phosphorous group, for example:





See: Malachowski, William P., Coward, James K., Journal of Organic Chemistry, 1994, 59 (25):7616
or:




Rc is alkyl, cycloalkyl, aryl, substituted aryl, aralkyl.
See: Aliouane, L., et. al, Tetrahedron Letters, 2011, 52(28):8681.
Haptens that bear aldehyde or ketone groups may be attached to spacers using methods including but not limited to reaction with a hydrazide group H2N—NH—C(O)— on the spacer to form an acylhydrazone, see: Chamow, S. M., Kogan, T. P., Peers, D. H., Hastings, R. C., Byrn, R. A. and Askenaszi, A., J. Biol. Chem., 1992, 267(22): 15916. Examples of bifunctional hydrazide spacer groups that allow attachment to a thiol group on the carrier are shown in Table 6.







TABLE 6













 















Haptens may also contain thiol groups which may be reacted with the carrier provided that the carrier has been modified to provide a group that may react with the thiol. Carrier groups may be modified by methods including but not limited to attachment of a group containing a maleimide functional group by reaction of an amino group on the carrier with N-Succinimidyl maleimidoacetate, (AMAS, CAS#55750-61-3), Succinimidyl iodoacetate (CAS#151199-81-4), or any of the bifunctional spacer groups shown in Table 1 to introduce a group which may undergo a reaction resulting in attachment of the hapten to the carrier.
The functional linking group capable of forming a bond with the carrier may be any group capable of forming a stable linkage and may be reactive to a number of different groups on the carrier. The functional linking group may preferably react with an amino group, a carboxylic acid group or a thiol group on the carrier, or derivative thereof. Non-limiting examples of the functional linking group are a carboxylic acid group, acyl halide, active ester (as defined previously), isocyanate, isothiocyanate, alkyl halide, amino group, thiol group, maleimide group, acrylate group (H2C═CH—C(O)—) or vinyl sulfone group H2C═CH—SO2—) See: Park, J. W., et. al., Bioconjugate Chem., 2012, 23(3): 350. The functional linking group may be present as part of a differentially activated spacer building block that may be reacted stepwise with the hapten and the resulting hapten derivative may then be reacted with the carrier. Alternatively, the hapten may be derivatized with a spacer that bears a precursor group that may be transformed into the functional linking group by a subsequent reaction. When the functional linking group on the spacer is an amine or a carboxylic acid group, the coupling reaction with the carboxylic acid group or amine on the carrier may be carried out directly through the use of peptide coupling reagents according to procedures in the references cited above for these reagents.
Particular disulfide groups, for example, pyridyldisulfides, may be used as the functional linking group on the spacer which may undergo exchange with a thiol group on the carrier to from a mixed disulfide linkage, see: Ghetie, V., et al., Bioconjugate Chem., 1990, 1:24-31. These spacers may be attached by reaction of the amine-bearing hapten with an active ester which is attached to a spacer bearing the pyridyldisulfide group, examples of which include but are not limited to those shown in Table 7







TABLE 7













 








 








 















Most often the carrier is a protein and the ε-amino groups of the lysine residues may be used for attachment, either directly by reaction with an amine-reactive functional linking group or after derivitization with a thiol-containing group, including N-Succinimidyl S-Acetylthioacetate, (SATA, CAS 76931-93-6), or an analogue thereof, followed by cleavage of the acetate group with hydroxylamine to expose the thiol group for reaction with the functional linking group on the hapten. Thiol groups may also be introduced into the carrier by reduction of disulfide bonds within protein carriers with mild reducing reagents including but not limited to 2-mercaptoethylamine, see: Bilah, M., et. al., Bioelectrochemistry, 2010, 80(1):49, phosphine reagents, see: Kirley, T. L., Analytical Biochemistry, 1989, 180(2):231 or dithioerythritol (DTT, CAS 3483-12-3) Cleland, W., Biochemistry, 1964, 3:480-482.
General Reaction Schemes
Representative compounds of the present invention can be synthesized in accordance with the general synthetic methods described below. Compounds of Formula (I) can be prepared by methods known to those who are skilled in the art. The following reaction schemes are only meant to represent examples of the invention and are in no way meant to be a limit of the invention.




Compounds of Formula I where R2 is CH2NHC(O)(CH2)mCO2H may be made according to Scheme 1. Reaction of (1-methyl-4-(2-methyl-10H-benzo[b]thieno[2,3-e][1,4]diazepin-4-yl)piperazin-2-yl)methanamine, prepared as described in Example 1, Step I, proceeds with a cyclic anhydride compound, such as succinic anhydride or glutaric anhydride, in a solvent such as pyridine, at temperatures ranging from room temperature to 60° C., for about 48 hours. Those skilled in the art will recognize that the same chemistry may be used to create compounds of Formula I where R1 is CH2NHC(O)(CH2)mCO2H.




Compounds of Formula I where R2 is




may be made according to Scheme 2. Compounds of Formula I, where R2 is CH2NHC(O)(CH2)mCO2H, prepared as described in Scheme 1, are treated with N-t-butoxycarbonylpiperazine, diethyl cyanophosphonate, and a base, such as diisopropylethylamine. The reaction is carried out in a solvent, such as dichloromethane, for about 2 hours at room temperature. Deprotection of the piperazinyl group is accomplished with trifluoroacetic anhydride as described in Scheme 2, followed by reaction with an appropriate anhydride, such as succinic anhydride or maleic anhydride, in the presence of a suitable base such as diisopropylethylamine. Those skilled in the art will recognize that the same chemistry may be used to create compounds of Formula I

where R1 is








Compounds of Formula I where R1 is




may be made according to Scheme 3. The maleimide may be introduced by any method known in the art. Maleimide functionalizing groups such as 2,5-dioxopyrrolidin-1-yl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetate where m is 1, may be used in a solvent such as DMF or CH2Cl2, and a base, such as tributylamine or triethylamine. Alternatively, the deprotected piperazinyl group described in Scheme 2 may be elaborated with a maleimide functionality, as described in Scheme 3 to give compounds of Formula I

where R1 is




Those skilled in the art will recognize that the same chemistry may be used to create compounds of Formula I where R2 is





Compounds in which the spacer and linking group are attached to the unsubstituted secondary nitrogen in the diazepine ring of olanzapine may be obtained by the reactions depicted in schemes 4 to 8. Acylation of the nitrogen is described by Su, J. et. al, Bioorganic and Med. Chem. Letters, 2006, 16:4548. Use of the mono ester mono acid chloride of succinic acid in the presence of a base, under anhydrous conditions in an aprotic solvent, provides an intermediate, the ester functionality of which may be hydrolyzed using standard conditions known to one skilled in the art, for example, aqueous base, to provide a hapten that may be further elaborated into an immunogen by methods previously described herein and illustrated by examples of this disclosure.




Su, et. al., above, also report preparation of sulfonamides. Through use of a functionalized sulfonylchloride in the presence of a base, under anhydrous conditions in an aprotic solvent, as shown in Scheme 5, a carboxy hapten may be prepared and transformed into an immunogen by methods previously described herein and illustrated by examples of this disclosure.




Su, et. al., above, also teach methods for preparation of a hydrazine as shown in Scheme 6, through diazotization of the ring nitrogen with a nitrite ester followed by reduction with zinc in acetic acid. The resulting hydrazine may be further functionalized in a number of ways as shown in Scheme 7. Reaction with a bifunctional spacer building block, for example AMAS, in the presence of an amine base, for example, tribuytlamine, in a solvent such as DMF as described elsewhere herein, may provide a maleimide hapten that may be attached to a carrier through reaction with a thiol group. Sulfonylation in the presence of base with a functionalized sulfonyl chloride, for example, m-carboxybenzenesulfonylchloride may provide a sulfonylhydrazide that bears a carboxy group for attachment to a carrier by methods previously described herein and illustrated by examples of this disclosure. Additionally, the hydrazine may be reacted with a functionalized aldehyde or ketone, for example, levulinic acid, as described in U.S. Pat. No. 4,022,780, with a catalytic amount of acid under conditions where water generated by the condensation is removed, to provide a hydrazone as shown in scheme 7. The hydrazone may be subsequently reduced using sodium cyanoborohydride in the method of Su, J. et al., previously referenced, to provide a saturated derivative.








Direct alkylation of the ring nitrogen as shown in Scheme 8, may also be accomplished using the method described in U.S. Pat. No. 6,034,078 to append an alkyl group directly to olanzapine. Though use of a functionalized alkyl halide, for example, 4-chloromethylbutyrate, one may obtain an intermediate which, through hydrolysis using standard conditions known to one skilled in the art, may provide a hapten that may be further elaborated into an immunogen by methods previously described herein and illustrated by examples of this disclosure.








Maleimide functionalized haptens wherein R1 or R2 is




may be conjugated to proteins according to the method shown in Scheme 9. Activation of protein lysine residues by acylation of the epsilon-nitrogen with N-succinimidyl S-acetylthioacetate (SATA), followed by subsequent hydrolysis of the S-acetyl group with hydroxylamine produces a nucleophilic sulfhydryl group. Conjugation of the sulfhydryl activated protein with the maleimide derivatized hapten (prepared as described in general scheme 3) proceeds via a Michael addition reaction. Suitable proteins are known to those skilled in the art and include keyhole limpet hemocyanin, bovine thyroglobulin, and ovalbumin. The same methodology may be used to conjugate proteins to maleimide functionalized haptens where R1 or R2 is









Carboxylic acid functionalized haptens, wherein R1 or R2 is CH2NHC(O)(CH2)mCO2H, may be conjugated to proteins according to the method shown in Scheme 10. Reaction with N-hydroxysuccinimide and a suitable coupling agent, such as dicyclohexylcarbodiimide, and a base, such as tributyl amine, in a solvent such as DMF, at a temperature of about 20° C., for about 18 hrs activates the carboxylic acid with the hydroxypyrrolidine-2,5-dione leaving group. The activated linker and hapten may then be conjugated to a protein in a solvent, such as a pH 7.5 phosphate buffer, at about 20° C., for about 2.5 hours. Suitable proteins are known to those skilled in the art and include keyhole limpet hemocyanin, bovine thyroglobulin, and ovalbumin. The same methodology may be used to conjugate proteins to carboxylic acid functionalized haptens where R1 or R2 is




Antibody Production

The conjugates above are useful for the production of antibodies which bind the anti-psychotic drug to which they were generated (olanzapine). These antibodies can be used in assays to detect the presence and/or amount of the anti-psychotic drug in patient samples. Such detection permits therapeutic drug monitoring enabling all of the benefits thereof. Detection of levels of anti-psychotic drugs may be useful for many purposes, including: detection in combination with the detection of other anti-psychotic drugs, including those selected from the group consisting of risperidone, paliperidone, quetiapine, aripiprazole, and metabolites thereof, such detection permitting the simultaneous measurement of these anti-psychotic drugs; determination of patient adherence or compliance with prescribed therapy; use as a decision tool to determine whether a patient should be converted from an oral anti-psychotic regimen to a long-acting injectable anti-psychotic regimen; use as a decision tool to determine if the dose level or dosing interval of oral or injectable anti-psychotics should be increased or decreased to ensure attainment or maintenance of efficacious or safe drug levels; use as an aid in the initiation of anti-psychotic drug therapy by providing evidence of the attainment of minimum pK levels; use to determine bioequivalence of anti-psychotic drug in multiple formulations or from multiple sources; use to assess the impact of polypharmacy and potential drug-drug interactions; and use as an indication that a patient should be excluded from or included in a clinical trial and as an aid in the subsequent monitoring of adherence to clinical trial medication requirements.
Having provided the conjugates of the subject invention, which comprise the compounds herein and an immunogenic carrier, antibodies can be generated, e.g., polyclonal, monoclonal, chimeric, and humanized antibodies, that bind to the anti-psychotic drug. Such antibodies that are particularly contemplated include monoclonal and polyclonal antibodies as well as fragments thereof, e.g., recombinant proteins, containing the antigen-binding domain and/or one or more complementarity determining regions of these antibodies. Preferably, the antibody will bind to the drug and any desired pharmacologically active metabolites. By altering the location of the attachment of an immunogenic carrier in a drug conjugate, selectivity and cross-reactivity with metabolites and/or related drugs can be engineered into the antibodies. For olanzapine, cross-reactivity with the related drug clozapine may or may not be desirable, and cross reactivity with olanzapine metabolites such as 10-N-gluronide or 4-N-desmethyl olanzapine may or may not be desirable. Antibodies may be generated that detect multiple ones of these drugs and/or metabolites, or antibodies may be generated that detect each separately (thus defining the antibody “specific binding” properties). An antibody specifically binds one or more compounds when its binding of the one or more compounds is equimolar or substantially equimolar.
Methods of producing such antibodies comprise inoculating a host with the conjugate (the compound and the immunogenic carrier being an immunogen) embodying features of the present invention. Suitable hosts include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, chickens, donkeys, horses, monkeys, chimpanzees, orangutans, gorillas, humans, and any species capable of mounting a mature immune response. The immunization procedures are well established in the art and are set forth in numerous treatises and publications including “The Immunoassay Handbook”, 2nd Edition, edited by David Wild (Nature Publishing Group, 2000) and the references cited therein.
Preferably, an immunogen embodying features of the present invention is administered to a host subject, e.g., an animal or human, in combination with an adjuvant. Suitable adjuvants include, but are not limited to, Freund's adjuvant, powdered aluminum hydroxide (alum), aluminum hydroxide together with Bordetella pertussis, and monophosphoryl lipid A-synthetic trehalose dicorynomycolate (MPL-TDM).
Polyclonal antibodies can be raised in a mammalian host by one or more injections of an immunogen which can optionally be administered together with an adjuvant. Typically, an immunogen or a combination of an immunogen and an adjuvant is injected into a mammalian host by one or multiple subcutaneous or intraperitoneal injections. Preferably, the immunization program is carried out over at least one week, and more preferably, over two or more weeks. Polyclonal antibodies produced in this manner can be isolated and purified utilizing methods well know in the art.
Monoclonal antibodies can be produced by the well-established hybridoma methods of Kohler and Milstein, e.g., Nature 256:495-497 (1975). Hybridoma methods typically involve immunizing a host or lymphocytes from a host, harvesting the monoclonal antibody secreting or having the potential to secrete lymphocytes, fusing the lymphocytes to immortalized cells, and selecting cells that secrete the desired monoclonal antibody.
A host can be immunized to elicit lymphocytes that produce or are capable of producing antibodies specific for an immunogen. Alternatively, the lymphocytes can be immunized in vitro. If human cells are desired, peripheral blood lymphocytes can be used, although spleen cells or lymphocytes from other mammalian sources are preferred.
The lymphocytes can be fused with an immortalized cell line to form hybridoma cells, a process which can be facilitated by the use of a fusing agent, e.g., polyethylene glycol. By way of illustration, mutant rodent, bovine, or human myeloma cells immortalized by transformation can be used. Substantially pure populations of hybridoma cells, as opposed to unfused immortalized cells, are preferred. Thus, following fusion, the cells can be grown in a suitable medium that inhibits the growth or survival of unfused, immortalized cells, for example, by using mutant myeloma cells that lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT). In such an instance, hypoxanthine, aminopterin, and thymidine can be added to the medium (HAT medium) to prevent the growth of HGPRT-deficient cells while permitting hybridomas to grow.
Preferably, immortalized cells fuse efficiently, can be isolated from mixed populations by selection in a medium such as HAT, and support stable and high-level expression of antibody following fusion. Preferred immortalized cell lines include myeloma cell lines available from the American Type Culture Collection, Manassas, Va.
Because hybridoma cells typically secrete antibody extracellularly, the culture media can be assayed for the presence of monoclonal antibodies specific for the anti-psychotic drug. Immunoprecipitation of in vitro binding assays, for example, radiioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA), can be used to measure the binding specificity of monoclonal antibodies.
Monoclonal antibody-secreting hybridoma cells can be isolated as single clones by limiting dilution procedures and sub-cultured. Suitable culture media include, but are not limited to, Dulbecco's Modified Eagle's Medium, RPMI-1640, and polypeptide-free, polypeptide-reduced, or serum-free media, e.g., Ultra DOMA PF or HL-1, available from Biowhittaker, Walkersville, Md. Alternatively, the hybridoma cells can be grown in vivo as ascites.
Monoclonal antibodies can be isolated and/or purified from a culture medium or ascites fluid by conventional immunoglobulin (Ig) purification procedures including, but not limited to, polypeptide A-SEPHAROSE, hydroxylapatite chromatography, gel electrophoresis, dialysis, ammonium sulfate precipitation, and affinity chromatography.
Monoclonal antibodies can also be produced by recombinant methods such as are described in U.S. Pat. No. 4,166,452. DNA encoding monoclonal antibodies can be isolated and sequenced using conventional procedures, e.g., using oligonucleotide probes that specifically bind to murine heavy and light antibody chain genes, preferably to probe DNA isolated from monoclonal antibody hybridoma cells lines secreting antibodies specific for anti-psychotic drugs.
Immunoassays
The antibodies thus produced can be used in immunoassays to recognize/bind to the anti-psychotic drug, thereby detecting the presence and/or amount of the drug in a patient sample. Preferably, the assay format is a competitive immunoassay format. Such an assay format and other assays are described, among other places, in Hampton et al. (Serological Methods, A Laboratory Manual, APS Press, St. Paul, Minn. 1990) and Maddox et al. (J. Exp. Med. 158:12111, 1983).
A reagent kit can also be provided comprising an antibody as described above. A representative reagent kit may comprise an antibody that binds to the anti-psychotic drug, olanzapine, a complex comprising an analog of an anti-psychotic drug or a derivative thereof coupled to a labeling moiety, and may optionally also comprise one or more calibrators comprising a known amount of an anti-psychotic drug or a related standard.
As noted above, reagent kits may comprise calibrators and/or control materials which comprise a known amount of the analyte to be measured. The concentration of the analyte can be calculated by comparing results obtained for a sample with resulted obtained for a standard. A calibration curve can be constructed and used for relating the sets of results and for determining the concentration of an analyte in a sample.
Any sample that is suspected of containing an analyte, e.g., an anti-psychotic drug, can be analyzed in accordance with the methods of the presently preferred embodiments. The sample can be pretreated if desired and can be prepared in any convenient medium that does not interfere with the assay. Preferably, the sample comprises an aqueous medium such as a body fluid from a host, most preferably plasma or serum.
Copending applications entitled “Haptens of Aripiprazole” (U.S. Provisional Patent Appl. No. 61/691,450, filed Aug. 21, 2012, and US 20140163206, filed Aug. 20, 2013), “Haptens of Olanzapine” (U.S. Provisional Patent Appl. No. 61/691,454, filed Aug. 21, 2012), “Haptens of Paliperidone” (U.S. Provisional Patent Appl. No. 61/691,459, filed Aug. 21, 2012, and US 20140213767, filed Aug. 20, 2013), “Haptens of Quetiapine” (U.S. Provisional Patent Appl. No. 61/691,462, filed Aug. 21, 2012, and US 20140221616, filed Aug. 20, 2013), “Haptens of Risperidone and Paliperidone” (U.S. Provisional Patent Appl. No. 61/691,469, filed Aug. 21, 2012, and US 20140155585, Aug. 20, 2013), “Antibodies to Aripiprazole Haptens and Use Thereof” (U.S. Provisional Patent Appl. No. 61/691,544, filed Aug. 21, 2012, and US 20140057299, filed Aug. 20, 2013), “Antibodies to Olanzapine Haptens and Use Thereof” (U.S. Provisional Patent Appl. No. 61/691,572, filed Aug. 21, 2012, US 20140057303, filed Aug. 20, 2013), “Antibodies to Paliperidone Haptens and Use Thereof” (U.S. Provisional Patent Appl. No. 61/691,634, filed Aug. 21, 2012, and US 20140057297, filed Aug. 20, 2013), “Antibodies to Quetiapine Haptens and Use Thereof” (U.S. Provisional Patent Appl. No. 61/691,598, filed Aug. 21, 2012, and US 20140057305, filed Aug. 20, 2013), “Antibodies to Risperidone Haptens and Use Thereof” (U.S. Provisional Patent Appl. No. 61/691,615, filed Aug. 21, 2012, and US 20140057301, filed Aug. 20, 2013), “Antibodies to Aripiprazole and Use Thereof” (U.S. Provisional Patent Appl. No. 61/691,522, filed Aug. 21, 2012, and US 20140057300, filed Aug. 20, 2013), “Antibodies to Olanzapine and Use Thereof” (U.S. Provisional Patent Appl. No. 61/691,645, filed Aug. 21, 2012, and US 20140057304, filed Aug. 20, 2013), “Antibodies to Paliperidone and Use Thereof” (U.S. Provisional Patent Appl. No. 61/691,692, filed Aug. 21, 2012, and US 20140057298, filed Aug. 20, 2013), “Antibodies to Quetiapine and Use Thereof” (U.S. Provisional Patent Application No. 61/691,659, filed Aug. 21, 2012, and US 20140057306, filed Aug. 20, 2013), “Antibodies to Risperidone and Use Thereof” (U.S. Provisional Patent Appl. No. 61/691,659, filed Aug. 21, 2012, and US 20140057306, filed Aug. 20, 2013), are all incorporated herein by reference in their entireties.
EXAMPLES
Representative compounds of the present invention can be synthesized in accordance with the general synthetic methods described below. Compounds of Formula (I) can be prepared by methods known to those who are skilled in the art. The following examples are only meant to represent examples of the invention and are in no way meant to be a limit of the invention.
Example 1
(1-Methyl-4-(2-methyl-10H-benzo[b]thieno[2,3-e][1,4]diazepin-4-yl)piperazin-2-yl)methanamine




Step A
tert-Butyl 3-cyanopiperazine-1-carboxylate




To a solution of tert-butyl 3-cyanopiperazine-1-carboxylate (21.1 g, 0.1 mol) and aqueous formaldehyde (24 g, 37% in water) in THF was added sodium cyanoborohydride (31.5 g, 0.5 mol) in small portions. The reaction mixture was aged at ambient temperature overnight then diluted with water and extracted with ethyl acetate. The organic phase was washed with saturated aqueous sodium chloride, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The crude product was purified by column chromatography to provide the title compound. 1H NMR (400 MHz, MeOD) δ 4.23-4.18 (m, 1H), 4.01-3.97 (br, 1H), 3.92-3.90 (br, 1H), 2.92-2.89 (br, 1H), 2.88-2.87 (br, 1H), 2.65-2.62 (m, 1H), 2.378 (s, 3H), 2.36-2.33 (m, 1H), 1.47 (s, 9H).
Step B
tert-Butyl 3-(aminomethyl)-4-methylpiperazine-1-carboxylate




To a solution of tert-butyl 3-cyano-4-methylpiperazine-1-carboxylate, prepared as described in Step A, (10.5 g, 47 mmol) in methanol (200 mL) was added metallic nickel (10 g) and triethylamine (5 mL). The mixture was stirred at ambient temperature overnight under atmosphere of hydrogen gas (50 psi). Upon consumption of tert-butyl 3-cyano-4-methylpiperazine-1-carboxylate, the mixture was filtered, and the filtrate was concentrated under vacuum to provide crude tert-butyl 3-(aminomethyl)-4-methylpiperazine-1-carboxylate used in the next step without purification.
Step C
tert-Butyl 3-((1,3-dioxoisoindolin-2-yl)methyl)-4-methylpiperazine-1-carboxylate




To a mixture of tert-butyl 3-(aminomethyl)-4-methylpiperazine-1-carboxylate, prepared as described in the previous step, (5.5 g, crude) and sodium bicarbonate (2.52 g, 30 mmol) in tetrahydrofuran (100 mL) was added a solution of 2H-isoindole-2-carboxylic acid, 1,3-dihydro-1,3-dioxo-, ethyl ester (6.59 g, 30 mmol) in tetrahydrofuran (20 mL) at ambient temperature. After stirring for 30 minutes, the suspension was filtered, and the filtrate was concentrated to give crude product which was purified by column chromatography to provide the title compound. 1H NMR (400 MHz, MeOD) δ 7.87-7.85 (m, 2H), 7.87-7.80 (m, 2H), 3.94-3.90 (m, 1H), 3.75-3.65 (br, 3H), 3.43-3.41 (br, 1H), 3.30-3.28 (m, 2H), 3.49 (s, 3H), 2.39-2.38 (m, 1H), 2.30-2.28 (m, 1H), 1.36 (s, 9H).
Step D
2-((1-Methylpiperazin-2-yl)methyl)isoindoline-1,3-dione




A solution of tert-butyl 3-1,3-dioxoisoindolin-2-yl)methyl)-4-methylpiperazine-1-carboxylate, prepared as described in the previous step, (8.6 g) in methanolic hydrogen chloride (20 mL) was stirred at room temperature for 1 hour. The solvent was removed under vacuum to provide 2-((1-methylpiperazin-2-yl)methyl)isoindoline-1,3-dione which was used in the next step without further purification. 1H NMR (400 MHz, MeOD) δ 7.88-7.86 (m, 2H), 7.82-7.80 (m, 2H), 3.99-3.95 (m, 1H), 3.77-3.73 (m, 1H), 3.24-3.23 (m, 1H), 3.29-3.23 (m, 1H), 3.17-3.14 (m, 1H), 3.04-2.84 (m, 2H), 2.81-2.78 (m, 1H), 2.55 (s, 3H), 2.46-2.40 (m, 1H).
Step E
5-Methyl-2-((2-nitrophenyl)amino)thiophene-3-carbonitrile




To a solution of 2-amino-5-methylthiophene-3-carbonitrile (13.8 g, 100 mmol) and 1-fluoro-2-nitrobenzene (16.92 g, 120 mmol) in dimethylsulfoxide was added potassium hydroxide (11.2 g, 200 mmol). The reaction mixture was stirred at room temperature overnight. The mixture was diluted with water, and the resulting suspension was filtered. The filtered cake was dried to give 5-methyl-2-((2-nitrophenyl)amino)thiophene-3-carbonitrile as a red solid used without further purification. 1H NMR: (400 MHz, CDCl3) δ 9.69 (s, 1H), 8.27-8.25 (m, 1H), 7.56-7.52 (m, 1H), 7.23-7.20 (m, 1H), 7.0-6.96 (m, 1H), 6.80 (s, 1H), 2.49 (s, 3H).
Step F
2-((2-Aminophenyl)amino)-5-methylthiophene-3-carbonitrile




To a solution of 5-methyl-2-((2-nitrophenyl)amino)thiophene-3-carbonitrile, prepared as described in the previous step, (43.3 g, 0.157 mol) in ethyl acetate (500 mL) was added 10% palladium on carbon (8 g). The black mixture was stirred at room temperature overnight under an atmosphere of hydrogen gas. When LCMS showed that most of 5-methyl-2-((2-nitrophenyl)amino)thiophene-3-carbonitrile was consumed completely, the mixture was filtered and the filtrate was concentrated to provide 2-((2-aminophenyl)amino)-5-methylthiophene-3-carbonitrile. 1H NMR (400 MHz, CDCl3) δ 7.29-7.21 (m, 1H), 7.11-7.10 (m, 1H), 6.86-6.79 (m, 2H), 6.48-6.47 (m, 1H), 6.42 (brs, 1H), 3.75-3.70 (br, 2H), 2.28 (s, 3H).
Step G
2-Methyl-10H-benzo[b]thieno[2,3-e][1,4]diazepin-4-amine




A mixture of 2-((2-aminophenyl)amino)-5-methylthiophene-3-carbonitrile, prepared as described in the previous step, (22.9 g, 100 mmol) in isopropanol (150 mL) and aqueous hydrochloric acid (50 mL, 18%) was heated at 80° C. for 3 hrs. The resulting suspension was filtered and the filter cake was dried to give the title compound as a red solid. 1H NMR (400 MHz CDCl3) δ 7.14-7.12 (t, 1H), 7.7.12-7.10 (t, 1H), 6.95-6.93 (d, J=8 MHz, 1H), 6.81-6.79 (d, J=8 MHz, 1H), 6.70 (s, 1H), 2.30 (s, 3H).
Step H
2-((1-Methyl-4-(2-methyl-10H-benzo[b]thieno[2,3-e][1,4]diazepin-4-yl)piperazin-2-yl)methyl)isoindoline-1,3-dione




A solution of 2-((1-methylpiperazin-2-yl)methyl)isoindoline-1,3-dione, prepared as described in step D, (100 mg, 0.38 mmol), 2-methyl-10H-benzo[b]thieno[2,3-e][1,4]diazepin-4-amine, prepared as described in step G, (150 mg, 0.52 mmol) and diisopropylethylamine (0.49 g, 3.8 mmol) in dimethylsulfoxide (0.5 mL) was stirred at 170° C. for 2 hrs. The reaction was diluted with water and extracted with ethyl acetate. The organic phase was concentrated and the residue purified by column to give 15 mg of 2-((1-methyl-4-(2-methyl-10H-benzo[b]thieno[2,3-e][1,4]diazepin-4-yl)piperazin-2-yl)methyl)isoindoline-1,3-dione. 1H NMR (400 MHz, CDCl3) δ 7.76-7.73 (m, 1H), 7.45-7.35 (m, 3H), 7.18-7.17 (m, 1H), 6.98-6.95 (m, 2H), 6.75-6.73 (m, 1H), 6.46 (s, 1H), 4.28-4.25 (m, 1H), 3.96-6.92 (m, 1H), 3.71-3.64 (m, 3H), 3.47-3.41 (m, 1H), 3.29-3.28 (m, 1H), 3.12-3.09 (m, 1H), 2.87-2.86 (m, 1H), 2.67-2.53 (m, 3H), 2.28 (s, 3H).
Step I
(1-Methyl-4-(2-methyl-10H-benzo[b]thieno[2,3-e][1,4]diazepin-4-yl)piperazin-2-yl)methanamine




A solution of 2-((1-methyl-4-(2-methyl-10H-benzo[b]thieno[2,3-e][1,4]diazepin-4-yl)piperazin-2-yl)methyl)isoindoline-1,3-dione, prepared as described in the previous step, (1.0 g) in ethanolic methylamine (20 mL) was stirred at ambient temperature overnight. The solvent was removed under vacuum and the residue purified by HPLC to give the hydrochloride salt of (1-methyl-4-(2-methyl-10H-benzo[b]thieno[2,3-e][1,4]diazepin-4-yl)piperazin-2-yl)methanamine as a red solid. 1H NMR (400 MHz, MeOD) δ 7.46-7.44 (m, 1H), 7.31-7.48 (m, 1H), 7.19-7.15 (m, 1H), 6.97-6.95 (m, 1H), 6.74 (s, 1H), 4.80-4.71 (br, 1H), 4.28-4.20 (br, 2H), 4.07-4.04 (br, 2H), 3.82-3.70 (br, 3H), 3.53-3.48 (m, 1H), 3.18 (s, 3H), 2.42 (m, 3H); ESI-MS (M+1): 342 calc. for C18H23N5S Exact Mass: 341.17.
Example 2
2-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((1-methyl-4-(2-methyl-10H-benzo[b]thieno[2,3-e][1,4]diazepin-4-yl)piperazin-2-yl)methyl)acetamide




To a solution of (1-methyl-4-(2-methyl-10H-benzo[b]thieno[2,3-e][1,4]diazepin-4-yl)piperazin-2-yl)methanamine, prepared as described in Example 1, (10.3 mg, 30.2 μmoles) in 570 μL of DMF and 13.3 μL of tributylamine was added 760 μL of a DMF solution of N-(α-maleimidoacetoxy) succinimide ester (AMAS, 10 mg/mL, 7.6 mg, 30.2 μmoles). The resulting solution was allowed to stir for 18 hours at 20° C., then used as such in conjugation reactions with thiol-activated protein.
Example 3
(2-Methyl-4-(4-methylpiperazin-1-yl)-10H-benzo[b]thieno[2,3-e][1,4]diazepin-7-yl)methanamine




Step A
2-(4-Cyano-2-nitro-phenylamino)-5-methyl-thiophene-3-carbonitrile




To a suspension of sodium hydride (60%, 0.58 g) in THF (2 mL), was added 4-fluoro-3-nitro-benzonitrile (1.33 g, 8.0 mmol) and 2-amino-5-methyl-thiophene-3-carbonitrile (1.10 g, 8.0 mmol) in THF (10 mL), dropwise. The mixture was stirred at room temperature overnight. Two more batches of sodium hydride (60%, 0.50 g and 0.4 g) were added over the next 6 hours. After stirring for 3 days, the mixture was poured into ice-water (20 mL) and acidified to pH 3 with 6N hydrochloric acid (7 mL). The precipitate was filtered and washed with water. The solid was extracted with dichloromethane (35 mL). The solution was concentrated to a solid, and used in the next step without additional purification. LC-MS: m/z 285 (M+1), 307 (M+23). 1H NMR (CDCl3, 400 MHz): δ (ppm) 9.76 (s, 1H), 8.59 (s, 1H), 7.70 (d, 1H), 7.14 (d, 1H), 6.87 (s, 1H), 2.52 (s, 1H).
Step B
10-Amino-2-methyl-4H-3-thia-4,9-diaza-benzo[f]azulene-7-carbonitrile hydrochloride




To a suspension of 2-(4-Cyano-2-nitro-phenylamino)-5-methyl-thiophene-3-carbonitrile, prepared as described in the previous step, (0.52 g) in ethanol (5 mL), was added tine chloride (1.36 g, 7.2 mmol) in 6 N HCl. The mixture was heated in an 85° C. oil bath for 3 hours and then cooled in ice bath. The solid was filtered, washed with water, and dried to brown give the title compound as a brown solid containing inorganic salt, which was used in the next step without additional purification. LC-MS: m/z 255 (M+1 of free base). 1H NMR (DMSO-d6, 400 MHz): δ (ppm) 11.18 (br, 1H), 10.09 (s, 1H), 9.35 (br, 1H), 8.94 (br, 1H), 7.54 (d, 1H), 7.27 (s, 1H), 6.95 (d, 1H), 2.26 (s, 3H).
Step C
2-Methyl-10-(4-methyl-piperazin-1-yl)-4H-3-thia-4,9-diaza-benzo[f]azulene-7-carbonitrile




To a solution of 10-amino-2-methyl-4H-3-thia-4,9-diaza-benzo[f]azulene-7-carbonitrile hydrochloride, prepared as described in the previous step, (0.6 g) in DMSO (6 mL) and toluene (6 mL), was added 1-methylpiperazine (4 mL). The mixture was heated in a 130° C. oil bath for 17 hours. The solution was concentrated, diluted with ethyl acetate (50 mL), washed with water (20 mL) and brine (20 mL), and then concentrated. The solid was dissolved in dichloromethane (10 mL) and treated with saturated sodium bicarbonate solution. The title compound was collected as a light yellow precipitate, washed with water and dichloromethane, dried, and used in the next step without additional purification. LC-MS: m/z 338 (M+1). 1H NMR (CD3OD, 400 MHz): δ (ppm) 7.19-7.15 (m, 2H), 6.74 (d, 1H), 6.37 (s, 1H), 3.51 (m, 4H), 2.53 (m, 4H), 2.34 (s, 3H), 2.32 (s, 3H).
Step D
(2-Methyl-4-(4-methylpiperazin-1-yl)-10H-benzo[b]thieno[2,3-e][1,4]diazepin-7-yl)methanamine




To a solution of 2-Methyl-10-(4-methyl-piperazin-1-yl)-4H-3-thia-4,9-diaza-benzo[f]azulene-7-carbonitrile, prepared as described in the previous step, (0.25 g) in methanol (90 mL) was added concentrated HCl (0.4 mL) and Pd black (57 mg). Hydrogenation was carried out at 50 psi for 1 h. More Pd black (147 mg) was added. The mixture was shaken at 50 psi for 22 h. The catalyst was filtered and washed with methanol. The filtrate was concentrated, treated with saturated sodium bicarbonate solution (5 mL), and concentrated to dryness. The product was purified by silica column. LC-MS: m/z 342 (M+1). 1H NMR (CD3OD, 400 MHz): δ (ppm) 6.89-6.85 (m, 2H), 6.64 (d, 1H), 6.34 (d, 1H), 3.66 (s, 2H), 3.46 (m, 4H), 2.54 (m, 4H), 2.34 (s, 3H), 2.30 (d, 3H).
Example 4
2-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((2-methyl-4-(4-methylpiperazin-1-yl)-10H-benzo[b]thieno[2,3-e][1,4]diazepin-7-yl)methyl)acetamide




To a solution of (2-methyl-4-(4-methylpiperazin-1-yl)-10H-benzo[b]thieno[2,3-e][1,4]diazepin-7-yl)methanamine, prepared as described in Example 3, (3.5 mg, 10.2 μmoles) in 185 μL of DMF and 4.5 μL of tributylamine was added 260 μL of a DMF solution of N-(α-maleimidoacetoxy) succinimide ester (AMAS, 10 mg/mL, 2.6 mg, 10.2 μmoles). The resulting solution was allowed to stir for 90 minutes at 20° C., then used as such in conjugation reaction with thiol-activated protein.
Example 5
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((2-methyl-4-(4-methylpiperazin-1-yl)-10H-benzo[b]thieno[2,3-e][1,4]diazepin-7-yl)methyl)hexanamide




To a solution of (2-methyl-4-(4-methylpiperazin-1-yl)-10H-benzo[b]thieno[2,3-e][1,4]diazepin-7-yl)methanamine, prepared as described in Example 3, (59 mg, 0.17 mmol) in dichloromethane (4 mL) was added triethylamine (0.048 mL, 0.34 mmol) and 6-maleimidohexanoic N-hydroxysuccinimide ester (53 mg, 0.17 mmol) in dichloromethane (1 mL). The solution was stirred at room temperature for 40 min, then loaded onto a silica column, eluted with 3-5% methanol/dichloromethane containing triethylamine. The title compound was obtained as a yellow solid. LC-MS: m/z 535 (M+1).
Example 6
N-[2-Methyl-10-(4-methyl-piperazin-1-yl)-4H-3-thia-4,9-diaza-benzo[f]azulen-7-ylmethyl]-succinamic acid




Step A
Succinic acid 2,5-dioxo-pyrrolidin-1-yl ester methyl ester




To a solution of 1-hydroxy-pyrrolidine-2,5-dione (1.23 mL, 10 mmol) in ethyl acetate (50 mL) was added 3-chlorocarbonyl-propionic acid methyl ester (1.15 g, 10 mmol). The mixture was cooled in an ice bath. Triethylamine (1.4 mL, 10 mmol) was added dropwise. The resulting suspension was stirred for 10 min in an ice bath and for 5 min without ice bath. The white solid was removed by filtration and washed with ethyl acetate (3×3 mL). The filtrate was concentrated to a white solid (2.32 g).
Step B
N-[2-Methyl-10-(4-methyl-piperazin-1-yl)-4H-3-thia-4,9-diaza-benzo[f]azulen-7-ylmethyl]-succinamic acid methyl ester




To a solution of (2-methyl-4-(4-methylpiperazin-1-yl)-10H-benzo[b]thieno[2,3-e][1,4]diazepin-7-yl)methanamine, prepared as described in Example 3, (40 mg, 0.12 mmol) in dichloromethane (2 mL) was added triethylamine (0.030 mL, 0.22 mmol) and succinic acid 2,5-dioxo-pyrrolidin-1-yl ester methyl ester, prepared as described in the previous step, (31 mg, 0.13 mmol). The solution was stirred at room temperature for 1 hour and concentrated. The crude was loaded onto a silica column, eluted with 3-5% methanol/dichloromethane containing ammonium hydroxide to give the title compound as a yellow solid. LC-MS: m/z 456 (M+1).
Step C
N-[2-Methyl-10-(4-methyl-piperazin-1-yl)-4H-3-thia-4,9-diaza-benzo[f]azulen-7-ylmethyl]-succinamic acid




To a suspension of N-[2-methyl-10-(4-methyl-piperazin-1-yl)-4H-3-thia-4,9-diaza-benzo[f]azulen-7-ylmethyl]-succinamic acid methyl ester, prepared as described in the previous step, (80 mg, 0.18 mmol) in THF (1.5 mL) was added LiOH (14 mg) in water (0.5 mL). The solution was stirred at room temperature for 3 h, acidified with dilute HCl, and concentrated to dryness. LC-MS: m/z 442 (M+1 of the parent).
Example 7
2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((1-methyl-4-(2-methyl-10H-benzo[b]thieno[2,3-e][1,4]diazepin-4-yl)piperazin-2-yl)methyl)acetamide-keyhole limpet hemocyanin-conjugate
Step A
To a 3.19 mL solution of keyhole limpet hemocyanin (KLH, 15.2 mg, 0.152 μmoles) in 100 mM phosphate buffer, 0.46M sodium chloride, at pH 7.4 was added 70.3 μL of a DMF solution of N-succinimidyl-5-acetylthioacetate (SATA, 25 mg/mL, 1.75 mg, 7.60 μmoles). The resulting solution was incubated at 20° C. for 1 hour on a roller mixer. To the reaction was added 319 μL of 2.5M hydroxylamine, 50 mM EDTA, pH 7.0 and the resulting solution was incubated at 20° C. for 25 min, on a roller mixer. The reaction was purified on a Sephadex G-25 column using 100 mM phosphate buffer, 0.46 M sodium chloride, 5 mM EDTA, at pH 6.0.
Step B
To the KLH-SH, prepared as described in the previous step, (4.29 mL, 12.7 mg 0.127 μmoles) was added an aliquot of the solution prepared in Example 2, (566.6 μL, 12.7 μmoles). The resulting cloudy mixture was incubated for 2 hours at 20° C. on a roller mixer. The reaction was filtered through a 20 μm syringe filter then purified on a Sephadex G-25 column using 100 mM phosphate buffer, 0.46M sodium chloride, at pH 7.4.
Example 8
2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((1-methyl-4-(2-methyl-10H-benzo[b]thieno[2,3-e][1,4]diazepin-4-yl)piperazin-2-yl)methyl)acetamide-bovine thyroglobulin-conjugate
Step A
To 2.0 mL of a solution of bovine thyroglobulin (BTG, 20.0 mg, 0.03 μmoles) in 100 mM phosphate buffer pH 7.5 was added 276.0 μL of a DMF solution of N-succinimidyl-S-acetylthioacetate (SATA, 25 mg/mL, 6.9 mg, 30.0 μmoles). The resulting solution was incubated at 20° C. for 1 hour on a roller mixer. To the reaction was added 230 μL of 2.5 M hydroxylamine, 50 mM EDTA, pH 7.0. The resulting solution was incubated at 20° C. for 15 minutes on a roller mixer. The reaction was purified on a Sephadex G-25 column using 100 mM phosphate buffer, 5 mM EDTA, at pH 6.0.
Step B
To the BTG-SH, prepared as described in the previous step, (4.73 mL, 14.3 mg, 0.022 μmoles) was added an aliquot of the solution prepared in Example 2, (969.6 μL, 21.7 μmoles). The resulting cloudy mixture was incubated for 3 hours at 20° C. on a roller mixer. The reaction was filtered through a 0.45 μm syringe filter, then purified on a Sephadex G-25 column using 100 mM phosphate buffer, 0.14M sodium chloride, at pH 7.4.
Example 9
2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((1-methyl-4-(2-methyl-10H-benzo[b]thieno[2,3-e][1,4]diazepin-4-yl)piperazin-2-yl)methyl)acetamide-ovalbumin-conjugate
Step A
To 1.2 mL of a solution of ovalbumin (12.0 mg, 0.27 μmoles) in 100 mM phosphate buffer pH 7.5 was added 50.1 μL of a DMF solution of N-succinimidyl-S-acetylthioacetate (SATA, 25 mg/mL, 1.25 mg, 5.42 μmoles). The resulting solution was incubated at 20° C. for 1 hour on a roller mixer. To the reaction was added 120 μL of 2.5M hydroxylamine, 50 mM EDTA, at pH 7.0. The resulting solution was incubated at 20° C. for 15 minutes on a roller mixer. The reaction was purified on a Sephadex G-25 column using 100 mM phosphate buffer, 5 mM EDTA, at pH 6.0.
Step B
To the ovalbumin-SH, prepared as described in the previous step, (4.2 mL, 8.0 mg, 0.18 μmoles) was added an aliquot of the solution prepared in Example 2, (200 μL, 4.5 μmoles). The resulting mixture was incubated for 3 hours at 20° C. on a roller mixer. The reaction was purified on a Sephadex G-25 column using 100 mM phosphate buffer, 0.14M sodium chloride, at pH 7.4.
Example 10
2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((2-methyl-4-(4-methylpiperazin-1-yl)-10H-benzo[b]thieno[2,3-e][1,4]diazepin-7-yl)methyl)acetamide-keyhole limpet hemocyanin-conjugate
To the KLH-SH, prepared as described in Example 7 Step A, (3.31 mL, 9.8 mg, 0.098 μmoles) was added a 300 μL aliquot of 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((2-methyl-4-(4-methylpiperazin-1-yl)-10H-benzo[b]thieno[2,3-e][1,4]diazepin-7-yl)methyl)acetamide solution, prepared as described in Example 4, (6.9 μmoles). The resulting cloudy mixture was incubated for 2.5 hours at 20° C. on a roller mixer. The reaction was filtered through a 0.2 μm syringe filter then purified on a Sephadex G-25 column using 100 mM phosphate buffer, 0.46 M sodium chloride, at pH 7.4.
Example 11
2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((2-methyl-4-(4-methylpiperazin-1-yl)-10H-benzo[b]thieno[2,3-e][1,4]diazepin-7-yl)methyl)acetamide-ovalbumin-conjugate
To the ovalbumin-SH, prepared as described in Example 9 Step A, (5.38 mL, 17.8 mg, 0.40 μmoles) was added a 200 μL aliquot of 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((2-methyl-4-(4-methylpiperazin-1-yl)-10H-benzo[b]thieno[2,3-e][1,4]diazepin-7-yl)methyl)acetamide solution, prepared as described in Example 4, (10.2 μmoles). The resulting mixture was incubated for 3 hours at 20° C. on a roller mixer. The reaction was filtered through a 0.45 μm syringe filter then purified on a Sephadex G-25 column using 100 mM phosphate buffer, 0.14 M sodium chloride, at pH 7.4.
Example 12
N-[2-Methyl-10-(4-methyl-piperazin-1-yl)-4H-3-thia-4,9-diaza-benzo[f]azulen-7-ylmethyl]-succinamic acid-bovine thyroglobulin-conjugate
Step A
A solution of N-[2-Methyl-10-(4-methyl-piperazin-1-yl)-4H-3-thia-4,9-diaza-benzo[f]azulen-7-ylmethyl]-succinamic acid, prepared as described in Example 6, (7.9 mg, 18.0 μmoles), N-hydroxysuccinimide (NHS, 8.3 mg, 72.0 μmoles) and N,N-dicyclohexylcarbodiimide (14.9 mg, 72.0 μmoles) in 500 μL of DMF and 5 μL of tributylamine was allowed to stir for 18 hours at 20° C., then used as such in conjugation with protein.
Step B
To 2.98 mL of a solution of bovine thyroglobulin (BTG, 14.9 mg, 0.023 μmoles) in 100 mM phosphate buffer pH 7.5 was added 500 μL of the solution prepared in Step A (18.0 μmoles). The resulting cloudy mixture was incubated at 20° C. for 2.5 hours on a roller mixer. The reaction was filtered through a 0.45 μm syringe filter then purified on a Sephadex G-25 column using 100 mM phosphate buffer, 0.14 M sodium chloride, at pH 7.4.
Example 13
Competitive Immunoassay for Olanzapine
Following a series of immunizations with olanzapine immunogens, mouse tail bleeds were tested for reactivity using an ELISA. Hybridoma supernatants were also tested, and the ELISA data shown in Tables 1 and 2 below shows reactivity of several hybridomas (fusion partner was NSO cells).







TABLE 1







Plate 2




















Dilution
1
2
3
4
5
6
7
8
9
10
11
12
























 1/400
25
26
27
28
29
30
31
21
33
34
35
36
Ag = Bt-


 1/1200












Compound#11


 1/3600















 1/10800















 1/400
37
38
39
40
41
42
43
44
45
46
47
48



 1/1200















 1/3600















 1/10800















 1/400
0.0136
0.0432
0.131
0.0654
0.4092
0.039
0.016
0.1408
0.0712
1.4854
2.0086
0.0861
Ag = Bt-


 1/1200
0.0113
0.0194
0.0477
0.0291
0.1293
0.031
0.012
0.0374
0.0126
0.4411
0.8874
0.0362
Compound#11


 1/3600
0.0092
0.0118
0.0233
0.0153
0.0462
0.013
0.009
0.0314
0.0275
0.2073
0.3555
0.0217



 1/10800
0.0105
0.0111
0.0159
0.0107
0.0224
0.012
0.009
0.0172
0.0168
0.0972
0.147
0.0141



 1/400
0.0333
0.1512
1.1412
1.0762
0.3042
0.04
0.449
0.1619
1.8038
0.0933
0.7666
1.258



 1/1200
0.0144
0.055
0.4575
0.3223
0.0907
0.016
0.144
0.0402
0.1536
0.0288
0.2956
0.4374



 1/3600
0.008
0.0333
0.2036
0.1077
0.0361
0.011
0.051
0.0206
0.708
0.0165
0.1212
0.2072



 1/10800
0.0109
0.0181
0.0885
0.0581
0.027
0.01
0.045
0.0217
0.5338
0.0132
0.0585
0.0954
















TABLE 2







Plate 3



















Dilution
1
2
3
4
5
6
7
8
9
10
11
12






















100
1B2
1C4
2B3
2G5
3A3
3E9
3F11
4G9
5D11
6G2
#13
Empty


100














300














300














900














900














2700














2700














100
0.0528
0.5634
2.9908
1.9083
0.7869
2.7564
2.296
1.027
0.1174
0.8223
0.041
0


100
0.0527
0.429
2.7862
1.3797
0.6534
2.3072
2.0249
0.934
0.1115
0.7692
0.0386
0.0057


300
0.0202
0.1452
1.3705
0.5961
0.2337
1.3963
0.8952
0.2999
0.0378
0.2486
0.0177
0.0031


300
0.0208
0.1408
1.3166
0.5235
0.2173
1.1112
0.9114
0.3116
0.0406
0.2483
0.0174
0.0052


900
0.0132
0.0242
0.4925
0.1967
0.0849
0.4472
0.2986
0.0896
0.0179
0.0851
0.012
0.0039


900
0.0148
0.0554
0.4551
0.1731
0.0839
0.4471
0.3499
0.0951
0.018
0.0863
0.0128
0.0055


2700
0.0109
0.0259
0.1877
0.0713
0.0334
0.1709
0.1381
0.0352
0.0111
0.036
0.0094
0.0041


2700
0.0122
0.028
0.1835
0.0903
0.0404
0.1924
0.1502
0.0409
0.0113
0.0325
0.0094
0.005









Supernatant was then tested by competition ELISA to determine if the signals were specific to olanzapine. FIGS. 1-3 show the results from three representative hybridomas resulting from mouse fusion 11.1. Data shows specific reactivity to olanzapine with varied reactivity to clozapine.
FIG. 4 shows the competitive immunoassay format used on a lateral flow assay device in which the capture antibody, an olanzapine clone, was deposited on a chip along with a detection conjugate consisting of olanzapine conjugated to a fluorophore. In this competitive format as show in FIG. 4, a low level of analyte (olanzapine) results in high signal, whereas a high level of analyte (olanzapine) results in low signal. The amount of olanzapine in the sample can be calculated from the loss of fluorescence compared to a control sample with no drug present. A typical dose response curve generated with olanzapine clone 35 is shown in FIG. 5, with olanzapine clone 61 is shown in FIG. 6, and with olanzapine clone 3F11 is shown in FIG. 7.

## Claims

1. The compound of Formula I:



wherein:
R1 is H,




 CH2NH2, or CH2NHC(O)(CH2)mCO2H;
R2 is H,




 CH2NH2, or CH2NHC(O)(CH2)mCO2H;
R3 is H; provided that either R1 or R2 must be H, and further provided that R1 and R2 may not all be H simultaneously;
m is 1, 2, 3, 4, or 5; and
n is 1, 2, 3, 4, or 5.

2. The compound of claim 1:
wherein:
R1 is




 CH2NH2, or CH2NHC(O)(CH2)mCO2H;
R2 is H;
R3 is H;
m is 1, 2, 3, 4, or 5; and
n is 1, 2, 3, 4, or 5.

3. The compound of claim 1
wherein:
R1 is H,
R2 is




 CH2NH2, or CH2NHC(O)(CH2)mCO2H;
R3 is H;
m is 1, 2, 3, 4, or 5; and
n is 1, 2, 3, 4, or 5.

4. The compound of claim 1
wherein:
R1 is H,




 CH2NH2, or CH2NHC(O)(CH2)mCO2H;
R2 is H,




 CH2NH2, or CH2NHC(O)(CH2)mCO2H; provided that either R1 or R2 must be H, and further provided that both R1 and R2 may not be H simultaneously;
R3 is H;
m is 1, 2, 3, 4, or 5; and
n is 1, 2, 3, 4, or 5.

5. The compound of claim 1
wherein:
R1 is H,




 CH2NH2, or CH2NHC(O)(CH2)mCO2H;
R2 is H,




 CH2NH2, or CH2NHC(O)(CH2)mCO2H; provided that either R1 or R2 must be H, and further provided that both R1 and R2 may not be H simultaneously;
R3 is H;
m is 1, 2, 3, 4, or 5; and n is 1, 2, 3, 4, or 5.

6. The compounds of claim 1, selected from the group consisting of:

7. The compound of claim 6 which is:

8. The compound of claim 6 which is:

9. A conjugate of the compound of claim 1 and an immunogenic carrier, wherein the immunogenic carrier is keyhole limpet hemocyanin, bovine thyroglobulin, or ovalbumin.

10. A conjugate of the compound of claim 2 and an immunogenic carrier, wherein the immunogenic carrier is keyhole limpet hemocyanin, bovine thyroglobulin, or ovalbumin.

11. A conjugate of the compound of claim 3 and an immunogenic carrier, wherein the immunogenic carrier is keyhole limpet hemocyanin, bovine thyroglobulin, or ovalbumin.

12. A conjugate of the compound of claim 4 and an immunogenic carrier, wherein the immunogenic carrier is keyhole limpet hemocyanin, bovine thyroglobulin, or ovalbumin.

13. A conjugate of the compound of claim 5 and an immunogenic carrier, wherein the immunogenic carrier is keyhole limpet hemocyanin, bovine thyroglobulin, or ovalbumin.

14. The conjugate of claim 9 wherein the compound is:

15. The conjugate of claim 9 wherein the compound is:

16. The conjugate of claim 9 wherein the compound is:

17. The conjugate of claim 9 wherein the compound is:

18. A product made by the process of contacting a compound of claim 1 with an immunogenic carrier, wherein the immunogenic carrier is keyhole limpet hemocyanin, bovine thyroglobulin, or ovalbumin.

19. A product made by the process of contacting a compound of claim 2 with an immunogenic carrier, wherein the immunogenic carrier is keyhole limpet hemocyanin, bovine thyroglobulin, or ovalbumin.

20. A product made by the process of contacting a compound of claim 3 with an immunogenic carrier, wherein the immunogenic carrier is keyhole limpet hemocyanin, bovine thyroglobulin, or ovalbumin.

21. A product made by the process of contacting a compound of claim 4 with an immunogenic carrier, wherein the immunogenic carrier is keyhole limpet hemocyanin, bovine thyroglobulin, or ovalbumin.

22. A product made by the process of contacting a compound of claim 5 with an immunogenic carrier, wherein the immunogenic carrier is keyhole limpet hemocyanin, bovine thyroglobulin, or ovalbumin.

23. The product of claim 18 wherein the compound is:

24. The product of claim 18 wherein the compound is:

25. The product of claim 18 wherein the compound is:

26. The product of claim 18 wherein the compound is:

27. The compound of claim 1
wherein:
R1 is H,




 CH2NH2, or CH2NHC(O)(CH2)mCO2H;
R2 is H,




 CH2NH2, or CH2NHC(O)(CH2)mCO2H; provided that either R1 or R2 must be H, and further provided that both R1 and R2 may not be H simultaneously;
R3 is H;
m is 1, 2, 3, 4, or 5; and
n is 1, 2, 3, 4, or 5.

28. A conjugate of the compound of claim 27 and an immunogenic carrier, wherein the immunogenic carrier is selected from keyhole limpet hemocyanin, ovalbumin or bovine thyroglobulin.

29. A product made by the process of contacting a compound of claim 27 with an immunogenic carrier, wherein the immunogenic carrier is selected from keyhole limpet hemocyanin, ovalbumin or bovine thyroglobulin.