JP2010508250A - Immunochemically equivalent HIV drug analogues - Google Patents

Abstract

  HIV protease inhibitor analogs and non-nucleoside reverse transcriptase inhibitor analogs that are immunochemically equivalent to the parent compound are provided. These analog compounds are useful as calibrators and positive controls in assays for measuring HIV protease inhibitors and non-nucleoside reverse transcriptase inhibitors in samples. A method for preparing a calibration curve using the analog and a test kit containing the analog are also provided.

Description

RELATED APPLICATION This application claims priority from US Provisional Application No. 60 / 863,442, filed Oct. 30, 2006.

The present invention relates to the field of therapeutic drug monitoring, and in particular to immunoassay methods for determining the presence or amount of HIV protease inhibitors or non-nucleoside reverse transcriptase inhibitors in a biological sample. More particularly, the present disclosure provides analogs of HIV protease inhibitors and non-nucleoside reverse transcriptase inhibitors that are useful as positive controls or as calibrators in immunoassays for measuring the amount of parent drug present in a sample. To do.

Clinical care of patients with background acquired immune deficiency syndrome (AIDS) has been significantly improved by the introduction of HIV protease inhibitors and HIV reverse transcriptase inhibitors.

  Currently, amprenavir, fosamprenavir, atazanavir, tipranavir, indinavir, darunavir, lopinavir, nelfinavir, ritonavir ) And saquinavir have been approved by the US Food and Drug Administration (FDA) for the treatment of AIDS patients. Combination therapy, including HIV protease inhibitors and HIV reverse transcriptase inhibitors, is the backbone of currently recommended therapies for HIV infection. HIV reverse transcriptase inhibitors fall into two general classes, depending on whether they are nucleosides or non-nucleoside analogs. Nucleoside reverse transcriptase inhibitors (NRTIs) are prodrugs that are converted intracellularly into their active nucleotide form. Non-nucleoside reverse transcriptase inhibitors (NNRTI), on the other hand, are the active drug form that is administered to patients. Currently, three NNRTIs have been approved by the US Food and Drug Administration for the treatment of AIDS patients: delavirdine, efavirenz, and nevirapine. Not all AIDS patients show the same optimal response to a combination therapy plan, and there is a large variation in drug response among individual patients. Accumulating clinical information supports the relationship between systemic exposure to protease inhibitors or NNRTIs and their antiviral effects. When a combination regimen is administered to a patient, potential pharmacokinetic drug-drug interactions may improve treatment or reduce its effectiveness. Patient compliance directly related to maintaining moderate drug levels can also affect treatment outcome.

  Therefore, it is desirable to measure the concentrations of HIV protease inhibitors and NNRTIs in patients to ensure that drug exposure is sufficient to maintain antiviral activity in AIDS patients. Such quantitative measurement methods need to be carefully calibrated and quality controlled using a calibrator or reference standard containing the parent drug. This is particularly true when the measurement method is an immunoassay. However, the problem is that such standards are often not commercially available or suitable for routine measurements. Surprisingly, in the case of immunoassays for detecting and measuring HIV drugs, an HIV protease that is immunochemically equivalent to the corresponding parent drug and can be used in place of the parent drug as a calibrator and positive control, and It has been found that analogs of NNRTI can be produced.

SUMMARY OF THE INVENTION As disclosed herein, an HIV protease inhibitor that exhibits an immunochemically equivalent binding affinity to the parent drug for antibodies raised against each of the parent HIV protease inhibitor and NNRTI compounds, and Provides analogs of NNRTI. Therefore, amprenavir, atazanavir, tipranavir, indinavir, darunavir, lopinavir, nelfinavir, ritonavir or saquinavir protease provides inhibitor analog wherein the hydroxyl groups of the central parent drug, -O (C ₁ -C ₁₀ alkyl), - OCONHR _3, groups selected from -OCH ₂ COOR ₃ and -OCH ₂ CONHR ₃ Wherein R ₃ is selected from the group consisting of H, C ₁ -C ₆ alkyl, phenyl, heteroaryl and — (CH ₂ ) _n —X, wherein n is 1-6 in and, X is COOR ₅ or NH-CH ₂ - phenyl -COOR _5, wherein, R ₅ is H or C ₁ -C ₆ alkyl. Accordingly, analogs of non-nucleoside reverse transcriptase inhibitors that are N-alkylated with C ₁ -C ₆ alkyl or (C ₁ -C ₆ alkyl) OH are also provided. Such HIV protease inhibitors and NNRTI analogs are immunochemically equivalent to the parent protease inhibitor and NNRTI compounds, respectively.

［式中、R₄は、C₁-C₁₀アルキル、-CH₂COOR₃、-CH₂CONHR₃および-CONHR₃からなる群から選ばれ、ここで、R₃は、H、C₁-C₆アルキル、フェニル、ヘテロアリールおよび-(CH₂)_n-Xからなる群から選ばれ、ここで、nは1〜6であり、XはCOOR₅またはNH-CH₂-フェニル-COOR₅であり、ここで、R₅はHまたはC₁-C₆アルキルである］
を有する。 More particularly, in one embodiment, an immunochemically equivalent analog of atazanavir (ATV) is provided, wherein the analog has the structure:

Wherein R ₄ is selected from the group consisting of C ₁ -C ₁₀ alkyl, —CH ₂ COOR ₃ , —CH ₂ CONHR ₃ and —CONHR ₃ , wherein R ₃ is H, C ₁ -C is selected from (CH ₂₎ group consisting of _n -X, wherein, n is 1 to 6, X is COOR ₅ or NH-CH ₂ - - ₆ alkyl, phenyl, heteroaryl and phenyl -COOR ₅ Where R ₅ is H or C ₁ -C ₆ alkyl]
Have

［式中、R₁は、Hおよび-CONHR₃からなる群から選ばれ、R₄は、C₁-C₁₀アルキル、-CH₂COOR₃、-CH₂CONHR₃および-CONHR₃からなる群から選ばれ、ここで、R₃は、H、C₁-C₆アルキル、フェニル、ヘテロアリールおよび-(CH₂)_n-Xからなる群から選ばれ、ここで、nは1〜6であり、XはCOOR₅またはNH-CH₂-フェニル-COOR₅であり、ここで、R₅はHまたはC₁-C₆アルキルである］
を有する。 In another specific embodiment, an immunochemically equivalent analog of lopinavir (LPV) is provided, wherein the analog has the structure:

Wherein R ₁ is selected from the group consisting of H and —CONHR ₃ and R ₄ is from the group consisting of C ₁ -C ₁₀ alkyl, —CH ₂ COOR ₃ , —CH ₂ CONHR ₃ and —CONHR ₃ Wherein R ₃ is selected from the group consisting of H, C ₁ -C ₆ alkyl, phenyl, heteroaryl and — (CH ₂ ) _n —X, wherein n is 1-6; X is COOR ₅ or NH-CH ₂ - phenyl -COOR _5, wherein, R ₅ is H or C ₁ -C ₆ alkyl]
Have

［式中、RはC₁-C₆アルキルまたは(C₁-C₆アルキル)OHである］
を有する。 In another specific embodiment, an immunochemically equivalent analog of efavirenz (EFV) is provided, wherein the analog has the structure:

[Wherein R is C ₁ -C ₆ alkyl or (C ₁ -C ₆ alkyl) OH]
Have

The present specification for establishing or creating a calibration curve for a HIV protease inhibitor or non-nucleoside reverse transcriptase inhibitor as a positive control for use in an immunochemical-based experiment or for use in an immunoassay. Also provided are methods of using the immunochemically equivalent analogs disclosed in the literature. In one particular embodiment, the protease inhibitor analogs disclosed herein are used to use amprenavir, atazanavir, tipranavir, indinavir, darunavir ( A calibration curve is generated for a protease inhibitor selected from the group consisting of darunavir, lopinavir, nelfinavir, ritonavir and saquinavir. A method for preparing such a calibration curve is an immunochemically equivalent analog of the protease inhibitor, wherein the central hydroxyl group of the protease inhibitor is -O (C ₁ -C ₁₀ alkyl),- Substituted with a group selected from OCONHR ₃ , —OCH ₂ CONHR ₃ and —OCH ₂ COOR ₃ , wherein R ₃ is H, C ₁ -C ₆ alkyl, phenyl, heteroaryl and — (CH ₂ ) _n —X selected from the group consisting of, n is 1 to 6, X is COOR ₅ or NH-CH ₂ - phenyl -COOR _5, R ₅ comprises a analogue is H or C ₁ -C ₆ alkyl, Preparing a series of samples. Each sample is then contacted with an amount of antibody and a labeled conjugate of the HIV drug. Competition occurs between the labeled conjugate and the HIV drug analog for a limited number of antibody binding sites. After a certain reaction time, the amount of labeled conjugate bound to the antibody is detected. (Alternatively, one skilled in the art will also recognize that the amount of labeled conjugate that remains free, i.e. unbound, can be detected.) The signal from the labeled conjugate and the amount of drug analog present. A calibration curve showing an inversely proportional relationship is established. Such a calibration curve is substantially equivalent to the curve obtained from the parent drug.

In another specific embodiment, a method is provided for establishing a calibration curve for efavirenz, an HIV non-nucleoside reverse transcriptase inhibitor. The method comprises immunochemically equivalent analogs of the non-nucleoside reverse transcriptase inhibitor at a range of concentrations that are N-alkylated with C ₁ -C ₆ alkyl or (C ₁ -C ₆ alkyl) OH. Preparing a series of samples or a set of calibrators. Each sample is then contacted with an amount of antibody and a conjugate comprising NNRTI and a signal generating moiety or label. Competition occurs between the labeled conjugate and the NNRTI drug analog for a limited number of antibody binding sites. After a certain reaction time, the labeled conjugate bound to the antibody is detected and measured. A calibration curve is then constructed that shows an inverse relationship between the signal from the labeled conjugate and the amount of drug analog present. Such a calibration curve is substantially equivalent to the curve obtained from the parent drug.

Further provided is a test kit for determining or measuring the amount of HIV protease inhibitor or non-nucleoside reverse transcriptase inhibitor present in a sample. The kit according to one embodiment comprises an antibody that specifically binds to an HIV protease inhibitor, and an immunochemically equivalent analog of the protease inhibitor, wherein the central hydroxyl group of the protease inhibitor is -O (C _1- C ₁₀ alkyl), -OCONHR ₃ , -OCH ₂ CONHR ₃ and -OCH ₂ COOR ₃ where R ₃ is H, C ₁ -C ₆ alkyl, phenyl, heteroaryl and - is selected from (CH ₂₎ group consisting of _n -X, wherein, n is 1 to 6, X is COOR ₅ or NH-CH ₂ - phenyl -COOR _5, wherein, R ₅ includes analogs that are H or C ₁ -C ₆ alkyl.

［式中、R₁は、Hおよび-CONHR₃からなる群から選ばれ、R₄は、C₁-C₁₀アルキル、-CH₂COOR₃、-CH₂CONHR₃および-CONHR₃からなる群から選ばれ、ここで、R₃は、H、C₁-C₆アルキル、フェニル、ヘテロアリールおよび-(CH₂)_n-Xからなる群から選ばれ、ここで、nは1〜6であり、XはCOOR₅またはNH-CH₂-フェニル-COOR₅であり、ここで、R₅はHまたはC₁-C₆アルキルである］を有する免疫化学的に同等なロピナビル類似体、
を含む。 More particularly, in one embodiment, a kit for measuring lopinavir in a sample is provided, wherein the kit specifically comprises an antibody that specifically binds to lopinavir, and a structure:

[Wherein R ₁ is selected from the group consisting of H and —CONHR ₃ and R ₄ is selected from the group consisting of C ₁ -C ₁₀ alkyl, —CH ₂ COOR ₃ , —CH ₂ CONHR ₃ and —CONHR ₃ Wherein R ₃ is selected from the group consisting of H, C ₁ -C ₆ alkyl, phenyl, heteroaryl and — (CH ₂ ) _n —X, wherein n is 1-6; X is COOR ₅ or NH-CH ₂ - phenyl -COOR _5, wherein, R ₅ is immunochemically equivalent lopinavir analogs with a a] H or C ₁ -C ₆ alkyl,
including.

［式中、R₄は、C₁-C₁₀アルキル、-CH₂COOR₃、-CH₂CONHR₃および-CONHR₃からなる群から選ばれ、ここで、R₃は、H、C₁-C₆アルキル、フェニル、ヘテロアリールおよび-(CH₂)_n-Xからなる群から選ばれ、ここで、nは1〜6であり、XはCOOR₅またはNH-CH₂-フェニル-COOR₅であり、ここで、R₅はHまたはC₁-C₆アルキルである］
を有する免疫化学的に同等なアタザナビル類似体、
を含む。 In another specific embodiment, a test kit for measuring atazanavir in a biological sample is provided, wherein the kit specifically comprises an antibody that specifically binds atazanavir, and a structure:

Wherein R ₄ is selected from the group consisting of C ₁ -C ₁₀ alkyl, —CH ₂ COOR ₃ , —CH ₂ CONHR ₃ and —CONHR ₃ , wherein R ₃ is H, C ₁ -C is selected from (CH ₂₎ group consisting of _n -X, wherein, n is 1 to 6, X is COOR ₅ or NH-CH ₂ - - ₆ alkyl, phenyl, heteroaryl and phenyl -COOR ₅ Where R ₅ is H or C ₁ -C ₆ alkyl]
An immunochemically equivalent atazanavir analog having
including.

［式中、RはC₁-C₆アルキルまたは(C₁-C₆アルキル)OHである］
を有するエファビレンツ類似体、を含む、サンプル中のエファビレンツの量を測定するためのキットを提供する。 The kit in another specific embodiment includes an antibody that specifically binds to a non-nucleoside reverse transcriptase inhibitor, and an immunochemically equivalent analog of the non-nucleoside reverse transcriptase inhibitor, wherein the analog The body is N-alkylated with C ₁ -C ₆ alkyl or (C ₁ -C ₆ alkyl) OH. In certain embodiments, an antibody that specifically binds to efavirenz, and a structure:

[Wherein R is C ₁ -C ₆ alkyl or (C ₁ -C ₆ alkyl) OH]
A kit for measuring the amount of efavirenz in a sample is provided.

FIG. 1 shows the structures of various HIV protease inhibitor compounds described herein. The arrow indicates the corresponding central hydroxyl group in each structure. FIG. 2 shows a schematic diagram of a synthetic scheme for the synthesis of lopinavir-O ^c -methyl ether ( 2 ). FIG. 3 shows a schematic diagram of a synthetic scheme for the synthesis of lopinavir-O ^c- (methoxycarbonylmethyl) ether ( 3 ). FIG. 4 shows a schematic diagram of a synthetic scheme for the synthesis of lopinavir-O ^c- (N-tert-butylcarbamoylmethyl) ether ( 6 ). FIG. 5 shows a schematic diagram of a synthetic scheme for the synthesis of lopinavir-O ^c -carbamate ( 8 ). FIG. 6 shows a schematic diagram of a synthetic scheme for the synthesis of N-carbamoyl-lopinavir-O ^c -carbamate ( 10 ). FIG. 7 shows a schematic diagram of a synthetic scheme for the synthesis of N-carbamoyl-lopinavir ( 13 ). FIG. 8 shows a schematic diagram of a synthetic scheme for the synthesis of N-alkylated efavirenz analogs 14 , 15 , 16 , 16a , 17 and 17a . FIG. 9 shows a schematic diagram of a synthetic scheme for the synthesis of N- (hydroxypropyl) efavirenz ( 19 ) and N- (hydroxyethyl) efavirenz ( 19a ). FIG. 10 shows atazanavir-O ^c -ethyl carbamate ( 21 ), atazanavir-O ^c -isopropyl carbamate ( 22 ), atazanavir-O ^c -tert-butyl carbamate ( 23 ), atazanavir-O ^c -carbamate ( 25 ) And atazanavir-O ^c- (3-pyridyl) carbamate ( 41 ). FIG. 11 is a schematic diagram of a synthetic scheme for the synthesis of atazanavir-O ^c -methyl ether ( 26 ) and atazanavir-O ^c- (hydroxyethyl) ether ( 28 ). FIG. 12a shows a calibration curve for the lopinavir antibody LPV 1.1.85 obtained using lopinavir and N-carbamoyl-lopinavir ( 13 ) as calibrators. FIG. 12b shows a calibration curve for the lopinavir antibody LPV 1.7.90 obtained using lopinavir and N-carbamoyl-lopinavir ( 13 ) as calibrators. FIG. 13 is a schematic diagram of a synthetic scheme for the synthesis of atazanavir-O ^c -carbonyl-aminobutyroyl-aminomethylbenzoic acid NHS ester ( 33 ). FIG. 14 is a schematic diagram of a synthetic scheme for the synthesis of 3- (2-iodo-acetamido) -propionic acid ethyl ester ( 34 ). FIG. 15 is a schematic diagram of a synthetic scheme for the synthesis of 4- [3- (Lopinavir-O ^c ) -acetamido) propionamido] methylbenzoic acid N-hydroxysuccinimide ester ( 40 ). FIG. 16 shows a calibration curve for atazanavir microparticle immunoassay using atazanavir and atazanavir-O ^c -carbamate as calibrators. FIG. 17 shows a spike recovery test for an atazanavir microparticle immunoassay using serum samples spiked with atazanavir and assayed against an atazanavir-O ^c -carbamate calibration curve. FIG. 18 shows a spike recovery test of a lopinavir microparticle immunoassay using serum samples spiked with lopinavir and assayed against an N-carbamoyl-lopinavir calibration curve. FIG. 19 is a schematic diagram of a synthetic scheme for the synthesis of efavirenz aminodextran conjugate ( 45 ).

DETAILED DESCRIPTION OF THE INVENTION The term “specifically binding” as used herein includes, for example, ligand / target pairs, enzyme / substrate pairs, receptor / agonist pairs, antibody / antigen pairs and lectin / sugar chain pairs. , Means binding with high avidity and / or high affinity between two paired species. The binding interaction can be mediated by covalent or non-covalent interactions, or a combination of covalent and non-covalent interactions.

As used herein, the term “antibody” refers to a polypeptide composed of at least one binding domain. From the folding of the variable domain of the antibody molecule to form a three-dimensional binding space with an internal surface shape and charge distribution that is complementary to the antigenic determinant characteristics of the antigen, allowing for an immune reaction with the antigen, from the antibody binding domain Is formed. Unless otherwise indicated, general references to antibodies include polyclonal and monoclonal antibodies. The term “antibody” also includes recombinant proteins comprising a binding domain, as well as fragments of antibodies, such as Fab, Fab ′, F (ab) ₂ and F (ab ′) ₂ .

  As used herein, the term “immunochemically equivalent” refers to two or more related compounds that bind to a single antibody with similar affinity, where the antibody is immunochemically Are produced for one member of the group of compounds equivalent to the above, and specifically bind to the member. More specifically, an immunochemically equivalent compound cross-reacts with the antibody in an immunoassay within a range of about 70% to about 130% as obtained using the compound from which the antibody is produced.

The term “C ₁ -C _n alkyl” represents a branched or straight chain alkyl group having from one to the specified number of carbon atoms. Typical C ₁ -C ₆ alkyl groups include methyl, ethyl, n-propyl, iso-propyl, butyl, iso-butyl, sec-butyl, tert-butyl, pentyl, hexyl, and the like. It is not limited.

  As used herein, the term “aryl” refers to monocyclic having one or two aromatic rings, including but not limited to phenyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl, and the like. Or refers to a bicyclic carbocyclic ring system. Aryl groups (including bicyclic aryl groups) may be unsubstituted or independently selected from lower alkyl, haloalkyl, alkoxy, amino, alkylamino, dialkylamino, hydroxyl, halo and nitro May be substituted with 1, 2 or 3 substituents. Substituted aryl groups include tetrafluorophenyl and pentafluorophenyl.

  As used herein, the term “heteroaryl” refers to a monocyclic or bicyclic ring system containing one or two aromatic rings and containing at least one nitrogen, oxygen or sulfur atom in the aromatic ring. Means. Heteroaryl groups (including bicyclic heteroaryl groups) may be unsubstituted or independently selected from lower alkyl, haloalkyl, alkoxy, amino, alkylamino, dialkylamino, hydroxyl, halo and nitro Optionally substituted by 1, 2 or 3 substituents. Specific examples of heteroaryl groups include, but are not limited to, pyridine, pyrazine, pyrimidine, pyridazine, pyrazole, triazole, thiazole, isothiazole, benzothiazole, benzoxazole, thiadiazole, oxazole, pyrrole, imidazole and isoxazole. Is not to be done.

  As used herein, the term “heteroalkyl” refers to an alkyl chain that contains one or more nitrogen, oxygen, or sulfur atoms in the backbone of the alkyl chain.

  Terms such as “preferably”, “generally” and “typically” are used herein to limit the scope of the claimed invention or certain features are claimed as It should be noted that it is not used to imply that it is critical, essential or even important to the structure or function of the invention being described. Rather, these terms are merely intended to highlight alternative or additional features that may or may not be used in certain embodiments of the invention.

  For purposes of describing and defining the present invention, the term “substantial” is used herein to refer to inherent uncertainties that may be attributed to any quantitative comparison, value, measurement, or other indication. Note that it is used to represent degrees. The term “substantial” is used herein to describe the degree to which a quantitative indication can vary from the indicated criteria without causing a change in the basic function of the object in question. Used.

  More specifically, the term “substantially equivalent” refers to the result obtained for an individual sample in a set of calibration materials, or the absorbance measured for an individual calibration material, or the measurement of such a result or absorbance. It means that the curve produced is within the accuracy limits of the assay. In particular, if the slope of the spike and recovery curve obtained using an analog calibration substance is within 10% of the ideal value, then the calibration or standard substance produced using the analog compound must be the parent drug. Considered to be substantially equivalent to the calibration or reference material produced using it. That is, if the slope of the curve generated using the parent drug is 1.0, the slope of the spike and recovery curve generated using the analog calibration material should be 0.9-1.1.

  As used herein, an “analog” of an HIV protease inhibitor or non-nucleoside reverse transcriptase inhibitor refers to the HIV protease inhibitor with respect to the binding affinity of an antibody that specifically binds to the HIV protease inhibitor or non-nucleoside reverse transcriptase inhibitor. Or a chemical compound derived from the HIV protease inhibitor or non-nucleoside reverse transcriptase inhibitor that behaves in substantially the same manner as the non-nucleoside reverse transcriptase inhibitor.

  The term “derivative” refers to a chemical compound or molecule made from a “parent” compound or molecule by one or more chemical reactions. In the present invention, the parent compound is an HIV protease inhibitor and a non-nucleoside reverse transcriptase inhibitor, and the analogs are derivatives of the HIV protease inhibitor and the non-nucleoside reverse transcriptase inhibitor, respectively.

As used herein, a “signal generating moiety” is an identifying tag that can be used to detect an analyte such as an HIV protease inhibitor or a non-nucleoside reverse transcriptase inhibitor when bound to a carrier material or molecule. Or a sign. The label can be bound to the carrier material directly or indirectly by a linking or bridging moiety. Specific examples of signal generating moieties include enzymes such as β-galactosidase and peroxidase, fluorescent compounds such as rhodamine and fluorescein isothiocyanate (FITC), luminescent compounds such as dioxetane and luciferin, radioisotopes such as ¹²⁵ I, and microparticles Is included.

  Any sample reasonably suspected of containing an HIV protease inhibitor or non-nucleoside reverse transcriptase inhibitor of interest can be analyzed by the methods of the invention. The sample is typically an aqueous solution, such as a body fluid from a host, such as urine, whole blood, plasma, serum, saliva, semen, feces, sputum, cerebrospinal fluid, tears, mucus, etc. Plasma or serum. If desired, the sample may be pretreated and can be prepared in any convenient medium that does not interfere with the assay. An aqueous medium is preferred.

  “Proofreading material” means any standard or reference material that contains a known amount of HIV protease inhibitor or non-nucleoside reverse transcriptase inhibitor to be measured. Samples suspected of containing the HIV protease inhibitor or non-nucleoside reverse transcriptase inhibitor of interest and generally a set of calibrators are assayed under similar conditions. The concentration of HIV protease inhibitor or non-nucleoside reverse transcriptase inhibitor in the sample is then determined by comparing the results obtained for the unknown sample with the results obtained for the calibration material or set of calibration materials. . This is typically done by creating a calibration curve such as in FIGS. 12a and 12b.

  A “positive control” is a sample containing a known amount of an HIV protease inhibitor or non-nucleoside reverse transcriptase inhibitor of interest. Samples suspected of containing the HIV protease inhibitor or non-nucleoside reverse transcriptase inhibitor of interest are assayed under similar conditions, and the accuracy of the assay results obtained for the unknown sample is determined by the recovery obtained in the positive control. decide.

  Abbreviations used herein for various HIV protease inhibitors are abbreviations that are currently becoming the standard used in the art, for example, ATV stands for atazanavir and LPV is lopinavir. EFV stands for efavirenz. Certain patent applications incorporated herein by reference may use old abbreviations for the same HIV protease inhibitor (eg, ATZ for atazanavir, LOPIN for lopinavir, and EFA for efavirenz). However, both the old and new abbreviations indicate the same HIV protease inhibitor.

  Another embodiment of the invention performs an assay for establishing a calibration curve for an HIV protease inhibitor or non-nucleoside reverse transcriptase inhibitor and for measuring an HIV protease inhibitor or non-nucleoside reverse transcriptase inhibitor in a sample It relates to a useful test kit. Reagents useful in the methods of the present invention are in liquid or lyophilized form, in the same or separate containers, in packaged combinations, such that the ratio of those reagents provides sufficient optimization of the method and assay. It can be provided conveniently. Each of these reagents may be present in a separate container, or various reagents may be combined in one or more containers depending on the cross-reactivity and stability of the reagents. The test kit generally includes appropriate calibrators, controls and instructions for use.

  In one embodiment, an analog of an HIV protease inhibitor drug that is immunochemically equivalent to the parent drug compound is provided. More particularly, in one embodiment, an HIV protease inhibitor analog that is immunochemically equivalent to the parent protease inhibitor is provided so that it can be used as a drug proofing substance and / or a positive control in an antibody-based immunoassay. . In one embodiment, the HIV protease inhibitor analog has about 80% to about 120% cross-reactivity in the immunoassay compared to that obtained using its parent compound. In another embodiment, the HIV protease inhibitor analog has about 90% to about 115% cross-reactivity in the immunoassay compared to that obtained using its parent compound. In another embodiment, the protease inhibitor is amprenavir, atazanavir, tipranavir, indinavir, darunavir, lopinavir, nelfinavir, ritonavir HIV protease inhibitor analogs are provided, selected from the group consisting of (ritonavir) and saquinavir, wherein the central hydroxyl group of the protease inhibitor is derivatized to form an ether or carbamate.

In another embodiment, amprenavir, atazanavir, tipranavir, indinavir, darunavir, lopinavir, nelfinavir, ritonavir and includes a protease inhibitor selected from the group consisting of saquinavir (saquinavir), the inhibitor is a hydroxyl group of the center -O (C ₁ -C _{10 alkyl), - CH 2 COOR 3,} -CONHR 3 or -CH ₂ CONHR ₃ are modified by substituting, where, R ₃ is, H, C ₁ -C ₆ alkyl, phenyl, heteroaryl, and - is selected from (CH ₂₎ group consisting of _n -X, wherein n is 1 to 6, X is COOR ₅ or NH-CH ₂ - phenyl -COOR _5, wherein, R ₅ is H or C ₁ -C ₆ alkyl.

In one embodiment, an immunochemically equivalent analog of lopinavir is provided. The structure of lopinavir is as follows.

［式中、R₁は、Hおよび-CONHR₃からなる群から選ばれ、R₄は、C₁-C₁₀アルキル、-CH₂COOR₃、-CH₂CONHR₃および-CONHR₃からなる群から選ばれ、ここで、R₃は、H、C₁-C₆アルキル、フェニル、ヘテロアリールおよび-(CH₂)_n-Xからなる群から選ばれ、ここで、nは1〜6であり、XはCOOR₅またはNH-CH₂-フェニル-COOR₅であり、ここで、R₅はHまたはC₁-C₆アルキルである］
を有する。 In one embodiment, the central hydroxyl group of lopinavir is derivatized to form an ether or carbamate and / or an N-carbamate group is added to a terminal cyclic urea. Immunochemically equivalent analogs are provided. In one embodiment, the immunochemically equivalent lopinavir analog has the structure:

Wherein R ₁ is selected from the group consisting of H and —CONHR ₃ and R ₄ is from the group consisting of C ₁ -C ₁₀ alkyl, —CH ₂ COOR ₃ , —CH ₂ CONHR ₃ and —CONHR ₃ Wherein R ₃ is selected from the group consisting of H, C ₁ -C ₆ alkyl, phenyl, heteroaryl and — (CH ₂ ) _n —X, wherein n is 1-6; X is COOR ₅ or NH-CH ₂ - phenyl -COOR _5, wherein, R ₅ is H or C ₁ -C ₆ alkyl]
Have

Specific examples of lopinavir analogs suitable for use as drug calibrators and / or positive controls for antibody-based assays for lopinavir include the following compounds:

In one embodiment, an immunochemically equivalent analog of atazanavir is provided. The structure of atazanavir is as follows:

［式中、R₄は、C₁-C₁₀アルキル、-CH₂COOR₃、-CH₂CONHR₃および-CONHR₃からなる群から選ばれ、ここで、R₃は、H、C₁-C₆アルキル、フェニル、ヘテロアリールおよび-(CH₂)_n-Xからなる群から選ばれ、ここで、nは1〜6であり、XはCOOR₅またはNH-CH₂-フェニル-COOR₅であり、ここで、R₅はHまたはC₁-C₆アルキルである］
を有する、アタザナビル（atazanavir）の免疫化学的に同等な類似体を提供する。別の実施形態においては、R₄は、-CH₂CONHR₃および-CONHR₃からなる群から選ばれ、ここで、R₃は、HまたはC₁-C₄アルキルである。別の実施形態では、R₄は、-CONHR₃であり、R₃は、HまたはCH₂CH₃である。 In one embodiment, the structure:

Wherein R ₄ is selected from the group consisting of C ₁ -C ₁₀ alkyl, —CH ₂ COOR ₃ , —CH ₂ CONHR ₃ and —CONHR ₃ , wherein R ₃ is H, C ₁ -C is selected from (CH ₂₎ group consisting of _n -X, wherein, n is 1 to 6, X is COOR ₅ or NH-CH ₂ - - ₆ alkyl, phenyl, heteroaryl and phenyl -COOR ₅ Where R ₅ is H or C ₁ -C ₆ alkyl]
An immunochemically equivalent analog of atazanavir is provided. In another embodiment, R ₄ is selected from the group consisting of —CH ₂ CONHR ₃ and —CONHR ₃ , wherein R ₃ is H or C ₁ -C ₄ alkyl. In another embodiment, R ₄ is —CONHR ₃ and R ₃ is H or CH ₂ CH ₃ .

［式中、R₃は、H、C₁-C₆アルキル、フェニル、ヘテロアリールおよび-(CH₂)_n-Xからなる群から選ばれ、ここで、nは1〜6であり、XはCOOR₅またはNH-CH₂-フェニル-COOR₅であり、ここで、R₅はHまたはC₁-C₆アルキルである］
を有する。 In another embodiment, an immunochemically equivalent analog of atazanavir is provided wherein the central hydroxyl group of atazanavir is derivatized to form a carbamate. In one embodiment, the immunochemically equivalent analog of atazanavir has the structure:

Wherein R ₃ is selected from the group consisting of H, C ₁ -C ₆ alkyl, phenyl, heteroaryl and — (CH ₂ ) _n —X, wherein n is 1-6 and X is COOR ₅ or NH-CH ₂ - phenyl -COOR _5, wherein, R ₅ is H or C ₁ -C ₆ alkyl]
Have

Specific examples of atazanavir analogs used as calibrators or positive controls in antibody-based assays for measuring atazanavir include the following compounds:

One embodiment provides an immunochemically equivalent analog of a non-nucleoside reverse transcriptase inhibitor (NNRTI). More particularly, in one embodiment, it is immunochemically equivalent to the parent NNRTI so that it can be used as a calibrator and / or positive control in an antibody-based immunoassay to measure NNRTI in a sample. NNRTI analogs are provided. In one embodiment, the NNRTI analog has about 80% to about 120% cross-reactivity in the immunoassay compared to that obtained using the parent NNRTI compound. In another embodiment, the NNRTI analog has about 90% to about 115% cross-reactivity in the immunoassay compared to that obtained using the parent NNRTI. In one embodiment, the NNRTI analog is efavirenz. In one embodiment, N-alkylated analogs of efavirenz are provided. The structure of efavirenz is as follows.

Specific examples of efavirenz analogs suitable for use as drug calibrators and / or positive controls in antibody-based assays to measure efavirenz in a sample include the following compounds:

The present invention also includes a test kit containing assay reagents for measuring HIV protease inhibitors and non-nucleoside reverse transcriptase inhibitors in a sample. The kit further includes various containers, such as vials, tubes, bottles, etc., in packaged combinations that contain the reagents used to perform the assay. Preferably, the kit also includes instructions for use. In one embodiment, the test kit comprises an antibody that specifically binds to the HIV drug to be detected in the sample and an immunochemically equivalent analog of the HIV drug to be detected. In one embodiment, the kit comprises an antibody that specifically binds lopinavir and lopinavir analog lopinavir-O ^c- (N-tert-butylcarbamoylmethyl) ether ( 6 ). In another embodiment, the kit comprises lopinavir antibody and the lopinavir analog N-carbamoyl-lopinavir ( 13 ). In one embodiment, the kit comprises an antibody that specifically binds atazanavir and the analog atazanavir-O ^c -carbamate ( 25 ). In another embodiment, the kit comprises an antibody specific for atazanavir and the analog atazanavir-O ^c -ethylcarbamate ( 21 ). Thus, the kits disclosed herein establish or create a calibration curve for HIV protease inhibitors or non-nucleoside reverse transcriptase inhibitors to be detected or measured using drug analogs supplied with the kit. Can be used. The analog may or may not have parent drug inhibitory activity. However, since the analog is immunochemically equivalent to the parent drug, the calibration curve established using the drug analog is based on the detection signal from the immunoassay and the concentration of that drug in a given sample. Can be used to determine concentration.

These protease inhibitors and NNRTI analogs and derivatives can be synthesized using the methods described below and methods known to those skilled in the art. In the case of lopinavir, the molecule also contains an acidic nitrogen atom on the cyclic tetrahydropyrimidinone moiety in addition to the central hydroxyl group. Selective modification with the nitrogen requires protection of the central hydroxyl group. A number of suitable protecting groups are well known in the art. For example, "Protective Groups in Organic Synthesis" , 2 nd edition, T. Greene and P. Wuts, see Wiley-Interscience, 1991.

The central hydroxyl group can be protected as a silyl protecting group, for example a TBDMS (t-butyldimethylsilyl) or TBDPS (t-butyldiphenylsilyl) group. An ester group can also be used to protect the central hydroxyl functionality of lopinavir, and an acetate group in the presence of a base (preferably pyridine) (see compound 11 in FIG. 7)] preferable. Protection of the central hydroxyl group of lopinavir is carried out in a solvent such as dimethylformamide (DMF), preferably tetrahydrofuran (THF), typically for a period ranging from 0.5 hours to 7 days. After protection of the central hydroxyl group, a protected isocyanate, most preferably trichloroacetyl isocyanate, may be used to modify the acidic nitrogen on the cyclic tetrahydropyrimidinone moiety. Trichloroacetyl isothiocyanate can also be used to produce lopinavir N-thiocarbamoyl analogs. Hydrolysis under basic conditions deprotects both the central acetate group as well as the protected N-carbamoyl group to give the desired lopinavir N-carbamoyl analog (compound 13 in FIG. 7).

In another embodiment, the disubstituted lopinavir analog protected N-carbamoyl lopinavir O ^c -carbamate is prepared by reacting lopinavir with an excess of protected isocyanate reagent, preferably trichloroacetyl isocyanate. . Hydrolysis under basic conditions (preferably using aqueous potassium carbonate) gives the desired N-carbamoyllopinavir O ^c -carbamate (compound 10 in FIG. 6).

In another embodiment, lopinavir protected O ^c -carbamate is produced by reacting lopinavir with a limited amount of trichloroacetyl isocyanate. Hydrolysis under basic conditions (preferably using aqueous potassium carbonate) gives the desired lopinavir O ^c -carbamate (compound 8 in FIG. 5).

In another embodiment, the central hydroxyl group of lopinavir can be modified by an ether linkage. Under controlled conditions, various haloalkylating agents are used, for example in the presence of a limited amount of a base (preferably sodium hydride) in a solvent (preferably DMF) at a temperature ranging from 0 ° C. to room temperature. It is possible to modify the central hydroxyl functionality of lopinavir using a limited amount of alkylating agent. A lopinavir analogs, see compound 3 Compound 2 and 3 in Figure 2.

Hydrolysis of the ester group of lopinavir analogues can be achieved under acidic or basic conditions, preferably basic conditions, for example under lithium hydroxide at 0 ° C. to room temperature (FIG. 4). The resulting acidic functionality can be activated by using various activators, preferably 1- (3- (dimethylamino) propyl) -3-ethyl-carbodiimide (EDC) or N-hydroxysuccinimide . The resulting activated ester can be reacted with an amine, preferably t-butylamine, to give lopinavir t-butylamide analog (compound 6 in FIG. 4).

Atazanavir also has a central hydroxyl group found in other protease inhibitors, but in atazanavir it has its specific structural properties, such as the sterically necessary phenyl-pyridyl moiety in its vicinity. (But not limited to), the reactivity is affected. Substitution at the hydroxyl group can be performed to give alkylated analogs such as methyl, ethyl or alkyl analogs. Such substitution may be achieved by reaction with an alkyl halide in the presence or absence of other appropriately protected functionalities present on the alkyl moiety, such as a catalyst or mediator, such as a transition metal salt, such as rhodium. It can be carried out in a suitable solvent (eg, DMF, THF, etc.) in the presence of a salt or silver oxide, typically at a temperature from room temperature to about 100 ° C. Alkylating agents such as appropriately protected α-haloacetate, alkyl azide, azidoacetate, azidoalkanoate, diazoacetate, diazoalkanoate, alkyltosylate, etc. can be envisaged, but other reagents will suggest to those skilled in the art Will be done. For example, reaction with an alkyl halide such as methyl iodide in a solvent such as DMF in the presence of silver oxide gives O ^c -methyl ether 26 and tetrahydropyran protected bromoethanol as shown in Example 20. Reaction with gives the O ^c -ethylene alcohol 28 as described in Example 21. Although less preferred, it is also possible to deprotonate the hydroxyl group using a base such as a metal hydride and then react with a suitable alkylating reagent such as an alkyl halide, azide, tosylate, etc. In this case, a by-product or a rearrangement product may be generated.

Another approach is to react the hydroxyl group of atazanavir or other protease inhibitors with an alkyl or aryl isocyanate or isothiocyanate to obtain a simple analog with a carbamate bond at the hydroxyl position. Yes, in this case the oxygen now contains part of the carbamate bond. A number of suitable isocyanates or isothiocyanates can be used for this purpose, such as C ₁ -C ₁₀ alkyl, phenyl and heteroaryl isocyanates or isothiocyanates. Preferably, C ₁ -C ₄ alkyl isocyanate is used. For example, reaction with ethyl isocyanate, isopropyl isocyanate or tert-butyl isocyanate provides the corresponding alkyl carbamates exemplified by compounds 21 , 22 and 23 as described in Examples 15, 16 and 17. . An unsubstituted carbamate, such as compound 25, is reacted with a protected isocyanate, such as, but not limited to, trichloroacetyl isocyanate, followed by mildly basic conditions (eg, a water-alcohol mixture). Can be synthesized by obtaining "bare" carbamates by deprotection with the addition of carbonate in). Alternative means may be used, for example, reacting the hydroxyl with phosgene to give the O ^c -carbonyl chloride, followed by reaction with ammonia to give the same analog, and also a phosgene equivalent such as nitro You may make it react with ammonia, after making it react with a phenyl chloroformate.

Alternatively, for example, as described in US 7,157,561 (the disclosure of which is incorporated herein by reference), substituted alkyl or aryl isocyanates or isothiocyanates can be used for further refinement thereof. It is possible to obtain analogs and derivatives having the carbamate linkage at the central hydroxyl position with alkyl or aryl linkers from functionalized carbamate groups. Preferably C ₁ -C ₁₀ alkyl, phenyl and heteroaryl isocyanates or isothiocyanates are used, more preferably C ₁ -C ₆ isocyanates, where the alkyl or aryl group is another suitable Has functionality, eg, a protected carboxy, protected amine, protected thiol, or maleimide group. Such a reaction can be carried out in any suitable solvent such as DMF or THF, with or without the addition of a base such as a tertiary amine such as triethylamine or diisopropylethylamine. Such reactions can typically be carried out at temperatures between 0 ° C. and 100 ° C., frequently at ambient temperatures to 60 ° C. Non-limiting examples of this approach include the synthesis of carbamate-butyrate derivatives ( 29 ) as described in Example 22, and NHS esters as described in Examples 23-26 and shown in FIG. The refinement to ( 33 ) is mentioned. Other applications of this approach to synthesize equivalent compounds will also suggest to those skilled in the art.

  Other carbamate forming equivalents may also be used. For example, reaction of the central hydroxyl of atazanavir with phosgene, carbonyldiimidazole, disuccinimidyl carbonate or nitrophenyl chloroformate provides the active intermediate, which is converted to the appropriate alkyl or aryl Upon reaction with an amine, the corresponding substituted carbamate is obtained. Other protease inhibitors could also be modified for derivatization in this way.

An activated derivative of lopinavir ( 40 ) was also prepared for conjugation to aminodextran. The lopinavir immunogen used to generate the lopinavir antibody was modified at the central hydroxyl group of lopinavir [compound 6F in US 7,193,065, the disclosure of which is incorporated herein by reference], which leaves the same position. It was preferred to have lopinavir label. In lopinavir derivative 40 , the central hydroxyl group of lopinavir was bonded to the linking group using an ether bond. Ether linkages are generally more stable than ester or carbamate linkages. The alkylation reaction of lopinavir was carried out in the presence of a base, preferably sodium hydride, at a temperature between 0 ° C. and room temperature, preferably in DMF, using a halo-alkylating agent. The halo-alkylating agent may contain C ₁ -C ₁₀ atoms containing one or more heteroatoms. Preferably, the alkylating agent is compound 34 , which can be prepared from iodoacetic acid (FIG. 14). The resulting lopinaviric acid derivative ( 35 ) can be converted to an active ester group after alkylation of the lopinavir after hydrolysis. The conversion of acid derivatives to activated esters is known to those skilled in the art and can preferably be accomplished using carbodiimides such as EDC.HCl and N-hydroxysuccinimide. The activated ester ( 37 ) can be further extended, for example by reaction with methylaminomethyl benzoate in the presence of a tertiary amine (FIG. 15) to give lopinavir methyl ester derivative 38 , It can be hydrolyzed under acidic or basic conditions. In this case, the hydrolysis was performed by basic hydrolysis using aqueous lithium hydroxide in a mixture of THF and methanol. As described above, lopinaviric acid ( 39 ) can be converted to an active ester by using carbodiimide and N-hydroxysuccinimide. Also, in order to obtain lopinavir derivative ( 40 ), various activators are used, and in this case, preferably O- (N-succinimidyl) -N, N, N ′, N′-tetra The active ester can be prepared by using methyluronium tetrafluoroborate in THF in the presence of a tertiary amine, preferably diisopropylethylamine.

Stable synthetic precursors to activated hapten derivatives are also suitable for use as analogs. For example, stable synthetic precursors that are isolated during the manufacture of labeled conjugates may serve a dual purpose as analogs for drug calibration and quality control. These stable synthetic precursors include carboxylic acids such as compounds 30 , 32 , 35 and 39 , and stable esters such as compounds 29 and 38 .

Synthesis of efavirenz analogs involves direct alkylation of efavirenz. The alkylation can be carried out by reaction with a haloalkyl reagent, in which case an alkali metal carbonate is used as a base in the presence of a phase transfer catalyst such as a crown ether. In one embodiment, potassium carbonate was used in combination with 18-crown-6. The reaction can be carried out in a solvent such as DMF at a temperature ranging from room temperature to 150 ° C. for 1 to 24 hours (see compounds 14 , 15 , 16 and 17 in FIG. 8).

In another embodiment, a hydroxylalkyl efavirenz analog is provided. The alkylation of efavirenz can be performed using a haloalkylating reagent having a protected hydroxyl functionality, preferably a silyl protecting group. The resulting silylated efavirenz intermediate can preferably be deprotected in the presence of tetrabutylammonium fluoride in THF (compound 19 , FIG. 9).

In one embodiment of the present invention, a method is provided for establishing or creating a calibration curve for HIV protease inhibitors, wherein the protease inhibitors are amprenavir, atazanavir, tipranavir (Tipranavir), indinavir, darunavir, lopinavir, nelfinavir, ritonavir, nerfinavir, and saquinavir. The method comprises preparing a set of proofreading materials comprising an immunochemically equivalent analog of the protease inhibitor, wherein in the analog, the central hydroxyl group of the protease inhibitor is Substituted with a group selected from -O (C ₁ -C ₁₀ alkyl), -OCONHR ₃ , -OCH ₂ CONHR ₃ and -OCH ₂ COOR ₃ , wherein R ₃ is H, C ₁ -C ₆ is selected from (CH ₂₎ group consisting of _n -X, wherein, n is 1 to 6, X is COOR ₅ or NH-CH ₂ - - alkyl, phenyl, heteroaryl and phenyl -COOR _5, Where R ₅ is H or C ₁ -C ₆ alkyl, where each calibrator contains one of the analogs in a range of concentrations. A sample from each calibrator in the set is then contacted with an antibody that specifically binds to the protease inhibitor analog. Each sample is then contacted with an amount of an antibody specific for the analog parent substance, and a conjugate comprising an HIV protease inhibitor and a signal generating moiety. Competition occurs between the HIV drug analog in the sample and the conjugate for a limited number of antibody binding sites. In one preferred form, the conjugate is an aminodextran-analog conjugate and the antibody is bound to microparticles. After a certain reaction time, the amount of signal resulting from the reaction between the antibody and the conjugate is measured. In the preferred particulate mode, the signal is caused by a change in absorbance in the visible region due to particle aggregation. A calibration curve is then constructed, for example by plotting the analog concentration along one axis of the graph and the signal along the other axis. This calibration curve will show an inverse relationship between the signal from the labeled conjugate and the amount of drug analog in the sample. Such a calibration curve is substantially equivalent to that curve obtained using the parent drug instead of the analog. The minimum number of samples in the set is 2, but in general it is necessary to add samples to increase the number of data points and thereby improve the accuracy of the calibration curve thus obtained. preferable. Typically, one of the sample concentrations is zero.

Similarly, a standard curve for HIV non-nucleoside reverse transcriptase inhibitors, including NNRTI efavirenz, can be generated. In this embodiment, a set of calibrators comprising a range of concentrations of immunochemically equivalent analogs of a non-nucleoside reverse transcriptase inhibitor of interest is provided, wherein the analogs are C containing ₁ -C ₆ alkyl or (C ₁ -C ₆ alkyl) OH in N- alkylated non-nucleoside reverse transcriptase inhibitors. The sample from each calibrator in the set is then contacted with a quantity of antibodies and analogs specific for the non-nucleoside reverse transcriptase inhibitor, and a conjugate comprising the non-nucleoside reverse transcriptase inhibitor and a signal generating moiety. Let For a limited number of antibody binding sites, competition occurs between the conjugate and the non-nucleoside reverse transcriptase inhibitor analog in the sample. In a preferred assay format, the conjugate is an aminodextran-NNRTI conjugate and the antibody is bound to microparticles. After a certain reaction time, the amount of signal resulting from binding of the antibody to the conjugate is detected. In this microparticle mode, the signal is due to a change in absorbance in the visible region due to particle aggregation, which can be detected by photometric measurement. A calibration curve is then constructed as described above, which shows an inverse relationship between the signal from the labeled conjugate and the amount of drug analog present in the sample. Such a calibration curve is substantially equivalent to that curve obtained from the parent drug.

  In another embodiment, the conjugate of the HIV drug (protease inhibitor or NNRTI) is bound to the solid phase in a heterogeneous immunoassay format, such as an enzyme linked immunosorbent assay or ELISA format. A series of samples containing various amounts of analogs of the HIV drug are then contacted with a fixed amount of antibody specific for the drug and the drug conjugate. Competition occurs between the conjugate and the HIV drug analog for a limited number of antibody binding sites. After a certain reaction time, the amount of antibody bound to the solid phase is detected using a secondary antibody labeled with a signal generating moiety. The secondary antibody specifically binds to the drug-specific antibody, thereby producing a detectable signal upon binding to the drug-specific antibody. A calibration curve is then constructed as described above, which shows an inverse relationship between the amount of antibody bound to the solid phase conjugate and the amount of drug analog present in the solution. Such a calibration curve is substantially equivalent to that curve obtained using the parent drug.

Specific Embodiments Reagents were obtained from Sigma-Aldrich Chemical Company unless otherwise indicated. Unless otherwise indicated, all solvents were obtained from JT Baker and were of ACS grade or higher than HPLC grade. Triethylamine was obtained from Fluka Chemical Co. Diisopropylethylamine (DIEA), dimethylformamide (DMF) and anhydrous dimethyl sulfoxide (DMSO) were obtained from Aldrich Chemical Co. Tetrahydrofuran (THF) was dried by boiling and distillation in the presence of sodium / benzophenone under argon. Methylene chloride (CH ₂ Cl ₂ ) was dried by boiling and distillation in the presence of calcium hydride under argon. Column chromatography was performed using EM Science flash grade silica gel (silica gel 60; 230-400 mesh ASTM) under positive pressure nitrogen. Thin layer chromatography (TLC) was performed using EM Science silica gel plates (thickness 0.025 cm). The mixed solvent is expressed as a volume expressed as volume% (eg, 10% MeOH-CHCl ₃ is chloroform containing 10% methanol by volume). HPLC analysis was performed on an Agilent 1100 LC / MS system configured with a diode array detector and a quaternary pump. LC analysis was performed using a Vydac 218TP54 column (RP-C18; 300 mm, 5μ) equipped with a Phenomenex guard module (Phenomenex KJO-4282 / C18 ODS 5μ) with a chromatographic flow entering the MS detector after the column. It was. The MSD used was operated in ES + mode (electrospray, positive mode). Unless otherwise indicated, the analysis was performed at a flow rate of 1 mL / min, 0.1% from 5% or 0% (0 min = starting) to 100% (20 min time point) in 0.1% TFA / H ₂ O. This was done using a TFA / MeCN gradient. Preparative RP-HPLC was performed on a Varian Dynamax (Rainin) system using two SD1 titanium head 2000 psi pumps equipped with a Varian Dynamax UV-C variable wavelength detector. Separation is achieved by a 250 x 21.4 mm (R00083221C; Microsorb 60-8, C18) column with a guard module (R00083221G; C18, 8μ) or a 250 x 41.4 mm column with a guard module (R00083241G; C18, 8μ) ( R00083241C; Microsorb 60-8, C18,) on a modular Varian Dynamax radial compression column. ¹ H-NMR spectra were obtained using a Varian Gemini 2000 at 200 MHz or a Varian XL-400 spectrometer at 400 MHz, each equipped with a Sun / Sparc station.

Example 1: Synthesis of lopinavir-O ^c -methyl ether ( 2 )
To 16 mg (0.040 mmol) sodium hydride (in oil, 60%) was added 3 mL of hexane and the supernatant was decanted off. To the residue was added 3 mL anhydrous THF (freshly distilled) followed by 50 mg (0.079 mmol) lopinavir as a solid. A stock solution of 45 μL iodomethane in 1 mL THF was prepared and 45 μL (0.31 mmol) of this iodomethane solution in THF was added to the reaction mixture at room temperature. The reaction mixture was stirred at room temperature for 30 minutes, 5 mL of 50 mM potassium phosphate (pH 7.5) was added and concentrated in a rotary evaporator to remove as much THF as possible. The aqueous residue was extracted with 6 × 10 mL of chloroform. The organic layers were combined, dried (anhydrous Na ₂ SO ₄ ) and concentrated. The residue was purified by preparative RP-HPLC using a gradient flow consisting of acetonitrile and water containing 0.1% trifluoroacetic acid. Fractions containing the desired product were concentrated in a rotary evaporator and lyophilized to give 7 mg (0.01 mmol, 14%) lopinavir-O ^c -methyl ether ( 2 ) as a white solid. LC / MS: M + H 643.3.

Example 2: lopinavir -O ^c - Synthesis of (methoxycarbonylmethyl) ether (3)
To 112 mg (2.8 mmol) sodium hydride (60% in oil) was added 10 mL hexane and the supernatant was decanted off. To the residue was added 10 mL anhydrous THF (freshly distilled) followed by 200 mg (0.31 mmol) lopinavir as a solid. The reaction mixture was stirred at room temperature for 15 minutes and 380 μL (4.0 mmol) of methyl bromoacetate was added. The reaction mixture was stirred at room temperature for 2 hours and the reaction was quenched with 20 mL of saturated ammonium chloride solution. The resulting reaction mixture was concentrated in a rotary evaporator to remove as much THF as possible and the aqueous residue was extracted with 6 × 30 mL of chloroform. The organic layers were combined, dried (anhydrous Na ₂ SO ₄ ) and concentrated. The residue was purified by preparative RP-HPLC using a gradient flow consisting of acetonitrile and water containing 0.1% trifluoroacetic acid. Fractions containing the desired product were concentrated in a rotary evaporator and lyophilized to give 61 mg (0.087 mmol, 27%) lopinavir-O ^c- (methoxycarbonylmethyl) ether ( 3 ) as a white solid Obtained. LC / MS: (M + H) 701.3.

Example 3: O ^c lopinavir 262 mg of sodium hydride (60% in oil) of (6.5 mmol) in the synthesis freshly distilled THF 5 mL of acetic acid (4) To a suspension of freshly distilled A solution of 250 mg (0.39 mmol) lopinavir in 5 mL of THF was added at room temperature. The mixture was stirred at room temperature for 1 hour. To the reaction mixture, 80 μL (0.84 mmol) of methyl bromoacetate was added and the resulting reaction mixture was stirred at room temperature for 3 days. To the reaction mixture, 5 mL of 50 mM potassium phosphate (pH 7.5) was added and 100 mg (2.3 mmol) of lithium hydroxide monohydrate was added as a solid. The resulting reaction mixture was stirred at room temperature for 18 hours and concentrated in a rotary evaporator. The aqueous residue was acidified with dilute phosphoric acid to pH 5-6 and extracted with 5 × 50 mL of chloroform. The combined organic layers were dried (anhydrous Na ₂ SO ₄ ) and concentrated to give a white gummy solid. This was purified by preparative thin layer chromatography using 20% methanol in chloroform to yield 43 mg (0.068 mmol) recovered lopinavir and 109 mg (0.16 mmol, 40%) O ^c lopinavir acetic acid ( 4 ). Was obtained as a white solid. LC-MS: (M + H) 687.4.

Example 4: lopinavir -O ^c - Synthesis of (N-tert-butylcarbamoyl) ether (6)
To a solution of 259 mg (0.37 mmol) lopinavir acetic acid ( 4 ) in 30 mL freshly distilled dichloromethane (distilled over CaH ₂ ) was added 152 mg (1.32 mmol) N-hydroxysuccinimide and 253 mg (1.32 mmol) of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC.HCl) was added. The reaction mixture was stirred at room temperature for 18 hours. The crude NHS ester ( 5 ) prepared in situ was used in the next step without isolation. To the reaction mixture was added a solution of 70 μL (0.65 mmol) t-butylamine in 930 μL freshly distilled dichloromethane (distilled over CaH ₂ ) and the reaction was stirred at room temperature for 2 hours. 30 mL of water was added to the reaction mixture and the organic layer was separated. The organic layer was washed with 30 mL saturated sodium bicarbonate, then 30 mL water, dried (anhydrous Na ₂ SO ₄ ) and concentrated. The residue was purified by preparative RP-HPLC using a gradient flow consisting of acetonitrile and water containing 0.1% trifluoroacetic acid. Fractions containing the desired product were concentrated in a rotary evaporator, lyophilized and 137 mg (0.18 mmol, 49%) lopinavir-O ^c- (N-tert-butylcarbamoylmethyl) ether ( 6 ) Was obtained as a white solid. LC / MS (M + H) 742.5.

Example 5: lopinavir -O ^c - solution of lopinavir carbamate 160 mg (0.25 mmol) in cooled freshly distilled dichloromethane (distilled over CaH ₂₎ that in the synthesis ice bath (8) To this was added 50 μL (0.42 mmol) of trichloroacetyl isocyanate. The resulting reaction mixture was stirred at 0 ° C. for 4 hours and concentrated under reduced pressure to give a crude product mixture containing lopinavir-O ^c -trichloroacetylcarbamate ( 7 ). This was used in situ in the next step without further purification. To the crude product 7 , 720 mg (5.2 mmol) of potassium carbonate, 7 mL of methanol and 2 mL of water were added at 0 ° C. The resulting reaction mixture was stirred at 0 ° C. for 1 hour and at room temperature for 18 hours. This was concentrated under reduced pressure, and 150 mL of chloroform and 50 mL of water were added to the residue. The organic layer was separated and the aqueous layer was extracted with 3 × 50 mL of chloroform. All organic layers were combined, dried (anhydrous Na ₂ SO ₄ ) and concentrated under reduced pressure. The residue was purified by preparative RP-HPLC using a gradient flow consisting of acetonitrile and water containing 0.1% trifluoroacetic acid. Fractions containing the desired product were concentrated in a rotary evaporator and lyophilized to give 130 mg (0.19 mmol, 76%) lopinavir-O ^c -carbamate ( 8 ). LC-MS: M + H 672.4.

Example 6: N-Karubamoru - lopinavir -O ^c - Synthesis of carbamate (10)
To a solution of 200 mg (0.32 mmol) lopinavir in 8 mL freshly distilled dichloromethane (distilled over CaH ₂ ), 1.08 g (7.8 mmol) potassium carbonate and 100 μL (0.83 mmol) trichloro Acetyl isocyanate was added. This was stirred at 0 ° C. for 3 hours and concentrated to give a crude reaction mixture containing N- (trichloroacetylamino-carbonyl) -lopinavir-O ^c -trichloroacetylcarbamate ( 9 ). This was used in situ in the next step without further purification. To the residue was added 16 mL methanol and 4 mL water at 0 ° C. This was stirred at 0 ° C. for 1 hour and at room temperature for 18 hours. The resulting reaction mixture was concentrated under reduced pressure. To the residue was added 150 mL chloroform and 50 mL water. The organic layer was separated and the aqueous layer was extracted with 3 × 50 mL of chloroform. All of the organic layers were combined, dried (anhydrous Na ₂ SO ₄ ) and concentrated under reduced pressure. The residue was purified by preparative RP-HPLC using a gradient flow consisting of acetonitrile and water containing 0.1% trifluoroacetic acid. Fractions containing the desired product were concentrated in a rotary evaporator and lyophilized to give 145 mg (0.20 mmol, 83%) of N-carbamoyl-lopinavir-O ^c -carbamate ( 10 ) as a white solid. It was. LC / MS: M + H 715.3.

Example 7: Synthesis of O ^c lopinavir acetate (11)
To 3.0 g (4.7 mmol) lopinavir was added 210 mg (1.71 mmol) 4- (dimethylamino) pyridine, followed by 90 mL freshly distilled THF (distilled over sodium and benzophenone). . The reaction mixture was cooled in an ice bath and 750 μL (9.2 mmol) of pyridine was added. This was followed by the addition of 750 μL (10.5 mmol) acetyl chloride. The reaction mixture was warmed to room temperature and stirred at room temperature for 72 hours. The reaction mixture became cloudy. This was concentrated and purified by preparative RP-HPLC using a gradient flow consisting of acetonitrile and water containing 0.1% trifluoroacetic acid. Fractions containing the desired product were concentrated in a rotary evaporator and lyophilized to give 2.48 g (3.69 mmol, 78%) of O ^c -lopinavir acetate ( 11 ) as a white solid. LC-MS: (M + H) 671.3.

Example 8: Synthesis of N-carbamoyllopinavir ( 13 )
To 2.48 g (3.69 mmol) lopinavir acetate ( 11 ) in 100 mL THF at 0 ° C. was added 1 mL (8.39 mmol) trichloroacetyl isocyanate. The mixture was stirred at 4 ° C. for 18 hours. The solution turned pale yellow. This was concentrated by a rotary evaporator to obtain a Hoff white solid. The reaction was monitored by LC / MS and the product was analyzed by LC / MS (M + H 858.3) as N- (trichloroacetylcarbamoyl) -lopinavir-O ^c -acetate as the desired intermediate product ( 12 ) Was identified. To the reaction mixture was added 160 mL methanol and 40 mL water followed by 4.8 g (36 mmol) potassium carbonate. The mixture was stirred at room temperature for 4 hours to give a white slurry. This was concentrated under reduced pressure and 400 mL of water was added. The resulting reaction mixture was extracted with 4 × 400 mL of ethyl acetate. The organic layers were combined, dried (Na ₂ SO ₄ ) and concentrated to give a white solid. This was purified by silica gel column chromatography by eluting with ethyl acetate to give a white solid. ¹ H NMR showed the presence of ethyl acetate even after drying under high vacuum. All of the substance 1: 1 was dissolved in water / acetonitrile and freeze-dried to give 1.8 g (2.6 mmol, 72% ) of N- carbamoyl lopinavir (13) as a white solid. LC / MS: (M + H) 672.3.

Example 9: Synthesis of N-methyl efavirenz ( 14 )
A solution of 40 mg (0.13 mmol) efavirenz in 2 mL anhydrous DMF was added to 33 mg (0.24 mmol) anhydrous potassium carbonate, 1 mg (3.78 × 10 ^-3 mmol) 18-crown-6 and 16 μL (0.25 mmol) of iodomethane was added. The reaction mixture was heated to 80 ° C. for 1.5 hours, then cooled and concentrated under reduced pressure. To the residue was added 10 mL of dichloromethane and the mixture was filtered. The filtrate was transferred to a separatory funnel and washed with 2 × 5 mL of water. The organic layer was dried (anhydrous Na ₂ SO ₄ ) and concentrated under reduced pressure. The residue was purified by preparative RP-HPLC using a gradient flow consisting of acetonitrile and water containing 0.1% trifluoroacetic acid. Fractions containing the desired product were concentrated in a rotary evaporator and lyophilized to give 30 mg (0.090 mmol, 73%) of N-methyl efavirenz ( 14 ). LC-MS: (M + H) 330.0.

Example 10: Synthesis of N-ethyl efavirenz ( 15 )
1.1 g (3.49 mmol) efavirenz, 40 mL anhydrous DMF, 40 mg (0.15 mmol) 18-crown-6, 2.65 g (19.2 mmol) anhydrous potassium carbonate, 210 mg (1.40 mmol) sodium iodide And 2.6 mL (34.7 mmol) of bromoethane was added. The reaction mixture was stirred at 125 ° C. for 2 hours. The color of the reaction mixture turned orange and a white solid formed at the bottom of the reaction flask. The reaction mixture was cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure. To the residue was added 200 mL of chloroform, washed with 2 × 100 mL of water, dried (MgSO ₄ ) and concentrated under reduced pressure to give 1.3 g of an orange oil. This was purified by preparative RP-HPLC using a gradient flow consisting of acetonitrile and water containing 0.1% trifluoroacetic acid. Fractions containing the desired product were concentrated in a rotary evaporator and lyophilized to give 884 mg (2.57 mmol, 74%) N-ethyl efavirenz ( 15 ) as a white powder. LC-MS: M + H 344.0.

Example 11: Synthesis of N-propyl efavirenz ( 16 )
To 60 mg (0.19 mmol) efavirenz, 2 mL anhydrous DMF was added, followed by 50 mg (0.36 mmol) anhydrous potassium carbonate, 10 mg (0.06 mmol) sodium iodide, 39 mg (0.31 mmol) 1- Bromopropane and 2 mg (7.5 × 10 ⁻³ mmol) 18-crown-6 were added. The resulting reaction mixture was stirred at 125 ° C. for 1 hour and then cooled to room temperature. This was filtered and the filtrate was concentrated under reduced pressure. 50 mL of chloroform was added to the residue and the organic layer was washed with 2 × 25 mL of water, dried (anhydrous Na ₂ SO ₄ ) and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using 10% ethyl acetate in hexane and then 40% ethyl acetate in hexane as eluent to give 32 mg (0.089 mmol, 49%) of N-propyl efavirenz ( 16 ) Was obtained as a white solid. LC-MS (M + H) 358.0.

Example 12: Synthesis of N-butyl efavirenz ( 17 )
A solution of 40 mg (0.12 mmol) efavirenz in 2 mL anhydrous DMF was added to 300 mg (2.1 mmol) anhydrous potassium carbonate, 1 mg (3.78 × 10 ^-3 mmol) 18-crown-6 and 68 μL (0.62 mmol) 1-bromobutane) was added. The mixture was heated to 60 ° C. for 4 hours and then to 120 ° C. for 1 hour. The resulting reaction mixture was concentrated under reduced pressure, 10 mL of dichloromethane was added and the mixture was filtered. The filtrate was transferred to a separatory funnel and washed with 2 × 5 mL of water. The organic layer was dried (anhydrous Na ₂ SO ₄ ) and concentrated under reduced pressure. The residue was purified by preparative RP-HPLC using a gradient flow consisting of acetonitrile and water containing 0.1% trifluoroacetic acid. Fractions containing the desired product were concentrated on a rotary evaporator and lyophilized to give 33 mg (0.088 mmol, 70%) of N-butyl efavirenz ( 17 ) as a colorless oil. LC / MS (M + H) 372.0.

Example 13: Synthesis of N- (tert-butyldimethylsilyloxypropyl) efavirenz ( 18 )
To a solution of 50 mg (0.16 mmol) efavirenz in 5 mL distilled acetone, 48 mg (0.19 mmol) (3-bromopropoxy) -tert-butyl-dimethylsilane, 32 mg (0.23 mmol) potassium carbonate, 3 mg (0.011 mmol) 18-Crown-6, 100 μL anhydrous DMF and 10 mg (0.06 mmol) sodium iodide were added. The reaction mixture was refluxed for 18 hours and the progress of the reaction was monitored by LC / MS. This showed only a trace amount of the desired product with unreacted starting efavirenz. The reaction mixture was concentrated under reduced pressure, 2 mL anhydrous DMF was added, followed by 35 mg (0.25 mmol) anhydrous potassium carbonate and 40 mg (0.15 mmol) (3-bromopropoxy) -tert-butyl-dimethylsilane. Was added. The resulting reaction mixture was heated at 125 ° C. for 2 hours, cooled to room temperature and filtered. The residue was washed with 50 mL ethyl acetate. The combined filtrate was concentrated, 50 mL of chloroform was added, and the organic phase was washed with 25 mL of water. The organic layer was dried (anhydrous Na ₂ SO ₄ ) and concentrated under reduced pressure to give a gummy solid. This was purified by silica gel column chromatography using 20% ethyl acetate in hexane to give 49 mg (0.10 mmol, 63%) of N- (tert-butyldimethylsilyloxypropyl) efavirenz ( 18 ) Obtained as a solid. LC-MS: M + H 488.0.

Example 14: Synthesis of N-hydroxypropyl efavirenz ( 19 )
To 43 mg (0.13 mmol) N- (tert-butyldimethylsilyloxypropyl) efavirenz ( 18 ) was added 100 μL freshly distilled THF and 500 μL (0.49 mmol) 1M tetrabutylammonium fluoride was added. The resulting reaction mixture was stirred at room temperature for 18 hours and concentrated under reduced pressure. 50 mL of chloroform was added to the residue, and the organic layer was washed with 2 × 25 mL of water. The organic layer was dried (anhydrous Na ₂ SO ₄ ) and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using 30% hexane in ethyl acetate. The isolated product showed some impurities and was further purified by preparative RP-HPLC using a gradient flow consisting of acetonitrile and water containing 0.1% trifluoroacetic acid. Fractions containing the desired product were concentrated on a rotary evaporator and lyophilized to give 30 mg (0.080 mmol, 93%) of N-hydroxypropyl efavirenz ( 19 ) as a white powder. LC-MS: M + H 374.0.

Example 15: atazanavir -O ^c - To a solution of 102 mg of atazanavir in 8.1 mL of dry dimethylformamide under argon ethyl carbamate (21) (DMF) (20 ), ethyl 22.5μL (2 eq) Isocyanide (Sigma-Aldrich) was added and the reaction was stirred at room temperature. After about 29 hours, an additional 11 μL of ethyl isocyanate was added the next day and stirring was continued overnight. Analysis by LC / MS showed that the reaction was complete. Solvent and volatiles were removed under vacuum (high vacuum rotovap), the residue was redissolved in 1: 1 acetonitrile (MeCN) / water (H ₂ O), filtered and the filtrate was added to 0.1% tri Purified by preparative RP-HPLC eluting with a gradient of fluoroacetic acid (TFA) /0.1% trifluoroacetic acid (TFA) / MeCN in water. The product fractions were collected, MeCN was removed at room temperature (rotovap), the residue was frozen and lyophilized to give 77 mg of product ( 21 ) as a partial TFA salt and fluffy white solid. LC / MS: M + H (parent) 776.3; HR-MS: calculated M + H (parent) 776.4342, observed 776.4330; ¹ H-NMR: compatible; microanalysis (MA): C ₄₁ H ₅₇ N ₇ O meet the ₈ · 0.33CF ₃ COOH.

Example 16: atazanavir -O ^c - to a solution of atazanavir 52 mg in 2 mL of dry DMF under argon isopropyl carbamate (22) (20), added DIEA in 14.2MyuL (about 1.1 equivalents), 8.6 μL (about 1.2 eq) isopropyl isocyanate (Sigma-Aldrich) was added and the reaction was stirred at room temperature overnight. The next day, an additional 3 μL of DIEA and 2 μL of isopropyl isocyanate were added and stirring was continued overnight. Analysis by LC / MS showed that the reaction was complete. Solvent and volatiles were removed under vacuum (high vacuum rotovap), the residue was redissolved in 1: 1 acetonitrile (MeCN) / water (H ₂ O), filtered and the filtrate was added to 0.1% tri Purified by preparative RP-HPLC eluting with a gradient of fluoroacetic acid (TFA) /0.1% trifluoroacetic acid (TFA) / MeCN in water. The product fractions were collected, MeCN was removed at room temperature (rotovap), the residue was frozen and lyophilized to give 53 mg of product ( 22 ) as a partial TFA salt and fluffy white solid. LC / MS: M + H (parent) 790.5; HR-MS: calculated M + H 790.4498, observed 790.4495; ¹ H-NMR: compatible; microanalysis (MA): C ₄₂ H ₅₉ N ₇ O ₈ , 1.25 Meets CF ₃ COOH.

Example 17: In addition of DIEA 28.5MyuL (about 2 equivalents) in a solution of 57 mg of atazanavir (20) in dry DMF 0.75 mL under argon atazanavir -O ^c-tert-butyl carbamate (23) Then 95.5 μL (about 10 equivalents) of tert-butyl isocyanate (Sigma-Aldrich) was added and the reaction was stirred at room temperature for 4 hours. Analysis by LC / MS showed that the reaction was complete. Solvents and volatiles were removed under vacuum (high vacuum rotovap). The residue was redissolved in 1: 1 acetonitrile (MeCN) / water (H ₂ O) and from a similar second reaction (using 53 mg atazanavir, 26.5 μL DIEA and 89 μL tert-butyl isocyanate) The residue was filtered and the filtrate was purified by preparative RP-HPLC eluting with a gradient of 0.1% trifluoroacetic acid (TFA) /0.1% trifluoroacetic acid (TFA) / MeCN in water. The product fractions were collected, MeCN was removed at room temperature (rotovap) and the residue was frozen and lyophilized to give 118 mg of product ( 23 ) as a partial TFA salt and fluffy white solid. LC / MS: M + H (parent) 804.4; HR-MS: calculated M + H (parent) 804.4655, observed 804.4646; ¹ H-NMR: fit; microanalysis (MA): C ₄₃ H ₆₁ N ₇ O Conforms to ₈・ 1.4CF ₃ COOH.

Example 18: atazanavir -O ^c - To a solution of 52 mg of atazanavir (20) in 1 mL of dry DMF under argon trichloroacetyl carbamate (24), trichloroacetyl isocyanate 70 [mu] L (about 8 eq) (Sigma-Aldrich) was added and the reaction was stirred at room temperature for about 1 hour. Analysis by LC / MS showed that the reaction was substantially complete. Solvents and volatiles were removed under vacuum (high vacuum rotovap). The residue was redissolved in 1: 1 acetonitrile (MeCN) / water (H ₂ O), filtered, and the filtrate was 0.1% trifluoroacetic acid (TFA) /0.1% trifluoroacetic acid (TFA) / MeCN in water. Purified by preparative RP-HPLC eluting with a gradient. Collect the central cut of the product fraction, remove MeCN at room temperature (rotovap), freeze the residue and lyophilize to give 52 mg of product ( 24 ) as a fluffy white solid assigned as TFA salt Obtained. LC / MS: M + H (parent) 892.2 (three chlorine isotope patterns); ¹ H-NMR: fit.

Example 19: atazanavir -O ^c - solution 1.345 mL of carbamate in 1.0 g of dry DMF in a 20 mL under argon (25) atazanavir (20) trichloroacetyl isocyanate (about 8 eq) (Sigma- Aldrich) was added and the reaction was stirred at room temperature for about 1 hour. Analysis by LC / MS showed that the reaction was substantially complete. Solvent and volatiles were removed under vacuum (high vacuum rotovap) and the residue was further dried under high vacuum to give crude product 24 . All of the crude product 24 was redissolved in 58 mL of methanol (MeOH), the solution was diluted with 14.5 mL of water, and then 4.96 g (> 25 eq) of potassium carbonate was added. The slightly heterogeneous reaction mixture was stirred vigorously under argon for about 4 hours. MeOH was removed under reduced pressure (rotovap), the aqueous residue was diluted with 50 mL of water, and the mixture was extracted with ethyl acetate (EtOAc) (2 × 150 mL). The extract is dried over sodium sulfate (Na ₂ SO ₄ ), filtered and evaporated to give the crude product which is redissolved in a small volume of EtOAc and eluted with EtOAc on flash grade silica gel. Purified by column chromatography. The product fractions (analysis by TLC) were combined and evaporated to give 888 mg of fairly pure product ( 25 ) as a white solid still containing EtOAc and as the free base. This material is redissolved in 5: 1 MeCN: H ₂ O and repurified by preparative RP-HPLC eluting with a gradient of 0.1% trifluoroacetic acid (TFA) /0.1% trifluoroacetic acid (TFA) / MeCN in water did. The main cut of the product peak was collected, MeCN was removed at room temperature (rotovap), the residue was frozen and lyophilized to give 870 mg of 25 as a white solid and as a TFA salt hydrate. LC / MS: M + H (parent) 748.3; HR-MS: calculated M + H 748.4029, observed 748.4031; ¹ H-NMR: compatible; microanalysis (MA): C ₃₉ H ₅₃ N ₇ O ₈・ 1.5 Meets CF ₃ COOH ・ 1.5H ₂ O.

Example 20: atazanavir -O ^c - of 10 mg of synthetic Teflon in dry DMF 0.8 mL in small reaction vial equipped with a lined screw cap (Wheaton Science Products) methyl ether (26) To a solution of atazanavir ( 20 ) was added 10 mg of silver (I) oxide followed by 10 μL (about 11.5 equivalents) of iodomethane (Sigma-Aldrich), the vial was capped and the reaction was stirred at room temperature for 2 days . An additional 21 mg of silver (I) oxide was added, the vial was capped and stirring was continued for another day. Analysis by LC / MS showed substantial consumption of atazanavir with the appearance of multiple product peaks (the main peak has the expected molecular ion and UV spectrum of O ^c -methyl ether). The reaction was diluted with a small amount of DMF and filtered from the silver salt. Solvent and volatiles are removed from the filtrate under vacuum (high vacuum rotovap), the residue is redissolved in MeCN / water, filtered, and the filtrate is 0.1% in 0.1% trifluoroacetic acid (TFA) / water. Purified by preparative RP-HPLC eluting with a gradient of% trifluoroacetic acid (TFA) / MeCN. A central cut of the most prominent product peak fractions was collected, MeCN was removed at room temperature (rotovap), the residue was frozen and lyophilized, and the resulting material was similarly treated with 0.1% trifluoroacetic acid (TFA) Re-purified by semi-preparative C18 RP-HPLC (Vydac 218TP510; 250 × 10 mm, 300Å, 5μ) eluting with a gradient of 0.1% trifluoroacetic acid (TFA) / MeCN in water. The product fractions were collected, MeCN was removed at room temperature (rotovap) and the residue was frozen and lyophilized to give a small amount of product 26 as an amorphous solid. LC / MS: M + H (parent) 719.3; Same UV spectrum as atazanavir.

Example 21: atazanavir -O ^c - (2'-hydroxyethyl) to 10 mg solution of atazanavir in 0.8 mL of dry DMF synthetic small reaction vial ether (28), 10 mg of silver (I) oxide Followed by 23 mL (about 11.5 eq) of 2- (2-bromoethoxy) -tetrahydro-2H-pyran (Sigma-Aldrich). After stirring at room temperature for 2 days, an additional 30 mg of silver (I) oxide was added along with 30 mg of sodium iodide, the vial was capped again, and the concentrated slurry was stirred at room temperature for an additional 2 days. LC / MS shows consumption of atazanavir and the appearance of three major product peaks (and other peaks), with the central major peak having an expected M + H and UV of 833.3 for the tetrahydropyran derivative ( 27 ) It was. The reaction mixture was diluted with DMF and filtered (0.45 m) [slow] from silver and sodium salts. The filtrate was further diluted with methylene chloride and refiltered (0.45μ). The filtrate was evaporated to dryness under reduced pressure. The residue was redissolved in 1: 1 MeCN / water and purified by preparative RP-HPLC eluting with a gradient of 0.1% trifluoroacetic acid (TFA) /0.1% trifluoroacetic acid (TFA) / MeCN in water. The desired peak was collected, MeCN was removed (rotovap), the residue was frozen and lyophilized to give 1.2 mg of amorphous semi-solid. This material was redissolved in 2 mL of 1: 1 MeCN / water 0.1% TFA and allowed to stand at room temperature for 5 hours, after which LC / MS showed substantially complete conversion to 28 . The solution was concentrated and similarly semi-preparative C18 RP-HPLC (Vydac 218TP510; 250 × 10 mm, eluting with a gradient of 0.1% trifluoroacetic acid (TFA) /0.1% trifluoroacetic acid (TFA) in water / MeCN. 300Å, 5μ). The product fractions were collected, MeCN was removed at room temperature (rotovap) and the residue was frozen and lyophilized to give 1.1 mg of product ( 28 ) as an amorphous solid. LC / MS: M + H (parent) 749.2; Same UV spectrum as atazanavir.

Example 22: Synthesis of 4-[(Atazanavir-O ^c ) -carbonylamino] butyric acid ethyl ester ( 29 )
To a solution of 97.4 mg atazanavir ( 20 ) in 2.5 mL dry DMF was added 55 μL (2.7 eq) ethyl 4-isocyanatobutyrate (Sigma-Aldrich) followed by 23 μL (1.2 eq) anhydrous triethylamine. (TEA) was added. The solution was stirred under argon and heated overnight with the oil bath maintained at about 60 ° C. The reaction was then cooled to room temperature, DMF was removed under vacuum (high vacuum rotovap) and the residue was washed with 0.1% trifluoroacetic acid (TFA) /0.1% trifluoroacetic acid (TFA) / MeCN in water. Purified by preparative RP-HPLC eluting with a gradient. Combine the main cuts of the product peak, remove MeCN (rotovap), freeze the residue, lyophilize to give 116 mg of product ( 29 ) as an amorphous slightly sticky solid and TFA salt hydrate Got as. LC / MS: M + H (parent) 862.5; HR-MS: Calculated M + H 862.4709, Found 862.4708; ¹ H-NMR: Fit; Microanalysis (MA): C ₄₅ H ₆₃ N ₇ O ₁₀ , 2CF ₃ Conforms to COOH · 2H ₂ O.

Example 23: Synthesis of 4-[(Atazanavir-O ^c ) -carbonylamino] butyric acid ( 30 )
To a solution of 40.6 mg of 29 in 1.25 mL of MeOH and 1.25 mL of water was added 21.2 mg (10.7 equiv) of lithium hydroxide monohydrate and the clear reaction solution was stirred at room temperature under argon for about 2 hours. LC / MS analysis indicated that the hydrolysis was complete. This was combined with another similarly completed reaction of 86.4 mg 29 and 42.2 mg LiOH.H ₂ O in 2.5 mL MeOH and 2.5 mL water. The reaction mixture was neutralized with 1N HCl to about pH 6-7 and the solvent was removed on a rotovap. The residue was redissolved in MeCN / water and purified by preparative RP-HPLC eluting with a gradient of 0.1% trifluoroacetic acid (TFA) /0.1% trifluoroacetic acid (TFA) / MeCN in water. The main cut of the product peak was combined, MeCN was removed (rotovap), the residue was frozen and lyophilized to give 124 mg of product ( 30 ) as a white solid and as a TFA salt hydrate. LC / MS: M + H (parent) 834.4; HR-MS: calculated M + H 834.4396, found 834.4386; ¹ H-NMR: fit.

Example 24 Synthesis of 4-[(Atazanavir-O ^c ) -carbonylamino] butyric acid N-hydroxysuccinimide ester ( 31 ) 123.4 mg of 30 agitation in 9 mL of freshly distilled dry methylene chloride under argon To the solution / suspension, 91.5 mg (6.1 eq) N-hydroxysuccinimide (NHS) was added followed by 151.9 mg (6.1 eq) 1-[(3-dimethylamino) propyl] -3-ethylcarbodiimide hydrochloride (EDC.HCl) was added and the clear reaction was stirred at room temperature for about 2 hours. Analysis by LC / MS showed a single atazanavir product peak with the desired M + H of m / z 931.5. The reaction is diluted with distilled CH ₂ Cl ₂ and washed sequentially and rapidly with 0.1N HCl (× 1), 0.1N NaOH (× 1), saturated brine (× 1), and the organic phase is dried (Na ₂ SO ₄ ), filtered and rotovap to give a crude but fairly pure NHS ester ( 31 ). The material was used immediately in the synthesis of 32 (Example 25).

In another implementation, the reaction of 34.5 mg 30 with 27.3 mg NHS and 42.4 mg EDC.HCl in 2 mL CH ₂ Cl ₂ was determined by LC / MS analysis using m + z 931.5 M + H The same product peak with Remove the solvent (rotovap), redissolve the residue in 2: 1 MeCN / water and elute with a gradient of 0.1% trifluoroacetic acid (TFA) /0.1% trifluoroacetic acid (TFA) / MeCN in water -Purified directly by HPLC. Freeze main cut of product peak immediately (dry ice / acetone), remove most of MeCN (sublimation on dry ice / acetone cooling fingers of high vacuum rotovap), freeze frozen residue below 20 ° C Dried to give 23.3 mg of purified NHS ester ( 31 ) as a white solid and as a TFA salt. LC / MS: M + H (parent) 931.5; HR-MS: calculated M + H 931.4560, found 931.4553; ¹ H-NMR: fit.

Example 25: Synthesis of 4- [4-[(Atazanavir-O ^c ) -carbonylamino] butyramide)] methylbenzoic acid ( 32 )
A solution of 22.1 mg 4- (aminomethyl) benzoic acid (Sigma-Aldrich) in 1.5 mL water (heated for dissolution and then cooled to room temperature) adjusted to pH ˜10 with a small volume of 0.1N NaOH did. This solution was then added to all of the crude NHS ester from the first paragraph of Example 24 ( 31 ) in 11 mL of freshly distilled dry tetrahydrofuran (THF) and the reaction mixture was allowed to reach room temperature under argon. And stirred overnight. Analysis by LC / MS showed consumption of NHS ester and formation of the desired product of M + H at m / z 967.5. The product mixture was neutralized with a small amount of 0.1N HCl and evaporated to dryness (high vacuum rotovap). The residue was taken up in 10% MeOH in ethyl acetate (EtOAc) and applied to the top of a silica gel column, which was then eluted with a gradient of 10-20% MeOH in EtOH. The product fractions were combined and evaporated (rotovap) and the residue was redissolved in dry CH ₂ Cl ₂ , filtered and re-evaporated (rotovap) to give 79 mg of product ( 32 ) as a white powder. . LC / MS: M + H 967.5; HR-MS: Calculated M + H 967.4924, Found 967.4921; ¹ H-NMR: Fit.

Example 26 Synthesis of 4- [4-[(Atazanavir-O ^c ) -carbonylamino] butyramide)] methylbenzoic acid N-hydroxysuccinimide ester ( 33 )
To a solution of 62 mg benzoic acid product ( 32 ) in 4.5 mL freshly distilled dry CH ₂ Cl ₂ was added 9.0 mg (1.12 eq) NHS followed by 15.4 mg (1.15 eq) EDC HCl. And the reaction was stirred at room temperature under argon. LC / MS analysis after about 4 hours indicated that the reaction was complete. The solvent was removed (rotovap). The residue was redissolved in 2: 1 MeCN / water and purified by preparative RP-HPLC eluting with a gradient of 0.1% trifluoroacetic acid (TFA) /0.1% trifluoroacetic acid (TFA) / MeCN in water. Freeze the main cut of the product peak immediately (dry ice / acetone), remove most of the MeCN (sublimation on dry ice / acetone cooling fingers of high vacuum rotovap) and remove the frozen residue below 20 ° C Lyophilization gave 40.2 mg of purified NHS ester ( 33 ) as a white solid and as a TFA salt. LC / MS: M + H (parent) 1064.5; ¹ H-NMR: Fit.

Example 27: Synthesis of 3- (2-iodo-acetamido) -propionic acid ethyl ester ( 34 )
7.4 g (39.7 mmol) iodoacetic acid in 90 mL distilled dichloromethane (distilled with CaH ₂ ) was cooled to 0 ° C. 4.4 mL (39.9 mmol) N-methylmorpholine was added to the reaction mixture, and 5.18 mL (39.9 mmol) isobutyl chloroformate was added. The reaction mixture was stirred at 0 ° C. for 20 minutes. The reaction mixture was stirred at 0 ° C. for 20 minutes. The reaction mixture was clear when N-methylmorpholine was added but turned dark yellow after addition of isobutyl chloroformate.

To the reaction mixture was added a mixed solution of β-alanine ethyl ester hydrochloride (6.14 g, 39.9 mmol, Sigma-Aldrich) in 60 mL dichloromethane and 4.4 mL (39.9 mmol) N-methylmorpholine over 5 minutes, The reaction mixture was stirred at 0 ° C. for 30 minutes. The reaction mixture was warmed to room temperature for 18 hours. The reaction mixture turned red. The reaction mixture was transferred into a separatory funnel and the organic layer was washed with 2 × 75 mL 20% citric acid, 2 × 75 mL saturated NaHCO ₃ and 1 × 50 mL saturated aqueous sodium chloride and dried (MgSO ₄ And filtered. To the filtrate was added 3 g of activated carbon, and the mixture was filtered through Celite (10 g). The resulting filtrate showed a pale yellow color. This was concentrated under reduced pressure to give a pale yellow oil. To this oil was added 2 × 20 mL of hexane, the mixture was agitated and allowed to settle, and the hexane layer was removed with a Pasteur pipette each time. To the resulting oil was added 40 mL of distilled acetone (distilled with anhydrous potassium carbonate) and 15 g (100 mmol) of sodium iodide. The reaction mixture was stirred at room temperature for 18 hours and then filtered. The filtrate was concentrated to give a yellow oil. This was dissolved in 90 mL of dichloromethane. The organic layer was washed with 30 mL water, 30 mL 2% sodium thiosulfate solution, 30 mL water and 30 mL saturated aqueous sodium chloride solution to give 8.7 g (30.5 mmol, 77%) of product ( 34 ) Was obtained as a pale yellow oil that solidified upon storage at 4 ° C. to a pale yellow solid. LC-MS (M + Na): 307.9.

Example 28: Synthesis of 3-[(Lopinavir-O ^c ) -acetamido] -propionic acid ( 35 )
To 500 mg (0.79 mmol) lopinavir was added 10 mL anhydrous toluene and the mixture was concentrated on a rotary evaporator. This process was repeated once. To the residue was added 4 mL of freshly distilled THF. To another flask was added 130 mg (3.25 mmol) sodium hydride (in oil, 60%) and 4 mL freshly distilled THF. To this stirred suspension of sodium hydride in THF was added dropwise a solution of lopinavir in THF (prepared above) and the reaction mixture was stirred at room temperature for 2 hours. A solution of iodoacetamide linker ( 34 ) (250 mg, 0.87 mmol) in 4 mL of freshly distilled THF was added to the reaction mixture and the reaction was stirred at room temperature for 3 hours. The reaction flask was cooled in ice and the reaction was quenched with 10 mL of 50 mM potassium phosphate (pH 7.5) and stirred at room temperature for 30 minutes. To the resulting mixture, 4 mL of water was added, followed by 250 mg (5.95 mmol) of LiOH.H ₂ O, and the mixture was stirred at 4 ° C. for 18 hours. The reaction mixture was concentrated on a rotary evaporator. The pH of the residue was adjusted to 3-4 and the resulting aqueous mixture was extracted with 6 × 50 mL of ethyl acetate. The organic layers were combined, dried (anhydrous Na ₂ SO ₄ ) and concentrated on a rotary evaporator to give a pale yellow oil. LC / MS indicated the presence of product. This oil was dissolved in 3: 1 acetonitrile / water and purified by preparative RP-HPLC to give 286 mg (0.37 mmol, 36%) lopinaviric acid derivative 35 and 83 mg recovered lopinavir starting material. The yield of lopinaviric acid derivative 35 was 53% based on the recovered starting lopinavir. LC / MS (M + H): 758.3.

Example 29: Synthesis of 4- [3- (Lopinavir-O ^c ) -acetamido) -propionamido] methylbenzoic acid methyl ester ( 38 )
A solution of 255 mg (0.33 mmol) lopinavir acid ( 35 ) in 19 mL dichloromethane was prepared. To this magnetically stirred reaction mixture was added 264 mg (1.37 mmol) of 1- [3- (dimethylamino) propyl] -3-ethylcarbodiimide hydrochloride (EDC · HCl), followed by 163 mg (1.4 mmol) of N-hydroxysuccinimide was added. The reaction mixture was stirred at room temperature for 18 hours. LC / MS analysis showed the formation of a (M + H) product with m / z 855.3. This activated ester ( 37 ) was used in the next step without isolation.

To a magnetically stirred solution of 210 mg (1.04 mmol) methyl 4- (aminomethyl) benzoate hydrochloride (Sigma-Aldrich) in 5.6 mL anhydrous DMF was added 0.76 mL (5.4 mmol) triethylamine, followed by A solution of lopinavir aliphatic NHS ester ( 37 ) prepared in situ from above was added. The reaction mixture was stirred at room temperature for 18 hours, then concentrated on a rotary evaporator and then under high vacuum. The residue was dissolved in 35 mL water and extracted with 4 × 100 mL ethyl acetate. The combined organic layers were dried (Na ₂ SO ₄ ) and concentrated. The residue was dissolved in 2: 1 acetonitrile / water and purified by preparative RP-HPLC to give 254 mg (0.28 mmol, 83%) of 38 as a white solid. LC / MS (M + H): 905.3.

Example 30: Synthesis of 4- [3- (Lopinavir-O ^c ) -acetamido) -propionamido] methylbenzoic acid ( 39 )
A solution of lopinavir methyl ester ( 38 , 302 mg, 0.33 mmol) in 10 mL methanol and 10 mL freshly distilled THF was prepared. To this was added a solution of 22 mg (0.52 mmol) of lithium hydroxide monohydrate in 22 mL of water. The reaction mixture was stirred at room temperature for 3 days. The resulting solution was concentrated under reduced pressure to adjust the pH to 3-4. The formation of a white solid was observed. The aqueous mixture was extracted with 4 × 60 mL of ethyl acetate. The organic layers were combined, dried (Na ₂ SO ₄ ) and concentrated. The residue was dissolved in 2: 1 acetonitrile / water and purified by preparative RP-HPLC to give 245 mg (0.27 mmol, 82%) lopinavir benzoic acid derivative 39 as a white solid. LC / MS (M + H): 891.3.

Example 31: 4- - Synthesis of [3- (lopinavir -O ^c) acetamido) propionamido] benzoate N- hydroxysuccinimide ester (40)
130 mg (0.43 mmol) O- (N-succinimidyl)-in a solution of 128 mg (0.14 mmol) lopinaviric acid 39 in 6.8 mL freshly distilled THF (distilled over sodium and benzophenone) N, N, N ′, N′-tetramethyluronium tetrafluoroborate (Sigma-Aldrich) and 75 μL (0.42 mmol) diisopropylethylamine were added. The reaction mixture was stirred at room temperature for 3 hours and then concentrated under reduced pressure. 5 mL of water was added to the residue, and the aqueous layer was extracted with 4 × 10 mL of ethyl acetate. The organic layers were combined, dried (anhydrous Na ₂ SO ₄ ) and concentrated under reduced pressure. The residue was dissolved in 2: 1 acetonitrile / water and purified by preparative RP-HPLC to give 102 mg (0.103 mmol, 72%) of lopinavir derivative 40 as a white solid. LC / MS (M + H): 988.3.

Example 32: atazanavir -O ^c - (3- pyridyl) Synthesis of the carbamate (41)
To a solution of 100 mg atazanavir ( 20 ) in 1.0 mL dry DMF, add 50 μL (just over 2 equivalents) of DIEA, followed by 45 mg (about 2.65 equivalents) of 3-isocyanatopyridine (Oakwood Products Inc., West Columbia, SC 29172; using material as received) was added and the reaction was stirred overnight at room temperature in a capped flask. An additional 125 mg of 3-isocyanatopyridine (about 7.35 equivalents; total = 10 equivalents) was added and the reaction mixture was warmed with a heat gun to cause reagent dissolution and the reaction was stirred overnight. Analysis by LC / MS showed an approximately 1: 1 mixture of product and starting material. Solvents and volatiles were removed under vacuum (high vacuum rotovap). The residue is redissolved in 1: 1 acetonitrile (MeCN) / water (H ₂ O), filtered, and the filtrate is gradient of 0.1% trifluoroacetic acid (TFA) /0.1% trifluoroacetic acid (TFA) / MeCN in water. Purified by preparative RP-HPLC eluting with The product fractions were collected, MeCN was removed (rotovap), the residue was frozen and lyophilized to give product 41 , assigned as a white solid and as a partial TFA salt. LC / MS: t _R about 10.9 minutes, M + H (parent) 825.4; ¹ H-NMR: compatible.

Example 33: 4- (6-Chloro-4-cyclopropylethynyl-2-oxo-4-trifluoromethyl-4H-benzo- [d] [1,3] oxazin-1-yl)-[4-butyl Synthesis of (amidomethyl) benzoic acid ( 43 )
The efavirenz derivative 42 was prepared by the method described in US 2004/0214251, the disclosure of which is incorporated herein by reference. To 91 mg (0.60 mmol) aminomethylbenzoic acid was added 3 mL of water, 650 μL of 1N NaOH, and then 10 mL of freshly distilled THF. To this reaction mixture was added a solution of 282 mg (0.56 mmol) of efavirenz NHS ester ( 42 ) in 7 mL of freshly distilled THF at room temperature. The pH of the reaction was maintained at 9 for 2 hours and concentrated to remove as much THF as possible. 150 mL of water was added to the residue, and the pH of the resulting solution was adjusted to pH 5. This was extracted with 2 × 100 mL of chloroform. The organic layers were combined, dried (anhydrous Na ₂ SO ₄ ) and concentrated. The residue was purified by preparative RP-HPLC using a gradient flow consisting of acetonitrile and water containing 0.1% trifluoroacetic acid. Fractions containing the desired product were concentrated on a rotary evaporator and lyophilized to give 74 mg (0.14 mmol, 24%) of efavirenz NHS ester ( 43 ) as a white powder. LC-MS (M + H) 535.2.

Example 34: 4- (6-Chloro-4-cyclopropylethynyl-2-oxo-4-trifluoromethyl-4H-benzo- [d] [1,3] oxazin-1-yl)-[4-butyl Synthesis of [amidomethyl] benzoic acid N-hydroxysuccinimide ester ( 44 )
To a solution of 63 mg (0.12 mmol) efavirenz derivative 43 in 6 mL dichloromethane was added 47 mg (0.25 mmol) 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide hydrochloride followed by 21 mg (0.18 mmol) N-hydroxysuccinimide was added. The reaction mixture was stirred at room temperature for 18 hours and 40 mL of dichloromethane was added. The organic layer was washed with 15 mL water and 15 mL saturated sodium bicarbonate and then with 15 mL water. This was dried (anhydrous Na ₂ SO ₄ ) and concentrated. The residue was purified by preparative RP-HPLC using a gradient flow consisting of acetonitrile and water containing 0.1% trifluoroacetic acid. Fractions containing the desired product were concentrated on a rotary evaporator and lyophilized to give 70 mg (0.11 mmol, 94%) of efavirenz NHS ester ( 44 ) as a white powder. LC-MS (M + H) 632.0.

Example 35: Efavirenz-aminodextran conjugate (45)
Aminodextran (MW 40,000) was prepared by the method described in US 6,653,456, the contents of which are incorporated herein by reference. 4.5 mL DMSO was added to 65 mg aminodextran at room temperature. The mixture was stirred at room temperature until all aminodextran was dissolved in the solution. To the reaction mixture was added 19.5 μL (0.138 mmol) of triethylamine. 24.6 mg (0.037 mmol) of efavirenz derivative 44 was dissolved in 3 mL anhydrous DMSO and 1 ml of this solution was added dropwise to the stirred aminodextran solution. The mixture is stirred at room temperature for 44 hours, transferred into a SPECTRAPOR dialysis tube (Spectrum Medical Industries, Molecular Weight Cutoff 2000) and dialyzed at room temperature according to the following plan (1 L volume, at least 8 hours each) (1 L for each dialysis) Of: 80% DMSO, 60% DMSO, 40% DMSO and 20% DMSO in deionized water followed by deionized water. The solution was removed from the dialysis tube and lyophilized to give 60 mg of efavirenz-dextran conjugate 45 as a white powder.

Example 36: Synthesis of N- (tert-butyldimethylsilyloxyethyl) efavirenz ( 18a )
408 μL (1.88 mmol) (2-bromoethoxy) -tert-butyl-dimethylsilane, 275 mg (1.98 mmol) potassium carbonate in a solution of 300 mg (0.95 mmol) efavirenz in 4 mL anhydrous DMF 5 mg of 18-crown-6 and 50 mg (0.33 mmol) of sodium iodide were added. The residue was washed with 2 × 5 mL of ethyl acetate and concentrated. The same reaction was repeated with 100 mg of efavirenz. The crude products from both batches were combined and dissolved in 150 mL ethyl acetate. The organic layer was washed with 3 × 50 mL of water, dried (anhydrous Na ₂ SO ₄ ) and concentrated. The residue was purified by silica gel column chromatography using 20% ethyl acetate in hexane to give 600 mg (1.22 mmol, 97%) N- (tert-butyldimethylsilyloxyethyl) efavirenz ( 18a ) as a colorless gum. Got as.

Example 37: Synthesis of N-hydroxyethyl efavirenz ( 19a )
To 380 mg (0.80 mmol) N- (tert-butyldimethylsilyloxyethyl) efavirenz ( 18a ) in 3 mL, add 3 mL of freshly distilled THF, then add 800 μL of 1M tetrabutylammonium fluoride. added. The resulting reaction mixture was stirred at room temperature for 18 hours and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using 50% hexane in ethyl acetate. The isolated product showed some impurities. It was further purified by preparative RP-HPLC using a gradient flow consisting of acetonitrile and water containing 0.1% trifluoroacetic acid. Fractions containing the desired product were concentrated on a rotary evaporator and lyophilized to give 180 mg (0.50 mmol, 63%) of N-hydroxyethyl efavirenz ( 19a ) as a white powder. LC-MS (M + H) 360.0.

Example 38: Synthesis of N-isopropyl efavirenz ( 16a )
To 300 mg (0.95 mmol) efavirenz was added 5 mL anhydrous DMF, followed by 263 mg (1.95 mmol) anhydrous potassium carbonate, 60 mg (0.040 mmol) sodium iodide, 178 μL (1.90 mmol) 2 -Bromopropane and 5 mg of 18-crown-6 were added. The resulting reaction mixture was stirred at 120 ° C. for 2 hours and then cooled to room temperature. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure. The same reaction was repeated with 100 mg of efavirenz. The crude products from both batches were combined and purified by preparative RP-HPLC using a gradient flow consisting of acetonitrile and water containing 0.1% trifluoroacetic acid. Fractions containing the desired product were concentrated on a rotary evaporator and lyophilized to give 295 mg (0.82 mmol, 87%) of N-isopropyl efavirenz ( 16a ). LC-MS (M + H) 358.0.

Example 39: Synthesis of N-isobutyl efavirenz ( 17a )
A solution of 300 mg (0.95 mmol) efavirenz in 5 mL anhydrous DMF was added to 272 mg (1.96 mmol) anhydrous potassium carbonate, 5 mg 18-crown-6 and 225 μL (1.94 mmol) 2- Bromo-2-methylpropane was added. The mixture was heated to 120 ° C. for 2 hours, cooled to room temperature and filtered. The residue was washed with 2 × 5 mL of ethyl acetate. All the filtrates were combined and concentrated under reduced pressure. The same reaction was repeated with 100 mg of efavirenz. The crude products from both batches were combined and the residue was purified by preparative RP-HPLC using a gradient flow consisting of acetonitrile and water containing 0.1% trifluoroacetic acid. Fractions containing the desired product were concentrated on a rotary evaporator and lyophilized to give 270 mg (0.72 mmol, 57%) of N-isobutyl efavirenz ( 17a ). LC / MS (M + H) 372.0.

Example 40: Production of antibodies against lopinavir Antibodies against lopinavir were produced as described in US 7,193,065.

Example 41: Preparation of lopinavir aminodextran conjugate
100 mg aminodextran (AMD) was dissolved in 10 mL anhydrous dimethyl sulfoxide (DMSO). To this solution, 30 μL of triethylamine was added. The aminodextran solution was continuously stirred. A solution of 19.8 mg lopinavir derivative 40 in anhydrous DMSO (1 mL) was slowly added to the aminodextran solution and the reaction mixture was stirred at room temperature for 48 hours. After 48 hours, the entire solution was transferred to a dialysis tube (molecular weight cut-off = 2000) and dialyzed against DMSO: water for 5 days at room temperature with decreasing amounts of DMSO. After final dialysis against water, lopinavir-aminodextran conjugate (LPV-AMD) was lyophilized to dryness. LPV: AMD stoichiometry was determined by UV-Vis spectroscopy using ε ₂₅₀ = 16,495 M ^-1 cm ^-1 and assuming that AMD did not significantly contribute to UV absorbance at that wavelength. . The ratio of LPV to AMD in the LPV-AMD conjugate was found to be 3.75.

Example 42: Production of lopinavir antibody microparticles Five monoclonal antibodies (LPV Ab) (1.1.85, 1.12.0, 1.7.90, 1.50.0 and 1.39.0) against lopinavir were used in the development of the immunoassay. Screened. Covalent binding of monoclonal lopinavir antibody to latex microparticles was performed as described below.

  1% latex fine particles (number of carboxy groups = 0.21 mmol / g latex, Seradyne Inc.) are washed with 50 mM 2-morpholino-ethanesulfonic acid (MES) (pH 5.5) to contain the original stock solution I tried not to. Weigh out the desired volume of 1% microparticles in 50 mM MES (pH 5.5), add N-hydroxysulfosuccinimide (sulfo-NHS), then immediately N-ethyl-N '-(3-dimethyl-aminopropyl) The microparticles were activated by adding carbodiimide hydrochloride (EDC · HCl). Both sulfo-NHS and EDC were added so that the number of moles of each reagent was 10 times the number of carboxylates present on the surface of the microparticles. After stirring at room temperature for 2 hours, the mixture was centrifuged (15,000 rpm, 4 ° C., 45-60 minutes) and the supernatant decanted. The activated microparticles were resuspended in 50 mM 3-morpholinopropanesulfonic acid (MOPS) (pH 6.4) by sonication to a concentration of 1%. Monodispersion after sonication was confirmed by light scattering measurement with a COBAS MIRA analyzer.

A solution of lopinavir monoclonal antibody in 50 mM MOPS buffer (pH 6.4) was added to the resuspended microparticles so that there was 10 μg of antibody per mg of latex. The antibody-latex mixture was stirred at room temperature for 2 hours. After 2 hours, a solution of 50 mg / mL BSA in 50 mM MOPS (pH 6.4) was added to the latex mixture (2.5 mg BSA / mg latex). After 2 hours, a solution of 840 mM 2-aminoethoxyethanol in 50 mM MOPS (pH 8.5) containing 0.09% (w / v) NaN ₃ was added to the latex mixture. After stirring overnight, the mixture is centrifuged (15,000 rpm, 4 ° C., 45-60 min) and the latex is stored in storage buffer (50 mM MOPS, pH 7.2, 0.1% bovine serum albumin (BSA), 0.09% azide ) Was resuspended by sonication. This is followed by further centrifugation and sonication to finally obtain 1% latex, which is stored at 2-8 ° C in storage buffer (50 mM MOPS, pH 7.2, 0.1% BSA, 0.09% azide). did.

Example 43: Cross-reactivity of lopinavir analogues to lopinavir antibody
Assays were performed at 37 ° C. on a Roche / Hitachi 917 clinical chemistry analyzer (Roche Diagnostics GmbH). The reaction mixture (296 μL) was 3 μL of lopinavir calibrator or sample (as described below), 0.05% (w / v) microparticles from Example 34, 0.4 μg / mL LPV-AMD conjugate, 0.6-0.8% ( w / v) polyacrylic acid (PAA), 50 mM KSCN, 5 mM triethanolamine, 1.25 mM ethylenediaminetetraacetic acid, 0.5% (w / v) BSA, 0.09% (w / v) NaN ₃ , 90 mM piperazine- It contained 1,4-bis (2-ethanesulfonic acid), 25 mM MOPS (pH 7.3). The assay was monitored spectrophotometrically by following the aggregation of the microparticles at a wavelength of 600 nm.

  Human serum for therapeutic drug monitoring (TDM serum) containing 0.1% (w / v) D-α-tocopheryl polyethylene glycol-1000 succinate (TPGS, Eastman Chemical Co) (Valley Biomedical Products and Services, Inc., Winchester, The reagent was calibrated against a multi-analyte set of calibrators containing lopinavir at concentrations of 0, 0.8, 1.6, 3.2, 6.4 and 12.8 μg / mL in (VA). Ethanol stock solutions of lopinavir, lopinavir M-1 metabolites and the respective lopinavir analogs were prepared gravimetrically. Samples for the assay were prepared by spiking each drug from the ethanol stock solution into TDM serum to concentrations of 0.8, 1.6, 3.2, 6.4 and 12.8 μg / mL. Each sample was assayed in triplicate. The cross-reactivity of the analog was calculated for lopinavir. The results obtained are shown in Table 1 below.

Alternatively, lopinavir immunoassay reagents containing lopinavir antibodies LPV 1.1.85 and LPV 1.7.90 were calibrated against a calibration material containing only the N-carbamoyl-lopinavir analog ( 13 ). The calibration curves for lopinavir antibodies LPV 1.1.85 and LPV 1.7.90 obtained when lopinavir or N-carbamoyl-lopinavir ( 13 ) are used as calibrators are shown in FIGS. 12a and 12b. Table 1 below shows the cross-reactivity of lopinavir analogs and metabolites to lopinavir antibodies when compared to lopinavir. The value shown is a percentage.

Example 44: Production of antibodies against atazanavir Antibodies against atazanavir were produced as described in patent application publication US 2005/0064517.

Example 45: Preparation of atazanavir-aminodextran conjugate
Aminodextran (AMD) was synthesized as described in US 6,653,456, the disclosure of which is incorporated herein by reference. The aminodextran had a molecular weight of about 40,000 and contained 6 amino groups per mole. ATV-N-hydroxysuccinimide (NHS) ester derivative 33 was synthesized as described in Example 26. ATV-AMD conjugates were prepared by reaction of AMD in anhydrous dimethyl sulfoxide (DMSO) with an 8-fold to 16-fold excess of ATV-NHS ester derivative 33 in the presence of triethylamine (0.3 μL TEA / 1 mg AMD). The reaction mixture was stirred at room temperature for 23 hours and then dialyzed against a DMSO-H ₂ O mixture containing a gradually decreasing DMSO content. The final aqueous solution was lyophilized to dryness. The ATV: AMD stoichiometry was determined by UV-Vis spectroscopy using ε ₂₅₀ = 16,495 M ⁻¹ cm ⁻¹ and assuming that AMD does not significantly contribute to UV absorbance at that wavelength. Lyophilization was performed with a Virtis Freezemobile 25ES lyophilizer. UV-Vis spectra were recorded on a Cary 50 Bio spectrophotometer (Varian).

Example 46: Preparation of Atazanavir Antibody Microparticles Carboxylated polystyrene microparticles (Part No. 83000520100390) were obtained as a 10% (w / v) aqueous suspension from Seradyn Inc. (Indianapolis, IN). The average particle diameter was about 200 nm, and the surface charge density of the carboxyl group was 0.29 mmol / g (fine particles). Centrifugation was performed at 4 ° C. in a DuPont Sorvall RC-5B centrifuge. The microparticles were thoroughly washed prior to use by centrifugation and at least 3 cycles of resuspension in water and subsequent 50 mM 2-morpholinoethanesulfonic acid (MES) (pH 5.5). . The Homogenizer 4710 Series sonicator (Cole-Parmer) was used at 25-45% power to resuspend the cooled microparticles with a 20-120 second burst of ultrasound. Determination of fine particle monodispersity and concentration was performed on a COBAS MIRA analyzer by light scattering at multiple wavelengths. Washed and resuspended microparticles were stored at 4 ° C. in 50 mM MES (pH 5.5) until needed.

ATV antibodies (from clones 5.1, 14.3, 28.3, 36.3, 62.1.1, 71.1.1) were covalently bound to the microparticle surface as follows. To 180 mL of 1.00% (w / v) microparticles in 50 mM MES (pH 5.5), 22.55 mL of 50 mg / mL N-hydroxysulfosuccinimide in the same buffer is added followed by 20.04 mL of 50 mg / mL aqueous N-ethyl-N ′-(3-dimethyl-aminopropyl) carbodiimide hydrochloride was added. The mixture was stirred at room temperature for 1 hour and then centrifuged (15,000 rpm, 4 ° C., 45 minutes). The microparticles were resuspended in 50 mM 3-morpholinopropane sulfonic acid (MOPS), 0.09% (w / v) NaN ₃ (pH 6.4) (buffer A) to 1.0% (w / v), and a 10 mL aliquot Divided into A 2 mL solution containing 0.25-1.5 mg ATV Ab in Buffer A was added to each aliquot. After stirring at room temperature for 1.5 hours, 5 mL of 50 mg / mL bovine serum albumin (BSA) in buffer A was added and the mixture was stirred at room temperature for an additional 1.5 hours before 50 mM MOPS, 0.09% (w / v ) 6.9 mL of 840 mM 2-aminoethoxyethanol in NaN ₃ (pH 8.5) was added. The mixture was stirred overnight at room temperature and then centrifuged (15,000 rpm, 4 ° C., 45 minutes). The microparticles are resuspended in 50 mM MOPS, 0.1% (w / v) BSA, 0.09% (w / v) NaN ₃ (pH 7.5) (buffer B) and centrifuged (15,000 rpm, 4 ° C., 45 minutes) ). The pellet was resuspended in buffer B to 1.0% (w / v) and stored at 4 ° C. until needed.

Example 47: Cross-reactivity of atazanavir analogs to atazanavir antibodies Stock solutions of atazanavir parent drug and analogs were prepared in EtOH and the concentration of each compound was ε ₂₅₀ = 14,520 M ^-1 cm ^-1 (free base) It confirmed using. Purchased therapeutic human serum containing 0.1% (w / v) D-α-tocopheryl polyethylene glycol-1000 succinate (TPGS) (Eastman Chemical Co., Kingsport, TN) using the EtOH stock Samples of 0.10-3.35 μg / mL (free base) of the compound in (TDM) (Valley Biomedical Products and Services, Inc., Winchester, VA) were prepared. A set of calibrators containing 0.00, 0.15, 0.33, 0.75, 1.50 and 3.00 μg / mL atazanavir parent drug was prepared in a similar manner from independently prepared ethanol stocks. Polyacrylic acid (PAA, molecular weight 225,000) was purchased from Polysciences Inc. (Warrington, PA). The assay was performed at 37 ° C. on a Roche / Hitachi 917 clinical chemistry analyzer. The reaction mixture (296 μL) contains 3 μL of ATV calibrator, atazanavir parent drug or atazanavir analog (prepared as above), 0.05% (w / v) sensitized microparticles, 0.05-0.15 μg / mL ATV -AMD conjugate, 0.5-1.0% (w / v) PAA, 50 mM KSCN, 5 mM triethanolamine, 1.25 mM ethylenediaminetetraacetic acid, 0.1% (w / v) BSA, 0.09% (w / v) NaN ₃ 90 mM piperazine-1,4-bis (2-ethanesulfonic acid) and 25 mM MOPS (pH 7.3). The assay was monitored spectrophotometrically by following microparticle aggregation at a wavelength of 600 nm. Cross-reactivity was determined by comparing the response of the analog to that of an equivalent concentration of the parent drug. Table 2 shows the cross-reactivity of atazanavir and atazanavir analogs for each antibody. The cross-reactivity is obtained from at least three independent sets of measurements. The cross-reactivity of the analog was calculated relative to the parent drug atazanavir. The value shown is a percentage.

Example 48: Microparticle immunoassay calibration curve using atazanavir or atazanavir-Oc-carbamate ( 25 ) Human serum for TDM containing 0.1% (w / v) TPGS as described in Example 47 was prepared calibrators carbamate (25) - atazanavir or atazanavir -O ^c in. The free base concentrations of atazanavir or atazanavir-O ^c -carbamate were 0.00, 0.15, 0.33, 0.75, 1.50 and 3.00 μgml ⁻¹ . An assay was performed as described in Example 47 using microparticles sensitized with antibody ATV5.1 to obtain a calibration curve as shown in FIG.

The nearly superimposed calibration curve obtained with both compounds confirms atazanavir-O ^c -carbamate cross-reactivity data for antibody ATV5.1 in Table 2 for calibration for atazanavir TDM immunoassays. The use of either atazanavir or atazanavir-O ^c -carbamate as a substance provides further confirmation that similar results are obtained when assaying clinical samples containing atazanavir.

Example 49: atazanavir -O ^c - carbamates using calibrators atazanavir microparticle immunoassay spike recovery test
Individual serum samples from 10 healthy blood donors were spiked with atazanavir from an ethanol stock solution to obtain 55 different serum samples containing atazanavir concentrations ranging from 0.04 to 3.00 μgml ^-1 . . Each sample was quantified by atazanavir microparticle immunoassay against atazanavir-O ^c -carbamate calibration curve obtained as described in Example 47. As described in Example 47 except that microparticles sensitized with antibody ATV 5.1 were used and the final reaction mixture further contained 0.07% (w / v) BRIJ-35 (Croda Uniqema Inc.). The assay was performed. A plot of measured concentration of atazanavir against spike concentration is shown in FIG. 17 along with a linear regression analysis of the data.

Linear regression analysis with both slope (0.91) and correlation coefficient (0.994) within the required ranges (0.90 to 1.10 and> 0.90, respectively) is a calibrator for quantifying atazanavir in human plasma or serum The suitability of the use of atazanavir-O ^c -carbamate has been demonstrated.

Example 50: Lopinavir microparticle immunoassay spike recovery test using N-carbamoyl-lopinavir ( 13 ) calibrator
Individual serum samples from 10 healthy blood donors were spiked with lopinavir from an ethanol stock solution to obtain 55 different serum samples containing lopinavir concentrations ranging from 0.21 to 12.80 μgml ^-1 . Each sample was quantified by lopinavir microparticle immunoassay against N-carbamoyl-lopinavir ( 13 ) calibration curve obtained as described in Example 43. The assay was performed as described in Example 43 except that microparticles sensitized with antibody LPV 1.7.90 were used and the final reaction mixture further contained 0.08% (w / v) BRIJ-35. went. A plot of measured concentration of lopinavir against spike concentration is shown in FIG. 18 along with a linear regression analysis of the data.

  Linear regression analysis with both slope (1.00) and correlation coefficient (0.994) within the required ranges (0.90 to 1.10 and> 0.90, respectively) is a calibrator for quantifying lopinavir in human plasma or serum N-carbamoyl-lopinavir has proven suitability for use.

Example 51: Production of antibodies against efavirenz
Sixteen week old female Balb / c mice were immunized with efavirenz-KLH immunogen 46 . The preparation of efavirenz-KLH immunogen 46 is described in EP 1470 825 A1, the disclosure of which is incorporated herein by reference. Initial immunization with 100 μg of immunogen emulsified in Freund's complete adjuvant was performed by intraperitoneal route. There was an approximately 3 week interval between immunizations. All subsequent immunizations were by the same dose emulsified in Freund's incomplete adjuvant, also by intraperitoneal injection. A total of 4 immunizations were performed.

Four days after the final fusion immunization, the mice were killed by cervical dislocation and the spleen was removed. The spleen was processed by breaking between sterile ground glass slides in warmed cell culture medium. Freed cells were taken into sterile 15 mL centrifuge tubes and large fragments were allowed to settle for 1-2 minutes. The suspended cells were pipetted into another sterile 15 mL centrifuge tube and a sample was taken for counting. F0 strain (ATCC) myeloma cells were added at a rate of 1 myeloma cell per 5 splenocytes, and the resulting mixture was centrifuged to sediment the cells into small pellets. Fusion consists of adding myeloma cells (1/5 of lymphocytes), washing by centrifugation, resuspension in serum-free, warm Iscove's Modified Dulbecco's Media (IMDM, Irvine Scientific), and resuspension. It included centrifugation. The centrifuge tube containing the resulting pellet was gently tapped to loosen the cells, and then 1 mL of warm PEG / DMSO solution (Sigma Chemicals) was added slowly with gentle mixing. The cells are kept warm for 1.5 minutes, then pre-warmed serum-free IMDM is added at a rate of 1 mL / min, 2 mL / min, 4 mL / min, and 10 mL / min, then the tube is added. Filled to 50 mL, sealed and incubated for 15 minutes. The cell suspension was centrifuged, the supernatant was decanted, and IMDM containing 10% fetal bovine serum (Summit Biologicals) was added. The cells were centrifuged again and resuspended in complete cloning medium. This contained IMDM, 10% FCS, 10% Condimed H1 (Roche Molecular Systems), 4 mM glutamine, 50 μM 2-mercaptoethanol, 40 μM ethanolamine and pen / strep antibiotics (all from Sigma). The cells were suspended at a density of 4 × 10 ⁵ lymphocytes / mL, dispensed at 100 μL / well into sterile 96-well microculture plates and incubated at 37 ° C. in 5% CO ₂ for 24 hours. The next day, 100 μL of HMT selection medium (cloning medium + 1: 25 HMT supplement (Sigma Chemicals)) was added. At the sixth day of incubation, approximately 150 μL of medium was withdrawn from each well using a sterile 8-place manifold connected to a low vacuum source. Then 150 μL of HT medium was added. This contained cloning medium + 1:50 HT supplement (Sigma Chemicals). The plates were returned to the incubator and observed daily for signs of growth. When growth was judged sufficient, wells were screened by ELISA for antibody production.

ELISA screening microtiter plates were coated with 100 μL of efavirenz-BSA conjugate 47 in 0.1 M carbonate buffer (pH 9.5) for 1 hour at 37 ° C. (humidified). The preparation of efavirenz-BSA conjugate 47 is described in EP 1470 825 A1. The plates were then emptied and filled with a post-coat solution consisting of Tris buffer, 1% gelatin hydrolyzate, 2% sucrose and 0.17% TWEEN 20 (all reagents were obtained from Sigma Chemicals). The plates were incubated for an additional hour at 37 ° C. (humidified) and then washed with phosphate buffered saline containing 0.1% TWEEN 20. The plates were then briefly filled with a 2% sucrose solution in 0.15M Tris (pH 7.2-7.4), then emptied and allowed to air dry at room temperature. Once dry, the plates were placed in a ZIPLOC bag containing some dry packaging, sealed and stored at 4 ° C. until use.

  When it was determined that the growing clones were sufficient for testing, 25 μL of the supernatant was taken from the wells and transferred to a 96-well flexible plate. Culture medium was added to each well to obtain a 1:10 dilution of the medium sample. 25 μL of PBS-TWEEN was added to each well of one coated plate, and 25 μL of 800 ng / mL chiral efavirenz solution in PBS-TWEEN was added to the wells of another coated plate. 25 μL of the diluted media sample was transferred to each of the coated plates. The plates were incubated for 1 hour at 37 ° C. in the covered state and then washed with PBS-TWEEN. The wells were then filled with 100 μL of goat anti-mouse IgG-HRP conjugate (Zymed Labs) diluted in PBS-TWEEN and the plates were reincubated for 1 hour. The plate was then rewashed and 100 μL of K-BLUE substrate (Neogen Corp) was added to each well. The color was developed over 5-15 minutes and the reaction was stopped by the addition of 100 μL 1N HCl. The color was read at 450 nm with a microplate reader and the results were collected by computer for analysis. The criteria for selection was binding to efavirenz-BSA conjugate coated wells that received the PBS-TWEEN and reduced binding to coated wells containing free efavirenz drug.

Example 52: Preparation of Efavirenz Antibody Microparticles Carboxylated polystyrene microparticles (Part No. 83000520100390) were obtained as a 10% (w / v) aqueous suspension from Seradyn Inc. (Indianapolis, IN). The average fine particle diameter was about 200 nm, and the surface charge density of the carboxyl group was 0.21 mmol / g (fine particles). Centrifugation was performed at 4 ° C. in a DuPont Sorvall RC-5B centrifuge. The microparticles were thoroughly washed prior to use by at least three cycles of centrifugation and subsequent resuspension in water and subsequent 50 mM 2-morpholinoethanesulfonic acid (MES) (pH 5.5). The Homogenizer 4710 Series sonicator (Cole-Parmer) was used at 25-45% power to resuspend the cooled microparticles with a 20-120 second burst of ultrasound. Determination of fine particle monodispersity and concentration was performed on a COBAS MIRA analyzer by light scattering at multiple wavelengths. Washed and resuspended microparticles were stored at 4 ° C. in 50 mM MES (pH 5.5) until needed.

Efavirenz (EFV) antibodies (clone 129.1.1.1, 137.1.1.1, 142.1.1.1.1, 143.1 and 149.1.3) were covalently bound to the microparticle surface as follows. To 140 mL of 1.00% (w / v) microparticles in 50 mM MES (pH 5.5), add 12.7 mL of 50 mg / mL N-hydroxysulfosuccinimide in the same buffer, and then 11.29 mL of 50 mg / mL aqueous N -Ethyl-N '-(3-dimethyl-aminopropyl) carbodiimide hydrochloride was added. The mixture was stirred at room temperature for 1 hour and then centrifuged (15,000 rpm, 4 ° C., 50 minutes). The microparticles 50 mM 3- morpholino acid (MOPS), and 0.09% (w / v) NaN 3 (pH 6.4) 1.00% was resuspended in (Buffer A) (w / v), an aliquot in 10 mL of Divided into A 2 mL solution containing 0.25-1.5 mg EFV Ab in buffer A was added to each aliquot. After stirring at room temperature for 1.5 hours, 5 mL of 50 mg / mL bovine serum albumin (BSA) in buffer A was added and the mixture was stirred at room temperature for an additional 1.5 hours before 50 mM MOPS, 0.09% (w / v ) 5.0 mL of 840 mM 2-aminoethoxyethanol in NaN ₃ (pH 8.5) was added. The mixture was stirred overnight at room temperature and then centrifuged (15,000 rpm, 4 ° C., 50 minutes). The microparticles are resuspended in 50 mM MOPS, 1.0% (w / v) BSA, 0.09% (w / v) NaN ₃ (pH 7.5) (buffer B) and centrifuged (15,000 rpm, 4 ° C., 50 minutes) ). The pellet was resuspended in buffer B to 1.00% (w / v) and stored at 4 ° C. until needed.

Example 53: Cross-reactivity of efavirenz analogs to efavirenz antibodies Stock solutions of efavirenz parent drug and analogs are prepared in EtOH, and the concentration of each compound is ε ₂₅₀ = 14,806 M ^-1 cm ^-1 (free base) It confirmed using. Purchased therapeutic human serum containing 0.1% (w / v) D-α-tocopheryl polyethylene glycol-1000 succinate (TPGS) (Eastman Chemical Co., Kingsport, TN) using the EtOH stock Samples of 1.00 to 7.00 μg / mL (free base) of the compound in (TDM) (Valley Biomedical Products and Services, Inc., Winchester, VA) were prepared. A set of calibrators containing 0.00, 0.60, 1.20, 2.40, 4.80 and 9.60 μg / mL efavirenz parent drug was prepared in a similar manner from independently prepared ethanol stocks. The assay was performed at 37 ° C. on a Roche / Hitachi 917 clinical chemistry analyzer. The reaction mixture (296 μL) consists of 3 μL of efavirenz calibrator, efavirenz parent drug or efavirenz analog (prepared as above), 0.05% (w / v) sensitized microparticles, 0.10-0.20 μg / mL EFV -Aminodextran conjugate 45 , 0.7-1.1% (w / v) polyacrylic acid (PAA, molecular weight 225,000, purchased from Polysciences Inc. (Warrington, PA)), 50 mM KSCN, 5 mM triethanolamine, 1.25 mM ethylenediamine Tetraacetic acid, 0.1% (w / v) BSA, 0.09% (w / v) NaN ₃ , 90 mM piperazine-1,4-bis (2-ethanesulfonic acid), 25 mM MOPS (pH 7.2), and Table 3 It contained a surfactant as shown in FIG. The assay was monitored spectrophotometrically by following microparticle aggregation at a wavelength of 600 nm. Cross-reactivity was determined by comparing the response of the analog to that of an equivalent concentration of the parent drug. Table 3 shows the cross-reactivity of efavirenz and efavirenz analogs to each antibody.

Claims (24)

The group consisting of amprenavir, atazanavir, tipranavir, indinavir, darunavir, lopinavir, nelfinavir, ritonavir, and saquinavir A compound comprising a selected HIV protease inhibitor, wherein the central hydroxyl group of the protease inhibitor is -O (C ₁ -C ₁₀ alkyl), -OCONHR ₃ , -OCH ₂ COOR ₃ and -OCH ₂ CONHR ₃ Wherein R ₃ is selected from the group consisting of H, C ₁ -C ₆ alkyl, phenyl, heteroaryl and — (CH ₂ ) _n —X, wherein R ₃ is substituted with a group selected from the group consisting of in, n is 1 to 6, X is COOR ₅ or NH-CH ₂ - phenyl -COOR _5, wherein, R ₅ is H or C ₁ -C ₆ alkyl, compounds. Construction:

Wherein R ₄ is selected from the group consisting of C ₁ -C ₁₀ alkyl, —CH ₂ COOR ₃ , —CH ₂ CONHR ₃ and —CONHR ₃ , wherein R ₃ is H, C ₁ -C is selected from (CH ₂₎ group consisting of _n -X, wherein, n is 1 to 6, X is COOR ₅ or NH-CH ₂ - - ₆ alkyl, phenyl, heteroaryl and phenyl -COOR ₅ Where R ₅ is H or C ₁ -C ₆ alkyl]
A compound having Construction:

Wherein R ₃ is selected from the group consisting of H, C ₁ -C ₆ alkyl, phenyl, heteroaryl and — (CH ₂ ) _n —X, wherein n is 1-6 and X is COOR ₅ or NH-CH ₂ - phenyl -COOR _5, wherein, R ₅ is H or C ₁ -C ₆ alkyl]
A compound having R ₃ is H or C ₁ -C ₄ alkyl The compound of claim 3, wherein. Construction:

Wherein R ₄ is selected from the group consisting of — (C ₁ -C ₁₀ alkyl), —CONHR ₃ , —CH ₂ COOR ₃ and —CH ₂ CONHR ₃ , wherein R ₃ is H, C ₁ -C ₆ alkyl, phenyl, heteroaryl, and - (CH ₂₎ selected from the group consisting of _n -X, wherein, n is 1 to 6, X is COOR ₅ or NH-CH ₂ - phenyl -COOR ₅ where R ₅ is H or C ₁ -C ₆ alkyl]
A compound having R ₄ is —CONHR ₃ or —CH ₂ COOR ₃ and R ₃ is selected from the group consisting of H, C ₁ -C ₆ alkyl, phenyl, heteroaryl and — (CH ₂ ) _n —X, where n is is 1 to 6, X is COOR ₅ or NH-CH ₂ - phenyl -COOR _5, R ₅ is H or C ₁ -C ₆ alkyl 6. the compound of claim 5, wherein. Construction:

Wherein R ₁ is selected from the group consisting of H and —CONHR ₃ and R ₄ is from the group consisting of C ₁ -C ₁₀ alkyl, —CH ₂ COOR ₃ , —CH ₂ CONHR ₃ and —CONHR ₃ Wherein R ₃ is selected from the group consisting of H, C ₁ -C ₆ alkyl, phenyl, heteroaryl and — (CH ₂ ) _n —X, wherein n is 1-6; X is COOR ₅ or NH-CH ₂ - phenyl -COOR _5, wherein, R ₅ is H or C ₁ -C ₆ alkyl]
A compound having R ₁ is H, R ₄ is selected from the group consisting of —CH ₂ COOR ₃ , —CH ₂ CONHR ₃ and —CONHR ₃ , and R ₃ is H or C ₁ -C ₆ alkyl. The described compound. 8. The compound of claim 7, wherein R ₁ is H, R ₄ is —CONHR ₃ or —CH ₂ COOR ₃ , and R ₃ is C ₁ -C ₆ alkyl. R ₁ is -CONHR _3, R ₄ is H, R ₃ is H or C ₁ -C ₆ alkyl, claim 7 compound according. R ₁ is -CONHR _3, R ₄ is -CONHR _2, R ₃ and R ₂ is C ₁ -C ₄ alkyl, respectively, according to claim 7 compound according. Construction:

[Wherein R is C ₁ -C ₆ alkyl or (C ₁ -C ₆ alkyl) OH]
A compound having A test kit for measuring the amount of protease inhibitor in a sample, wherein the protease inhibitor is present in the sample amprenavir, atazanavir, tipranavir, indinavir, A kit selected from the group consisting of darunavir, lopinavir, nelfinavir, ritonavir and saquinavir, wherein the kit is packaged as the following:
An antibody that specifically binds to the protease inhibitor;
An analog of the protease inhibitor that is immunochemically equivalent to the protease inhibitor, wherein the central hydroxyl group of the protease inhibitor is -O (C ₁ -C ₁₀ alkyl), -OCONHR ₃ , -OCH ₂ COOR Substituted with a group selected from the group consisting of ₃ and —OCH ₂ CONHR ₃ , wherein R ₃ is from H, C ₁ -C ₆ alkyl, phenyl, heteroaryl and — (CH ₂ ) _n —X. is selected from the group consisting, wherein, n is 1 to 6, X is COOR ₅ or NH-CH ₂ - phenyl -COOR _5, wherein, R ₅ is H or C ₁ -C ₆ alkyl , Analogs of the protease inhibitor, and instructions for measuring the amount of the protease inhibitor in the sample,
A test kit comprising: The protease inhibitor is atazanavir and the analog has the structure:

Wherein R ₃ is selected from the group consisting of H, C ₁ -C ₆ alkyl, phenyl, heteroaryl and — (CH ₂ ) _n —X, wherein n is 1-6 and X is COOR ₅ or NH-CH ₂ - phenyl -COOR _5, wherein, R ₅ is H or C ₁ -C ₆ alkyl]
14. The test kit according to claim 13, comprising: R ₃ is H or C ₁ -C ₄ alkyl, claim 14 test kit according. The protease inhibitor is lopinavir and the analog has the structure:

[Wherein R ₄ is —CONHR ₃ or —CH ₂ COOR ₃ , and R ₃ is selected from the group consisting of H, C ₁ -C ₆ alkyl, phenyl, heteroaryl and — (CH ₂ ) _n —X. is, where, n is 1 to 6, X is COOR ₅ or NH-CH ₂ - phenyl -COOR _5, wherein, R ₅ is H or C ₁ -C ₆ alkyl]
14. The test kit according to claim 13, comprising: A test kit for measuring the amount of efavirenz in a sample, the kit being packaged as follows:
An antibody that specifically binds to efavirenz,
Efavirenz analogues, including efavirenz derivatives N-alkylated with C ₁ -C ₆ alkyl or (C ₁ -C ₆ alkyl) OH, which are immunochemically equivalent to efavirenz, and efavirenz in the sample Instructions for measuring the quantity,
A test kit comprising: The analog has the structure:

[Wherein R is C ₁ -C ₆ alkyl or (C ₁ -C ₆ alkyl) OH]
18. The test kit according to claim 17, comprising: The group consisting of amprenavir, atazanavir, tipranavir, indinavir, darunavir, lopinavir, nelfinavir, ritonavir, and saquinavir A method for creating a calibration curve for a selected HIV protease inhibitor comprising:
Preparing a set of calibrators, each calibrator containing one of a range of concentrations of an immunochemically equivalent analog of the protease inhibitor, wherein the analog is includes derivatives of the protease inhibitor, wherein the hydroxyl groups of the center of the protease inhibitors, -O (C ₁ -C ₁₀ alkyl), - OCONHR _3, the group consisting of -OCH ₂ CONHR ₃ and -OCH ₂ COOR ₃ Wherein R ₃ is selected from the group consisting of H, C ₁ -C ₆ alkyl, phenyl, heteroaryl and — (CH ₂ ) _n —X, wherein n is 1 to 6, X is COOR ₅ or NH-CH ₂ - phenyl -COOR _5, wherein, R ₅ is H or C ₁ -C ₆ alkyl, step,
Contacting each of the calibrators with a monoclonal antibody that specifically binds to the protease inhibitor and analog, and a conjugate comprising the protease inhibitor and a moiety that generates a signal upon binding of the antibody to the analog The process of
Measuring the signal produced by each calibrator, and establishing a calibration curve by plotting the analog concentration against the signal measured for each calibrator;
Comprising a method. The protease inhibitor is atazanavir and the analog has the structure:

Wherein R ₃ is selected from the group consisting of H, C ₁ -C ₆ alkyl, phenyl, heteroaryl and — (CH ₂ ) _n —X, wherein n is 1-6 and X is COOR ₅ or NH-CH ₂ - phenyl -COOR _5, wherein, R ₅ is H or C ₁ -C ₆ alkyl]
20. The method of claim 19, comprising: R ₃ is H or C ₁ -C ₄ alkyl, The method of claim 20, wherein. The protease inhibitor is lopinavir and the analog has the structure:

[Wherein R ₄ is —CONHR ₃ or —CH ₂ COOR ₃ , and R ₃ is selected from the group consisting of H, C ₁ -C ₆ alkyl, phenyl, heteroaryl and — (CH ₂ ) _n —X. is, where, n is 1 to 6, X is COOR ₅ or NH-CH ₂ - phenyl -COOR _5, wherein, R ₅ is H or C ₁ -C ₆ alkyl]
20. The method of claim 19, comprising: A method for establishing a calibration curve for efavirenz, including:
Preparing a set of calibrators, wherein each calibrator contains one of a range of concentrations of an immunochemically equivalent analog of efavirenz, wherein the analog is C Comprising a derivative of efavirenz that is N-alkylated with ₁ -C ₆ alkyl or (C ₁ -C ₆ alkyl) OH,
Contacting each of the calibrators with a monoclonal antibody that specifically binds to efavirenz and the analog, and a conjugate comprising efavirenz with a moiety that generates a signal upon binding of the antibody to the analog;
A method comprising: measuring a signal generated by each calibration substance; and establishing a calibration curve by plotting the concentration of the analog against the signal measured for each calibration substance. The analog has the structure:

[Wherein R is C ₁ -C ₆ alkyl or (C ₁ -C ₆ alkyl) OH]
24. The method of claim 23, comprising:

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