Haptens Of Risperidone And Paliperidone

  *US09012648B2*
  US009012648B2                                 
(12)United States Patent(10)Patent No.: US 9,012,648 B2
  et al. (45) Date of Patent:Apr.  21, 2015

(54)Haptens of risperidone and paliperidone 
    
(75)Inventors: Janssen Pharmaceutica NV,  Beerse (BE); 
  Ortho-Clinical Diagnostics, Inc.,  Raritan, NJ (US) 
(73)Assignee:Janssen Pharmaceutica NV,  Beerse (BE), Type: Foreign Company 
(*)Notice: Subject to any disclaimer, the term of this patent is extended or adjusted under 35 U.S.C. 154(b) by 0 days. 
(21)Appl. No.: 13/970,673 
(22)Filed: Aug.  20, 2013 
(65)Prior Publication Data 
 US 2014/0155585 A1 Jun.  5, 2014 
 Related U.S. Patent Documents 
(60)Provisional application No. 61/691,469, filed on Aug.  21, 2012.
 
Jan.  1, 2013 C 07 K 16 44 F I Apr.  21, 2015 US B H C Jan.  1, 2013 C 07 D 487 04 L I Apr.  21, 2015 US B H C Jan.  1, 2013 C 07 D 471 04 L I Apr.  21, 2015 US B H C
(51)Int. Cl. C07D 211/00 (20060101); C07D 413/00 (20060101); C07K 016/44 (20060101); C07D 487/04 (20060101); C07D 471/04 (20060101)
(52)U.S. Cl. 546/184; 548/241
(58)Field of Search  548/241; 546/184

 
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     Primary Examiner —James O Wilson
     Assistant Examiner —Oluwafemi Masha
     Art Unit — 1624
     Exemplary claim number — 1
 
(74)Attorney, Agent, or Firm — Peter Herridge

(57)

Abstract

The invention relates to compounds of Formula I, wherein R1 and R2 are defined in the specification, useful for the synthesis of novel conjugates and immunogens derived from risperidone and paliperidone. The invention also relates to conjugates of a risperidone or paliperidone hapten and a protein.
[see pdf for image]
18 Claims, 5 Drawing Sheets, and 5 Figures


CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority of the benefits of the filing of U.S. Provisional Application Ser. No. 61/691,469, 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

[0002] The invention relates to the field of immunoassays for determining the presence of risperidone and paliperidone in human biological fluids.

BACKGROUND OF THE INVENTION

[0003] 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.
[0004] 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.
[0005] 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.
[0006] Risperidone is:
[0007]  [see pdf for image]
[0008] Paliperidone is:
[0009]  [see pdf for image]

SUMMARY OF THE INVENTION

[0010] The subject invention provides compounds and conjugates that permit such an improved method for determining the levels of the anti-psychotic drug risperidone.
[0011] The invention comprises the compound of Formula I
[0012]  [see pdf for image]
wherein:
R1 is H, or OH;
R2 is O(CH2)rNH2,
[0013]  [see pdf for image]
O(CH2)rNHC(O)(CH2)mCO2H, or Z—(Y)p-G;
wherein:
Z is selected from the group consisting of:
—N(R4)—, —O—, —S—, -heteroalkyl-;
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;
r is 1, 2, 3, 4, or 5;
m is 1, 2, 3, 4, or 5;
n is 1, 2, 3, 4, or 5.
[0014] 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

[0015] FIGS. 1 and 2 show Competitive ELISA results generated with hybridoma 5-9;
[0016] FIG. 3 shows Competitive ELISA results generated with risperidone clone 2A5;
[0017] FIG. 4 shows the competitive immunoassay format used on a lateral flow assay device; and
[0018] FIG. 5 shows a typical dose response curve generated with risperidone clone 5-9.

DETAILED DESCRIPTION OF THE INVENTION

[0019] 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.
[0020] 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).
[0021] The invention comprises the compound of Formula I
[0022]  [see pdf for image]
wherein:
R1 is H, or OH;
R2 is O(CH2)rNH2,
[0023]  [see pdf for image]
O(CH2)rNHC(O)(CH2)mCO2H, or Z—(Y)p-G;
wherein:
Z is selected from the group consisting of:
—N(R4)—, —O—, —S—, -heteroalkyl-;
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;
r is 1, 2, 3, 4, or 5;
m is 1, 2, 3, 4, or 5;
n is 1, 2, 3, 4, or 5.
[0024] The invention comprises the compound of Formula I

wherein:

R1 is H, or OH;

R2 is O(CH2)rNH2,

[0025]  [see pdf for image]
O(CH2)rNHC(O)(CH2)mCO2H, or Z—(Y)p-G;
wherein:
Z is O;
Y is an organic spacer group;
G is a functional linking group capable of binding to a carrier;
p is 0, or 1;
r is 1, 2, 3, 4, or 5;
m is 1, 2, 3, 4, or 5;
n is 1, 2, 3, 4, or 5.
[0026] The invention comprises the compound of Formula I

wherein:

R1 is H, or OH;

R2 is O(CH2)rNH2,

[0027]  [see pdf for image]
O(CH2)rNHC(O)(CH2)mCO2H, or Z—(Y)p-G;
wherein:
Z is O(CH2)rNH;
Y is an organic spacer group;
G is a functional linking group capable of binding to a carrier;
p is 0, or 1;
r is 1, 2, 3, 4, or 5;
m is 1, 2, 3, 4, or 5;
n is 1, 2, 3, 4, or 5.
[0028] The invention comprises the compound of Formula I

wherein:

R1 is H, or OH;

R2 is O(CH2)rNH2,

[0029]  [see pdf for image]
O(CH2)rNHC(O)(CH2)mCO2H, or Z—(Y)p-G;
wherein:
Z is O(CH2)rNH;
Y is an organic spacer group;
G is a functional linking group capable of binding to a carrier;
p is 1;
r is 2;
m is 1, 2, 3, 4, or 5;
n is 1, 2, 3, 4, or 5.
[0030] The invention comprises the compound of Formula I

wherein:

R1 is H, or OH;

R2 is O(CH2)rNH2,

[0031]  [see pdf for image]
or
O(CH2)rNHC(O)(CH2)mCO2H;
wherein:
r is 2;
m is 1, 2, 3, or 4;
n is 1, or 2.
in another embodiment of the invention:
R1 is H, or OH; and
R2 is O(CH2)rNH2, or
[0032]  [see pdf for image]
wherein r is 2;
wherein m is 1.
[0033] Another embodiment of the invention is the compound
[0034]  [see pdf for image]
[0035] The invention further provides conjugates of the above compounds with an immunogenic carrier, conjugates of the above compounds with proteins and conjugates of the above compounds with the proteins keyhole limpet hemocyanin, ovalbumin and bovine thyroglobulin in addition to products produced by the process of contacting the compounds of the invention with immunogenic carriers.
[0036] The invention comprises a conjugate of the compound of Formula I
[0037]  [see pdf for image]
wherein:
R1 is H, or OH;
R2 is O(CH2)rNH2,
[0038]  [see pdf for image]
O(CH2)rNHC(O)(CH2)mCO2H, or Z—(Y)p-G;
wherein:
Z is selected from the group consisting of:
—N(R4)—, —O—, —S—, -heteroalkyl-;
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;
r is 1, 2, 3, 4, or 5;
m is 1, 2, 3, 4, or 5;
n is 1, 2, 3, 4, or 5; with an immunogenic carrier.
[0039] The invention comprises a conjugate of the compound of Formula I

wherein:

R1 is H, or OH;

R2 is O(CH2)rNH2,

[0040]  [see pdf for image]
O(CH2)rNHC(O)(CH2)mCO2H, or Z—(Y)p-G;
wherein:
Z is O;
Y is an organic spacer group;
G is a functional linking group capable of binding to a carrier;
p is 0, or 1;
r is 1, 2, 3, 4, or 5;
m is 1, 2, 3, 4, or 5;
n is 1, 2, 3, 4, or 5; with an immunogenic carrier.
[0041] The invention comprises a conjugate of the compound of Formula I

wherein:

R1 is H, or OH;

R2 is O(CH2)rNH2,

[0042]  [see pdf for image]
O(CH2)rNHC(O)(CH2)mCO2H, or Z—(Y)p-G;
wherein:
Z is O(CH2)rNH;
Y is an organic spacer group;
G is a functional linking group capable of binding to a carrier;
p is 0, or 1;
r is 1, 2, 3, 4, or 5;
m is 1, 2, 3, 4, or 5;
n is 1, 2, 3, 4, or 5; with an immunogenic carrier.
[0043] The invention comprises a conjugate of the compound of Formula I

wherein:

R1 is H, or OH;

R2 is O(CH2)rNH2,

[0044]  [see pdf for image]
O(CH2)rNHC(O)(CH2)mCO2H, or Z—(Y)p-G;
wherein:
Z is O(CH2)rNH;
Y is an organic spacer group;
G is a functional linking group capable of binding to a carrier;
p is 1;
r is 2;
m is 1, 2, 3, 4, or 5;
n is 1, 2, 3, 4, or 5;
[0045] The invention comprises a conjugate of the compound of Formula I

wherein:

R1 is H, or OH;

R2 is O(CH2)rNH2,

[0046]  [see pdf for image]
or
O(CH2)rNHC(O)(CH2)mCO2H;
wherein:
r is 2;
m is 1, 2, 3, or 4;
n is 1, or 2; with an immunogenic carrier.
[0047] The invention comprises a conjugate of the compound of Formula I

R1 is H, or OH; and

R2 is O(CH2)rNH2, or

[0048]  [see pdf for image]
wherein r is 2;
wherein m is 1; with an immunogenic carrier.
[0049] The invention comprises a conjugate of the compound
[0050]  [see pdf for image]
with an immunogenic carrier.
[0051] Another embodiment of the invention is a conjugate of any of the above compounds of Formula I and protein.
[0052] Another embodiment of the invention is a conjugate any of the above compounds of Formula I and a protein wherein the protein is keyhole limpet hemocyanin, ovalbumin, or bovine thyroglobulin.
[0053] The invention also provides products formed from the process of contacting the above compounds with an immunogenic carrier.
[0054] Another embodiment of the invention is thus the product formed from the process of contacting a compound of Formula I
[0055]  [see pdf for image]
wherein:
R1 is H, or OH;
R2 is O(CH2)rNH2,
[0056]  [see pdf for image]
O(CH2)rNHC(O)(CH2)mCO2H, or Z—(Y)p-G;
wherein:
Z is selected from the group consisting of:
—N(R4)—, —O—, —S—, -heteroalkyl-;
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;
r is 1, 2, 3, 4, or 5;
m is 1, 2, 3, 4, or 5;
n is 1, 2, 3, 4, or 5; with an immunogenic carrier.
[0057] Another embodiment of the invention is the product formed from the process of contacting a compound of Formula I

wherein:

R1 is H, or OH;

R2 is O(CH2)rNH2,

[0058]  [see pdf for image]
O(CH2)rNHC(O)(CH2)mCO2H, or Z—(Y)p-G;
wherein:
Z is O;
Y is an organic spacer group;
G is a functional linking group capable of binding to a carrier;
p is 0, or 1;
r is 1, 2, 3, 4, or 5;
m is 1, 2, 3, 4, or 5;
n is 1, 2, 3, 4, or 5; with an immunogenic carrier.
[0059] Another embodiment of the invention is the product formed from the process of contacting a compound of Formula I

wherein:

R1 is H, or OH;

R2 is O(CH2)rNH2,

[0060]  [see pdf for image]
O(CH2)rNHC(O)(CH2)mCO2H, or Z—(Y)p-G;
wherein:
Z is O(CH2)rNH;
Y is an organic spacer group;
G is a functional linking group capable of binding to a carrier;
p is 0, or 1;
r is 1, 2, 3, 4, or 5;
m is 1, 2, 3, 4, or 5;
n is 1, 2, 3, 4, or 5; with an immunogenic carrier.
[0061] Another embodiment of the invention is the product formed from the process of contacting a compound of Formula I

wherein:

R1 is H, or OH;

R2 is O(CH2)rNH2,

[0062]  [see pdf for image]
O(CH2)rNHC(O)(CH2)mCO2H, or Z—(Y)p-G;
wherein:
Z is O(CH2)rNH;
Y is an organic spacer group;
G is a functional linking group capable of binding to a carrier;
p is 1;
r is 2;
m is 1, 2, 3, 4, or 5;
n is 1, 2, 3, 4, or 5; with an immunogenic carrier.
[0063] Another embodiment of the invention is the product formed from the process of contacting a compound of Formula I
[0064] wherein:
R1 is H, or OH;
R2 is O(CH2)rNH2,
[0065]  [see pdf for image]
or
O(CH2)rNHC(O)(CH2)mCO2H;
wherein:
r is 2;
m is 1, 2, 3, or 4;
n is 1, or 2; with an immunogenic carrier.
[0066] Another embodiment of the invention is the product formed from the process of contacting a compound of Formula I

R1 is H, or OH; and

R2 is O(CH2)rNH2, or

[0067]  [see pdf for image]
wherein r is 2;
wherein m is 1; with an immunogenic carrier.
[0068] Another embodiment of the invention is contacting the compound
[0069]  [see pdf for image]
with an immunogenic carrier.
[0070] A preferred embodiment of the invention is the product formed from the process of contacting any of the above compounds of Formula I with an immunogenic carrier wherein the immunogenic carrier is a protein.
[0071] A more preferred embodiment of the invention is the product formed from the process of contacting any of the above compounds of Formula I with an immunogenic carrier wherein the immunogenic carrier is a protein and wherein the protein is keyhole limpet hemocyanin, ovalbumin, or bovine thyroglobulin.

ABBREVIATIONS

[0072] Herein and throughout the application, the following abbreviations may be used.
[0073] AMAS N-(α-maleimidoacetoxy) succinimide ester
[0074] BTG bovine thyroglobulin
[0075] Bu3N tributylamine
[0076] DCC dicyclohexylcarbodiimide
[0077] DCM dichloromethane
[0078] DIEA diisopropylethylamine
[0079] DMF N,N-dimethylformamide
[0080] EDTA ethylenediaminetetraaceticacid
[0081] KLH keyhole limpet hemocyanin
[0082] SATA N-succinimidyl S-acetylthioacetate
[0083] TEA triethylamine
[0084] THF tetrahydrofuran
[0085] TFA trifluoroacetic acid
[0086] Et3N triethylamine
[0087] TBDMS t-butyldimethylsilyl
[0088] DIC diisopropylcarbodiimide
[0089] DMAP N,N-dimethyl-4-aminopyridine
[0090] EDC 1-ethyl-3(3-dimethylaminopropyl) carbodiimidehydrochloride
[0091] NHS N-hydroxysuccinimide
[0092] TFP Tetrafluorophenyl
[0093] PNP p-nitrophenyl
[0094] TBTU O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate
[0095] HOBT N-Hydroxybenzotriazole
[0096] DEPBT 3-(diethoxyphosphoryloxy)-1,2,3-benzotrazi-4(3H)-one
[0097] BOP-Cl Bis(2-oxo-3-oxazolidinyl)phosphonic chloride
[0098] DTT dithioerythritol

DEFINITIONS

[0099] 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 a protein. In the conjugate the small molecule maybe joined at one or more active sites on the large molecule.
[0100] 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.
[0101] The term “immunogen” refers to a substance capable of eliciting, producing, or generating an immune response in an organism.
[0102] 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.
[0103] 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.
[0104] Immunogenic carriers can also include poly amino-polysaccharides, which are a high molecular weight polymer 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.
[0105] 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.
[0106] 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.
[0107] The term “derivative” refers to a chemical compound or molecule made from a parent compound by one or more chemical reactions.
[0108] 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.
[0109] 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 or antibody. 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.
[0110] 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.
[0111] 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.
[0112] 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.
[0113] 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.
[0114] 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.
[0115] 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.
[0116] The term “alkyl” refers to both 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.
[0117] 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.
[0118] 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.
[0119] 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.
[0120] The term “hydroxyalkyl” refers to at least one hydroxyl group bonded to any carbon atom along an alkyl chain.
[0121] The term “aminoalkyl” refers to at least one primary or secondary amino group bonded to any carbon atom along an alkyl chain.
[0122] The term “alkoxyalkyl” refers to at least one alkoxy group bonded to any carbon atom along an alkyl chain.
[0123] The term “polyalkoxyalkyl” refers to long-chain alkoxy compounds and includes polyethylene glycols of discreet or monodispersed sizes.
[0124] 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.
[0125] 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.
[0126] The term “alkylcarbonyl” refers to a group that has a carbonyl group bonded to any carbon atom along an alkyl chain.
[0127] 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.
[0128] The term “heteroaralkyl” refers to a C1-6 alkyl group having a heteroaryl substituent. Examples include furylethyl and 2-quinolinylpropyl.
[0129] 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 methoxy, ethoxy, propoxy, isopropoxy and butoxy.
[0130] 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.
[0131] The term “aralkyl” refers to a C1-6 alkyl group containing an aryl substituent. Examples include benzyl, phenylethyl or 2-naphthylmethyl.
[0132] The term “heteroaralkyl” refers to a C1-6 alkyl group containing a heteroaryl substituent. Examples include furylmethyl and pyridylpropyl.
[0133] The term “aryloxy” refers to an oxygen atom bound to an aryl substituent. Examples include phenoxy and benzyloxy.
[0134] The term “arylalkoxy” refers to an alkoxy group bound to an aryl substituent. Examples include phenylmethyl ether.
[0135] The term “acyl” refers to the group —C(O)Ra, where Ra is alkyl, aryl, aralkyl, heteroaryl and heteroaralkyl. An “acylating agent” adds the —C(O)Ra group to a molecule.
[0136] The term “sulfonyl” refers to the group —S(O)2Ra, where Ra is hydrogen, alkyl, cycloalkyl, haloalkyl, aryl, aralkyl, heteroaryl and heteroaralkyl. A “sulfonylating agent” adds the —S(O)2Ra group to a molecule.
[0137] 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.
[0138] 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, NY, 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.
[0139] 
[00001] [TABLE-US-00001]
  TABLE 1
 
[see pdf for image]
 
[see pdf for image]
 
[see pdf for image]
 
[see pdf for image]
 
[see pdf for image]
 
[see pdf for image]
 
[see pdf for image]
 
[see pdf for image]
 
[see pdf for image]
 
[see pdf for image]
 
[see pdf for image]
 
  Reasonable values for m and n are between 1 and 10
[0140] 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 0-(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.
[0141] 
[00002] [TABLE-US-00002]
  TABLE 2
 
[see pdf for image]
 
[see pdf for image]
 
[see pdf for image]
 
  reasonable values for n is between 1-10
[0142] 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.
[0143] 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.
[0144] 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.
[0145] 
[00003] [TABLE-US-00003]
  TABLE 3
 
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[see pdf for image]
 
[see pdf for image]
 
[see pdf for image]
 
[0146] 
[00004] [TABLE-US-00004]
  TABLE 4
 
[see pdf for image]
 
[see pdf for image]
 
[see pdf for image]
 
[see pdf for image]
 
[0147] 
[00005] [TABLE-US-00005]
  TABLE 5
 
[see pdf for image]
 
[see pdf for image]
 
[see pdf for image]
 
[0148] Other electrophilic groups may be present on the hapten to attach the spacer, for example, a sulfonyl halide
[0149]  [see pdf for image]
or electrophilic phosphorous group, for example:
[0150]  [see pdf for image]
See: Malachowski, William P., Coward, James K., Journal of Organic Chemistry, 1994, 59 (25):7616
or:
[0151]  [see pdf for image]
Rc is alkyl, cycloalkyl, aryl, substituted aryl, aralkyl.
See: Aliouane, L., et. al, Tetrahedron Letters, 2011, 52(28):8681.
[0152] 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.
[0153] 
[00006] [TABLE-US-00006]
  TABLE 6
 
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[see pdf for image]
 
[0154] 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.
[0155] 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.
[0156] 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.
[0157] 
[00007] [TABLE-US-00007]
  TABLE 7
 
[see pdf for image]
 
[see pdf for image]
 
[see pdf for image]
 
[see pdf for image]
 
[0158] 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 5-Acetylthioacetate, (SATA, CAS 76931-93-6), or an analogue thereof, followed by cleavage of the actetate 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

[0159] 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.
[0160] Attachment of a spacer to the parent ring structure of risperidone may be accomplished through use of the silyl-protected starting compound shown in Scheme 1, the preparation of which is described in Example 1. Alkylation with an N-protected haloalkyl derivative is also described in Example 1. N-protected haloalkyl derivatives of varying chain lengths are commercially available or my be made by standard organic reactions known to one skilled in the art. Preferred values for r are between 1 and 5. Deprotection as described in Example 1 may provide the amino compound which may be further elaborated to attach additional spacer atoms or may be linked directly to the carrier. Derivatives of the amino compound which lack the hydroxyl group in final product may be made as described in Example 3.
[0161]  [see pdf for image]
[0162] Alkylation of risperidone may also be accomplished using a thiol, for example, 3-mercaptomethylpropionate, using the method of Wang, J., L., et. al., Bioorganic and Med. Chem. Letters, 2010, 20:7159, using K2CO3 in DMF followed by hydrolysis with NaOH in aqueous THF, as shown in Scheme 2, to provide an analogue of the hapten that bears a thioalkyl linkage terminating in a carboxy group that may be attached directly to a carrier or further elaborated to extend the spacer portion. Alkylation with an amine may be carried out also as shown in Scheme 2, according to the method used to make intermediate 535 in US20110245224. Versions of the aminoalkyl or thioalkyl analogues of Scheme 2 in which R1 is either OH or H may be made through routine optimization of the chemical procedures taught in the aforementioned references by a skilled chemist.
[0163]  [see pdf for image]
[0164] The phenolic hydroxyl group of the starting compound shown in Scheme 3, wherein R1 is either H or a silyl-protected alcohol, may be the site of introduction of a group that bears a functional linking group for attachment to a carrier. The phenolic compound may be reacted directly with succinic anhydride as shown in Scheme 3 and described in US20060251592 to provide a carboxy-bearing intermediate, or may be reacted with an isocyanate bifunctional spacer, as shown in Scheme 4, according to the Annunziato reference provided elsewhere in this disclosure to provide a hapten that bears a thiol-reactive functional linker. Deprotection as described in subsequent examples is required when R1 is a silyl-protected alcohol.
[0165]  [see pdf for image]
[0166]  [see pdf for image]
[0167]  [see pdf for image]
[0168] Scheme 5 illustrates how haptens with spacers which terminate in an alkyl amine group, such as Examples 1 and 2, may be further functionalized with a maleimide group. The maleimide may be introduced by any method known in the art. For example, reaction with N-maleoyl-substituted alkyl amino acid in a solvent such as dichlorormethane and coupling reagents such as diisopropylethylamine and diethyl cyanophosphonate gives the maleimide functionalized spacer on the hapten. Reaction of the risperidone derived amine with alkyl-maleimide functionalizing group, such as 2,5-dioxopyrrolidin-1-yl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetate, in a solvent such as DMF, in the presence of a base, such as tributylamine, at room temperature for about an hour, generates haptens with a maleimide functionalized spacer.
[0169]  [see pdf for image]
[0170] Haptens with spacers which terminate in an alkyl amine group may be elaborated by reaction with a cyclic anhydride compound, such as succinic anhydride or glutaric anhydride, as shown in Scheme 6. The reaction may be carried out in a solvent such as THF, at room temperature, overnight.
[0171]  [see pdf for image]
[0172] Maleimide functionalized haptens may be conjugated to proteins according to the method shown in Scheme 7. 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 5) 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.
[0173]  [see pdf for image]
[0174] Carboxylic acid functionalized haptens may be conjugated to proteins according to the method shown in Scheme 8. Reaction with N-hydroxysuccinimide and a suitable coupling agent, such as dicyclohexylcarbodiimide (DCC), and a base, such as tributylamine, in a solvent such as DMF at a temperature of about 20° C., for about 18 hours, activates the carboxylic acid with the N-hydroxysuccinimide leaving group. The activated spacer and hapten may then be conjugated to a protein in a solvent such as 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.

Antibody Production

[0175] The conjugates above are useful for the production of antibodies which bind the anti-psychotic drug to which they were generated (risperidone). 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 paliperidone, quetiapine, olanzapine, 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.
[0176] 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 risperidone, cross-reactivity with risperidone metabolites such as 9-hydroxyrisperidone (paliperidone, which is also administered as an anti-psychotic drug), 7-hydroxyrisperidone, and N-dealkylrisperidone may or may not be desirable. An antibody that cross-reacts with risperidone and paliperidone may be desirable, which does not react with 7-hydroxyrisperidone or N-dealkylrisperidone, thus detecting risperidone and its major pharmacologically active metabolite. Alternatively, it may be desirable to detect the pharmacologically active metabolites, risperidone and paliperidone, separately, while still not detecting the inactive metabolites, 7-hydroxyrisperidone and N-dealkylrisperidone. 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.
[0177] 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.
[0178] 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).
[0179] 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.
[0180] 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.
[0181] 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.
[0182] 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.
[0183] 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.
[0184] 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.
[0185] 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.
[0186] 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.
[0187] 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

[0188] 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).
[0189] 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, risperidone, 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.
[0190] 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.
[0191] 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.
[0192] Copending applications, Application Nos. 61/691,450 filed Aug. 21, 2012 and 13/970,650 filed Aug. 20, 2013 entitled “Haptens of Aripiprazole”; Application Nos. 61/691,454 filed Aug. 21, 2012 and 13/970,653 filed Aug. 20, 2013 entitled “Haptens of Olanzapine”; Application Nos. 61/691,459 filed Aug. 21, 2012 and 13/970,660 filed Aug. 20, 2013 entitled “Haptens of Paliperidone”; Application No. 61/691,462 filed Aug. 21, 2012 and 13/970,667 filed Aug. 20, 2013 entitled “Haptens of Quctiapinc”; Application Nos. 61/691,469 filed Aug. 21, 2012 and 13/970,673 filed Aug. 20, 2013 entitled “Haptcas of Risperidone and Paliperidone”Application Nos. 61/691,544 filed Aug. 21, 2012 and 13/971,429 filed Aug. 20, 2013 entitled “Antibodies to Aripiprazole Haptens and Use Thereof”; Application Nos. 61/691,572 filed Aug. 21, 2012 and 13/971,519 filed Aug. 20, 2013 entitled “Antibodies to Olanzapine Haptens and Use Thereof”; Application Nos. 61/691,634 filed Aug. 21, 2012 and 13/971,387 filed Aug. 20, 2013 entitled “Antibodies to Paliperidone Haptens and Use Thereof”Application Nos. 61/691,59S filed Aug. 21, 2012 and 13/971,546 filed Aug. 20, 2013 entitled “Antibodies to Quctiapine Haptens and Use Thereof”; Application Nos. 61/691,615 filed Aug. 21, 2012 and 13/971,475 filed Aug. 20, 2013 entitled “Antibodies to Risperidone Haptens and Use Thereof”; Application Nos. 61/691,522 filed Aug. 21, 2012 and 13/971,448 filed Aug. 20, 2013 entitled “Antibodies to Aripiprazolc and Use Thereof”; Application Nos. 61/691,645 filed Aug. 21, 2012 and 13/971,536 filed Aug. 20, 2013 entitled “Antibodies to Olanzapine and Use Thereof”; Application Nos. 61/691,692 filed Aug. 21, 2012 and 13/971,416 filed Aug. 20, 2013 entitled “Antibodies to Paliperidone and Use Thereof”; Application Nos. 61/691,659 filed Aug. 21, 2012 and 13/971,562 filed Aug. 20, 2013 entitled “Antibodies to Quetiapine and Use Thereof”; and Application Nos. 61/691,675 filed Aug. 21, 2012 and 13/971,499 filed Aug. 20, 2013 entitled “Antibodies to Risperidone and Use Thereof”, all filed concurrently herewith are all incorporated herein by reference in their entireties.

EXAMPLES

[0193] 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

Step A

9-((tert-butyldimethylsilyl)oxy)-3-(2-chloroethyl)-2-methyl-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-4-one

[0194]  [see pdf for image]
[0195] A solution of 3-(2-chloroethyl)-9-hydroxy-2-methyl-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-4-one (1.0 g, 4.12 mmol) in DMF (5 mL) was treated with 1H-imidazole (701.24 mg, 64.66 mmol), followed by a solution of t-butyldimethylchlorosilane (683.12 mg, 4.53 mmol) in DMF (1 mL). After stirring for 18 h at room temperature, the solvents were removed under vacuum and the residue was taken up in dichloromethane/water (10 mL/10 mL) with addition of a spatula of potassium carbonate. The aqueous layer was extracted with dichloromethane (three times 10 mL). The combined organic fractions were dried over Na2SO4, filtered, and the solvent was removed under vacuum. The crude mixture was used without further purification in the next step. (ESI-MS (M+1) 357)

Step B

9-((tert-butyldimethylsilyl)oxy)-3-(2-(4-(6-hydroxybenzo[d]isoxazol-3-yl)piperidin-1-yl)ethyl)-2-methyl-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-4-one

[0196]  [see pdf for image]
[0197] A solution of 9-((tert-butyldimethylsilyl)oxy)-3-(2-chloroethyl)-2-methyl-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-4-one, prepared as described in the previous step, (0.5 g, 1.40 mmol) in methanol (25 mL) and diisopropylethylamine (732.83 μL, 4.20 mmol) was treated with 3-(piperidin-4-yl)benzo[d]isoxazol-6-ol hydrochloride salt (374.62 mg, 1.47 mmol), and the reaction mixture was stirred for 17 h at 60° C. under argon. Diisopropylethylamine (732.83 μL, 4.20 mmol) was added and the mixture was stirred additionally for 4 h at 60° C. The reaction mixture was evaporated under vacuum and the residue was taken up in water (25 mL), extracted with chloroform (3×25 mL). The combined organic layers were dried over MgSO4, filtered, and the solvent was removed under vacuum. The residue was purified by silica gel chromatography (elution with dichloromethane/methanol (98/2) to give the title compound (ESI-MS (M+1) 539).

Step C

tert-butyl (2-((3-(1-(2-(9-((tert-butyldimethylsilyl)oxy)-2-methyl-4-oxo-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-3-yl)ethyl)piperidin-4-yl)benzo[d]isoxazol-6-yl)oxy)ethyl)carbamate

[0198]  [see pdf for image]
[0199] A solution of 9-((tert-butyldimethylsilyl)oxy)-3-(2-(4-(6-hydroxybenzo[d]isoxazol-3-yl)piperidin-1-yl)ethyl)-2-methyl-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-4-one, prepared as described in the previous step, (50 mg, 0.093 mmol) in acetone (0.5 mL) and DMF (0.5 mL) was treated with potassium carbonate (33.3 mg, 0.24 mmol) and N-Boc-2-bromoaminoethane (27 mg, 0.12 mmol), and the reaction mixture was stirred for 17 h at 60° C. under argon. The reaction mixture was evaporated at 40° C. under reduced pressure and dissolved in water (10 mL) and extracted with dichloromethane (3×10 mL). The organic layers were combined, dried over Na2SO4, filtered, and the solvent was evaporated yield the crude title compound. (ESI-MS (M+1) 682)

Step D

3-(2-(4-(6-(2-aminoethoxy)benzo[d]isoxazol-3-yl)piperidin-1-yl)ethyl)-9-hydroxy-2-methyl-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-4-one

[0200]  [see pdf for image]
[0201] A solution of tert-butyl (2-((3-(1-(2-(9-((tert-butyldimethylsilyl)oxy)-2-methyl-4-oxo-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-3-yl)ethyl)piperidin-4-yl)benzo[d]isoxazol-6-yl)oxy)ethyl)carbamate, prepared as described in the previous step, (70 mg, 0.103 mmol) in HCl/isopropanol (10 mL, 5 N) was stirred for 1 h at 60° C. The reaction mixture was evaporated at 40° C. under reduced pressure and dissolved carefully in aqueous saturated sodium bicarbonate solution (5 mL) and extracted with dichloromethane (3×10 mL). The combined organic layers were dried over Na2SO4, filtered, and evaporated under reduced pressure at 40° C. The aqueous layer still contained product, which was recovered by evaporating the aqueous layer to dryness at 40° C. under reduced pressure. The resulting residue from the aqueous layer was re-dissolved in water and brought over a conditioned Waters Oasis SPE (6 cc) column and afterwards eluted with methanol. The methanol elution fraction was combined with the residue of the dichloromethane extraction and evaporated to dryness at 40° C. under reduced pressure to yield the title compound along with a side product (ESI-MS (M+1) 468; side product being 5% 9-hydroxy-3-(2-(4-(6-hydroxybenzo[d]isoxazol-3-yl)piperidin-1-yl)ethyl)-2-methyl-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-4-one (M+1) 425). The mixture was used in the next step without additional purification.

Example 2

2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(2-((3-(1-(2-(9-hydroxy-2-methyl-4-oxo-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-3-yl)ethyl)piperidin-4-yl)benzo[d]isoxazol-6-yl)oxy)ethyl)acetamide

[0202]  [see pdf for image]
[0203] To a solution of 3-(2-(4-(6-(2-aminoethoxy)benzo[d]isoxazol-3-yl)piperidin-1-yl)ethyl)-9-hydroxy-2-methyl-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-4-one, prepared as described in Example 1, (4.0 mg, 8.5 μmoles) in 215 μL of DMF and 4.3 μL of tributylamine was added 214 μL of a DMF solution of N-(α-maleimidoacetoxy) succinimide ester (AMAS, 10 mg/mL, 2.1 mg, 8.5 μmoles). The resulting solution was allowed to stir for 60 minutes at 20° C., then used as such in conjugation reaction with thiol-activated protein.

Example 3

Step A

3-(2-(4-(6-hydroxybenzo[d]isoxazol-3-yl)piperidin-1-yl)ethyl)-2-methyl-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-4-one

[0204]  [see pdf for image]
[0205] A solution of 3-(2-chloroethyl)-2-methyl-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-4-one (14.4 g, 0.05 mmol), 3-(piperidin-4-yl)benzo[d]isoxazol-6-ol (14.0 g, 0.05 mmol), sodium carbonate (16.0 g, 0.15 mmol) and potassium iodide (spatula point) in DMF (150 mL) was stirred for 5 h at 80° C. The mixture was allowed to cool down to room temperature and water was added. The precipitate was removed by filtration, and the filtrate was extracted with chloroform (3×100 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated. The residue was crystallized with isopropyl alcohol (70 mL), filtered, and washed with isopropanol/diisopropyl ether 50/50 mixture (10 mL). The residue was dried overnight at 100° C. yielding the title compound and was used without further purification in the next step.

Step B

3-(2-(4-(6-(2-aminoethoxy)benzo[d]isoxazol-3-yl)piperidin-1-yl)ethyl)-2-methyl-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-4-one

[0206]  [see pdf for image]
[0207] A solution of 3-(2-(4-(6-hydroxybenzo[d]isoxazol-3-yl)piperidin-1-yl)ethyl)-2-methyl-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-4-one, prepared as described in the previous step, (6.6 g, 0.015 mmol) in DMF (50 mL) and acetone (50 mL) was treated with potassium carbonate (3.0 g, 0.03 mmol) and ethyl (2-bromoethyl)carbamate (2.4 g, 0.015 mmol). After stirring overnight at 60° C., the reaction mixture was poured in water (150 mL), extracted with chloroform (3×100 mL). The combined organic layers were dried over Na2SO4, filtered, concentrated, and purified by silica gel chromatography (elution with dichloromethane/methanol (90/10). The combined fractions were treated with HBr (150 mL, 48%) and heated to reflux for 30 min. The mixture was allowed to cool down to room temperature and made basic with ammonium hydroxide (28% NH3 in H2O) and extracted with chloroform (3×100 mL). The combined organic layers were dried over Na2SO4, filtered, concentrated, and purified by silica gel chromatography (gradient elution with dichloromethane/methanol (90/10 to 50/50) resulting in a solid which was dissolved in isopropanol (50 mL) and treated with isopropanol/HCl. The precipitate was removed by filtration and washed with iPrOH/diisopropyl ether (50/50, 3×20 mL). The precipitate was dried under vacuum to yield the title compound ESI-MS (M+1) 452. 1H NMR (360 MHz, DMSO-d6) δ ppm 1.76-1.85 (m, 1H) 1.87-1.96 (m, 1H) 2.19 (d, J=12.81 Hz, 1H) 2.37-2.48 (m, 4H) 2.98-3.10 (m, 3H) 3.10-3.28 (m, 5H) 3.37-3.46 (m, 3H) 3.72 (d, J=11.34Hz, 3H) 3.79-3.85 (m, 2H) 4.31 (t, J=4.94Hz, 1H) 7.05 (dd, J=8.78, 1.83 Hz, 1H) 7.35-7.39 (m, 1H) 8.08 (d, J=8.78 Hz, 1H).

Example 4

2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(2-((3-(1-(2-(2-methyl-4-oxo-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-3-yl)ethyl)piperidin-4-yl)benzo[d]isoxazol-6-yl)oxy)ethyl)acetamide

[0208]  [see pdf for image]
[0209] To a solution of 3-(2-(4-(6-(2-aminoethoxy)benzo[d]isoxazol-3-yl)piperidin-1-yl)ethyl)-2-methyl-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-4-one, prepared as described in Example 3, (3.4 mg, 7.58 μmoles) in 185 μL of DMF and 3.7 μL of tributylamine was added 190 μL of a DMF solution of N-(α-maleimidoacetoxy) succinimide ester (AMAS, 10 mg/mL, 1.9 mg, 7.58 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

2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(2-((3-(1-(2-(9-hydroxy-2-methyl-4-oxo-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-3-yl)ethyl)piperidin-4-yl)benzo[d]isoxazol-6-yl)oxy)ethyl)acetamide-keyhole limpet hemocyanin-conjugate

Step A

[0210] To a 4.22 mL solution of keyhole limpet hemocyanin (KLH, 18.0 mg, 0.18 μmoles) in 100 mM phosphate buffer, 0.46M sodium chloride, at pH 7.4 was added 83.2 μL of a DMF solution of N-succinimidyl-S-acetylthioacetate (SATA, 25 mg/mL, 2.1 mg, 9.0 μmoles). The resulting solution was incubated at 20° C. for 1 hour 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

[0211] To 9.37 mL of KLH-SATA solution, prepared as described in Step A, (17.1 mg, 0.171 μmoles) was added 937 μL of 2.5 M hydroxylamine, 50 mM EDTA, at pH 7.0. The resulting solution was incubated at 20° C. for 40 minutes on a roller mixer. The reaction was used as such in conjugation reaction with maleimide-activated hapten.

Step C

[0212] To an aliquot of the resulting KLH-SH solution, prepared as described in Step B, (3.4 mL, 0.058 μmoles) was added an aliquot of 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(2-((3-(1-(2-(9-hydroxy-2-methyl-4-oxo-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-3-yl)ethyl)piperidin-4-yl)benzo[d]isoxazol-6-yl)oxy)ethyl)acetamide solution, prepared as described in Example 2, (282.8 μL, 5.0 μmoles). The resulting cloudy mixture was incubated for 3 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, 0.46M sodium chloride, at pH 7.4.

Example 6

2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(2-((3-(1-(2-(9-hydroxy-2-methyl-4-oxo-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-3-yl)ethyl)piperidin-4-yl)benzo[d]isoxazol-6-yl)oxy)ethyl)acetamide-bovine thyroglobulin-conjugate

Step A

[0213] To 1.0 mL of a solution of bovine thyroglobulin (BTG, 9.3 mg, 0.014 μmoles) in 100 mM phosphate buffer at pH 7.5 was added 132 μL of a DMF solution of N-succinimidyl-S-acetylthioacetate (SATA, 25 mg/mL, 3.3 mg, 14.1 μmoles). The resulting solution was incubated at 20° C. for 1 hour 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

[0214] To 2.11 mL of BTG-SATA solution, prepared as described in Step A, (7.4 mg, 0.011 μmoles) was added 211 μL of 2.5 M hydroxylamine, 50 mM EDTA, at pH 7.0. The resulting solution was incubated at 20° C. for 60 minutes on a roller mixer. The reaction was used as such in conjugation reaction with maleimide-activated hapten.

Step C

[0215] To an aliquot of the resulting BTG-SH solution, prepared as described in Step B, (2.3 mL, 0.011 μmoles) was added an aliquot of 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(2-((3-(1-(2-(9-hydroxy-2-methyl-4-oxo-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-3-yl)ethyl)piperidin-4-yl)benzo[d]isoxazol-6-yl)oxy)ethyl)acetamide solution, prepared as described in Example 2, (280.4 μL, 5.5 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, 0.14M sodium chloride, at pH 7.4.

Example 7

2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(2-((3-(1-(2-(2-methyl-4-oxo-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-3-yl)ethyl)piperidin-4-yl)benzo[d]isoxazol-6-yl)oxy)ethyl)acetamide-keyhole limpet hemocyanin-conjugate

[0216] To an aliquot of KLH-SH solution, prepared as described in Example 5 Step B, (1.5 mL, 0.025 μmoles) was added an aliquot of 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(2-((3-(1-(2-(2-methyl-4-oxo-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-3-yl)ethyl)piperidin-4-yl)benzo[d]isoxazol-6-yl)oxy)ethyl)acetamide solution, prepared as described in Example 4, (113 μL, 2.26 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, and 0.46M sodium chloride, at pH 7.4.

Example 8

2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(2-((3-(1-(2-(2-methyl-4-oxo-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-3-yl)ethyl)piperidin-4-yl)benzo[d]isoxazol-6-yl)oxy)ethyl)acetamide-bovine thyroglobulin-conjugate

[0217] To an aliquot of BTG-SH solution, prepared as described in Example 6 Step B, (0.63 mL, 0.0033 μmoles) was added an aliquot of 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(2-((3-(1-(2-(2-methyl-4-oxo-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-3-yl)ethyl)piperidin-4-yl)benzo[d]isoxazol-6-yl)oxy)ethyl)acetamide solution, prepared as described in Example 4 (80 μL, 1.6 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, and 0.14M sodium chloride, at pH 7.4.

Example 9

Competitive Immunoassay for Risperidone

[0218] Following a series of immunizations with risperidone 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). As shown in Table 2, reactivity of hybridomas 2A5 and 5G11 was seen.
[0219] 
[00008] [TABLE-US-00008]
  TABLE 1
 
  Dilution   1   2   3   4   5   6   7   8   9   10   11   12  
 
  400   1   5   14   39   41   47   58   62   67   72   76   Blank   Ag = Bt-
  1200                           Compound#1
  3600                          
  10800                          
  400   1   5   14   39   41   47   58   62   67   72   76    
  1200                          
  3600                          
  10800
 
  Dilution   1   2   3   4   5   6  
 
  400   3.2562   3.2897   3.3148   3.6038   0.6857   3.3976   Ag = Bt-
  1200   1.3591   1.4605   1.521   2.3063   0.1476   1.9245   Cmpd#1
  3600   0.3745   0.4617   0.3733   0.7613   0.038   0.6163  
  10800   0.0918   0.1149   0.0908   0.1919   0.0156   0.1834
 
  Dilution   7   8   9   10   11   12  
 
  400   1.3444   2.8639   0.5676   3.5993   2.5144   0.0143   Ag = Bt-
  1200   0.2841   1.0387   0.1158   2.6921   0.8711   0.0142   Cmpd#1
  3600   0.0689   0.2742   0.0304   0.9549   0.2236   0.0115  
  10800   0.0199   0.0639   0.013   0.2766   0.056   0.0099
 
  Dilution   1   2   3   4   5   6  
 
  400   3.1217   3.1103   3.1532   3.633   0.6089   3.5705   Ag = Bt-
  1200   1.2607   1.4817   1.3412   2.1411   0.1327   1.9831   Cmpd#1
  3600   0.3281   0.4159   0.3819   0.7373   0.0361   0.593  
  10800   0.0879   0.1127   0.0929   0.1949   0.0156   0.189
 
  Dilution   7   8   9   10   11   12  
 
  400   1.1067   2.4001   0.4963   3.4172   2.2432   0.0095   Ag = Bt-
  1200   0.2691   0.961   0.1027   2.5321   0.7418   0.0098   Cmpd#1
  3600   0.0723   0.292   0.0284   0.8426   0.2024   0.0079  
  10800   0.0229   0.0722   0.0141   0.2393   0.052   0.0086
 
[0220] 
[00009] [TABLE-US-00009]
  TABLE 2
 
  Plate 1
    Dilution   1   2   3
 
    neat   Blank   1C4   6 E6
    neat     2A5   7A7
    neat     2G10   Empty
    neat     3B7  
    neat     4D8  
    neat     5A12  
    neat     5G11  
    neat     6C1  
    neat   0.0072   0.038    0.0309
    neat   0.0077   3.9563   0.1163
    neat   0.0069   0.0093   0.0086
    neat   0.0076   0.0753   0.0108
    neat   0.0114   0.1139   0.0084
    neat   0.009    0.0193   0.0123
    neat   0.0087   0.2503   0.0085
    neat   0.0092   0.086    0.0121
 
[0221] After clones were identified via ELISA reactivity, competition ELISAs were run to approximate affinity and cross-reactivity with similar compounds. FIGS. 1 and 2 show the ELISA cross-reactivity results from hybridoma subclone 59. Data shows reactivity to risperidone, as well as its metabolites paliperidone and 7-hydroxyrisperidone.
[0222] Supernatants were also tested by competition ELISA to determine if the signals were specific to either risperidone or paliperidone. FIG. 3 shows the results from hybridoma subclone 2A5. Data shows reactivity to both risperidone and paliperidone. FIG. 4 shows the competitive immunoassay format used on a lateral flow assay device in which the capture antibody, risperidone clone 5-9, was deposited on a chip along with a detection conjugate consisting of risperidone conjugated to a fluorophore. In this competitive format as show in FIG. 4, a low level of analyte (risperidone) results in high signal, whereas a high level of analyte (risperidone) results in low signal. The amount of risperidone 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 risperidone clone 5-9 is shown in FIG. 5.
(57)

Claims

1. The compound of Formula I
[see pdf for image]
wherein:
R1 is H, or OH;
R2 is O(CH2)rNH2,
[see pdf for image]
O(CH2)rNHC(O)(CH2)mCO2H, or Z—(Y)p-G;
wherein:
Z is selected from the group consisting of:
—N(R4)—, —O—, —S—, -heteroalkyl-;
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;
r is 1, 2, 3, 4, or 5;
m is 1, 2, 3, 4, or 5;
n is 1, 2, 3, 4, or 5.
2. The compound of claim 1
wherein:
R1 is H, or OH;
R2 is O(CH2)rNH2,
[see pdf for image]
O(CH2)rNHC(O)(CH2)mCO2H, or Z—(Y)p-G;
wherein:
Z is O;
Y is an organic spacer group;
G is a functional linking group capable of binding to a carrier;
p is 0, or 1;
r is 1, 2, 3, 4, or 5;
m is 1, 2, 3, 4, or 5;
n is 1, 2, 3, 4, or 5.
3. The compound of claim 1
wherein:
R1 is H, or OH;
R2 is O(CH2)rNH2,
[see pdf for image]
O(CH2)rNHC(O)(CH2)mCO2H, or Z—(Y)p-G;
wherein:
Z is O(CH2)rNH;
Y is an organic spacer group;
G is a functional linking group capable of binding to a carrier;
p is 0, or 1;
r is 1, 2, 3, 4, or 5;
m is 1, 2, 3, 4, or 5;
n is 1, 2, 3, 4, or 5.
4. The compound of claim 3
wherein:
p is 1;
r is 2,
m is 1, 2, 3, 4, or 5;
n is 1, 2, 3, 4, or 5.
5. The compound of claim 3
wherein
R1 is H, or OH;
R2 is O(CH2)rNH2,
[see pdf for image]
or
O(CH2)rNHC(O)(CH2)mCO2H;
wherein:
r is 2;
m is 1, 2, 3, or 4; and
n is 1, or 2.
6. The compound of claim 5, wherein
R1 is H, or OH; and
R2 is O(CH2)rNH2, or
[see pdf for image]
r is 2;
m is 1.
7. The compound of claim 1, selected from the group consisting of
[see pdf for image]
8. A conjugate of a compound of claim 1 and an immunogenic carrier.
9. The conjugate of claim 8, where said immunogenic carrier is a protein.
10. The conjugate of claim 9, where said protein is keyhole limpet hemocyanin, ovalbumin or bovine thyroglobulin.
11. A conjugate of the compound of claim 2 and an immunogenic carrier.
12. A conjugate of the compound of claim 3 and an immunogenic carrier.
13. A conjugate of the compound of claim 4 and an immunogenic carrier.
14. A conjugate of the compound of claim 5 and an immunogenic carrier.
15. A conjugate of the compound of claim 6 and an immunogenic carrier.
16. The conjugate of claim 8 wherein the compound is
[see pdf for image]
17. The conjugate of claim 16, where said immunogenic carrier is a protein.
18. The conjugate of claim 17, where said protein is keyhole limpet hemocyanin, ovalbumin or bovine thyroglobulin.
*****

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