HK1134498A

HK1134498A - Saquinavir derivatives useful in immunoassay

Info

Publication number: HK1134498A
Application number: HK09111765.1A
Authority: HK
Inventors: Raymond A. Hui; Gerald F. Sigler; Richard T. Root; Wie Yuan
Original assignee: F. Hoffmann-La Roche Ag
Priority date: 2004-12-10
Filing date: 2005-12-08
Publication date: 2010-04-30

Description

Saquinavir derivatives for immunoassay

Technical Field

The present invention relates to novel protease inhibitor derivatives for use in immunoassays. More particularly, the present invention relates to novel derivatives useful for generating immunogens to the HIV protease inhibitor saquinavir and to novel immunogens useful for the preparation of antibodies to saquinavir useful in immunoassays for the determination of saquinavir in biological samples.

Background

HIV protease inhibitors are an important new class of drugs that have had a significant impact on AIDS patient health care since the first such drug, saquinavir, was marketed in 1995. Examples of other protease inhibitors include amprenavir, nelfinavir, lopinavir, ritonavir, and atazanavir. They are particularly effective in combination with other anti-HIV drugs such as reverse transcriptase inhibitors or with other HIV protease inhibitors. Despite the significant success achieved with these new treatment regimens, there is strong evidence that the effect would be much improved if therapeutic drug detection methods were available to detect protease inhibitor concentrations. Not all patients respond optimally to protease inhibitor combination therapy. Even those who respond may subsequently develop resistance due to the very high rate of mutation of the HIV virus. However, it has been shown that there is a clear relationship between plasma levels of protease inhibitors and therapeutic efficacy based on reduced viral load and increased CD4 cell numbers. One problem that exists is that the drugs are extensively metabolized and are subject to complex drug-drug interactions. The result is a very complex pharmacokinetics and the presence of an unpredictable, strong factor between dose and resulting drug level at any particular time for any particular patient. With therapeutic drug monitoring, drug dosages can vary from patient to patient and the likelihood of maintaining viral control will be higher. Conventional therapeutic drug monitoring of protease inhibitors requires the use of simple automated assays suitable for high throughput clinical analyzers. Currently, most reports on the monitoring of protease inhibitor therapeutic agents employ HPLC methods, which are slow, labor intensive, and expensive. There have been recent reports of Radioimmunoassay (RIA) methods for saquinavir (Wiltshire et al, Analytical Biochemistry281, 105-. However, such methods are not suitable for high-throughput therapeutic drug monitoring and, like all RIA methods, suffer from regulatory, safety and waste disposal issues related to the radioisotope label used in the assay. The most desirable assay format for therapeutic drug monitoring is the non-isotopic immunoassay, and such methods have not been reported to date for monitoring HIV protease inhibitors.

Saquinavir was developed by Hoffmann LaRoche and approved for use in 1995 under the trade name Invirase. Currently, a more recent form of saquinavir is marketed under the trade name Fortovase, which is better absorbed by the body and thus has a more potent anti-HIV effect than Invirase.

As noted above, HPLC has been the method of choice for monitoring HIV protease inhibitors. Two recent reports in the literature describe HPLC detection methods for the simultaneous determination of protease inhibitors in human plasma, Poirier et al, Therapeutic Drug Monitoring22, 465-473, 2000 and Remmel et al, Clinical Chemistry 46, 73-81, 2000.

Chemical and biological detection methods typically involve contacting an analyte of interest with a predetermined amount of one or more detection reagents, measuring one or more properties of the resulting product (detection product), and correlating the measured value with the amount of analyte present in the original sample, typically by using a relationship determined from a standard or standard sample containing a known amount of the analyte of interest within a desired range of the sample to be detected. Typically, the detection product incorporates one or more detectable labels, which are provided by one or more detection reagents. Examples of commonly used labels include functionalized microparticles, radioisotope labels such as¹²⁵I and³²p, enzymes such as peroxidase and beta-galactosidase and enzyme substrate labels, fluorescent labels such as fluorescein and rhodamine, electron spin resonance labels such as nitroxide radicals, immunologically active labels such as antibodies and antigens, labels that are one member of a binding pair such as biotin-avidin and biotin-streptavidin, and electrochemiluminescent labels such as those comprising a ruthenium bipyridine moiety. Sandwich assays (Sandwich assays) generally involve the formation of a complex in which an analyte of interest is sandwiched between one detection reagent, e.g., an antibody, antigen or one of the members of a binding pair, that is ultimately used for separation, and a second detection reagent that provides a detectable label. Competitive assays (competitive assays) generally involve a system in which both an analyte of interest and an analog of the analyte compete for a binding site on another reagent, such as an antibody, wherein one of the analyte, analog or binding reagent has a detectable label.

Co-pending U.S. patent application serial No. 09/712,525, filed on EP 1207394 at 11/14/2000, having the same patent assignee as the present application and published on 22/5/2002, describes a non-isotopic immunoassay for HIV protease inhibitors comprising culturing a sample comprising the inhibitor with a receptor specific for the inhibitor or for a metabolite of the inhibitor and further with a conjugate comprising an analog of the inhibitor and a non-isotopic signal generating moiety. Determining the signal generated as a result of the binding of the receptor to the inhibitor and correlating it to the presence or amount of protease inhibitor in the original sample. The protease inhibitor conjugates of the invention are particularly useful for such assays.

Co-pending U.S. patent application serial No. 10/192,052, having the same patent assignee as the present application and filed on 23/1/2003 with 2002, 7/10, published as WO03/006506, describes derivatives of saquinavir derived from the central hydroxyl group of the molecule, which when used as a marker or as a hapten for forming an immunogen, produce drug conjugates and antibodies that allow for the formation of immunoassays with good dose-response curves for saquinavir. However, high cross-reactivity to saquinavir metabolites was seen.

Among other problems, there remains a need for improved activated haptens, derivatives and conjugates of the HIV protease inhibitor saquinavir, which can be used in immunoassays for determination of saquinavir in biological samples. There is also a need for immunogens that allow the development of antibodies specific for saquinavir with low cross-reactivity to saquinavir metabolites. The present invention addresses these and other issues.

Summary of The Invention

Against the above background, the present invention provides advantages and advances over the prior art that are not obvious. In particular, the present inventors have recognized a need for improvements in saquinavir derivatives for use in immunoassays.

The present invention relates to saquinavir derivatives having the structure:

wherein X is a heterocyclic aromatic structure, L is a linking group comprising 0 to 40 carbon atoms in a linear or branched arrangement, is saturated or unsaturated and comprises at most two ring structures and 0-20 heteroatoms, with the proviso that at most two heteroatoms may be connected in sequence, and a is an activated functional group selected from the group consisting of active esters, isocyanates, isothiocyanates, thiols, imidates, anhydrides, maleimides, thiolactones (thiolactones), diazonium groups and aldehydes.

In one embodiment of the invention, X is selected from the group consisting of a pyridine ring structure and a quinoline ring structure.

Also within the scope of the present invention is a process for preparing saquinavir derivatives having the structure:

One embodiment of the present invention includes a pyridyl analog of saquinavir having the structure:

wherein A and L are as defined above.

Another embodiment of the present invention includes derivatives of saquinavir having the structure:

wherein A and L are as defined above.

In a preferred embodiment of the invention, a is an active ester.

The present invention also relates to coupled derivatives of saquinavir having the structure:

wherein X is a heterocyclic aromatic structure, L is a linking group comprising 0 to 40 carbon atoms in a linear or branched arrangement, is saturated or unsaturated and comprises at most two ring structures and 0-20 heteroatoms, with the proviso that at most two heteroatoms may be linked in sequence, and Q is selected from the group consisting of polypeptides, polysaccharides, synthetic polymers and non-isotopic labels, and Q is a number per kilodalton molecular weight of 1 to 50.

In one embodiment of the invention, n is a number from 1 to 20 per kilodalton molecular weight of Q, more preferably a number from 1 to 10 per kilodalton molecular weight of Q, most preferably a number from 1 to 5 per kilodalton molecular weight of Q.

In another embodiment of the invention, Q is selected from the group consisting of bovine serum albumin, keyhole limpet hemocyanin, and aminodextran (aminodextran).

Also within the scope of the present invention is a process for preparing saquinavir conjugate derivatives having the structure:

One embodiment of the present invention includes a conjugated derivative of saquinavir having the structure:

wherein L and Q are as defined above.

Another embodiment of the present invention includes coupled derivatives of saquinavir having the structure:

wherein L and Q are as defined above.

Another aspect of the invention includes antibodies generated in response to an immunogen having the structure:

wherein X is a heterocyclic aromatic structure, L is a linking group comprising 0 to 40 carbon atoms in a linear or branched arrangement, is saturated or unsaturated and comprises at most two ring structures and 0-20 heteroatoms, with the proviso that at most two heteroatoms may be linked in sequence, P is a polypeptide, and n is a number of 1 to 50 per kilodalton molecular weight of P.

Methods of preparing antibodies generated in response to an immunogen having the structure:

One embodiment of the invention includes antibodies generated in response to an immunogen having the structure:

another embodiment of the invention includes an antibody produced in response to an immunogen having the structure:

monoclonal antibodies specific for saquinavir are also within the scope of the invention having less than 1% cross-reactivity with nelfinavir and with saquinavir metabolites M4 and M6.

The invention also relates to murine hybridoma SAQ 137.3.3 having ATCC number PTA-6329.

These and other features and advantages of the present invention will be more fully understood from the following detailed description of the invention taken together with the accompanying claims. It is noted that the scope of the claims is defined by the recitations therein and not by the specific discussion of features and advantages set forth in the present description.

Brief description of the drawings

The following detailed description of embodiments of the present invention can be best understood when read in conjunction with the following drawings, where like structure is indicated with like reference numerals and in which:

FIG. 1 shows compounds as described in examples 1-75a、8aAnd8bschematic diagram of the synthesis method of (1).

FIG. 2 shows compounds as described in examples 8-1012Schematic diagram of the synthesis method of (1).

FIG. 3 shows compounds as described in examples 11 and 1215Schematic diagram of the synthesis method of (1).

FIG. 4 shows compounds as described in examples 13 and 1418Schematic diagram of the synthesis method of (1).

FIG. 5 shows a compound as described in example 1519Schematic diagram of the synthesis method of (1).

FIG. 6 shows compounds as described in examples 16-1824Schematic diagram of the synthesis method of (1).

FIG. 7 is a schematic representation of saquinavir-KLH conjugate as described in example 1925And saquinavir-BSA conjugate as described in example 2026Schematic diagram of the synthesis method of (1).

FIG. 8 is a schematic representation of saquinavir-BSA conjugate as described in example 2127Schematic diagram of the synthesis method of (1).

FIG. 9 is a schematic view showingCompounds described in examples 24 and 2530Schematic diagram of the synthesis method of (1).

FIG. 10 shows a compound as described in examples 26-2833Schematic diagram of the synthesis method of (1).

FIG. 11 shows compounds as described in examples 29-3139Schematic diagram of the synthesis method of (1).

Detailed Description

It is noted that terms like "preferably," "commonly," and "generally" are not utilized herein to limit the scope of the claimed invention or to imply that certain features are critical, essential, or even important to the structure or function of the claimed invention. Rather, these terms are merely intended to highlight alternative or additional features that may or may not be utilized in a particular embodiment of the present invention.

For the purposes of describing and defining the present invention it is noted that the term "substantially" is utilized herein to represent the inherent degree of uncertainty that may be attributed to any quantitative comparison, value, measurement, or other representation. The term "substantially" is also utilized herein to represent the degree by which a quantitative representation may vary from a stated reference without resulting in a change in the basic function of the subject matter at issue.

Having described the invention in detail and by reference to specific embodiments thereof, it will be apparent that modifications and variations are possible without departing from the scope of the invention defined in the appended claims. More specifically, although some aspects of the present invention are identified herein as preferred or particularly advantageous, it is contemplated that the present invention is not necessarily limited to these preferred aspects of the invention.

Saquinavir is represented by the following molecular structure:

wherein L is a linking group comprising from 0 to 40 carbon atoms in a linear or branched arrangement, is saturated or unsaturated and comprises up to two ring structures and from 0 to 20 heteroatoms, with the proviso that up to two heteroatoms can be attached in sequence, and a is an activated functional group selected from the group consisting of active esters, isocyanates, isothiocyanates, thiols, imidates, anhydrides, maleimides, thiolactones, diazonium groups and aldehydes.

In one embodiment of the invention, the compound has the structure

In another embodiment, the compound is a conjugate of succinimidyl-oxycarbonyl-ethylamino-glycyl-glutaryl-aminomethyl- (pyr) saquinavir with KLH (25)。

In another embodiment, the compound is a conjugate of succinimidyl-oxycarbonyl-ethylamino-glycyl-glutaryl-aminomethyl- (pyr) saquinavir with BSA: (26)。

In another embodiment, the compound is a conjugate of succinimidyl-benzoyl-aminocaproyl-aminomethyl- (pyr) saquinavir with BSA (b)27)。

wherein L is a linking group comprising 0 to 40 carbon atoms in a linear or branched arrangement, saturated or unsaturated and comprising up to two ring structures and 0-20 heteroatoms, with the proviso that up to two heteroatoms may be linked sequentially, and Q is selected from the group consisting of polypeptides, polysaccharides, synthetic polymers and non-isotopic labels, and n is a number per kilodalton molecular weight of from 1 to 50.

Another embodiment of the invention includes an antibody derived from an immunogen having the structure:

wherein L is a linking group comprising 0 to 40 carbon atoms in a linear or branched arrangement, is saturated or unsaturated and comprises up to two ring structures and 0-20 heteroatoms, and with the proviso that up to two heteroatoms can be connected in sequence, P is a polypeptide, and n is a number of 1 to 50 per kilodalton molecular weight of P.

Another embodiment of the present invention includes the derivative saquinavir having the structure:

Another embodiment of the present invention includes coupled derivatives having the structure:

Throughout this specification, bold and underlined type numbers are used to refer to chemical structural formulas illustrated in the drawings.

As used herein, "analyte" refers to a substance or group of substances, the presence or amount of which is to be determined.

"antibody" refers to a specific binding partner for the analyte and is any substance or group of substances that has a specific binding affinity for the analyte to substantially exclude other unrelated substances. The term includes polyclonal antibodies, monoclonal antibodies, and antibody fragments.

Haptens are partial or incomplete antigens. They are protein-free substances, mainly low molecular weight substances, which do not stimulate antibody production, but which react with antibodies. The latter is formed by coupling the hapten to a high molecular weight carrier and injecting the coupled product into a human or animal. Examples of haptens include therapeutic drugs such as digoxin and theophylline, drugs of abuse such as morphine and LSD, antibiotics such as gentamicin and vancomycin, hormones such as estrogen and progesterone, vitamins such as vitamin B12 and folic acid, thyroxine, histamine, serotonin, epinephrine, and others.

Activated hapten means a hapten derivative that has been provided with available reactive sites, for example by attachment or assembly of activated groups for synthetically derivatized conjugates.

The term "linker" refers to a chemical moiety that links the hapten to a carrier, immunogen, label, tracer or another linker. The linker may be a straight or branched, saturated or unsaturated carbon chain. They may also contain one or more heteroatoms within or at the end of the chain. Heteroatom means an atom other than carbon, selected from oxygen, nitrogen and sulfur. The use of a linker may or may not be beneficial or desirable depending on the particular hapten and carrier pair.

When "carrier" is used herein as a term, it is an immunogenic substance, usually a protein, which can be conjugated to a hapten, thereby enabling the hapten to stimulate an immune response. Carrier materials include proteins, glycoproteins, complex polysaccharides and nucleic acids which are recognized as foreign and thereby elicit an immune response in the host.

As used herein, the terms "immunogen" and "immunogenic" refer to a substance that is capable of producing or generating an immune response in an organism.

The terms "conjugate" and "derivative" refer to a compound or molecule that is prepared from a parent compound or molecule by one or more chemical reactions.

As used herein, a detector molecule, label or tracer is an identifying label, when usedWhen attached to a carrier substance or molecule, can be used to detect an analyte. The label may be directly or indirectly attached to its carrier substance by means of a linking or bridging moiety. Examples of labels include enzymes such as beta-galactosidase and peroxidase, fluorescent compounds such as rhodamine and Fluorescein Isothiocyanate (FITC), luminescent compounds such as dioxetanes and luciferin, and radioisotopes such as radioisotopes¹²⁵I。

The term "active ester" within the meaning of the present invention includes activated ester groups which can react with free amino groups of nucleophiles such as, but not limited to, peptides, polyamino acids, polysaccharides or labels under conditions effective to occur without interfering side reactions with other reactive groups of the species carried by the nucleophile.

In general, the compounds of the invention may be prepared by coupling of the saquinavir moiety of the precursor moiety, as exemplified by amine intermediate 14 (see, e.g., EP 1207394) and as shown in figures 3, 5, 9 and 11, with an appropriately substituted pyridine or quinoline intermediate, any of which also carries a moiety capable of readily reacting with said intermediate14Or an activated form thereof. For example, compounds of formula I can be prepared by reacting intermediates14Coupled with a pyridine intermediate substituted firstly at the ortho position to the ring nitrogen by a carboxylic ester or activated carboxylic ester group and secondly by another group G. The coupling may be achieved by means of a suitable coupling agent, such as but not limited to carbodiimide, when the coupling is carried out with carboxylic acids. Alternatively, the active ester may be derived from a carboxylic acid and a permissible or intermediate14Ester formation by reaction of the amine of (a). The second substituent group G of the pyridine may be located in any other free position of the ring nucleus and may comprise carbon atoms or heteroatoms or both in a linear or branched arrangement, as long as it carries a suitable activated functional group itself, or may be converted to its active formCan be used to react with other suitable moieties to produce the entire linker L carrying the final reactive functional group a, as shown in figure 1. This method can also be advantageously described as linker extension and many variations of the method are well known to those skilled in the art. In most cases, the group G may comprise the linker L with an already present activated functional group a or masked equivalent, e.g., an alkyl ester, which may then be hydrolyzed to an acid and converted to an active ester using methods well known in the art. In a similar manner, intermediates can be prepared14Coupling with a quinoline intermediate substituted in the same manner as described above for the pyridine intermediate gives compounds of structure III.

Preferred groups G are those that can be converted, e.g., to an amine or thiol functional group that exceeds the central hydroxyl group of the core structure of saquinavir or saquinavir-pyridyl analog, preferentially reacting with an additional suitable bifunctional moiety. For example, amines and especially alkylamines will be preferentially at the central hydroxyl group but have greater reactivity with the active ester or haloalkyl moiety, and coupling of the amine to the bifunctional moiety can be achieved with minimal reaction with the central hydroxyl group. Particularly preferred are alkylamino groups and their protected equivalents, e.g. aminomethyl groups and tertiary BOC-protected aminomethyl groups, e.g. compounds8aAnd8band the compounds obtained subsequently15. Examples of preferred bifunctional moieties that can be used to extend the linker moiety include aminoalkanoic esters, such as aminocaproic esters in which the amino group is masked by a protecting group that can be easily removed under mild conditions; or a short peptide, such as glycyl- β -alanine as exemplified in figure 7; or an alkanedioic acid chloride active ester, such as the compound of FIG. 1034The glutarate linker exemplified; or aryl diacid (aryldioic acid) active esters, e.g. the compounds of FIG. 628As exemplified; or peptide-alkanoate moieties, e.g. the compound of FIG. 412As illustrated.

In most cases, greater steric hindrance at the central hydroxyl group is sufficient to allow useful reactions to occur on the group G without protecting the central hydroxyl group. In some cases, such as when group G is hydroxy or hydroxyalkyl, where reactivity can be expected to be about the same as the central hydroxy group of the saquinavir core moiety, selectivity can be obtained by protecting the central hydroxy group, for example as benzoyl ester or trichloroacetate or the like, the linker is then extended by reaction with a suitable bifunctional moiety, such as a moiety containing an acyl chloride to form an ester linkage, or a moiety containing an isocyanate to form a carbamate linkage, followed by deprotection of the central hydroxy group of the saquinavir or saquinavir-pyridyl core moiety, prior to deprotection of the hydroxy or hydroxyalkyl group in group G.

The sequence of such protection and deprotection steps is well known to those skilled in the art and many examples of such steps and other suitable protecting groups for amines, hydroxyls and thiols can be found in the relevant literature, for example in "Protective groups in Organic Synthesis", second edition, T.Greene & P.Wuts, Wiley-Interscience, 1991. Protecting groups which are removed under mild basic or acidic conditions are preferred so as not to affect the integrity of the bonds or other moieties in the saquinavir or saquinavir-pyridyl group. An example of an N-protecting group that is removed under mild basic conditions is Fluorenylmethoxycarbonyl (FMOC). An example of an N-protecting group that is readily removed with an acid is t-Butyloxycarbonyl (BOC). Many other suitable N-protecting groups as well as O-protecting groups and S-protecting groups are well known in the art and are exemplified by Greene & Wuts.

For example, as shown in FIG. 1, compounds8aCarrying a masked amine function corresponding to the group G as described above, and a carboxylic acid group. Coupling to said intermediate14Followed by unmasking of the amine contained in G and then providing the compound15Then it is mixed with anotherA suitable moiety-in this case a protected peptide12-reaction to give a compound of structure I bearing the entire linker L with a protected or masked terminal reactive functional group, which is then itself unmasked and converted into a reactive functional group a using methods well known to those skilled in the art and as exemplified in figures 3 and 4 and examples 15 to 18. Other non-limiting examples of groups G may include haloalkyl, cyanoalkyl, alkylcarboxylate esters, or other groups bearing an alkyl or aryl group, such as a group selected from a.

Another example is a compound in which the group G is contained and15the exemplified aminomethyl or aminoalkyl groups may be reacted with a bifunctional moiety, such as an aminocaproate ester active ester in which the amine is protected, followed by deprotection of the terminal amine and reaction with another bifunctional moiety, such as di- (N-hydroxysuccinimide) phthalate diester, to form an extended linker L bearing a terminal reactive functional group A28And as compounds24As illustrated and as shown in figure 6 and in embodiments 16-18. Other suitable functional groups will be readily suggested by those skilled in the art. By way of further illustration, the haloalkyl group G can be reacted with an amine-containing moiety bearing an additional functional group, such as an alkyl ester, thereby forming an extended linker comprising L and an ester and the ester is hydrolyzed to an acid and converted to an active ester to form a. The cyanoalkyl group may be hydrogenated to form an amine, or hydrolyzed to an imidoester (iminoester), which may then be reacted with additional moieties such as the amine-alkyl ester moieties described in the previous sentence and transferred to the compound comprising L and a. Another example of a group G may be a nitro group which may then be reduced to an amine which may then be reacted with a suitable difunctional moiety, for example comprising an acyl group, under reducing conditions, for example in the presence of a borohydride, to provide an intermediate with an extended linkerA chloro group, or an alkyl halide wherein the halogen is bromine or iodine, or a bifunctional moiety of an aldehyde. Yet another example of group G may include a masked thiol, such as an acetylthioalkyl group, which is then deprotected under mild basic conditions and the free thiol is reacted with a bifunctional moiety comprising a maleimide or another thiol-reactive functional group to provide an extended linker. Alternatively, G may comprise a maleimido group, which may then be reacted with another bifunctional moiety comprising a thiol to provide an extended linker. Still further examples may include a hydroxy or hydroxyalkyl group which may be protected by a suitable group such as a t-butyldimethylsilyl group or the like, which is then deprotected, and then coupled to another bifunctional moiety such as one comprising an acid chloride, to provide an extended linker. Other suitable transformations and couplings will be apparent to those skilled in the art.

Still further examples of such transformations can be seen in FIGS. 9 and 10, wherein the nitro-containing group G is present in a quinoline derivative which is coupled to a compound14Thereby forming compound 29. The nitro group is then reduced to an amine which is then reacted with a suitable difunctional moiety such as glutaryl chloride N-hydroxysuccinimide ester to form a compound comprising a compound of formula III bearing a linker L having a reactive functional group A31. As also illustrated in FIG. 10, such compounds may be extended with yet another bifunctional linker, such as the peptide shown, followed by conversion of the carboxylate ester to an active ester at the terminus, thereby providing the compound33Also included are compounds of structural formula III bearing a linker L having a reactive functional group a, as shown in fig. 10.

The linker L serves the purpose of providing additional spacing between the terminally activated functional group a and saquinavir or a saquinavir-pyridyl analog moiety. It is well known to those skilled in the art that linker length and composition have an effect on the immunogenic response and the performance of the conjugate. There are many examples of commercially available or readily synthesized linkers in the literature that can be used in this case to extend the group G to provide the final linker L. For a good discussion of this problem, the reader is referred to Bioconjugate Techniques, G.Hermanson, Academic Press, 1996. In some cases, linker L may be omitted and the saquinavir or saquinavir-pyridyl analog moiety is directly linked to activated functional group a.

The compounds of formulae I or III can then be coupled via their active functional groups a to a detector molecule or label, for example an enzyme such as galactosidase or peroxidase, or to a fluorescent label such as fluorescein or rhodamine or Cy-5 or the like, or to a luminescent label such as fluorescein or the like, or to a polysaccharide such as aminodextran or the like, or to other synthetic polymers such as polylysine or the like, to provide compounds of formulae II and IV. In addition, these compounds can be coupled via their reactive functional groups A to other carrying radioisotopes such as¹²⁵I or the like, to provide compounds similar to structural formulae II and IV but now having isotopic labels. Typically, such coupling is achieved by using complementary reactive functional groups, such as the reaction of the amino group of lysine of the enzyme with a compound in which a is an active ester to form a stable amide bond, or with a compound in which a is an isothiocyanate or an isocyanate to form the corresponding thiourea or urea bond. Other suitable combinations will be apparent to those skilled in the art. These reactions can generally be carried out under relatively mild conditions, for example in a suitable organic solvent such as DMSO or DMF or the like with or without the addition of an aqueous component as a co-solvent, and at mild temperatures, typically from 0 ℃ to less than 100 ℃ and generally around room temperature.

Similarly, the compounds of structural formula I, which in the present case may also be designated activated haptens, can then be coupled via their active functional groups a to polypeptides, such as proteins, to provide compounds of structural formula V. In a similar manner, the compounds of structural formula III can be coupled to similar components to produce similar compounds as previously described. Typically, such coupling is achieved by using complementary reactive functional groups, such as reacting the amino group of a lysine of the protein with a compound in which a is an active ester to form a stable amide bond. Conjugation to polypeptides, particularly proteins that are well known as immunogen carrier materials, provides conjugates that are useful as immunogens. These immunogens are then used to immunize animals, such as mice, to obtain antibodies using methods well known in the art. The immunogen-carrier is generally a polypeptide or polysaccharide having a molecular weight greater than 10 kD. Preferred immunogenic carriers are polypeptides with a molecular weight greater than 100 kD. Examples of preferred carrier substances are Keyhole Limpet Hemocyanin (KLH), limulus hemocyanin (LPH) and Bovine Thyroglobulin (BTG). Other useful polypeptides include proteinases, such as Bovine Serum Albumin (BSA), which provide conjugates useful in screening assays, such as ELISA assays, or even as immunogens, depending on the amount of drug moiety conjugated to the polypeptide. The reaction between the activated hapten and the amino group on the support is generally carried out at room temperature in a buffered mixture of water and a water-soluble organic solvent for 0.5 to 5 days. Typically, the pH of the buffer is between 6 and 8 for active esters, isocyanates and isothiocyanates, or between 7 and 10 for imidates, and is adjusted according to the known reactivity of the carrier amino groups with the activated functional groups. In the case where the terminal group a is maleimide, the reactive group on the support is a thiol. These thiol groups either occur naturally to the carrier or may be introduced with a thiolating agent such as 2-IT or SATP. The optimum pH for maleimide coupling to a thiol group to give a thioether is typically between 5 and 7. After the reaction, the immunogen is dialyzed or subjected to size exclusion chromatography, thereby removing the unconjugated hapten and the organic solvent. An alternative method of obtaining an immunogen is to react an activated hapten, wherein a is an aldehyde, with an amino group of a carrier protein or polypeptide to form a schiff base, which is then reduced with a weak reducing agent such as cyanoborohydride to form a stable amine linkage. Variations on this last method aspect will also be suggested to those skilled in the art, and the invention is intended to fall within this field. Other suitable combinations will be apparent to those skilled in the art. Examples of coupling of compounds of structural formula I to immunogenic carrier materials can be found in FIGS. 7 and 8.

Detailed description of the preferred embodiments

In the following embodiments, bold-type, underlined numbers refer to corresponding structures in the drawings.

Flash chromatography was performed on silica gel 60 (230-. Thin layer chromatography was performed on silica gel plates (0.25mm, EM Science, Cat. # 5717-5) and observed under an ultraviolet lamp.

Solvents were obtained from j.t.baker Company unless otherwise noted. Ethyl acetate (EtOAc), hexane (hex), methanol (MeOH), and dichloromethane (CH)₂Cl₂) The obtained product was used for chromatography and reaction treatment. Dry CH is obtained by boiling over calcium hydride under argon and under reflux₂Cl₂. Dry Tetrahydrofuran (THF) was obtained by boiling over sodium-benzophenone under argon and under reflux. In Sure/Seal^TMDried Dimethylformamide (DMF) and dried Dimethylsulfoxide (DMSO) in bottles were obtained from Aldrich Chemical Company.

Unless otherwise noted, reagents and Chemicals were obtained from Sigma-Aldrich chemical company or from Fluka Chemicals.

Proton nuclear magnetic resonance spectra were obtained on a Varian Gemini 2000(200MHz) equipped with a Sun/spark workstation (S) ((S))¹H-NMR). All NMR chemical shift values are reported in delta units (ppm) for the control remaining solvent. Abbreviations used are: s, singlet; d, double peak; t, triplet; br, broad peak.

Liquid chromatography mass spectra (LC-MS) were obtained on an Agilent HPl 100 LC/MS system equipped with a diode array detector and a quaternary pump to load the chromatography stream from the column back into the MSD detector. Unless otherwise stated, the analytical column used was a Vydac 218TP54 analytical column (300) equipped with a Phenomenex guard module (KJO-4282)5 μ). Unless otherwise stated, TFA-H at 0.1% was used₂0.1% TFA-MeCN (C) in O (A), and the concentration of TFA-H in 0.1%₂0.1% TFA-MeCN (C) in O (A) has a solvent gradient of 5% (0 min) to 100% (20 min) to 5% (25 min) of (C) in (A).

Preparative reverse phase HPLC (RP-HPLC) was performed using two Varian/Rainin SD-I pumps with Varian/Dynamax radial compression columns (C18, Microorb 60-8). A gradient of (C) in (A) at a flow rate of 15% (0 min) to 85% (20 min) of 20-40mL/min was used.

In order that the invention may be more readily understood, reference is made to the following examples, which are intended to be illustrative of the invention and are not intended to limit its scope.

Example 1.

Compound (I)2Synthesis of (2)

HCl gas was slowly bubbled through a suspension of 2, 6-pyridinedicarboxylic acid (25g, 0.15mol, Aldrich Chemical Company) in 150mL of methanol at room temperature. As heat is released, the suspension slowly transforms into a clear solution. The HCl gas bubbling was stopped and the reaction flask was tightly sealed with a septum and stirred at room temperature in a well-ventilated hood. A white precipitate formed after about 40 minutes. After stirring at room temperature for 12 hours, the reaction mixture was concentrated under reduced pressure to give a white solid residue. The white solid was dissolved in dichloromethane (150ml) and the organic layer was washed with saturated NaHCO₃The solution (2 × 50ml) was washed and dried (MgSO₄) And concentrated to give the product as a white solid2(28.8g，98.5％)：TLC：R_f0.36 (50% EtOAc in hexanes); NMR (CDCl)₃，200MHz)δ 4.02(s，6H)，8.01(d，J＝8Hz，0.5H)，8.04(q，J＝8Hz，0.5H)，8.32(d，J＝8Hz，2H)。

Example 2.

Compound (I)3Synthesis of (2)

The monoalcohols were prepared from the diesters according to the literature (Liren Huang, James C.Quada, Jr. and William Lown, Bioconjugate Chemistry, 1995, 6, 21-33). Diester in 200ml methanol: (214.1g, 72.3mmol) with NaBH₄(4.2g, 0.11mol) the reaction gave 8.3g (69%) of the product as a white powder3。TLC：R_f 0.36(EtOAc)；NMR(CDCl₃，200MHz)δ 3.42(br s，1H)，3.99(s，1H)，4.85(s，2H)，7.52(d，J＝7.2，1H)，7.84(t，J＝7.6Hz，1H)，8.03(d，J＝7.2Hz，1H)。

Example 3.

Compound (I)4Synthesis of (2)

The reaction product of alcohol and alcohol is reacted with alcohol,3(3.4g, 20mmol) to a solution in 50ml dry DMF was added imidazole (1.9g, 28mmol, 1.4 equiv.) followed by tert-butyldimethylsilyl chloride (3.4g, 22.6mmol, 1.13 equiv.). The reaction was stirred at room temperature for 12 hours. The reaction mixture was diluted with EtOAc, the organic layer was washed with water (5 × 30ml) and concentrated to give the crude product as a viscous oil. Purification by flash column (chromatography) on silica gel (20% EtOAc in hexane) then afforded the product as a white solid4(5.3g, 95%) having: TLC: r_f0.48 (25% EtOAc in hexanes); NMR (CDCl)₃，200MHz)δ 0.11(s，6H)，0.95(s，9H)，3.99(s，3H)，4.93(s，2H)，7.75(dd，J＝0.6，7.8Hz，1H)，7.85(t，J＝7.8Hz，1H)，8.00(dd，J＝0.6，8Hz，IH)。

Example 4.

Compound (I)5And5asynthesis of (2)

To the direction of4(1.75g, 6.2mmol) to a stirred solution in 20ml THF/methanol (2/1) was added LiOH (monohydrate, 294mg, 7mmol, 1.1 equiv. dissolved in-1 ml water and heated to dissolve). The reaction was stirred at room temperature for 2 hours at which time TLC showed the reaction was complete. The solvent was removed under vacuum, using 10ml of 1M H₃PO₄(pH 3) the residue was treated, the mixture was shaken and the pH of the solution was carefully readjusted to 3 with 1N HCI. The solution was extracted with dichloromethane (4 × 20 mL). The organic extracts were combined and dried (MgSO)₄) And concentrated to give the protected acid as a clear oil (5) (1.4g, 85%, slightly impure). The crude protected acid (380mg, 1.4mmol) was dissolved in 10ml of dry dichloromethane and then N-hydroxysuccinimide (NHS) (242mg, 2.1mmol, 1.5 equiv.) was added to the stirred solution at room temperature followed by 1- (3-dimethyl-aminopropyl) -1-ethylcarbodiimide hydrochloride (EDC. HCl) (355mg, 1.85mmol, 1.3 equiv.). The reaction was stirred at room temperature for 12 hours. The reaction mixture was concentrated to a volume of-2 ml, then loaded directly onto a silica gel column and eluted with 40% ethyl acetate in hexane. NHS ester(s) (yield white solid)5a) (210mg, 41%) having: TLC: r_f0.55 (50% EtOAc in hexanes); NMR (CDCl)₃，200MHz)δ 0.12(s，6H)，0.95(s，9H)，2.91(br s，4H)，4.93(s，2H)，7.82-7.96(m，2H)，8.08(d，J＝8.0Hz，1H)。

Example 5.

Compound (I)6Synthesis of (2)

Compound 6 was prepared analogously to the methods described in the literature (A.J.Y.lan, R.O.Heuckeroth, and P.S.Mariano, J.Am.chem.Soc, 1987, 109, 2738-2745).

To the alcohol being stirred at room temperature,3(4.1g, 24.6mmol) in 100ml dichloromethane and diethyl ether (1/1) triphenylphosphine (7.1g, 27.06mmol, 1.1 equiv.) was added followed by carbon tetrabromide. The reaction was stirred at room temperature for 30 minutes. TLC showed complete reaction. The reaction mixture was concentrated to remove most of the solvent. The residue was treated with EtOAc to precipitate triphenylphosphine oxide, after which the EtOAc layer was decanted. The procedure was repeated two more times. The EtOAc layers were combined and concentrated to give crude product as an off-white syrup. Recycling the residueDissolved and purified by silica gel column chromatography to give the title compound 6(5.4g, 89%) as a white solid: TLC: r_f0.55 (50% EtOAc in hexanes); NMR (CDCl)₃，200MHz)δ 4.00(s，3H)，4.63(s，2H)，7.68(dd，J＝1，7.8Hz，1H)，7.86(t，J＝8Hz，1H)，8.06(dd，J＝1，7.8Hz，1H)。

Example 6.

Compound (I)7Synthesis of (2)

Potassium tert-butoxide (1.12g, 10mmol) is added to a stirred solution of di-tert-butyl iminodicarboxylate (2.13g, 9.8mmol, Fluka) in 10ml of dry DMF at room temperature under argon. The solution was stirred at room temperature for 30 minutes. To the stirred solution at room temperature was added the compound in 8ml dry DMF6(2.1g, 8.5 mmol). After stirring for two hours at 50 ℃, the reaction mixture was concentrated under vacuum to remove most of the DMF. The residue was purified by silica gel column chromatography (30% EtOAc in hexanes) to give the product as a pale yellow oil (2.8g, 86%) which solidified after 10 hours at room temperature: TLC: r_f0.66 (50% EtOAc in hexanes); NMR (CDCl)₃，200MHz)δ1.42(s，18H)，3.98(s，3H)，5.01(s，2H)，7.3(d，J＝7.8Hz，1H)，7.80(t，J＝7.8Hz，1H)，8.00(d，J＝1，7.6Hz，1H)。

Example 7.

Compound (I)8aAnd8bsynthesis of (2)

To the stirred compound7(1.80g, 4.7mmol) in 20ml THF/methanol (1/4) LiOH (monohydrate, 220mg, 5.2mmol, 1.1 equiv. dissolved in-1 ml water was added and heated to dissolve). The reaction was stirred at room temperature for 3 hours, after which time TLC showed the reaction was complete. The solvent was removed in vacuo and 8ml of 1M H was used₃PO₄(pH 3) the residue was treated and the resulting solution was extracted with dichloromethane (5 × 20 ml). The organic extracts were combined and dried (MgSO)₄) And concentrated to give the product as an oil (1.45g). NMR showed that it was a mono-BOC compound (C)8a) (-80%) with a di-BOC compound (b)8b) (-20%) mixture: delta 4.51(d, J ═ 6.2Hz, BOC-NH-CH₂-)，δ 4.97(s，(BOC)₂N-CH₂-). The mixture was used in the next step without further purification.

Example 8.

Compound (I)10Synthesis of (2)

To a stirred BOC-glycyl-OH in a 50-mL round bottom flask,9(2.5g, 10.8mmol, Bachem America, Cat. A-1750) to a solution of N-hydroxysuccinimide (NHS) (1.49g, 12.9mmol) in 10mL dry DMF was added 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl) (2.2g, 11.3mmol, Aldrich). The reaction was stirred at room temperature for 3 hours. To the reaction mixture was added beta-alanine benzyl ester p-toluenesulfonate (3.9g, 11mmol, Sigma), followed by dry diisopropylethylamine (1.55g, 2.1ml, 12 mmol). The reaction was stirred at room temperature for 14 hours. Most of the solvent (DMF) was removed under reduced pressure leaving a clear oil, which was partitioned between 100ml ethyl acetate and 20ml water. The organic layer was separated and then washed sequentially with water (1X 20ml), saturated NaHCO₃Solution, 1N HCl (2 × 15ml) and water (1 × 10ml) wash; with MgSO₄Dried and concentrated to dryness to give the product as a white solid10(3.8g，90％)：TLC：R_f0.67(1:2:2 MeOH/CH₂Cl₂/EtOAc)；NMR(CDCl₃，200MHz)δ 1.44(s，9H)，2.59(t，J＝6.2Hz，2H)，3.54(q，J＝6.2Hz，2H)，3.80(d，J＝5.8Hz，2H)，3.89(d，J＝5.6Hz，2H)，5.12(s，2H)，6.8(br s，1H)，6.85(br s，1H)，7.35(m，5H)。

Example 9.

Compound (I)11Synthesis of (2)

Protected dipeptides10(2.1g, 5.3mmol) was dissolved in 80ml THF/EtOAc (1: 1). Then bubbling HCl gas into the mixture at room temperatureThe solution is continued for about 5-10 minutes and the resulting mixture is held at room temperature for 12-14 hours. The resulting white solid product (1.67g, 95%) was filtered off, air dried, and then further dried under vacuum. The substance has: NMR (D2O, 200MHz) δ 2.62(t, J ═ 6.4Hz, 2H), 3.47(t, J ═ 6.6Hz, 2H), 3.83(s, 2H), 3.84(s, 2H), 5.14(s, 2H), 7.41(brs, 5H).

Example 10.

Compound (I)12Synthesis of (2)

To a stirred dipeptide HCl salt at room temperature (b)111.67g, 5.1mmol) in 15ml dry THF and 3ml dry pyridine was added penta dianhydride (600mg, 4.9mmol, Aldrich). The suspension was stirred at room temperature for 12 hours. The reaction mixture remained as a suspension throughout. The reaction mixture was concentrated to remove most of the THF, after which the residue was treated with 3ml of water to give a clear solution. The solution was acidified with 6N HCl (approximately 6ml) whereupon the product precipitated as a white solid. The solid was collected by filtration, washed with water (1 × 5ml), then dried under high vacuum overnight to give the title compound (b) as a white powder12) (1.77g, 86%) having: NMR (CD)₃OD，200MHz)δ 1.90(m，2H)，2.33(t，J＝7Hz，2H)，2.34(t，J＝7.2Hz，2H)，2.60(t，J＝6.8Hz，2H)，3.82(d，J＝5.8Hz，4H)，5.12(s，2H)，7.35(br s，5H)，LC-MS：t_R8.98 minutes; m + H408 was measured.

Example 11.

Compound (I)14Synthesis of (2)

The compounds are obtained in a manner analogous to that described in European patent application EP 1207394A 214。

Compounds in 100ml of methanol containing 0.16g palladium on carbon (10% Pd-C, Aldrich) were added at room temperature and atmospheric pressure13(1.5g, 2.3mmol, U.S. Pat. No. 5,196,438) was hydrogenated for 4 hours. The reaction mixture was filtered through a pad of CELITE in a fritted glass funnel. The filtrate was concentrated in vacuo to give the compound as a white solid14(1.15g，97％)：TLC：R_f 0.25(1:2:2MeOH/CH₂Cl₂EtOAc), then co-eluted with authentic material.

Example 12.

Compound (I)15Synthesis of (2)

To the stirred compound8aAnd8bto a solution of the-80: 20 mixture (0.19g, 0.75mmol) in 15ml of dry dichloromethane were added NHS (115mg, 1mmol) and EDC. HCl (175mg, 0.91mmol) in that order. The reaction mixture was stirred at room temperature for 40 minutes. Adding a compound to the reaction mixture14(260mg, 0.5mmol) followed by the addition of a catalytic amount of 4-dimethylaminopyridine (DMAP, 10 mg). After stirring at room temperature for 4 hours, the reaction was concentrated and the residue was dissolved in-50 ml EtOAc. With water (1X 10ml), saturated NaHCO₃The organic layer was washed with solution (2 × 10ml) and then concentrated to give crude product. Column chromatography over silica gel (70% EtOAc in hexane, then 10:40:50 MeOH/EtOAc/CH)₂Cl₂) Purification afforded a mixture of intermediate N-protected product [. about.260 mg, mono-Boc (80%) and di-Boc (20%) as a white solid]：TLC R_f0.62 (mono-BOC) and 0.69 (di-BOC), 1:2:2MeOH/CH₂Cl₂EtOAc). The protected product was dissolved in 10ml dichloromethane/TFA (1:1) and the solution was then stirred at room temperature for 2 hours. The reaction was concentrated in vacuo, after which the residue was taken up in 20ml dichloromethane and 10ml saturated NaHCO₃The solutions were partitioned. The layers were separated and the aqueous layer was further extracted with dichloromethane (5 × 10 mL). The organic extracts were combined and dried (MgSO)₄) And concentrated to give the product as a white solid15(～210mg)：TLC R_f0.50[70:28:2CH₂Cl₂/MeOH/NH4OH (28% aq)]；LC-MS t_R10.13 min, M + H650 was measured.

Example 13.

Compound (I)17Synthesis of (2)

To the stirred compound12(150mg, 0.37mmol) and NHS (56mg, 0.48mmol) in 2.5ml dry DMF was added EDC. HCl (85mg, 0.4 mmol). After stirring at room temperature for 5 hours, using15The reaction mixture was treated with a solution of DMAP (2mg) in 4ml of THF (150mg, 0.23mmol) and then stirred at room temperature for a further 12 hours. TLC indicated starting material15Disappear and the compound16Formed and confirmed by LC-MS (t)_R12.0 min, M + H1039.5 was measured). Small amounts of residual reagents and by-products were also observed. Will contain crude compounds16The reaction mixture of (2) was diluted with methanol (3 × 30ml) and transferred to a 250ml round bottom flask, after which the components were hydrogenated in the presence of palladium on carbon (80mg, 10% Pd-C) at room temperature and atmospheric pressure for 4 hours. The catalyst was removed by filtration through a pad of CELITE in a fritted glass funnel (4 μm). The filtrate was evaporated under reduced pressure and then under high vacuum to remove the solvent. The residue was purified by preparative RP-HPLC, after which the product fractions were freeze-dried to give the compound as an off-white solid17(95mg), determined as trifluoroacetate salt. Similar experiments were carried out in a similar manner to obtain equivalent substances17Which has: LC-MS: t is t_R10.5 min, M + H949.5 (parent) was determined.

Example 14.

Compound (I)18Synthesis of (2)

To the acid being stirred17(85mg,. about.0.09 mmol) in 1ml DMF and 2ml acetonitrile NHS (62mg, 0.54mmol, 6 equiv.) and EDC. HCl (103mg, 0.54mmol, 6 equiv.) were added. The reaction was stirred at room temperature overnight. LC-MS showed disappearance of the starting material and formation of the product18. The product mixture was directly purified by preparative RP-HPLC. The product containing eluates were combined and frozen, acetonitrile was removed by high vacuum rotary evaporation (sublimation) onto a dry ice/acetone finger condenser, after which the residue was freeze dried to give the product as an off-white solid18(39mg), determined as trifluoroacetate salt.

Similar experiments were performed in analogy to the above and the same product was obtained after RP-HPLC purification and freeze-drying as above18Which has: LC-MS t_R10.6 min, M + H1046.6 (parent) was determined; HR-ES MS: m + H (parent) 1046.5306 was calculated and 1046.5293 was measured.

Example 15.

Compound (I)19Synthesis of (2)

To the stirred compound5a(200mg, 0.55mmol) in 10ml dry dichloromethane was added14(210mg, 0.4mmol) followed by the addition of a catalytic amount of DMAP (. about.10 mg). After stirring at room temperature for 12 hours, the reaction mixture was concentrated. The residue was dissolved in 25ml EtOAc, then washed sequentially with water (2 × 10ml), saturated NaHCO₃The organic layer was washed with solution (2 × 10ml) and concentrated. Purification by silica gel column chromatography then afforded the intermediate TBDMS protected product as a white solid (250mg, 82%): TLC: r_f0.75(1:2:2MeOH/CH₂Cl₂EtOAc). The white solid was dissolved in 10ml of acetonitrile, and then 0.5ml of 48% aqueous HF solution was added to the solution. After stirring at room temperature for 4 hours, the reaction mixture was diluted with 20ml dichloromethane and saturated NaHCO₃The solution (2 × 10ml) was washed. The organic layer was concentrated and purified by silica gel column chromatography to give the product as a white solid19(180mg, 85%) having: TLC: r_f0.63(1:2:2MeOH/CH₂Cl₂/EtOAc)；LCMS t_R11.2 min, M + H651.3; HR-ES MS: m + H651.3865 was calculated and 651.3872 was measured.

Example 16.

Compound (I)22Synthesis of (2)

To 6- (FMOC) aminocaproic acid20(Advanced ChemTech, Louisville, KY, USA; Cat. FX2650) (36mg, 0.102mmol) in dry dichloromethane (3ml) NHS (13.1mg) was added followed by EDC. HCl (20.5mg), and thenThe reaction was stirred at room temperature under argon overnight. LC-MS detection of the reaction mixture indicated formation of the corresponding NHS ester product21Which is a single main peak (t)_R15.7 min, M + H451.2) was determined. The reaction mixture was split in half. Adding a compound to half of the mixture15(45mg,. about.0.05 mmol, as the di-trifluoroacetate salt) followed by triethylamine (21. mu.l,. about.15 mg,. about.0.15 mmol) and dry DMF (0.5mL) to help dissolve the reaction. Detection of the reaction by LC-MS after 1.5h indicated formation of the desired product while both starting materials were still present. After stirring overnight at room temperature, LC-MS indicated the product was formed with side products. Using 30-40ml of CH₂Cl₂The reaction was diluted sequentially with 0.1N HCl (twice), water (once), saturated NaHCO₃Washed (once) with saturated NaCl (once) and dried (Na)₂SO₄) Filtered and evaporated under reduced pressure. Redissolving the residual liquid in chloroform (CHCl)₃) 5% MeOH in, then column chromatography on silica gel (in CHCl)₃Gradient of 5% to 10% MeOH) to give a colorless/white glassy product22(20mg, 40%). The substance has: TLC R_f0.42 (in CHCl)₃10% MeOH in (g); LC-MS t_R15.1 min, M + H985.5 was measured.

Example 17.

Compound (I)23Synthesis of (2)

To the compound22(20mg, 0.0203mmol) in dry CH₂Cl₂(3.6ml) to the solution was added piperidine (0.4ml) and the reaction was stirred at room temperature under argon. Detection by TLC after 1.5h indicated completion of the reaction. Volatiles were removed at room temperature under reduced pressure and then under high vacuum to give a residual white solid. This material was redissolved in acetonitrile (MeCN) -water with a small amount of trifluoroacetic acid (TFA) added, filtered (0.45m) and subjected to preparative RP-HPLC [ at 0.1% TFA/H ]₂Gradient of 5% (at 0 min) to 100% (at 20 min) of 0.1% TFA/MeCN in O]And (5) purifying. The product fractions were combined, MeCN was removed under reduced pressure, and the aqueous residue was frozen and freeze-driedThus, the product was obtained as a white solid23And was identified as di-TFA salt. The substance has: LC-MS t_R10.3 min, M + H763.4 (parent) was determined.

Example 18.

Compound (I)24Synthesis of (2)

To a stirred bis-N-hydroxysuccinimide terephthalate28[ Ghoshal et al, EP 1,148,339A 2](3.2mg, 0.0089mmol) and 4. mu.L of triethylamine in 1.5mL dry CH₂Cl₂To the semi-solution/suspension of (A) is added dropwise23A solution of (8.8mg, 0.009mmol) in 1:2 chloroform-DMSO (0.75mL) was then used to stir the mixture for an additional 1.5 hours. Detection by LC-MS indicated that the reaction was substantially complete. Volatiles were removed under high vacuum. The residue was dissolved in a small amount of MeCN/water and purified by preparative RP-HPLC [ C18; gradient of 5% (at 0 min) to 100% (at 20 min) of 0.1% TFA/MeCN in 0.1% TFA/water]And (5) purifying. The main product peak was collected, immediately frozen, acetonitrile was sublimed off (high vacuum rotary evaporator, dry ice/acetone finger condenser) and the residue was freeze dried to yield product 24 containing a small amount of terephthaloyl bisamide product. The substance has: NMR: the consistency is achieved; LC-MS: t is t_RAt 12.4 min, M + H1008.4 was measured. (bis-product at t_RAt 13.0 min, found M + H1656.1)

Example 19.

Conjugates of succinimidyl-oxycarbonyl-ethylamino-glycyl-glutaryl-aminomethyl-pyr saquinavir (pyrsoquinavir) with KLH25Synthesis of (2)

To 20mg of keyhole limpet hemocyanin (KLH, Pierce Biotechnology, inc., Rockford, IL, USA) reconstituted in 1.5mL of 50mM potassium phosphate buffer (KPi) pH7.5 was added dropwise 0.5mL of dimethyl sulfoxide (DMSO) while cooling in an ice-water bath with stirring. One quarter (0.5mL) of the resulting solution (equivalent to 5mg of KLH) was withdrawn for use as a standard/control. To the remaining protein solution (15mg KLH) was added lysisCompounds in a total of 150. mu.L DMSO18(6.5mg, 5.1. mu. mol of the bis-TFA salt). The cooling bath was removed and the mixture was stirred in a capped vial overnight. The mixture was transferred to a dialysis cassette (dialysis cassette) (Pierce Biotechnology, Inc, 10K molecular weight sieve cut-off membrane; product number 66425) and dialyzed sequentially against 30% (two changes) in 50mM KPi pH7.5, followed by 20%, then 10% DMSO at room temperature, followed by 50mM KPi pH7.5, and then 50mM KPi pH7.5 (three changes) at 4 ℃. The KLH standard/control (see above) was similarly transferred to a smaller dialysis cassette (Pierce Biotechnology, Inc.) and dialyzed in the same manner. Removing the retentate from the dialysis cartridge to obtain the conjugate25And both the standard/control, as milky gray solutions. By using A₂₈₀The concentration of KLH standard/control was determined at 1mg/mL UV ═ 1.77. The KLH standard/control was used to establish a standard curve and the Coomassie blue protein assay (modified Bradford) on the conjugate solution indicated 4.5mg/mL protein. Trinitrobenzenesulfonic acid test (TNBS test) on the conjugate solution using the KLH standard/control as a control sample showed that-34% of the available lysine was modified.

Example 20.

Succinimidyl-oxycarbonyl-ethylamino-glycyl-glutaryl-aminomethyl-pyr saquinavir conjugate with BSA26Synthesis of (2)

To a solution of 120mg of Bovine Serum Albumin (BSA) (interben Company, commercially available, NY, USA, Cohn Fraction V modified powder) in 4.0mL of 50mM potassium phosphate buffer (KPi) pH7.5 was slowly added dropwise 0.5mL of dimethyl sulfoxide (DMSO). 0.75mL of the resulting solution (equivalent to 20mg BSA) was withdrawn for use as a standard/control. To the remaining protein solution (100mg BSA) was added compound 18(3.1mg, 2.4. mu. mol, as di-TFA salt) dissolved in a total of 300. mu.L DMSO. The flask was capped and the reaction mixture was stirred overnight. Transferring the mixture toDialysis cassettes (Pierce Biotechnology, Inc; 10K molecular weight sieve cut-off membranes) and dialyzed sequentially against 20% DMSO (one variation) in 50mM KPi pH7.5, then 10% DMSO, at room temperature, then 50mM KPi pH7.5 (one variation), then 50mM KPi pH7.5 (three variations) at 4 ℃. The BSA standard/control (see above) was similarly transferred to a smaller dialysis cartridge and dialyzed in the same manner. Removing the retentate from the dialysis cartridge to obtain the conjugate26And both the standard/control, are clear solutions. By using A₂₈₀The concentration of BSA standard/control was determined at 1mg/mL UV-0.6. A standard curve was established using the BSA standard/control, and coomassie blue protein assay (modified Bradford) on the conjugate solution indicated 11.2mg/mL protein.

Example 21.

Succinimidyl-benzoyl-aminocaproyl-aminomethyl-pyr saquinavir conjugates with BSA27Synthesis of (2)

To a solution of 100mg Bovine Serum Albumin (BSA) (interben Company, commercially available, NY, USA, Cohn Fraction V modified powder) in 2.0mL50mM potassium phosphate buffer (KPi) pH7.5 was slowly added 0.2mL dimethyl sulfoxide (DMSO). 0.44mL of the resulting solution (equivalent to 20mg BSA) was withdrawn for use as a standard/control. To the remaining protein solution (80mg BSA) was added compound 24(2.7mg, 2.4. mu. mol as TFA salt) dissolved in a total of 750. mu.L DMSO. The flask was capped and the reaction mixture was stirred overnight. The mixture was transferred to a dialysis cassette (Pierce Biotechnology, Inc; 10K molecular weight sieve cut-off membrane; product #66425) and dialyzed sequentially against 30% DMSO (two changes), then 20% DMSO, then 10% DMSO in 50mM KPi pH7.5 at room temperature, then 50mM KPi pH7.5 (five changes) at 4 ℃. The BSA standard/control (see above) was similarly transferred to a smaller dialysis cartridge and dialyzed in the same manner. Removing the retentate from the dialysis cassetteOpening to obtain the conjugate27And both the standard/control, are clear solutions. By using A₂₈₀The concentration of BSA standard/control was determined at 1mg/mL UV-0.6. A standard curve was established using the BSA standard/control, and coomassie blue protein detection (modified Bradford) on the conjugate solution indicated 14.5mg/mL protein. Purple heterodyne spectroscopy (relative to BSA standard/control) indicated the presence of the hapten.

Example 22.

Antibody development

All mice were housed in clear plastic cages with stainless steel wire grid lids, one cage per 5. The bottom of the cage was covered with granular dry corn cob bedding. Mice food was supplied ad libitum with dry pellet feed when drinking water. Periodically, the water was replaced with grapefruit juice.

Female Balb/c mice of at least 3 months of age were used for immunization. Conjugate of saquinavir with KLH from example 19 (c)25) Emulsified in 50% complete Freund's adjuvant and 50% saline at a final concentration of 100. mu.g/ml. Each mouse was injected 100. mu.l into the peritoneal cavity. Thirty-five days later, similar injections were given by the same route with Freund's incomplete adjuvant and the same concentration. Twenty-five days later, a third immunization identical to the second formulation was given. The mice were allowed to rest (without further immunization) for approximately 3 months. Mice selected for fusion were given enhanced immunity with the same saquinavir-KLH conjugate for the second and third injections. Four days later, the mice were used for cell fusion to develop hybridomas secreting monoclonal antibodies.

Mice selected for fusion were killed via exsanguination, spleens were harvested post-operatively and ground between two sterile glass slides to release the thymus-dependent cells. The resulting lymphocyte suspensions were used for fusion with FO myeloma cell lines (purchased from ATCC).

The fusion consists of the following steps: adding myeloma cellsThe cells (1/5 for lymphocyte number), were washed by centrifugation, resuspended in warm Iscove's medium without serum modified Dulbecco's medium (IMDM), and re-centrifuged. The centrifuge tube containing the resulting pellets was gently tapped to disperse the cells, and then 1ml of warmed PEG/DMSO solution (Sigma Chemicals) was added slowly with gentle stirring. The cells were incubated for 1.5 minutes, after which pre-warmed serum-free IMDM was added at the following rate: 1mL/min, 2mL/min, 4mL/min, 10mL/min, then the tube was filled to 50mL, sealed and incubated for 15 minutes. The cell suspension was centrifuged, the supernatant decanted and thereafter IMDM containing 10% fetal bovine serum was added. The cells were again centrifuged and resuspended in complete cloning medium. It consisted of IMDM, 10% FCS, 10% conditioned H1(Roche molecular Systems), 4mM glutamine, 50. mu.M 2-mercaptoethanol, 40. mu.M ethanolamine, and penicillin/streptomycin antibiotics. Subjecting the cells to a reaction at 4 x10⁵Individual thymus-dependent cells/ml were suspended at density, dispensed into sterile 96-well microplates at 100 μ l/well and incubated at 37 ℃ in 5% CO₂And culturing for 24 hours. The next day, 100. mu.l of HMT selective medium (cloning medium +1:25 HMT supplement from Sigma Chemicals) was added. On day 6 of culture, approximately 150. mu.l of medium was withdrawn from each well using a sterile 8-channel manifold connected to a light vacuum source. One hundred fifty microliters of HT medium was then added. It consisted of cloning medium +1:50HT supplement (Sigma Chemicals). The plates were returned to the incubator and checked daily for signs of growth. When growth was judged to be sufficient, wells were screened for antibody production by ELISA.

The microplate was coated with 100 μ l of saquinavir-BSA conjugate from example 20 (26) at 1 μ g/ml in 0.1M carbonate buffer, pH 9.5 at 37 ℃ for 1 h (to allow wetting). The plates were then emptied and filled with a post-coating solution consisting of tris buffer, 1% gelatin hydrolysate, 2% sucrose and 0.17% TWEEN20 (all reagents from Sigma Chemicals). The plates were incubated at 37 ℃ for another 1 hour (to allow wetting) and thereafter washed with phosphate buffered saline containing 0.1% TWEEN 20. The plates were then simply filled with a 2% sucrose solution in 0.15Mtris, pH 7.2-7.4, then emptied and allowed to air dry at room temperature. When dry, the plates were packed into zip-lock bags containing several desiccant pillows, sealed and stored at 4 ℃ until use.

When the growing clones were judged to be ready for testing, 25. mu.l of supernatant was removed from each well and transferred to 96-well flexible plates. Media was added to each well to provide a 1:10 dilution of the media sample.

Two saquinavir-BSA conjugate-coated wells were used for each culture well tested. One well contained 50 μ l of PBS buffer and the other well contained 50 μ l of PBS containing saquinavir drug at a concentration of 800 ng/mL. Fifty microliters of the diluted sample was transferred into each of the two coated wells above. The plates were incubated at 37 ℃, covered for 1 hour, and then washed with PBS-TWEEN. The wells were then filled with 100. mu.l of goat anti-mouse IgG-HRP conjugate (Zymed Labs) diluted 1:5000 in PBS-TWEEN, after which the plates were incubated for an additional 1 hour. The plates were then washed again and 100. mu. l K-Blue substrate (Neogen Corp) was added. It was allowed to proceed for 5-15 minutes, after which the reaction was stopped by adding 100. mu.l of 1N HCl. The color was read at 450nm with the aid of a microplate reader and collected by the computer for analysis. Due to the free drug, the selected standard binds to the saquinavir-BSA conjugate and effectively inhibits binding in the second well.

Table 1: representative fractions of plate screens

Culture well	OD₄₅₀In the absence of free drug	OD₄₅₀In the presence of free drug
Culture well	OD₄₅₀In the absence of free drug	OD₄₅₀In the presence of free drug	19 F2(SAQ 134)	1.500	0.121
22 G5(SAQ 137)	1.526	0.083	19 F2(SAQ 134)	1.500	0.121
22 G5(SAQ 137)	1.526	0.083	47 H1(SAQ 158)	1.627	0.076
15 H6(SAQ 221)	1.699	0.295	47 H1(SAQ 158)	1.627	0.076
15 H6(SAQ 221)	1.699	0.295	27 G10(SAQ 300)	1.542	0.104

After selecting a clone from the fusion plate, the cells were subjected to stringent cloning by limiting dilution. Subclones grown from those wells in which single cells have been verified by microscopy were then retested by the method described above. Stability of antibody expression was judged based on the number of wells showing antibody, the level of binding, and the presence of any wells showing growth but little antibody. If any of the latter is found, the wells showing high antibody secretion are then used for repeated compact subcloning. This was repeated as necessary to obtain 100% subclones secreting equal amounts of antibody. Cells from the selected wells are then expanded in culture and used to prepare a primary cell bank. Supernatants from those cultures were then subjected to specific analysis.

The antibody-containing culture supernatant from the amplification culture was subjected to specificity analysis as follows. First, the titer suitable for the analysis was determined by dilution analysis. The dilution of antibody that provided approximately 50% of the maximum binding was selected for the next step. Second, binding to the saquinavir-BSA conjugate was tested in the presence of varying amounts of the structurally most closely related HIV protease inhibitor drug (nelfinavir) and two metabolites of saquinavir, M4 and M6, at the above antibody dilutions. The data were analyzed by fitting a nonlinear regression curve to a 4-parameter logistic function. The description corresponds to 50% binding in the absence of free drugThe parameter of free drug concentration of (a) is named ED of the drug₅₀. The specificity of the antibody can thus be determined by comparing the ED of the related drug, saquinavir, according to the following equation₅₀Or saq ED₅₀Described with another value of other drugs fitted from those data (for this example, nelfinavir data were used):

the four-parameter logic function used isWherein S is a curvature parameter, OD_maxOptical Density at zero drug concentration, OD_minOptical Density and OD of background of Instrument_xIs the optical density measured at the drug concentration X in moles per liter (M/L). The cross-reactivity of the two saquinavir antibodies by this assay is given in table 2.

Table 2: specificity of antibodies

Example 23.

Response to concentration

Antibody SAQ 137.3.3 was used to demonstrate the concentration response of saquinavir in an ELISA assay format. The assay was performed as described in the specific assay, with the modification that a greater number of concentrations were assayed and no other drugs were added. Table 3 lists the dose response, which is the optical density at 450nm provided by each concentration of saquinavir.

Table 3: concentration response using SAQ 137.3

Concentration of saquinavir M/L	OD₄₅₀
Concentration of saquinavir M/L	OD₄₅₀	2.98 x 10^-5	0.069
9.93 x 10^-6	0.078	2.98 x 10^-5	0.069
9.93 x 10^-6	0.078	3.31 x 10^-6	0.082
1.10 x 10^-6	0.100	3.31 x 10^-6	0.082
1.10 x 10^-6	0.100	3.68 x 10^-7	0.145
1.23 x 10^-7	0.240	3.68 x 10^-7	0.145
1.23 x 10^-7	0.240	4.09 x 10^-8	0.423
1.36 x 10^-8	0.670	4.09 x 10^-8	0.423
1.36 x 10^-8	0.670	4.54 x 10^-9	0.902
1.50 x 10^-9	1.029	4.54 x 10^-9	0.902
1.50 x 10^-9	1.029	5.05 x 10^-10	1.580
1.68 x 10^-10	2.192	5.05 x 10^-10	1.580
1.68 x 10^-10	2.192	5.61 x 10^-11	2.824
1.87 x 10^-11	2.967	5.61 x 10^-11	2.824
1.87 x 10^-11	2.967	6.23 x 10^-12	3.091

Murine hybridoma SAQ 137.3.3 was deposited at the American Type Culture Collection (ATCC) on 23.11.2004 at the American Type Culture Collection, and was registered as ATCC No. PTA-6329.

Example 24.

Compound (I)29Synthesis of (2)

To a stirred solution of 5-nitroquinaldinic acid (210mg, 0.96mmol) in 20mL CH under argon at room temperature₂Cl₂To a solution in acetonitrile (3:1) was added NHS (220mg, 1.92mmol) followed by EDC & HCl (236mg, 1.16 mmol). After stirring for 1.5h, the compound was added14(420mg, 0.80mmol) and the resulting mixture was stirred overnight. The reaction was diluted with 40mL EtOAc, then sequentially with water (2X 20mL) and saturated NaHCO₃Washed with aqueous solution (2 × 10mL) and dried (MgSO)₄) Filtered and evaporated under reduced pressure. The residue was redissolved and purified by addition of MeOH-EtOAc-CH₂Cl₂(1:6:6) purification by column chromatography on silica gel eluting, dividing from the center of the main product band and after evaporation of the solvent and drying under high vacuum, gave compound 29(302mg, 44%) as a pale yellow solid.¹H-NMR: and (4) the consistency is achieved. LC/MS: t is t_R13.5 min, M + H716.3 was measured.

Example 25.

Compound (I)30Synthesis of (2)

Treatment of the compound with 10% Palladium on carbon (10% Pd-C) (165mg)29(282.5mg, 0.395mmol) in 30ml MeOH, after which the mixture is hydrogenated at about 35psi at room temperature with vigorous stirring for about 3 hours. By passing through sintered glass funnelsThe catalyst was removed by filtration and washed with MeOHAnd washing the filter cake. The combined filtrates were evaporated to dryness and purified by addition of MeOH-EtOAc-CH₂Cl₂(1:4:5) purifying the residue by silica gel column chromatography to obtain a solid compound by dividing the product band from the center30(170mg，63％)。¹H-NMR: and (4) the consistency is achieved. LC-MS: t is t_RM + H686.3 was measured for 12.3 minutes (0% to 100% 0.1% TFA/MeCN in 0.1% TFA/water over 20 minutes; 1 mL/min).

In another procedure, 200mg of the compound29And hydrogenation of 110mg of 10% Pd-C in 20mL MeOH, after filtering off the catalyst, evaporation and purification of the residue by preparative RP-HPLC to give the compound as the trifluoroacetate salt30(60mg)。LC-MS：t_R11.4 min, M + H686 was measured.

Example 26.

Compound (I)31Synthesis of (2)

Preparation of Succinimidyl-oxycarbonyl-butyryl chloride, i.e. 5- (2, 5-dioxo-1-pyrrolidinyl-oxy) -5-oxo-valeryl chloride, according to EP 0503454 to Antonian et al34。

To the compound30In dry CH (TFA salt) (15mg, 0.0164mmol)₂Cl₂(3mL) and dried DMF (0.1mL) solution was added to the bifunctional linker compound34(4.3mg,. about.3 mol. eq.) and then the reaction was stirred at room temperature under argon for about 3 hours. Analytical RP-HPLC analysis showed that the product was associated with a considerable amount of the faster-running material and lesser amounts of other materials. The solvent was removed under high vacuum (rotary evaporator) and the residue was redissolved in a small amount of MeCN/water (1:1) and purified by RP-HPLC. The product peaks (second main peak) were combined, frozen immediately (dry ice/acetone bath), acetonitrile was sublimed off on a high vacuum rotary centrifugal evaporator equipped with a dry ice/acetone finger condenser, and the still mostly frozen aqueous residue was freeze-dried in general to give the product compound 31(4.2mg) as a pale yellow solid.¹H-NMR: and (4) the consistency is achieved. LC-MS: t is t_RAt 12.4 min, M + H897.4 was measured.

Example 27.

Compound (I)32Synthesis of (2)

The compound is reacted at room temperature under argon30(25mg, 0.036mmol) and a compound34(14mg, 0.057mmol) in about 2mL dry CH₂Cl₂The mixture of (1) was stirred for about 3 hours and then kept at 4 ℃ overnight. The reaction was warmed to room temperature and the solvent was removed under reduced pressure to yield a crude compound containing31The residue of (2).

The material was redissolved in dry DMF (5mL) and solid glycyl- β -alanine (Bachem California Inc., Torrance, Calif., USA; Cat # H-3295) (15mg, 0.074mmol) was added. The stirred mixture was heated at 80 ℃ overnight under reflux condenser and under argon. Analytical RP-HPLC indicated that the desired product was formed as the main peak. The solvent was removed under high vacuum (rotary centrifugal evaporator) and the residue was redissolved in approximately 1.5mL of MeCN/water and purified by RP-HPLC. The product fractions were combined, MeCN was removed under reduced pressure, and the aqueous residue was frozen and freeze-dried to give the product as a solid32(23mg, 64% overall).¹H-NMR: and (4) the consistency is achieved. LC-MS: t is t_R10.5 min, M + H985.5 was measured.

Example 28.

Compound (I)33Synthesis of (2)

To the stirred compound32(18.1mg, 0.0184mmol) in 1mL dry CH₂Cl₂And 1mL of dry DMF was added NHS (2.6mg, 0.0226mmol) and EDC & HCl (3.9mg, 0.0203mmol), after which the reaction was stirred at room temperature under argon, followed by analytical RP-HPLC. After stirring overnight, additional NHS (2.6mg) and EDC & HCl (3.9mg) were added, after which the reaction was stirred at room temperature for an additional 4 hours. Analytical RP-HPLC indicated essentially complete reaction, formation of the desired product and small amounts of starting acid remained. Removing under high vacuumThe solvent was removed and the residue redissolved in 1:1 MeCN/water and purified by preparative RP-HPLC to give the product peak: t is t_R11.0 min, M + H1082.6; is sensitive to hydrolysis.

Example 29.

Compound (I)35Synthesis of (2)

Reduction of 2-methoxycarbonyl-4-chloro-6-nitroquinoline with stannous chloride in ethanol containing aqueous HCl according to literature procedures (Royer, R.J.chem.Soc, 1949, 1803; Bellamy, F.D. and Ou, K.tetrahedron Letters, 1984, 25,839-34(Maybridge CombiChem, Maybridge PLC, Tintagel, Cornwall, United Kingdom; catalog number SEW 05145) to give compound 35 as a dark red solid, which was used without further purification.

Example 30.

Compound (I)38Synthesis of (2)

Subjecting the crude compound35(0.68g) was dissolved in 10ml of 50% methanol in ethyl acetate, and then (BOC) was added to the solution₂O (865mg, 4mmol) followed by DMAP (40mg, 0.3mmol) was added. After stirring at room temperature for 8 hours, the reaction mixture was concentrated and then directly purified by silica gel column chromatography eluting with EtOAc/hexanes (1:1) to give the mono-BOC compound36a(100mg) and a di-BOC compound36b(120mg)。TLC：R_f0.33 (Compound)36aAnd 0.5936b50% EtOAc in hexanes).¹H-NMR: both are identical.

Mono-and di-BOC protected compounds36aAnd36b(remixed mixture, 160mg) was dissolved in 10mL of methanol, followed by saponification with LiOH (43mg, monohydrate, 1mmol, dissolved in 0.5mL of water) for 4 hours. The reaction mixture was concentrated, the residue treated with 2.5mL 1N HCl, and extracted with EtOAc (2 × 10 mL). The extracts were combined and dried (MgSO)₄) And concentrated in vacuo to afford the product (150mg) as a yellow solid, which contained¹H-NMR and LC-MS analysis showed mono-BOC compounds as a 2:1 mixture37aAnd di-BOC compounds37b. The mixture was used in the next step without further purification. LC-MS: compound (I)37a：t_R13.7 min, M + H323.1; compound (I)37b：t_R15.6 min, M + H423.1 was measured.

Will come from above37aAnd37bthe mixture of (3) was dissolved in 5mL of dry THF, and NHS (58mg, 0.5mmol) was then added to the stirred solution, followed by EDC. HCl (83mg, 0.43 mmol). After stirring at room temperature for 2h, the reaction mixture was washed with14(247mg, 0.38mmol) and DMAP (10mg) were treated the reaction mixture containing the corresponding NHS ester. The resulting mixture was further stirred at room temperature for 12 hours, and then concentrated under reduced pressure. The residue was treated with 10mL TFA and the solution was stirred at room temperature for 4 hours. TFA was removed in vacuo and the residue was then purified by preparative RP-HPLC to give the compound as a pale brown solid38(34mg)。¹H-NMR: and (4) the consistency is achieved. LC-MS: t is t_R12.7 min, M + H720.3 was measured.

Example 31.

Compound (I)39Synthesis of (2)

In analogy to the synthesis of Compounds31The method of (1) Compound38And compounds34Reacted and purified in a similar manner to give the compound39。

Claims

1. A compound having the structure

2. The compound of claim 1, wherein a is an active ester.

3. The compound of claim 1 having the structure

4. A compound having the structure

5. The compound of claim 4 wherein Q is selected from the group consisting of bovine serum albumin, keyhole limpet hemocyanin, and aminodextran.

6. Conjugate of the compound succinimidyl-oxycarbonyl-ethylamino-glycyl-glutaryl-aminomethyl- (pyr) saquinavir with KLH: (25)

7. (ii) a conjugate of the compound succinimidyl-oxycarbonyl-ethylamino-glycyl-glutaryl-aminomethyl- (pyr) saquinavir with BSA26)

8. The compound succinimidyl-benzoyl-aminocaproyl(ii) conjugates of methyl- (pyr) saquinavir with BSA27)。

9. A compound having the structure

10. The compound of claim 9, wherein a is an active ester.

11. A compound having the structure

12. The compound of claim 11 wherein Q is selected from the group consisting of bovine serum albumin, keyhole limpet hemocyanin, and aminodextran.

13. An antibody produced in response to a compound having the structure:

wherein L is a linking group comprising 0 to 40 carbon atoms in a linear or branched arrangement, is saturated or unsaturated and comprises up to two ring structures and 0-20 heteroatoms, with the proviso that up to two heteroatoms can be connected in sequence, P is a polypeptide, and n is a number of 1 to 50 per kilodalton molecular weight of P.

14. A monoclonal antibody specific for saquinavir having less than 1% cross-reactivity with nelfinavir and with saquinavir metabolites M4 and M6.

15. Murine hybridoma SAQ 137.3.3, having ATCC number PTA-6329.