U.S. patent application number 11/420497 was filed with the patent office on 2006-11-23 for protease inhibitor conjugates and antibodies useful in immunoassay.
Invention is credited to Ina Deras, Mitali Ghoshal, Erasmus Huber, Raymond A. Hui, Peter Kern, Sigrun Metz, Richard Terry Root, Gerald F. Sigler, Herbert W. Von Der Eltz.
Application Number | 20060264618 11/420497 |
Document ID | / |
Family ID | 34194810 |
Filed Date | 2006-11-23 |
United States Patent
Application |
20060264618 |
Kind Code |
A1 |
Deras; Ina ; et al. |
November 23, 2006 |
PROTEASE INHIBITOR CONJUGATES AND ANTIBODIES USEFUL IN
IMMUNOASSAY
Abstract
Monoclonal antibodies specific for lopinavir having less than
10% cross-reactivity with saquinavir, nelfinavir, amprenavir,
ritonavir, and indinavir, and a murine hybridoma producing said
antibodies are described.
Inventors: |
Deras; Ina; (San Diego,
CA) ; Sigler; Gerald F.; (Carmel, IN) ; Hui;
Raymond A.; (Indianapolis, IN) ; Root; Richard
Terry; (Fishers, IN) ; Ghoshal; Mitali;
(Noblesville, IN) ; Huber; Erasmus; (Finning,
DE) ; Von Der Eltz; Herbert W.; (Weilheim, DE)
; Metz; Sigrun; (Munich, DE) ; Kern; Peter;
(Penzberg, DE) |
Correspondence
Address: |
Roche Diagnostics Corporation, Inc.
9115 Hague Road
PO Box 50457
Indianapolis
IN
46250-0457
US
|
Family ID: |
34194810 |
Appl. No.: |
11/420497 |
Filed: |
May 26, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10669831 |
Sep 24, 2003 |
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11420497 |
May 26, 2006 |
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10192052 |
Jul 10, 2002 |
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10669831 |
Sep 24, 2003 |
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60305192 |
Jul 13, 2001 |
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Current U.S.
Class: |
530/388.1 ;
435/326 |
Current CPC
Class: |
A61P 43/00 20180101;
C07K 16/38 20130101; C07K 16/44 20130101; A61P 31/18 20180101 |
Class at
Publication: |
530/388.1 ;
435/326 |
International
Class: |
C07K 16/18 20060101
C07K016/18; C12N 5/06 20060101 C12N005/06 |
Claims
1. A monoclonal antibody specific for lopinavir having less than
10% cross-reactivity with saquinavir, nelfinavir, amprenavir,
ritonavir, and indinavir.
2. Murine hybridoma <LOPIN> M 1.1.85 having DSMZ No. ACC
2611.
Description
RELATED APPLICATIONS
[0001] This application divisional of U.S. Ser. No. 10/669,831,
filed Sep. 24, 2003, which is a continuation-in-part of U.S. patent
application Ser. No. 10/192,052 filed Jul. 10, 2002, which claims
priority to U.S. Provisional Application No. 60/305,192 filed Jul.
13, 2001.
FIELD OF THE INVENTION
[0002] This invention relates to novel protease inhibitor
conjugates and antibodies useful in immunoassay. More specifically,
this invention relates to novel activated haptens useful for
generating immunogens to HIV protease inhibitors, to novel
immunogens useful for producing antibodies to HIV protease
inhibitors, and to novel antibodies and labeled conjugates useful
in immunoassays for HIV protease inhibitors.
BACKGROUND OF THE INVENTION
[0003] HIV protease inhibitors are an important new class of drugs
which have made a significant impact on the health care of AIDS
patients since the first one, saquinavir, was introduced to the
marketplace in 1995. Examples of other protease inhibitors include
amprenavir, indinavir, nelfinavir, lopinavir, ritonavir, and
atazanavir. They are especially effective in combination with other
anti-HIV drugs such as reverse transcriptase inhibitors or with
other HIV protease inhibitors. In spite of remarkable success with
these new therapeutic regimens, there are strong indications that
results would be much improved if therapeutic drug testing methods
were available for monitoring the concentrations of protease
inhibitors. Not all patients respond optimally to protease
inhibitor combination therapies. Even those who do respond can
subsequently develop drug resistance due to the notoriously high
rate of mutation of the HIV virus. However, it has been shown that
there is a clear relationship between plasma levels of the protease
inhibitors and therapeutic efficacy based upon decreased viral load
and increased CD4 cell count. One problem lies in the fact that the
drugs are metabolized extensively and are subject to complex
drug-drug interactions. The results are extremely complex
pharmacokinetics and a strong element of unpredictability between
dosage and resultant drug levels at any particular time for any
particular patient. With therapeutic drug monitoring, drug dosages
could be individualized to the patient, and the chances of keeping
the virus in check would be much higher. But routine therapeutic
drug monitoring or protease inhibitors would require the
availability of simple automated tests adaptable to high throughput
clinical analyzers. Currently most reports on therapeutic drug
monitoring of protease inhibitors have used HPLC methods which are
slow, labor-intensive, and expensive. Recently there was a report
of a radioimmunoassay (RIA) method for saquinavir (Wiltshire et
al., Analytical Biochemistry 281, 105-114, 2000). However, such a
method would not be adaptable to high-throughput therapeutic drug
monitoring and, like all RIA methods, suffers from the
disadvantages of having regulatory, safety and waste disposal
issues related to the radioactive isotope label used in the assay.
The most desirable assay formats for therapeutic drug monitoring
are non-isotopic immunoassays, and such methods have heretofore
been unknown for monitoring HIV protease inhibitors.
[0004] As indicates above, HPLC has been the method of choice for
monitoring HIV protease inhibitors. Two recent reports in the
literature describe HPLC assays for the simultaneous determination
of several protease inhibitors in human plasma, Poirier et al.,
Therapeutic Drug Monitoring 22, 465-473, 2000 and Remmel et al.,
Clinical Chemistry 46, 73-81, 2000.
[0005] Chemical and biological assays generally involve contacting
the analyte of interest with a pre-determined amount of one or more
assay reagents, measuring one or more properties of a resulting
product (the detection product), and correlating the measured value
with the amount of analyte present in the original sample,
typically by using a relationship determined from standard or
calibration samples containing known amounts of analyte of interest
in the range expected for the sample to be tested. Typically, the
detection product incorporates one or more detectable labels which
are provided by one or more assay reagents. Examples of commonly
used labels include functionalized microparticles, radioactive
isotope labels such as .sup.125I and .sup.32P, enzymes such as
peroxidase and beta-galactosidase and enzyme substrate labels,
fluorescent labels such as fluoresceins and rhodamines,
electron-spin resonance labels such as nitroxide free radicals,
immunoreactive labels such as antibodies and antigens, labels which
are one member of a binding pair such as biotin-avidin and
biotin-streptavidin, and electrochemiluminescent labels such as
those containing a ruthenium bipyridyl moiety. Sandwich assays
typically involve forming a complex in which the analyte of
interest is sandwiched between one assay reagent which is
ultimately used for separation, e.g., antibody, antigen, or one
member of a binding pair, and a second assay reagent which provides
a detectable label. Competition assays typically involve a system
in which both the analyte of interest and an analog of the analyte
compete for a binding site on another reagent, e.g., an antibody,
wherein one of the analyte, analog or binding reagent possesses a
detectable label.
[0006] Copending U.S. patent application Ser. No. 09/712,525 filed
Nov. 14, 2000 having the same assignee as the present application
and published as EP 1 207 394 on May 22, 2002, describes a
non-isotopic immunoassay for an HIV protease inhibitor comprising
incubating a sample containing the inhibitor with a receptor
specific for the inhibitor or for a metabolite of said inhibitor
and further with a conjugate comprising an analog of the inhibitor
and a non-isotopic signal generating moiety. Signal generated as a
result of binding of the inhibitor by the receptor is measured and
correlated with the presence or amount of protease inhibitor in the
original sample. The protease inhibitor conjugates of the present
invention are especially useful in such an assay.
SUMMARY OF THE INVENTION
[0007] The present invention relates to novel activated haptens
useful for generating immunogens to HIV protease inhibitors. These
activated haptens have the general structure:
I--X--(C.dbd.Y).sub.m-L-A wherein I is an HIV protease inhibitor
radical, X is O or NH, Y is O, S or NH, m is 0 or 1, L is a linker
consisting of from 0 to 40 carbon atoms arranged in a straight
chain or a branched chain, saturated or unsaturated, and containing
up to two ring structures and 0-20 heteroatoms, with the proviso
that not more than two heteroatoms may be linked in sequence, and A
is an activated functionality chosen from the group consisting of
active esters, isocyanates, isothiocyanates, thiols, imidoesters,
anhydrides, maleimides, thiolactones, diazonium groups and
aldehydes.
[0008] The present invention also relates to novel immunogens
having the following structure:
[I--X--(C.dbd.Y).sub.m-L-Z].sub.n--P wherein I is an HIV protease
inhibitor radical, X is O or NH, Y is O, S, or NH, m is 0 to 1, L
is a linker comprising 0 to 40 carbon atoms arranged in a straight
chain or a branched chain, saturated or unsaturated, and containing
up to two ring structures and 0-20 heteroatoms, with the proviso
that not more than two heteroatoms are linked in sequence, Z is a
moiety selected from the group consisting of --CONH--, --NHCO--,
--NHCONH--, --NHCSNH--, --OCONH--, --NHOCO--, --S--,
--NH(C.dbd.NH)--, --N.dbd.N--, --NH--, and ##STR1##
[0009] P is a polypeptide, a polysaccharide, or a synthetic
polymer, and n is a number from 1 to 50 per 50 kilodaltons
molecular weight of P.
[0010] The present invention also relates to novel labeled
conjugates having the following structure:
[I--X--(C.dbd.Y).sub.m-L-Z].sub.n-Q wherein I is an HIV protease
inhibitor radical, X is O or NH, Y is O, S, or NH. m is 0 or 1, L
is a linker comprising 0 to 40 carbon atoms arranged in a straight
chain or a branched chain, saturated or unsaturated, and containing
up to two ring structures and 0-20 heteroatoms, with the proviso
that not more than two heteroatoms are linked in sequence, Z is a
moiety selected from the group consisting of --CONH--, --NHCO--,
--NHCONH--, --NHCSNH--, --OCONH--, --NHOCO--, --S--,
--NH(C.dbd.NH)--, --N.dbd.N--, --NH--, and ##STR2##
[0011] Q is a non-isotopic label, and n is a number from 1 to 50
per 50 kilodaltons molecular weight of Q.
[0012] The present invention also comprises specific monoclonal
antibodies to saquinavir, nelfinavir, indinavir, amprenavir,
lopinavir, and ritonavir having less than 10% cross-reactivity to
other protease inhibitors. Finally, the present invention comprises
antibodies generated from the immunogens of the invention as well
as immunoassay methods and test kits which incorporate the
antibodies and labeled conjugates of the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 illustrates a scheme for synthesis of O-acylated
ritonavir activated haptens, LPH immunogen and BSA conjugate.
[0014] FIG. 2 illustrates a scheme for synthesis of O-acylated
saquinavir activated haptens, KLH immunogen and BSA conjugates.
[0015] FIG. 3 illustrates a scheme for synthesis of O-acylated
amprenavir activated haptens, KLH immunogen and BSA conjugate.
[0016] FIG. 4 illustrates a scheme for synthesis of O-acylated
indinavir activated haptens, KLH immunogen and BSA conjugate.
[0017] FIG. 5 illustrates a scheme for synthesis of O-acylated
nelfinavir activated haptens, KLH immunogen and BSA conjugate.
[0018] FIG. 6 illustrates a scheme for synthesis of O-acylated
lopinavir activated haptens, KLH immunogen and BSA conjugate.
[0019] FIG. 7 illustrates a scheme for synthesis of an alternative
O-acylated saquinavir and ritonavir activated haptens and an
alternative ritonavir immunogen.
[0020] FIG. 8 illustrates a scheme for synthesis of N-acylated
amprenavir immunogen.
[0021] FIG. 9 illustrates a scheme for synthesis of O-alkylated
nelfinavir immunogen.
[0022] FIGS. 10(a) and 10(b) illustrate a scheme for synthesis of
O-carbamylated saquinavir activated haptens.
[0023] FIG. 11 illustrates a scheme for synthesis of O-carbamylated
nelfinavir activated haptens.
[0024] FIG. 12 illustrates a scheme for synthesis of O-acylated
saquinavir maleimide activated hapten.
[0025] FIG. 13 illustrates a scheme for synthesis of O-acylated
saquinavir activated haptens with peptide linkers and maleimide end
groups. Also illustrated is KLH immunogen and BSA conjugate derived
from the latter activated haptens.
[0026] FIG. 14 illustrates a scheme for synthesis of fluorescein
conjugates of saquinavir and ritonavir and of a biotin conjugate of
indinavir.
[0027] FIG. 15 is a chart showing antibody titers generated in
Example 77 using conjugates 2G, 2W, 2D and 2S.
[0028] FIG. 16 illustrates the structures of the conjugates used in
Example 77.
[0029] FIG. 17 are graphs showing the cross-reaction of monoclonal
antibody <INDIN> M 1.158.8 and monoclonal antibody
<INDIN> M 1.003.12 with indinavir, nelfinavir, ritonavir,
saquinavir and amprenavir as described in Example 80.
[0030] FIG. 18 illustrates a scheme for synthesis of O.sup.ar-MEM
O.sup.c-succinimido-oxycarbonylmethyl-nelfinavir ether.
[0031] FIG. 19 illustrates a scheme for synthesis of
O.sup.c-succinimido-oxycarbonylmethyl-saquinavir ether.
[0032] FIG. 20 is a graph showing the cross-reaction of monoclonal
antibody <AMPREN> M 1.1.52 with indinavir, saquinavir,
ritonavir, lopinavir, and nelfinavir as described in Example
81.
[0033] FIG. 21 is a graph showing the cross-reaction of monoclonal
antibody <LOPIN> M 1.1.85 with indinavir, saquinavir,
ritonavir, amprenavir, and nelfinavir as described in Example
82.
[0034] FIG. 22 is a graph showing the cross-reaction of monoclonal
antibody <RITON> M 1.5.44 with indinavir, saquinavir,
amprenavir, lopinavir, and nelfinavir as described in Example
83.
[0035] FIG. 23 illustrates a scheme for synthesis of O-acylated
atazanavir activated haptens, KLH immunogen, and BSA conjugate.
[0036] Throughout the specification, numbers in boldface type are
used refer to chemical structures illustrated in the drawings.
DETAILED DESCRIPTION OF THE INVENTION
[0037] As used herein, analyte refers to a substance, or group of
substances, whose presence or amount thereof is to be
determined.
[0038] Antibody means a specific binding partner of the analyte and
is any substance, or group of substances, which has a specific
binding affinity for the analyte to the essential exclusion of
other unrelated substances. The term includes polyclonal
antibodies, monoclonal antibodies and antibody fragments.
[0039] Haptens are partial or incomplete antigens. They are
protein-free substances, mostly low molecular weight substances,
which are not capable of stimulating antibody formation, but which
do react with antibodies. The latter are formed by coupling a
hapten to a high molecular weight carrier and injecting this
coupled product into humans or animals. Examples of haptens include
therapeutic drugs such as digoxin and theophylline, drugs of abuse
such as morphine and LSD, antibiotics such as geniamicin and
vancomycin, hormones such as estrogen and progesterone, vitamins
such as vitamin B12 and folic acid, thyroxin, histamine, serotonin,
adrenaline and others.
[0040] An activated hapten refers to a hapten derivative that has
been provided with an available site for reaction, such as by the
attachment of, or furnishing of, an activated group for
synthesizing a derivative conjugate.
[0041] The term linker refers to a chemical moiety that connects a
hapten to a carrier, immunogen, labels, tracer or another linker.
Linkers may be straight or branched, saturated or unsaturated
carbon chains. They may also include one or more heteroatoms within
the chain or at termini of the chains. By heteroatoms is meant
atoms other than carbon which are chosen from the group consisting
of oxygen, nitrogen and sulfur. The use of a linker may or may not
be advantageous or needed, depending on the specific hapten and
carrier pairs.
[0042] A carrier, as the term is used herein, is an immunogenic
substance, commonly a protein, that can join with a hapten, thereby
enabling the hapten to stimulate an immune response. Carrier
substances include proteins, glycoproteins, complex polysaccharides
and nucleic acids that are recognized as foreign and thereby elicit
an immunologic response from the host.
[0043] The terms immunogen and immunogenic as used herein refer to
substances capable of producing or generating an immune response in
an organism.
[0044] The terms conjugate and derivative refer to a chemical
compound or molecule made from a parent compound or molecule by one
or more chemical reactions.
[0045] As used herein, a detector molecule, label or tracer is an
identifying tag which, when attached to a carrier substance or
molecule, can be used to detect an analyte. A label may be attached
to its carrier substance directly or indirectly by means of a
linking or bridging moiety. Examples of labels include enzymes such
as .beta.-galactosidase and peroxidase, fluorescent compounds such
as rhodamine and fluorescein isothiocyanate (FITC), luminescent
compounds such as dioxetanes and luciferin, and radioactive
isotopes such as .sup.125I.
[0046] The term active ester within the sense of the present
invention encompasses activated ester groups which can react with
nucleophiles such as, but not limited to, free amino groups of
peptides, polyaminoacids, polysaccharides or labels under such
conditions that no interfering side reactions with other reactive
groups of the nucleophile-carrying substance can usefully
occur.
[0047] An object of the present invention is to provide novel
activated haptens that can be used to generate immunogens to HIV
protease inhibitors. These activated haptens take the general
structure: I--X--(C.dbd.Y).sub.m-L-A where I is an HIV protease
inhibitor radical, X is O or NH, Y is O, S or NH, m is 0 or 1, L is
a linker consisting of from 0 to 40 carbon atoms arranged in a
straight chain or a branched chain, saturated or unsaturated, and
containing up to two ring structures and 0-20 heteroatoms, with the
proviso that not more than two heteroatoms may be linked in
sequence, and A is an activated functionality chosen from the group
consisting of active esters, isocyanates, isothiocyanates, thiols,
imidoesters, anhydrides, maleimides, thiolactones, diazonium groups
and aldehydes.
[0048] As used herein, an HIV protease inhibitor radical is the
intact drug lacking only a hydroxyl group or an amino group, XH,
where X is O or NH. The X and C.dbd.Y moieties include, but are not
limited to, esters (where X is O, Y is O, and m is 1), amides
(where X is NH, Y is O, and m is 1), urethanes (where X is O, Y is
O, m is 1, and the first atom in L adjacent to C.dbd.Y is N), ureas
(where X is NH, Y is O, m is 1, and the first atom in L adjacent to
C.dbd.Y is N), thioureas (where X is NH, Y is S, m is 1, and the
first atom in L adjacent to C.dbd.Y is N), amidines (where X is NH,
Y is NH, and m is 1), ethers (where X is O, and m is 0) and amines
(where X is NR wherein R is H or lower alkyl, and m is 0). "Lower
alkyl" means methyl, ethyl, propyl and isopropyl groups. Preferred
activated haptens are esters or urethanes formed with the central,
non-terminal hydroxyl group common to all HIV protease inhibitors.
This central hydroxyl group is functionally important for the
therapeutic activity of the protease inhibitors but also provides a
convenient handle for derivatization and linker attachment.
Moreover, generally the metabolism of the protease inhibitors takes
place at terminal residues, and therefore the central hydroxyl
groups are attractive sites for immunogens designed to generate
antibodies which discriminate between parent drug and metabolites.
As used here, this central hydroxyl group is designated as
HO.sup.c. When the hydrogen of the central hydroxyl group is
replaced by a (C.dbd.Y).sub.m-L-A group, the residual bonded oxygen
is shown as O.sup.c.
[0049] The linker L serves the purpose of providing an additional
spacer between the terminal activated functionality A and the HIV
protease inhibitor radical, the first spacer being the X and
C.dbd.Y groups. Linker length and composition are well known to
those skilled in the art to have important effects on immunogen
response and conjugate performance. There are may examples of
commercially available or easily synthesized linkers in the
literature for attachment to hydroxyl and amino groups. For a good
treatise on this subject, the reader is referred to Bioconjugate
Techniques, G. Hermanson, Academic Press, 1996. In some cases the
additional linker L is dispensed with and the C.dbd.Y moiety is
directly attached to an activated functionality A. An example of a
preferred linker moiety L is --(CH.sub.2).sub.x--NH-- where x is
1-12. Particularly preferred is x=5 in combination with C.dbd.Y
where Y is O (i.e., aminocaproyl esters). Such linkers are formed
by acylation of an HIV protease inhibitor with an N-protected amino
acid (i.e., aminocaproic acid). The protecting group is preferably
one which is removed under mildly basic or acidic conditions to as
not to affect the integrity of the X--C.dbd.Y bonds or other
moieties in the HIV protease inhibitor radical. An example of an
N-protecting group removed under mildly basic conditions is
fluorenylmethyloxycarbonyl (FMOC). An example of an N-protecting
group easily removed with acid is t-butyloxycarbonyl (BOC). Many
other suitable N-protecting groups are well known in the art (see
"Protective Groups" in Organic Synthesis, 2nd, edition, T. Greene
and P. Wuts, Wiley-Interscience, 1991).
[0050] The acylation reaction of HIV protease inhibitor hydroxyl or
amino groups with N-protected amino acids is accomplished by using
condensation reagents such as carbodiimides with or without a
catalyst. A preferred combination is dicyclohexylcarbodiimide with
dimethylaminopyridine as catalyst. The acylation reaction is
carried out in a suitable solvent such as methylene chloride at
0-35.degree. C. for a time which typically ranges from 0.5 to 7
days. Following isolation of the product, the N-protecting group is
removed. For the preferred FMOC protecting group, this is
accomplished by treatment with a solution of 10% piperidine in
methylene chloride for 0.5 to 2 hours. The amino group of the
resultant aminoacyl-protease inhibitor is amenable to acylation
reactions with a wide variety of carboxyl activated linker
extensions or labels which are well known to those skilled in the
art to which the present invention belongs. Liner extension is
often performed at this stage to generate terminal activated groups
A such as active esters, isocyanates and maleimides. For example,
reaction of aminoacyl-protease inhibitor with one end of
homobifunctional N-hydroxysuccinimide esters of bis-carboxylic
acids such as terephthalic acid will generate stable
N-hydroxysuccinimide ester terminated linker adducts which are
useful for conjugation to amines of polypeptides, polysaccharides,
and labels. Linker extension can also be accomplished with
heterobifunctional reagents such as maleimido alkanoic acid
N-hydroxysuccinimide esters to generate terminal maleimido groups
for subsequent conjugation to thiol groups on polypeptides and
labels. Alternatively, an amino-terminated linker can be extended
with a heterobifunctional thiolating reagent which reacts to form
an amide bond at one end and a free or protected thiol at the other
end. Some examples of thiolating reagents of this type which are
well known in the art are 2-iminothiolane (2-IT), succinimidyl
acetylthiopropionate (SATP) and succinimido
2-pyridyldithiopropionate (SPDP). The incipient thiol group is then
available, after deprotection, to form thiol ethers with maleimido
or bromoacetylated modified immunogens or labels. Yet another
alternative is to convert the amino group of the amino-terminated
linker into a diazonium group and hence the substance into a
diazonium salt, for example, by reaction with an alkali metal
nitrite in the presence of acid, which is then reactive with a
suitable nucleophilic moiety, such as, but not limited to, the
tyrosine residues of peptides, proteins, polyaminoacids and the
like. Examples of suitable amino-terminated linkers for conversion
to such diazonium salts include aromatic amines (anilines), but may
also include the aminocaproates and similar substances referred to
above. Such anilines may be obtained by substituting into the
coupling reaction between the hydroxyl of a protease inhibitor and
an N-protected amino acid, as discussed above, the corresponding
amino acid wherein the amino group is comprised of an aromatic
amine, that is, an aniline, with the amine suitably protected, for
example, as an N-acetyl or N-trifluoracetyl group, which is then
deprotected using methods well-known in the art. Other suitable
amine precursors to diazonium salts will be suggested to one
skilled in the art of organic synthesis.
[0051] Another favored type of heterobifunctional linker is a mixed
active ester-acid chloride such as succinimido-oxycarbonyl-butyryl
chloride. The more reactive acid chloride end of the linker
preferentially acylates amino or hydroxyl groups on the HIV
protease inhibitor to give N-hydroxysuccinimidyl ester linker
adducts directly (see Examples 40 for amprenavir and 8 for
ritonavir).
[0052] Yet another type of terminal activated group useful in the
present invention is an aldehyde group. Aldehyde groups may be
generated by coupling the hydroxyl of the protease inhibitor with
an alkyl or aryl acid substituted at the omega position (the distal
end) with a masked aldehyde group such as an acetal group, such as
1,3-dioxolan-2-yl or 1,3-dioxan-2-yl moieties, in a manner similar
to that described previously, followed by unmasking of the group
using methods well-known in the art. (See, e.g., T. Greene and P.
Wuts, supra). Alternatively, alkyl or aryl carboxylic acids
substituted at the omega position with a protected hydroxy, such
as, for example, an acetoxy moiety, may be used in the coupling
reaction, followed by deprotection of the hydroxy and mild
oxidation with a reagent such as pyridinium dichromate in a
suitable solvent, preferably methylene chloride, to give the
corresponding aldehyde. Other methods of generating
aldehyde-terminated substances will be apparent to those skilled in
the art.
[0053] In certain cases, it is desirable to introduce polarity into
the linker composition to improve solubility or performance
characteristics in the assay of interest. Particularly useful in
this regard are peptide linkers, which offer a wide diversity of
possibilities for optimization and are readily accessible by solid
phase peptide synthesis or by other means.
[0054] Another approach which is particularly useful for generating
acylated HIV protease inhibitors with urethane, urea or thiourea
bonds at the point of attachment to the protease inhibitor is to
react the hydroxyl or amino group of the protease inhibitor with a
linker isocyanate or a linker isothiocyanate. For example, a
carboxyalkylisocyanate with or without a protecting group on the
carboxyl group may be reacted directly with the target hydroxyl
group on a protease inhibitor to give a protected
carboxyalkylurethane or a carboxyarylurethane. The protected
carboxy is preferably an ester which is removed under basic or
acidic conditions. Once freed, the carboxyl group may be activated
to give an active ester for subsequent conjugation or which may be
directly conjugated to polypeptides, polysaccharides and labels.
Alternatively, a preactivated carboxyalkylisocyanate or
carboxyarylisocyanate such as
N-hydroxysuccinimidyl-isocyanatobenzoate may be reacted directly
with protease inhibitor hydroxyl or amine groups to give
linker-acylated protease inhibitor with an active ester
terminus.
[0055] Yet another approach for generating urethane, urea and
thiourea bonds at the point of attachment to the HIV protease
inhibitor is to first treat the target hydroxyl or amine function
with phosgene or thiophosgene to give an oxycarbonyl chloride or
oxythiocarbonyl chloride. The latter intermediates react readily
with amines to give urethanes, ureas or thioureas. Alternative
phosgene equivalents such as carbonyldiimidazole or
disuccinimidyl-carbonate will react similarly.
[0056] Another approach is also useful for generating alkylated
derivatives of HIV protease inhibitors out of the central hydroxyl
group. For example, a protease inhibitor (or properly protected
protease inhibitor) can be reacted with a strong base under
suitable conditions to deprotonate the central hydroxyl group. This
can be reacted with a variety of halo alkyl reagents bearing a
protected carboxylic acid or appropriately protected functionality
such as an amino group protected as the phthalimide to form ether
linkages. The protected carboxyl group is preferably an ester which
is removed under acid or basic conditions. The free carboxylic acid
group may be activated to give an active ester for subsequent
conjugation to polypeptides, polysaccharides and labeling groups.
The free amino groups, after deprotection, can also be extended
using a bi-functional linker with an activated carboxylic acid
group or it can be coupled to a polypeptide by means of a urea
linkage or similar group.
[0057] For generation of amidine adducts, the amine of an HIV
protease inhibitor is reacted with an imidoester, many of which are
known in bioconjugate chemistry as linkers (see Hermanson,
ibid.)
[0058] Alternatively, protease inhibitors derivatized with linkers
bearing an imidate moiety (imido ester; or iminium group) as the
activated group may be obtained by, for example, using a linker
carrying a suitable precursor group, for example, a terminal
nitrile group, when appropriately functionalizing a protease
inhibitor. For example, an O.sup.c-alkylated derivative, or an
O.sup.ar-alkyl derivative, for example, of nelfinavir, or
N.sup.ar-alkyl derivative, for example, of amprenavir, carrying a
terminal nitrile may be synthesized in a manner analogous to that
described above, followed by conversion of the nitrile to an
imidate group by methods known in the art, for example, by
treatment with hydrogen chloride in an alcohol. See also:
Hermanson, ibid; and Jerry March, Advanced Organic Chemistry,
3.sup.rd Ed., John Wiley & Sons, 1985. Other methods of
obtaining imido esters will be suggested to one skilled in the
art.
[0059] In certain protease inhibitors with multiple hydroxy groups,
i.e., indinavir and nelfinavir, or hydroxy groups and amino groups
in the same protease inhibitor, i.e., amprenavir, it may be
necessary to protect one of the groups in order to effect clean
reaction at the other functional group. For example, the indinavir
indane hydroxyl group can be protected with an isopropylidine group
bridging to the adjacent amide nitrogen (see compound 4A, Example
4). For the purposes of this application the indane hydroxyl group
is labeled as HO.sup.in to distinguish it from HO.sup.c. the
isopropylidine protected indinavir HO.sup.in by extension is
designated as O.sup.inN.sup.inisopropylidinyl.
[0060] In another example, nelfinavir aromatic hydroxyl (HO.sup.ar
as used herein) is protected with a t-butyldimethylsily (TBDMS)
group before reaction with the central hydroxyl group, HO.sup.c
(see compound 5A, Example 5). Nelfinavir aromatic hydroxyl is also
protected with a methoxy ethoxymethyl ether (MEM) group (see
compound 5M, Example 31). Many other suitable protecting groups for
alcohols and phenols are known in the art, and the reader is again
referred to Greene and Wuts, ibid. for further examples.
[0061] In other cases, adjustment of the reaction conditions will
allow for selection of one functional group over another, and
protection will not be needed. An example of the latter approach is
the selective acylation of amprenavir hydroxyl group or amino group
(see Examples 3 and 40). Another example is the selective
alkylation of nelfinavir phenolic hydroxyl group (HO.sup.ar) in the
presence of unprotected aliphatic central hydroxyl group (HO.sup.c,
see Example 36).
[0062] From the description above, it is evident that there are
many variations of linker technology which will provide an
activated terminal group A in the HIV protease inhibitor hapten
compositions of interest. Some of these variations will not be
described in more detail. Active esters are the most preferred A
group. Active esters of the invention are reactive with
nucleophiles, especially primary amines, at relatively low
temperatures, generally 0-100.degree. C. in a variety of aqueous
and non-aqueous solvent mixtures. Typical conditions for active
ester couplings with primary or secondary amines to give amides are
reaction in dipolar aprotic solvents such as N,N-dimethylformamide
(DMF) or dimethylsulfoxide (DMSO) with or without added water at
room temperature. A buffer or a tertiary amine is often added to
maintain the basic pH needed to keep the primary amine reactant in
a deprotonated state. Typical active esters are p-nitrophenyl
esters, N-hydroxysulfosuccinimidyl esters, N-hydroxysuccinimidyl
esters, 1-hydroxybenzotriazolyl esters and pentafluorophenyl
esters. Especially preferred are the N-hydroxysuccinimidyl esters
because of their balance of stability, reactivity and the easy
removal of side product N-hydroxysuccinimide. Other active esters
are well known to those skilled in the art and may be used
similarly.
[0063] An alternative activation method for protease inhibitor
linkers terminated with carboxylic acids is in situ preparation of
anhydrides. Particularly preferred are the mixed carbonic
anhydrides formed with alkylchloroformates such as
isobutylchloroformate. These mixed anhydrides are readily formed at
temperature typically ranging from -30.degree. C. to +30.degree.
C., usually -20.degree. C. to 0.degree. C., by the reaction of
carboxylic acid and alkylchloroformate in the presence of a
tertiary amine such as triethylamine or N-methylmorpholine is
solvents such as DMF or tetrahydrofuran (THF) for 5 minutes to 1
hour. The mixed anhydride is then reacted with amino groups on
labels, immunogens and carriers, typically for 5 minutes to 1 hour
at 0.degree. C., to +30.degree. C., to give stable amide
conjugates. Also symmetrical anhydrides may be formed by reaction
of two equivalents of a protease inhibitor linker carboxylic acid
group with carbodiimides such as dicyclohexylcarbodiimides (DDC) or
ethyl-dimethylaminopropyl-carbodiimide (EDAC) in a variety of
solvents such as THF, DMF or dichloromethane. The activation and
coupling to amines is typically carried out under similar
conditions as the mixed anhydride coupling above.
[0064] Yet another activation method for protease inhibitor linkers
terminated with carboxylic acids is conversion to masked thiol
groups, such as thiolactones, by coupling of the carboxylic acid
group with a substance such as homocysteine thiolactone. (See,
e.g., U.S. Pat. No. 5,302,715). The resulting linker-thiolactone
may then be unmasked with mild base to give a terminal thiol which
is then reactive with moieties like maleimido groups or bromoacetyl
or iodoacetyl groups, such as on maleimido- or haloacetyl-modified
peptides, polysaccharides, polyaminoacids, labels and the like, to
give thio-maleimido or thio-acetyl adducts in a similar manner to
that described previously.
[0065] Other useful A groups are isothiocyanate or isocyanate
moieties. Isothiocyanates also react readily with nucleophiles such
as primary amines to give thioureas under conditions similar to the
active ester reaction described above, while isocyanates react
similarly to give ureas. An added advantage of the isothiocyanate
or isocyanate reaction is that it is an addition rather than a
substitution, and therefore there is no side-product to be
concerned about as in the case of active esters. Isocyanate
equivalents, such as, for example, p-nitrophenyloxycarbonylamino
moieties react similarly with primary amines to give ureas.
[0066] Finally, when the target nucleophile is a thiol group,
maleimides are especially preferred because of their rapid
formation of thiol ethers under very mild conditions, i.e., ambient
temperature and neutral pH. Alternatively, active haloalkyl A
groups such as iodoacetyl or bromoacetyl also react readily to form
stable thiol ethers.
[0067] Another object of the invention is to provide novel
immunogens with the following structure:
[I--X--(C.dbd.Y).sub.m-L-Z].sub.n--P where I is an HIV protease
inhibitor radical, X is O or NH, Y is O, S, or NH, m is 0 or 1, L
is a linker consisting of from 0 to 40 carbon atoms arranged in a
straight chain or a branched chain, saturated or unsaturated, and
containing up to two ring structures and 0-20 heteroatoms, with the
proviso that not more than two heteroatoms are linked in sequence,
Z is a moiety chosen from the group consisting of --CONH--,
--NHCO--, --NHCONH--, --NHCSNH--, --OCONH--, --NHOCO--, --S--,
--NH(C.dbd.NH)--, --N.dbd.--, --NH--, and ##STR3##
[0068] P is a polypeptide, a polysaccharide or a synthetic polymer,
and n is a number from 1 to 50 per 50 kilodaltons molecular weight
of P.
[0069] For immunogens, the preferred mode of the invention is to
link from the central hydroxyl group common to all HIV protease
inhibitors by an acylation reaction to form an ester bond (i.e., X
is O, m is 1 and Y is O). A wide variety of linkers L and activated
functionalities A may be used as described above. Thus an activated
hapten of the type I--X--(C.dbd.Y)m-L-A is constructed and reacted
with an immunogenic carrier substance. The immunogenic carrier is
typically a polypeptide or a polysaccharide with a molecular weight
more than 10 kD. Preferred immunogenic carriers are polypeptides
with a molecular weight more than 100 kD. Examples of preferred
carrier substances are keyhole limpet hemocyanin (KLH), Limulus
polyphemus hemocyanin (LPH) and bovine thyroglobulin (BTG). The
reaction between the activated hapten and amino groups on the
carrier is typically carried out in a buffered mixture of water and
a water miscible organic solvent such as DMSO at room temperature
for 0.5 to 5 days. The pH of the buffer is typically between 6 and
8 for active esters, isocyanates, and isothiocyanates, or between 7
and 10 for imidates, and is adjusted according to the known
reactivity of the carrier amino groups and the activated
functionality. In the case where the terminal group A is a
maleimide, the reactive groups on the carrier are thiols. These
thiol groups are either native to the carrier or may be introduced
using thiolating reagents such as 2-IT or SATP. The optimum pH for
the conjugation of maleimides to thiol groups to give thioethers is
typically between 5 and 7. Following the reaction, the immunogen is
dialyzed or subjected to size exclusion chromatography in order to
remove unconjugated hapten and organic solvent.
[0070] An alternative method of obtaining immunogens is to react an
activated hapten wherein A is aldehyde with the amino groups of a
carrier protein or polypeptide to form a Schiff base, followed by
reduction with mild reducing agents such as a cyanoborohydride, to
form a stable amine bond. Variations on this last approach will
also be suggested to those skilled in the art to which the present
invention belongs.
[0071] Yet another object of the present invention is to provide
antibodies to HIV protease inhibitors generated from the immunogens
of the invention. In order to generate antibodies, the immunogen
can be prepared for injection into a host animal by the rehydrating
lyophilized immunogen to form a solution or suspense of the
immunogen. Alternatively, the immunogen may be used as a previously
prepared liquid solution or as a suspension in buffer. The
immunogen solution is then combined with an adjuvant such as
Freund's to form an immunogen mixture. The immunogen may be
administered in a variety of sites, at several doses, one or more
times, over many weeks.
[0072] Preparation of polyclonal antibodies using the immunogens of
the invention may follow any of the conventional techniques known
to those skilled in the art. Commonly, a host animal such as a
rabbit, goat, mouse, guinea pig, or horse is injected with the
immunogen mixture. Further injections are made, with serum being
assessed for antibody titer until it is determined that optimal
titer has been reached. The host animal is then bled to yield a
suitable volume of specific antiserum. Where desirable,
purification steps may be taken to remove undesired material such
as nonspecific antibodies before the antiserum is considered
suitable for use in performing assays.
[0073] Monoclonal antibodies may be obtained by hydridizing mouse
lymphocytes, from mice immunized as described above, and myeloma
cells using a polyethylene glycol method such as the technique
described in Methods in Enzymology 73 (Part B), pp. 3-46, 1981.
[0074] In the case of ELISA assays, protease inhibitor derivatives
coupled to bovine serum albumin (BSA) are preferred for coating of
microtiter plates.
[0075] Another object of the invention is to provide novel labeled
conjugates with the following structure:
[I--X--(C.dbd.Y).sub.m-L-Z].sub.n-Q wherein I is an HIV protease
inhibitor radical, X is O or NH, Y is O, S, or NH, m is 0 or 1, L
is a linker consisting of from 0 to 40 carbon atoms arranged in a
straight chain or a branched chain, saturated or unsaturated, and
containing up to two ring structures and 0-20 heteroatoms, with the
proviso that not more than two heteroatoms are linked in sequence,
Z is a moiety chosen from the group consisting of --CONH--,
--NHCO--, --NHCONH--, --NHCSNH--, --OCONH--, --NHOCO--, --S--,
--NH(C.dbd.NH)--, --N.dbd.N--, --NH--, and ##STR4##
[0076] Q is a non-isotopic label, and n is a number from 1 to 50
per 50 kilodaltons molecular weight of Q.
[0077] For the synthesis of conjugates of HIV protease inhibitors
and non-isotopic labels, similar procedures as for the preparation
of immunogens are employed.
[0078] Alternatively, the activated haptens may be conjugated to
amino or thiol groups on enzymes to prepare labels for ELISA
application. Some examples of useful enzymes for ELISA for which
conjugates are well-known in the art are horseradish peroxidase
(HRP), alkaline phosphatase and .beta.-galactosidase. Conjugates of
proteins including enzymes are typically prepared in a buffered
mixture of water and water miscible organic solvents followed by
dialysis analogous to the conditions for preparation of immunogens.
In the case of latex agglutination assays, conjugates with aminated
dextran carriers having molecular weights between 10 kD and 300 kD,
preferably 40 kD, are especially useful. These conjugates are
prepared in buffered solvent mixtures as above or in an anhydrous
organic solvent such as DMSO containing a tertiary amine such as
triethylamine to promote the reaction. In the case of labels of
small molecular weight, i.e., less than 1 kD, reaction conditions
are adjusted according to the nature of the label. One label which
is particularly preferred is biotin in combination with labeled
avidin or streptavidin. The versatility of (strept)avidin/biotin
systems for non-isotopic detection is well known in the art of
bio-conjugate chemistry (see Hermanson, ibid.). A variety of
enzyme- and fluorophore-labeled conjugates of avidin and
streptavidin are commercially available to detect biotin-labeled
substances in a high affinity interaction. Furthermore, a variety
of biotinylating agents are commercially available to react with
activated functionalities A. For example, a biotin-amine derivative
may be reacted with activated haptens of the invention in which A
is an active ester, isocyanate or isothiocyanate to give biotin
amide, urea and thiourea conjugates respectively. These coupling
reactions are typically carried out in a dipolar aprotic solvent
such as DMF or DMSO containing an organic base such as
triethylamine at room temperature for 0.5 to 5 days. The biotin
conjugates are preferentially isolated by chromatographic methods
such as reversed phase HPLC.
[0079] Other preferred labels are fluorophores such as fluorescein,
rhodamine, TEXAS RED fluorescent dye (Molecular Probes, Inc.),
dansyl, and cyanine dyes, e.g., Cy-5, of which many activated
derivatives are commercially available. Generally, these conjugates
may be prepared similarly as biotin conjugates in a dipolar aprotic
solvent containing a tertiary amine followed by chromatographic
isolation.
[0080] It is also possible to use a reporter group as label which
is indirectly coupled to a detection system. One example is biotin
as described above. Another example is mycophenolic acid
derivatives for inhibition of inosine monophosphate dehydrogenase
as described in PCT publication WO 200101135, published Jan. 4,
2001.
[0081] It will be obvious to those skilled in the art that there
are other possibilities for non-isotopic labels including
electrochemiluminescent labels such as ruthenium bipyridyl
derivatives, chemiluminescent labels such as acridinium esters,
electrochemical mediators, and a variety of microparticles and
nanoparticles which can be used for the invention after suitable
introduction of suitable nucleophilic groups on the label, e.g.,
amines or thiols, for reaction with activated groups A on the HIV
protease inhibitor activated hapten.
SPECIFIC EMBODIMENTS
[0082] In the examples that follow, numbers in boldface type refer
to the corresponding structure shown in the drawings. These
examples are presented for illustration only without any intent to
limit the invention.
O-Acylation of Protease Inhibitors
EXAMPLE 1
Synthesis of O.sup.c-(N-FMOC-aminocaproyl)-ritonavir (1A)
[0083] Ritonavir (1, 0.3605 g), FMOC-aminocaproic acid (0.1944 g,
Advanced ChemTech, Louisville, Ky.), dimethylaminopyridine (0.0672
g, Aldrich Chemical Co., Milwaukee, Wis.) and
dicyclohexylcarbodiimide (0.1238 g, Fluke Chemical Corp.,
Milwaukee, Wis.) were stirred overnight in anhydrous methylene
chloride (5 mL) at room temperature. The mixture was filtered, and
the filtrate was evaporated to dryness under reduced pressure and
directly purified by silica gel (EM Science Cat. No. 9385-9, silica
gel 60, 230-400 mesh ASTM) chromatography under a positive pressure
of nitrogen (3% methanol in chloroform elution) to yield
O.sup.c-(N-FMOC-aminocaproyl)-ritonavir (1A) as a white solid
(0.5023 g, 95%). M+H 1056.2.
EXAMPLE 2
Synthesis of O.sup.c-(N-FMOC-aminocaproyl)-saquinavir (2A)
[0084] O.sup.c-(N-FMOC-aminocaproyl)-saquinavir (2A) was prepared
from saquinavir methanesulfonate (2, 0.1917 g) following the
conditions described in Example 1, except more methylene chloride
(75 mL) was used and the reaction was stirred for 2 days. (A.
Farese-Di Giorgio et. al., Antiviral Chem. and Chemother. 11,
97-110, 2000) (0.2354 g, 94%). M+H 1006.2
EXAMPLE 3
Synthesis of O.sup.c-(N-FMOC-aminocaproyl)-amprenavir (3A)
[0085] O.sup.c-(N-FMOC-aminocaproyl)-amprenavir (3A) was prepared
from amprenavir (3) (0.1517 g) following the conditions described
in Example 1 (0.2248 g; 89%). M+H 841
EXAMPLE 4
Synthesis of
O.sup.c-(N-FMOC-aminocaproyl)-O.sup.in,N.sup.inisopropylidinyl-indinavir
(4B)
[0086] Indinavir sulfate (4, 03559 g), camphorsulfonic acid (0.1401
g, Aldrich Chemical Co.), and magnesium sulfate (4 mg) were
refluxed overnight in dimethoxypropane (5 mL, A. Farese-Di Giorgio
et al., Antiviral Chem. and Chemother, 11, 97-100, 2000). The
mixture was partitioned between methylene chloride and saturate
aqueous sodium bicarbonate. The organic layer was evaporated to
dryness under reduced pressure and directly purified by silica gel
chromatography (4% methanol in chloroform elution) to yield
O.sup.in,N.sup.in-isopropylidyl-indinavir (4A) as a colorless oil
(0.2350 g; 72%). M+H 654.4.
[0087]
O.sup.c-(N-FMOC-aminocaproyl)-O.sup.in,N.sup.in-isopropylidinyl-in-
dinavir (4B) was prepared from
O.sup.in,N.sup.in-isopropylidyl-indinavir (4A, 0.1317 g) following
the conditions described in Example 1 (0.1742 g; 87%). M+H
989.4
EXAMPLE 5
Synthesis of
O.sup.c-(N-FMOC-aminocaproyl)-O.sup.ar-TBDMS-nelfinavir (5B)
[0088] Nelfinavir (5, 0.2839 g) and sodium hydride (18 mg) were
stirred in DMF (3 mL) for 15 minutes. t-Butyldimethylsilyl (TBDMS)
chloride (0.1130 g) was added and the reaction was stirred
overnight. The mixture was evaporated to dryness under reduced
pressure and directly purified by silica gel chromatography (3%
methanol in chloroform elution) to yield O.sup.ar-TBDMS-protected
nelfinavir (5A) as a white foam (0.2857 g; 84%). M+H 682.4.
[0089] O.sup.c-(N-FMOC-aminocaproyl)-O.sup.ar-TBDMS-nelfinavir (5B)
was prepared from O.sup.ar-TBDMS protected nelfinavir (5A, 0.3297
g) following the conditions described in Example 1 (0.3385 g; 69%).
M+H 1017.7
EXAMPLE 6
Synthesis of O.sup.c-(N-FMOC-aminocaproyl)-lopinavir (6A)
[0090] O.sup.c-(N-FMOC-aminocaproyl)-lopinavir (6A) was prepared
from lopinavir (6, 0.712, g) following the conditions described in
Example 1 (0.500 g; 45%). M+H 964.4
EXAMPLE 7
Synthesis of O.sup.c-[3-(4'-carboxyphenyl)-propionyl)]-saquinavir
(2H)
[0091] 3-(4'-Carboxyphenyl)-propionyl-saquinavir (2H) was prepared
from saquinavir methanesulfonate (2, 0.1534 g) and
3-(4'-carboxyphenyl)-propionic acid (0.0485 g, Lancaster Synthesis
Inc., Windham, N.H.) following the conditions described in Example
1 (0.1041 g; 61%). N+H 847.4. Spectral data (.sup.1H-NMR) for the
product was compatible with esterification at the alkyl carboxy
rather than the aryl carboxy.
EXAMPLE 8
Synthesis of O.sup.c-(succinimido-oxycarbonyl-butyryl)-ritonavir
(1G)
[0092] Succinimido-oxycarbonyl-butyryl chloride, i.e.,
5-(2,5-dioxo-1-pyrrolidinyl-oxy)-5-oxo-pentanoyl chloride, is
prepared according to Antonian et al., EP 0 503 454. Ritonavir (1,
0.2163 g) and succinimido-oxycarbonyl-butyryl chloride (0.0817 g)
were stirred overnight in anhydrous DMF (3 mL) at 50.degree. C. The
mixture was evaporated to dryness under reduced pressure and
directly purified by silica gel chromatography (30% tetrahydrofuran
in ethyl acetate elution) to yield
O.sup.c-(succinimido-oxycarbonyl-butyryl)-ritonavir (1G) as a white
solid (0.1220 g, 44%). M+H 931.8.
Deprotection of O-Acylated Protease Inhibitors
EXAMPLE 9
Synthesis of O.sup.c-(aminocaproyl)-ritonavir (1B)
[0093] O.sup.c-(N-FMOC-aminocaproyl)-ritonavir (1A) from Example 1
(0.2113 g) was stirred 1 hour in 10% piperidine in anhydrous
methylene chloride (4 mL) at room temperature. The mixture was
evaporated to dryness under reduced pressure and directly purified
by silica gel chromatography (20-25% methanol in chloroform
gradient elution) to yield O.sup.c-(aminocaproyl)-ritonavir (1B) as
a white solid (0.1525 g, 91%). M+H 834
EXAMPLE 10
Synthesis of O.sup.c-(aminocaproyl)-saquinavir (2B)
[0094] O.sup.c(aminocaproyl)-saquinavir (2B) was prepared from
O-(N-FMOC-aminocaproyl)-saquinavir (2A) of Example 2 (0.7547 g)
following the conditions described in Example 9 (0.5253 g: 89%).
M+H 784.3
EXAMPLE 11
Synthesis of O.sup.c-(aminocaproyl)-amprenavir (3B)
[0095] O.sup.c-(aminocaproyl)-amprenavir (3B) was prepared from
O-(N-FMOC-aminocaproyl-amprenavir (3A) of Example 3 (0.2523 g)
following the conditions described in Example 9 (0.1160 g; 63%).
M+H 619.3
EXAMPLE 12
Synthesis of O.sup.c-(aminocaproyl)-indinavir (4D)
[0096]
O.sup.c-(N-FMOC-aminocaproyl)-O.sup.in,N.sup.in-isopropylidinyl-in-
dinavir (4B) synthesized as in Example 4 (0.5869 g) was stirred
overnight in 50% trifluoroacetic acid in anhydrous methylene
chloride (6 mL) at room temperature to remove the isopropylidinyl
protecting group. The mixture was evaporated to dryness under
reduced pressure, the residue partitioned between methylene
chloride and saturated aqueous sodium bicarbonate. The organic
layer was separated, dried (sodium sulfate), and evaporated to a
light yellow foam (0.5329 g). The foam was dissolved in 5%
piperidine in anhydrous methylene chloride (5 mL) and stirred
overnight. Solvent was removed and the off-white residue purified
by silica gel chromatography (eluting with 5:1 chloroform/methanol
containing 1% concentrated aqueous ammonium hydroxide) to give
O.sup.c-(aminocaproyl)-indinavir (4D) as a colorless oil (0.2866 g:
66% overall). M+H 727.5
[0097] In another run,
O.sup.c-(N-FMOC-aminocaproyl)-O.sup.in,N.sup.in-isopropylidinyl-indinavir
(4B) from Example 4 (0.2301 g) was stirred 2 hours in 50%
trifluoracetic acid in anhydrous methylene chloride (3 mL) at room
temperature to remove the isopropylidinyl protecting group. The
mixture was evaporated to dryness under reduced pressure and
directly purified by silica gel chromatography (5% methanol in
chloroform elution) to yield
O.sup.c-(N-FMOC-aminocaproyl)-indinavir (4C) as a white foam
(0.1603 g, 70%). M+H 949.3.
EXAMPLE 13
Synthesis of O.sup.c-(aminocaproyl)-nelfinavir (5C)
[0098] O.sup.c-(N-FMOC-aminocaproyl)-O.sup.ar-TBDMS-nelfinavir (5B)
from Example 5 (0.1752 g) and tetraethylammonium fluoride (0.2092
g) were stirred for 2 hours in anhydrous THF (10 mL) at room
temperature to remove both the TBDMS and FMOC protecting groups in
one step. The mixture was evaporated to dryness under reduced
pressure, redissolved in methylene chloride, washed with water then
saturated aqueous sodium chloride (brine) and evaporated to
dryness. The residue was purified by silica gel chromatography
(elution with 2% to 10% methanol in chloroform gradient to remove
front-running material, then 100% methanol to elute product) to
yield O.sup.c-(aminocaproyl)-nelfinavir (5C) as a white foam
(0.0711 g, 75%). M+H 681.3
EXAMPLE 14
O.sup.c-(aminocaproyl)-lopinavir (6B)
[0099] O.sup.c-(aminocaproyl)-lopinavir (6B) was prepared from
O.sup.c-(N-FMOC-aminocaproyl)-lopinavir (6A, 0.100 g) of Example 6
following the conditions described in Example 9, except for
purification by silica gel chromatography (10% methanol in
chloroform containing 2% ammonium hydroxide) to yield product 6B
(0.043 g; 56%). M+H 742.2
[0100] Another reaction, 0.300 g of (6A), performed in 10%
piperidine in water instead of methylene chloride gave produce
(0.150 g; 65%) after evaporation and silica gel chromatography as
described above.
Linker Extension of O-Acylated Protease Inhibitors to Generate
Activated Haptens
EXAMPLE 15
Synthesis of
O.sup.c-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-ritonavir
(1C)
[0101] O.sup.c-(aminocaproyl)-ritonavir (1B) from Example 9 (60.9
mg), triethylamine (10 .mu.L), and succinimido-oxycarbonyl butyryl
chloride (Antonian, ibid., 17.5 mg) were stirred 2 hours in
anhydrous THF (6 mL) at 0.degree. C. The mixture was evaporated to
dryness under reduced pressure and directly purified by silica gel
chromatography (30% THF in ethyl acetate elution) to yield
O.sup.c-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-ritonavir as
a white solid (38.8 mg, 51%). M+H 1045.2
EXAMPLE 16
Synthesis of
O.sup.c-[4'(succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-ritonavir
(1D)
[0102] First, disuccinimidyl terephthate was prepared by the method
of Kopia et al., U.S. Pat. No. 5,667,764. to a stirring solution of
disuccinimidyl terephthalate (21.6 mg) and triethylamine (8 .mu.L)
in anhydrous methylene chloride (8 mL) was slowly added a solution
of O.sup.c-(aminocaproyl)-ritonavir (1B) from Example 9 (48.0 mg)
in anhydrous methylene chloride (8 mL). The mixture was stirred 4
hours at room temperature under argon. The mixture was evaporated
to dryness under reduced pressure and directly purified by silica
gel chromatography (30%) THF in ethyl acetate elution) to yield
O.sup.c-[4'-(succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-ritonavir
as a white solid (41.6 mg, 67%). M+H 1079
EXAMPLE 17
Synthesis of
O.sup.c-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-saquinavir
(2C)
[0103]
O.sup.c-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-saquinavir
(2C) was prepared from O.sup.c-(aminocaproyl)-saquinavir (2B) of
Example 10 (52.8 mg) following the conditions described in Example
15, except that a gradient of 5% to 10% methanol in chloroform was
used as the eluent in the silica gel chromatographic purification
(48 mg; 72%). M+H 995.3
EXAMPLE 18
Synthesis of
O.sup.c-[4'-(succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-saquinavir
(2F)
[0104]
O.sup.c-[4'-(succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-saquin-
avir (2F) was prepared from O.sup.c(aminocaproyl)-saquinavir (2B)
of Example 10 (11 mg) following the conditions described in Example
16, but using 2% methanol in chloroform as the eluent in the silica
gel chromatographic purification (12 mg; 83%). M+H 1029.3
EXAMPLE 19
Synthesis of
O.sup.c-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-amprenavir
(3C)
[0105]
O.sup.c-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-amprenavir
(3C) was prepared from O.sup.c-(aminocaproyl)-amprenavir (3B) of
Example 11 (104.0 mg) following the conditions described in Example
15, but with stirring for 6 hours and with the use of 5% methanol
in chloroform as the eluent in the silica gel chromatographic
purification (80 mg; 57%). M+Na 852.4.
EXAMPLE 20
Synthesis of
O.sup.c-[4'-(succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-amprenavir
(3D)
[0106]
O.sup.c-[4'-(succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-ampren-
avir (3D) was prepared from O.sup.c-(aminocaproyl)-amprenavir (3B)
of Example 11 (86.5 mg) following the conditions described in
Example 16, but using 4% methanol in chloroform as the eluent in
the silica gel chromatographic purification (70.3 mg; 58%). M+Na
886.4
EXAMPLE 21
Synthesis of
O.sup.c-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-indinavir
(4E)
[0107]
O.sup.c-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-indinavir
(4E) was prepared from O.sup.c-(aminocaproyl)-indinavir (4D) of
Example 12 (80.0 mg) following the conditions described in Example
15, but with stirring for 6 hours and with the use of a 5% rising
to 17% methanol in chloroform gradient as the eluent in the silica
gel chromatographic purification (37.4 mg; 36%). M+H 938.6
EXAMPLE 22
Synthesis of
O.sup.c-[4'-(succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-indinavir
(4F)
[0108]
O.sup.c-[4'-(succinimido-oxycarbonyl-benzoyl)-aminocaproyl]-indina-
vir (4F) was prepared from O-(aminocaproyl)-indinavir (4D) of
Example 12 (90.0 mg) following the conditions described in Example
16, except that 5% methanol in chloroform was used as the eluent in
the silica gel chromatographic purification (61.8 mg; 51%). M+H
972.6
EXAMPLE 23
Synthesis of
O.sup.c-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-nelfinavir
(5D)
[0109]
O.sup.c-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-nelfinavir
(5D) was prepared from O.sup.c-(aminocaproyl)-nelfinavir (5C) of
Example 13 (60.0 mg) following the conditions described in Example
15, except that a 2% rising to 5% methanol in chloroform gradient
was used as the eluent in the silica gel chromatographic
purification (67.2 mg; 85%). M+H 892.5
EXAMPLE 24
Synthesis of
O.sup.c-[4'-(succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-nelfinavir
(5E)
[0110]
O.sup.c-[4'-(succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-nelfin-
avir (5E) was prepared from O-(aminocaproyl)-nelfinavir (5C) of
Example 13 (61.8 mg) following the conditions described in Example
16, except that 5% methanol in chloroform was used as the eluent in
the silica gel chromatographic purification (43.3 mg; 52%). M-H
926.6
EXAMPLE 25
Synthesis of
O.sup.c-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-lopinavir
(6C)
[0111]
O.sup.c-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-lopinavir
(6C) was prepared from O.sup.c-(aminocaproyl)-lopinavir (6B) of
Example 14 (86 mg) following the conditions described in Example
15, except for purification by silica gel chromatography (5%
methanol in chloroform) (68 mg; 62%). M+H 953.4
EXAMPLE 26
Synthesis of
O.sup.c-[4'-(succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-lopinavir
(6D)
[0112]
O.sup.c-[4'-(succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-lopina-
vir (6D) is prepared from O-(aminocaproyl)-lopinavir (6B) of
Example 14 (80 mg) following the conditions described in Example
16, except for purification by silica gel chromatography (50%
tetrahydrofuran in ethyl acetate) (35 mg; 33%). M+H 987.3
EXAMPLE 27
Synthesis of
O.sup.c-3-[4'-(succinimido-oxycarbonyl)-phenyl-propionyl]-saquinavir
(2I).
[0113]
O.sup.c-3-[4'-(succinimido-oxycarbonyl)-phenyl-propionyl]-saquinav-
ir was prepared from
O.sup.c-[3-(4'-carboxyphenyl)-propionyl)]-saquinavir (2H) of
Example 7 following the conditions described in Example 38 (96%).
M+H 944.5
EXAMPLE 28
Synthesis of
N-maleimidopropionyl-L-glutamyl-(gamma-O.sup.c-saquinavir)-L-alanine
(2P)
[0114] Boc-L-Glu(OBzl)OSu (Bachem), 434 mg (1 mmol) is reacted with
L-Ala-O.sup.1Bu. HCl, 182 mg (1 mmol) in 10 mL DMF containing
triethylamine (202 mg). After stirring for 16 hours at room
temperature, the reaction mixture is rotary evaporated to dryness
and the residue is redissolved in methylene chloride, washed with
water, dried over sodium sulfate and evaporated to dryness. The
residue is redissolved in methanol, 50 mL, and transferred to a
Parr flask. 10% Pd/C catalyst (Aldrich), 50 mg, is added and the
flask is charged with 40 psi hydrogen gas on a Parr shaker. The
mixture is shaken for 2 hours at room temperature or until no
further consumption of hydrogen is noted. The Parr flask is
evacuated and charged with argon gas. The mixture is filtered
through Celite, and the filtrate is rotary evaporated to give crude
Boc-L-Glu-L-Ala-O.sup.1Bu.
[0115] Saquinavir (335 mg), Boc-L-Glu-L-Ala-O.sup.1Bu (187 mg),
dicyclohexylcarbodiimide (103 mg), hydroxybenzotriazole (67.5 mg),
N-ethylmorpholine (57.5 mg), and dimethylaminopyridine (61 mg) were
stirred overnight in anhydrous THF (5 mL). The reaction was diluted
with ethyl acetate and filtered. The filtrate was washed with 2 M
HCl, saturated aqueous sodium bicarbonate, and brine. The organic
layer was evaporated to dryness under reduced pressure and directly
purified by silica gel chromatography (5% methanol) in methylene
chloride elution) to yield
N-t-butyloxycarbonyl-L-glutamyl-(gamma-O.sup.c-saquinavir-L-alanine
t-butyl ester (2N) as an off-white foam (384 mg, 75%). M+H 1027
[0116]
N-t-butyloxycarbonyl-L-glutamyl-(gamma-O.sup.c-saquinavir)-L-alani-
ne t-butyl ester (2N, 3.0 mg) was stirred 1 hour in 50%
trifluoroacetic acid in anhydrous methylene chloride (0.05 mL) and
evaporated to dryness under reduced pressure. The residue was
dissolved in anhydrous methylene chloride (0.1 mL) and stirred 30
minutes with triethylamine (1 .mu.L) and succinimidyl
maleimidopropionate (synthesized by the method of Ede, Tregear and
Haralambidis, Bioconjugate Chem. 5, 373-378, 1994; 0.9 mg). The
mixture was evaporated to dryness under reduced pressure and
directly purified by preparative TLC (25% methanol in chloroform
development) to yield
N-maleimidopropionyl-L-glutamyl-(gamma-O.sup.c-saquinavir)-L-alanin-
e (2P) as a white solid (1.7 mg, 57%). M+H 1022.3
EXAMPLE 29
Synthesis of
N-maleimidopropionyl-L-Ala-L-Glu-(gamma-O.sup.c-saquinavir)
(2Q)
[0117] Boc-L-Ala-L-Glu-O.sup.1Bu is first synthesized using the
procedure for Boc-L-Glu-L-Ala-O.sup.1Bu in Example 29 substituting
L-Glu(OBzl)-O.sup.1Bu (Bachem) for L-Ala-O.sup.1Bu and
Boc-L-Ala-OSu (Bachem) for Boc-L-Glu(OBzl)-OSu.
Boc-L-Ala-L-Glu(gamma-Oc-saquinavir)-O.sup.1Bu (2O) was prepared
from saquinavir (335 mg) and Boc-L-Ala-L-Glu-O.sup.1Bu (187 mg)
following the conditions described in Example 28 for intermediate
(2N) (84%). M+H 1027
[0118] N-maleimidopropionyl-L-Ala-L-Glu-(gamma-O.sup.c-saquinavir)
(2Q) was prepared from
N-t-Boc-L-Ala-L-Glu-(gamma-O.sup.c-saquinavir)-O.sup.1Bu (2O, 3.0
mg) following the conditions described in Example 28 (57%). M+H
1022.3
EXAMPLE 30
Synthesis of O.sup.c-(maleimido-propionyl-aminocaproyl)-saquinavir
(2M)
[0119] O.sup.c-(aminocaproyl)-saquinavir (2B) from Example 10
(0.1098 g), succinimidyl maleimidopropionate (0.048 g), and
triethylamine (20 .mu.L) were stirred 45 minutes in anhydrous
methylene chloride (1.5 mL). The mixture was evaporated to dryness
under reduced pressure and directly purified by silica gel
chromatography (4% methanol in chloroform elution) to yield
O.sup.c-(maleimido-propionyl-aminocaproyl)-saquinavir (2M) as a
colorless oil (0.0647 g, 49%). M+H 935.5
Alkylation of Protease Inhibitors at the Central Hydroxyl
EXAMPLE 31
Synthesis of O.sup.ar-methoxyethoxymethyl-nelfinavir (5M)
[0120] To 28 mg (0.70 mmol) of NaH (60% in oil) was added 1 mL of
hexane. The mixture was allowed to stir for 2-3 minutes under argon
at room temperature and hexane was decanted. To the residue was
added 1 mL of freshly distilled THF and 0.5 mL of anhydrous DMF
followed by 50 mg (0.075 mmol) of nelfinavir mesylate as a solid in
several portions. The mixture was heated at 50.degree. C. for 45
minutes under argon and allowed to cool to room temperature. To the
reaction mixture was added 12.5 .mu.L (0.10 mmol) of
2-methoxyethoxymethyl chloride (MEM chloride) and allowed to stir
at room temperature under argon for 18 hours. To the reaction
mixture was added 1 mL of 50 mM potassium phosphate (pH 7.5) and
the mixture was concentrated under reduced pressure. To the reside
were added 25 mL of CHCl.sub.3 and 15 mL of 50 mM potassium
phosphate (pH 7.5). The organic layer was separated and the aqueous
layer was extracted with additional 4.times.25 mL of CHCl.sub.3.
All the organic extracts were combined, dried (anhydrous
Na.sub.2SO.sub.4) and concentrated. The crude product was purified
by preparative thin layer chromatography (silica gel, EM Science
Cat. No. 5717-7) using 20:1 CHCl.sub.3:MeOH as eluting solvent to
give 43 mg (0.065 mmol, 88%) of
O.sup.ar-methoxyethoxymethyl-nelfinavir (5M) as a white solid. M+H
656.
EXAMPLE 32
Synthesis of O.sup.ar-MEM-O.sup.ccarboxymethyl-nelfinavir (5N)
[0121] To 14 mg (0.35 mmol) of NaH (60% in oil) was added 1 mL of
hexane. The mixture was allowed to stir at room temperature under
argon for 2-3 minutes and hexane was decanted. To the residue 2 mL
of freshly distilled THF and 1 mL of anhydrous DMF was added. A
solution of 23 mg (0.035 mmol) of 5M in 1 mL of freshly distilled
THF was added to the reaction mixture. The reaction mixture was
heated at 50.degree. C. under argon for 1 hour and allowed to cool
to room temperature. To the reaction mixture was added a solution
of 6.5 .mu.L (0.043 mmol) of t-butyl bromoacetate (Aldrich Chemical
Co.) in 500 .mu.L of freshly distilled THF, and the reaction
mixture was allowed to stir at room temperature for 18 hours under
argon. To the reaction mixture was added 1 mL of water and the
mixture was concentrated under reduced pressure. To the residue
were added 20 mL of CHCl.sub.3 and 15 mL of water. The organic
layer was separated and the aqueous layer was extracted with
additional 4.times.20 mL of CHCl.sub.3. All the organic extracts
were combined, dried (Na.sub.2SO.sub.4) and concentrated. The crude
product was purified by preparative thin layer chromatography
(silica gel) using 20% methanol in chloroform as eluent to give 22
mg (0.031 mmol, 88%) of
O.sup.ar-MEM-O.sup.c-carboxymethyl-nelfinavir (5N) as a white solid
M+H 714.
EXAMPLE 33
Synthesis of
O.sup.ar-MEM-O.sup.c-(succinimido-oxycarbonyl-methyl)-nelfinavir
(5O)
[0122] The activated ester (5O) is prepared from (5N) by following
the procedure described in Example 38.
EXAMPLE 34
Synthesis of O.sup.c-(carboxymethyl)-saquinavir (2AA)
[0123] To 65 mg (1.6 mmol) of NaH (60% in oil) was added 2 mL of
hexane. The mixture was allowed to stir at room temperature under
argon for 2-3 minutes and hexane was decanted. To the residue 2 mL
of freshly distilled THF and 1 mL of anhydrous DMF was added.
Saquinavir mesylate (2, 112 mg, 0.14 mmol) was added to the
reaction mixture as a solid in several portions. The reaction
mixture was heated at 50.degree. C. for 1 hour and allowed to cool
to room temperature. To the reaction mixture a solution of 30 .mu.L
(0.203 mmol) of t-butyl bromo acetate in 500 .mu.L of freshly
distilled THF was added and the reaction was allowed to stir at
room temperature under argon for 18 hours. To the reaction mixture
1 mL of water was added and the mixture was concentrated under
reduced pressure. To the residue 20 mL of water was added and the
pH of the reaction was adjusted to 6 with 5% phosphoric acid. To
the reaction mixture 25 mL of CHCl.sub.3 was added. The organic
layer was separated and the aqueous layer was extracted with
additional 4.times.25 mL of CHCl.sub.3. All the organic extracts
were combined, dried (Na.sub.2SO.sub.4) and concentrated. The
residue was purified by flash column chromatography (silica gel)
using 20:1 CHCl.sub.3:MeOH as eluent to give 68 mg (0.093 mmol,
64%) of O.sup.c(carboxy-methyl)-saquinavir (2AA) as a white solid.
M+H 729
EXAMPLE 35
Synthesis of O.sup.c-(succinimido-oxycarbonyl-methyl)-saquinavir
(2BB)
[0124] The activated ester (2BB) is prepared from (2AA) by
following the procedure described in Example 38.
Derivatization of Protease Inhibitors at Positions Other than the
Central Hydroxyl
EXAMPLE 36
Synthesis of ethyl O.sup.ar-carboxypropyl-nelfinavir (5H)
[0125] Nelfinavir (5) phenol (OH.sup.ar) was selectively alkylated
as follows: nelfinavir (62.5 mg) and sodium hydride (2.8 mg) were
stirred 15 minutes in anhydrous DMF (1 mL) at room temperature.
Ethyl 4-bromobutyrate (27.6 mg, Fluka Chemical Corp.) was added and
the mixture was stirred 3 hours at room temperature. The mixture
was evaporated to dryness under reduced pressure and directly
purified by silica gel chromatography (3% methanol in chloroform
elution) to yield ethyl O.sup.ar-carboxypropyl-nelfinavir (5H) as a
white solid (74.7 mg, 95%). M+H 682.4
EXAMPLE 37
Synthesis of O.sup.ar-carboxypropyl-nelfinavir (5I)
[0126] Ethyl O.sup.ar-carboxypropyl-nelfinavir (5H) from Example 31
(0.1440 g) and lithium hydroxide (0.0960 g) were stirred overnight
in 50% aqueous THF (10 mL). The reaction mixture was allowed to
settle (two layers), the organic layer separated and evaporated to
dryness under reduced pressure. A sample was purified by
preparative RP-HPLC (C18; 45% acetonitrile-water containing 0.1%
trifluoroacetic acid) to give the analytical sample. The remainder
was dried to yield O.sup.ar-carboxypropyl-nelfinavir (5I) as a
white solid, shown by .sup.1H-NMR spectroscopy to be fairly clean
material (0.1234 g, 89%) M+H 654.3
EXAMPLE 38
Synthesis of O.sup.ar-(succinimido-oxycarbonyl-propyl)-nelfinavir
(5J)
[0127] O.sup.ar-carboxypropyl-nelfinavir (5I) from Example 37
(0.1210 g, 0.185 mmol), N-hydroxysuccinimide (0.0426 g, 0.37 mmol,
2 mol. equiv.; Aldrich Chemical Co.) and ethyl diethylaminopropyl
carbodiimide hydrochloride (0.0170 g, 0.37 mmol, 2 mol. equiv.;
Sigma Chemical Co) was stirred 2 hours in 10% anhydrous
DMF-methylene chloride (9 mL). The mixture was evaporated to
dryness under reduced pressure and purified by silica gel
chromatography (3% methanol in chloroform elution) followed by
preparative RP-HPLC (C18; 45% acetonitrile-water containing 0.1%
trifluoroacetic acid) to yield
O.sup.ar-(succinimido-oxycarbonyl-propoxy)-nelfinavir (5J, 0.0681
g, 49%). M+H 751.3
[0128] Another reaction performed as above but using 5I (0.2764 g)
followed by silica gel chromatography (3% methanol in chloroform
elution) gave crude but fairly clean product (5J, 0.3526 g) as an
oil.
EXAMPLE 39
Synthesis of
O.sup.ar-(succinimido-oxycarbonyl-propylamino-.sup.co-glycyl-glycyl-propy-
l)-nelfinavir (5K)
[0129]
O.sup.ar-(succinimido-oxycarbonyl-propylamino-.sup.co-glycyl-glycy-
l-propyl)-nelfinavir (5K) was prepared from
O.sup.ar-(succinimido-oxycarbonyl-propoxy)-nelfinavir (5J, 0.32 g)
of Example 38 following the conditions of Example 41 (0.0657 g;
32%). M+H 950.4
EXAMPLE 40
Synthesis of N-(succinimido-oxycarbonyl-butyryl)-amprenavir
(3G)
[0130] Amprenavir (3, 0.1517 g) and succinimido-oxycarbonyl butyryl
chloride (0.0817 g) were stirred overnight in anhydrous DMF (3 mL)
at 50.degree. C. The mixture was evaporated to dryness under
reduced pressure and directly purified by silica gel chromatography
(15% THF in ethyl acetate elution) to yield
N-(succinimido-oxycarbonyl-butyryl)-amprenavir (3G) as a white
solid (0.1395 g, 61%). M+Na 739.2. Spectral data (.sup.1H-NMR) was
compatible with functionalization at the aniline nitrogen.
EXAMPLE 41
Synthesis of
N-(succinimidyl-oxycarbonyl-propylamino-.sup.co-glycyl-glycyl-glutaryl)-a-
mprenavir (3H)
[0131] (a) N-(succinimido-oxycarbonyl-propionyl)-amprenavir (3G)
from Example 40 (131.5 mg) and glycyl-glycyl-4-aminobutyric acid
(43.4 mg, Bachem California Inc., Calif.) were stirred 7 hours in
25% aqueous borate (pH 10) in THF (5 mL). The mixture was
evaporated to dryness under reduced pressure and directly purified
by preparative RP-HPLC (C18; 45% acetonitrile-water containing 0.1%
trifluoroacetic acid) to yield
N-(3-carboxypropylamino-.sup.co-glycyl-glycyl-glutaryl)-amprenavir
as a white solid (98.2 mg, 65%). M-H 817.4
[0132] (b)
N-(4-carboxypropylamino-.sup.coglycyl-glycyl-glutaryl)-amprenavir
(40.9 mg), N-hydroxysuccinimide (11.5 mg), and ethyl
dimethylaminopropyl carbodiimide (19.2 mg) were stirred 5 hours in
20% anhydrous DMF in methylene chloride (2.5 mL). The mixture was
evaporated to dryness under reduced pressure and directly purified
by silica gel chromatography (12% methanol in chloroform elution)
to yield
N-(succinimidyl-oxycarbonyl-propylamino-.sup.co-glycyl-glycyl-glutaryl)-a-
mprenavir (3H) as a white foam (37.9 mg, 83%). M+H 938.4
Urethane Derivatization of Protease Inhibitors
EXAMPLE 42
Synthesis of ethyl O.sup.c-(carboxymethylaminocarbonyl)-saquinavir
(2J)
[0133] Saquinavir methanesulfonate (2, 76.7 mg), ethyl
isocyanatoacetate (23.0 mg, Aldrich Chemical Co.), and
triethylamine (30 .mu.L) were stirred 5 days in anhydrous DMF (1
mL) at 50.degree. C. The mixture was evaporated to dryness under
reduced pressure and directly purified by silica gel chromatography
(5% methanol in chloroform elution) to yield ethyl
O.sup.c-(carboxymethylaminocarbonyl)-saquinavir (2J) as a white
solid (32.3 mg, 40%). M+H 800.4
EXAMPLE 43
Synthesis of O.sup.c-(carboxymethylaminocarbonyl)-saquinavir
(2K)
[0134] Ethyl O.sup.c-(carboxymethylaminocarbonyl)-saquinavir (2J)
from Example 36 (0.1600 g) and lithium hydroxide (0.0960 g) were
stirred 1 hour in 50% aqueous THF (10 mL). The organic layer was
isolated, dried with anhydrous sodium sulfate, and evaporated to
dryness under reduced pressure to yield
O.sup.c-(carboxymethylaminocarbonyl)-saquinavir (2K) as a white
foam (0.1403 g, 91%). M+H 772.3.
EXAMPLE 44
Synthesis of
O.sup.c-(succinimido-oxycarbonyl-methylaminocarbonyl)-saquinavir
(2L)
[0135] O.sup.c-(carboxymethylaminocarbonyl)-saquinavir (2K) of
Example 43 (0.1930 g), succinimidyl tetramethyluronium
tetrafluoroborate (0.1882 g, Aldrich Chemical Co.) and
diisopropylethylamine (0.15 mL) were stirred overnight in anhydrous
THF (10 mL). HPLC-MS showed 80% complete reaction, product peak
(2L) M+H 869.3
EXAMPLE 45
Synthesis of
O.sup.c-[(4-methoxycarbonylphenyl)-methylamino-.sup.co-glycyl-carbonyl]-s-
aquinavir (2W)
[0136] O.sup.c-(carboxymethylaminocarbonyl)-saquinavir (2K) from
Example 43 (0.1929 g) and succinimidyl tetramethyluronium
tetrafluoroborate (0.1505 g) were stirred overnight in anhydrous
tetrahydrofuran (10 mL) containing diisopropylethylamine (0.15 mL)
to give (2L) in situ. Methyl-4-aminomethylbenzoate hydrochloride
(0.1008 g, Aldrich Chemical Co.) and diisopropylethylamine (0.15
mL) were added and stirred 3 hours. The mixture was evaporated to
dryness under reduced pressure and directly purified by silica gel
preparative TLC (50% ethyl acetate and 2% methanol in chloroform)
to yield 2W as a white solid (0.1905 g, 83%). M+H 919.4
EXAMPLE 46
Synthesis of
O.sup.c-[(4-carboxyphenyl)-methylamino-.sup.co-glycyl-carbonyl]-saquinavi-
r (2X)
[0137]
O.sup.c-[(4-methoxycarbonylphenyl)-methylamino-.sup.coglycyl-carbo-
nyl]-saquinavir (2W) from Example 45 (0.232 g) was dissolved in
methanol (10 mL). Lithium hydroxide (0.154 g) and water (2.5 mL)
were added and the reaction was stirred overnight. The reaction
mixture was extracted with methylene chloride, and the organic
layer was dried with anhydrous sodium sulfate and evaporated to
dryness under reduced pressure. The residue was purified by silica
gel chromatography (10% methanol in chloroform containing 2% acetic
acid) to yield 2X as a white solid (0.100 g, 44%). M-H 772.3
EXAMPLE 47
Synthesis of
O.sup.c-[4-(succinimido-oxycarbonyl-phenyl)-methylamino-.sup.coglycyl-car-
bonyl]-saquinavir (2Y)
[0138]
O.sup.c-[(4-succinimido-oxycarbonyl-phenyl)-methylamino-.sup.co-gl-
ycyl-carbonyl]-saquinavir (2Y) was prepared from
O.sup.c-[(4-carboxyphenyl)-methylamino-.sup.co-glycyl-carbonyl]-saquinavi-
r (2X) from Example 46 (85 mg) following the conditions described
in Example 38. M+H 1002.3
EXAMPLE 48
Synthesis of
O.sup.c-[4'-(succinimido-oxycarbonyl)-phenyl-aminocarbonyl]-saquinavir
(2U).
[0139] 50 mg (65.2 .mu.mol) of saquinavir mesylate (2) in 5 mL
freshly distilled DMF and 9 .mu.L (65.2 .mu.mol) triethylamine were
stirred for about 10 minutes at ambient temperature until a clear
solution was obtained. 236.1 mg (1.3 mmol) 4-isocyanatobenzoyl
chloride were added and the mixture turned red instantly. After
standing at room temperature for 2 hours a 1 .mu.L sample of the
solution was injected into analytical HPLC (Vydac C18 column, 300
.ANG., 5 .mu.m, 4.6.times.250 mm; eluent A: Millipore water/0.1%
trifluoroacetic acid, eluent B: acetonitrile/0.1% trifluoracetic
acid; gradient of 0% B in A, rising to 60% B in A, over 60
minutes). The chromatography profile at 226 nm showed nearly
complete derivatization of educt (t.sub.r=45.1 minutes) and
formation of urethane (t.sub.r=48.3 min.) together with some
by-products.
[0140] 22 mg of crude produce was isolated from the mixture by
preparative HPLC (Vydac C18 column, 300 .ANG., 15-20 .mu.m,
50.times.250 mm; eluent A: Millipore water/0.1% trifluoroacetic
acid, eluent B: 80% acetonitrile/0.1% trifluoroacetic acid;
gradient of 0% of B in A, rising to 70% B in A, over 140 minutes).
The appropriate fractions eluting at about 62-65% B were pooled,
lyophilized and subjected to a second chromatography step (modified
gradient: gradient of 0% B in A, rising to 75B in A, over 120
minutes). 10 mg (18%) of slightly red pure product were obtained
from fractions 16 and 17. MALDI-TOF MS of purified carboxylic acid
intermediate 2T. M+H 834, M+Na856
[0141] 10 mg (12 .mu.mol) of
O.sup.c-(4-carboxyphenylaminocarbonyl)-saquinavir (2T) was
dissolved in 500 .mu.L freshly distilled DMF and 1.7 mg (15
.mu.mol) N-hydroxysuccinimide (NHS) and 2.9 mg (15 .mu.mol)
ethyl-dimethylaminopropyl carbodiimide (EDC) were added. The
solution was stirred 5 hours at room temperature under argon, then
again 1.7 mg (15 .mu.mol) NHS and 2.9 mg EDC were added. The
mixture was stirred further and allowed to react 2.5 days at room
temperature. HPLC showed formation of NHS ester 2U, which was not
isolated but used in situ for further reactions.
EXAMPLE 49
Synthesis of ethyl
O.sup.c-(carboxymethylaminocarbonyl)-O.sup.ar-TBDMS-nelfinavir
(5P)
[0142] O.sup.ar-TBDMS-Nelfinavir (5A) of Example 5 (0.102 g), ethyl
isocyanatoacetate (42 .mu.L), and triethylamine (55 .mu.L) were
stirred 3.5 days in anhydrous DMF (2 mL) at 50.degree. C. The
mixture was evaporated to dryness under reduced pressure and
purified first by silica gel chromatography (2% methanol in
chloroform) followed by preparative RP-HPLC (C18) (60%
acetonitrile-water containing 0.1% trifluoroacetic acid/30 minutes
rising to 70% acetonitrile-water containing 0.1% trifluoracetic
acid over 30 minutes) to give recovered starting material 5A
(0.0503 g; 43%) followed by the product 5P (0.0623 g; 45%) after
lyophilization of the appropriate fractions. M+H 811.4
EXAMPLE 50
Synthesis of O.sup.c-(carboxymethylaminocarbonyl)-nelfinavir
(5Q)
[0143] Ethyl
O.sup.c-(carboxymethylaminocarbonyl)-O.sup.ar-TBDMS-nelfinavir (5P)
of Example 49 (56.5 mg) in 3.5 mL of 1:1 tetrahydrofuran-water was
treated with 50 mg of lithium hydroxide monohydrate and the
reaction stirred for 4 hours. The layers were allowed to settle,
the organic layer isolated, dried with sodium sulfate and
evaporated. The residue was largely redissolved in acetonitrile (5
mL), filtered and purified by preparative RP-HPLC (C18) (35%
acetonitrile in water containing 0.1% trifluoroacetic acid) to give
the O.sup.ar-deprotected protect 5Q (24.3 mg; 52%) M+H 669.2
EXAMPLE 51
Synthesis of
O.sup.c-[(3-carboxypropyl)amino-.sup.co-glycyl-glycyl-glycyl-carbonyl)-ne-
lfinavir (5R)
[0144] O.sup.c-(carboxymethylaminocarbonyl)-nelfinavir (5Q) of
Example 50 (20.4 mg), succinimidyl tetramethyluronium
tetrafluoroborate (12.0 mg), and diisopropylethylamine (8 .mu.L)
were stirred overnight in anhydrous THF (1.5 mL) for 5.5 hours.
LC/MS showed the presence of the corresponding NHS ester together
with some starting material. Glycyl-glycyl-4-aminobutyric acid (7.0
mg) was added followed by 50 mM phosphate buffer (pH 10) until a
clear solution was obtained. After stirring overnight the reaction
was concentrated to .about.1 mL, the milky residue diluted with
acetonitrile and sonicated to give a clear solution which was
purified by preparative RP-HPLC (C18)(30% acetonitrile in water for
30 minutes, 30% to 45% acetonitrile in water over 30 minutes, 45%
to 90% acetonitrile in water over 30 minutes, all containing 0.1%
trifluoroacetic acid) to give the product 5R from the main peak
after lyophilization. (12.6 mg, 48%) M+H 868.4
EXAMPLE 52
Synthesis of
O.sup.c-(succinimido-oxycarbonyl-propylamino-.sup.co-glycyl-glycyl-glycyl-
-carbonyl)-nelfinavir (5S)
[0145]
O.sup.c-(Succinimido-oxycarbonyl-propylamino-.sup.co-glycyl-glycyl-
-glycyl-carbonyl)-nelfinavir (5S) is synthesized from
O.sup.c-[(3-carboxypropyl)amino-.sup.co-glycyl-glycyl-glycyl-carbonyl)-ne-
lfinavir (5R) of Example 51 following the conditions of Example
41(b). M+H 964.4
Conjugation of Protease Inhibitors to Small Molecular Weight
Labels
EXAMPLE 53
Synthesis of
O.sup.c-(fluoresceinyl-glycinamidyl-butyryl-aminocaproyl)-saquinavir
(2V)
[0146]
O.sup.c-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-saquinavir
(2C) from Example 17 (10.0 mg) and fluoresceinyl glycinamide (5.0
mg, Molecular Probes, OR) is stirred overnight in 3%
triethylamine-pyridine (0.1 mL). The mixture was evaporated to
dryness under reduced pressure and directly purified by preparative
RP-HPLC (C18; 50% acetonitrile-water containing 0.1% trifluoracetic
acid) to yield
O.sup.c-(fluoresceinyl-glycinamidyl-butyryl-aminocaproyl)-saquinavir
(2V; 7.6 mg, 75%). M+H 1284.6
EXAMPLE 54
Synthesis of O.sup.c-(fluoresceinyl-glycinamidyl-butyryl)-ritonavir
(1I)
[0147] O.sup.c-(fluoresceinyl-glycinamidyl-butyryl)-ritonavir (1I)
was prepared from
O.sup.c-(succinimido-oxycarbonyl-butyryl)-ritonavir (1G) of Example
8 following the conditions described in Example 53 (8.4 mg; 69%).
M+H 1221.4
EXAMPLE 55
Synthesis of
O.sup.c-[4'-(1-biotinyl-amino-3,6-dioxa-octylamino)-terephthaloyl-aminoca-
proyl]-indinavir (4I)
[0148] 5.0 mg of
O.sup.c-[4'-(succinimido-oxycarbonyl)-benzoyl-aminocaproyl-indinavir
(4F) from Example 22 were dissolved in 5.0 mL freshly distilled
DMF. 13.6 mg of 1-biotinylamino-3,6-dioxa-octaneamine
(biotin-DADOO, Roche Applied Science, Cat. No. 1112074-103) and 5.6
.mu.L triethylamine were added, and the resulting clear solution
was stirred under argon overnight. HPLC control showed complete
reaction after 20 hours. DMF was removed on a rotavapor (high
vacuum, much less than 1 Torr pressure, water bath 30.degree. C.).
The remaining oily product was dissolved in 0.5 mL DMSO, filtered
and injected into a preparative HPLC system (Vydac C18 column, 300
.ANG., 15-20 .mu.m, 50.times.250 mm; eluent A: Millipore water/0.1%
trifluoracetic acid, eluent B: 80% acetonitrile/0.1%
trifluoroacetic acid; gradient of 0% B in A rising to 70% B in A
over 140 minutes), the appropriate fractions containing pure
product were pooled and lyophilized. Structure was confirmed by
MALDI-TOF-MS (M=1231). Yield: 3.5 mg (2.84 .mu.mol, 55% of
theoretical yield)
EXAMPLE 56
Synthesis of
O.sup.c-[4'-(1-biotinyl-amino-3,6-dioxa-octylamino)-terephthaloyl-aminoca-
proyl]-amprenavir (3J)
[0149] An amprenavir-biotin conjugate (3J,
O.sup.c-[4'-(1-biotinyl-amino-3,6-dioxa-octylamino)-terephthaloyl-aminoca-
proyl]-amprenavir) was synthesized using the activated hapten
O.sup.c-[4'-(succinimido-oxycarbonyl)-benzoyl-aminocaproyl-amprenavir
(3D) from Example 20 in the procedure described in Example 55
above. Structure was confirmed by MALDI-TOF-MS (M=1123). Yield: 1.8
mg (1.60 .mu.mol, 31% of theoretical yield) ##STR5##
EXAMPLE 57
Synthesis of
O.sup.c-[4'-(1-biotinyl-amino-3,6-dioxa-octylamino)-terephthaloyl-aminoca-
proyl]-lopinavir (6G)
[0150] A lopinavir-biotin conjugate (6G,
O.sup.c-[4'-(1-biotinyl-amino-3,6-dioxa-octylamino)-terephthaloyl-aminoca-
proyl]-lopinavir) was synthesized using the activated hapten
O.sup.c-[4'-(succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-lopinavir
(6D) from Example 26 in the procedure described in Example 55
above. Structure was confirmed by MALDI-TOF-MS (M=1246). Yield: 0.6
mg (0.48 .mu.mol, 10% of theoretical yield) ##STR6##
EXAMPLE 58
Synthesis of
O.sup.c-[4'-(1-biotinyl-amino-3,6-dioxa-octylamino)-terephthaloyl-aminoca-
proyl]-ritonavir (1J)
[0151] A ritonavir-biotin conjugate (1J,
O.sup.c-[4'-(1-biotinyl-amino-3,6-dioxa-octylamino)-terephthaloyl-aminoca-
proyl]-ritonavir) was synthesized using the activated hapten
O.sup.c-[4'-(succinimido-oxycarbonyl)-benzoyl-aminocaproyl-ritonavir
(1D) from Example 22 in the procedure described in Example 55
above. Structure was confirmed by MALDI-TOF-MS (M=1338). Yield: 5.4
mg (4.03 .mu.mol, 87% of theoretical yield) ##STR7##
Conjugation of Protease Inhibitors to Proteins
EXAMPLE 59
Synthesis of conjugate of 2S of
N-maleimidopropionyl-L-alanyl-L-(gamma-O.sup.c-saquinavir)-glutamic
acid with 2-IT modified bovine serum albumin
[0152] Bovine serum albumin (30 mg) and 2-iminothiolane (2-IT)
hydrochloride (0.5 mg, Pierce Biotechnology Inc., Ill.) were
allowed to stand 1 hour in the dark in 10 mM potassium phosphate,
0.1 M sodium chloride, 1 mM EDTA, pH 8.0 (3 mL). The mixture was
desalted by gel filtration on a PD-10 column (Amersham-Pharmacia,
N.J.) eluting with 10 mM potassium phosphate, 0.1 M sodium
chloride, 1 mM EDTA, pH 8.0 The appropriate fractions were
collected, adjusted to pH 7.2, and
N-maleimidopropionyl-L-alanyl-L-(gamma-O.sup.c-saquinavir)-glutamic
acid (2Q) from Example 29 (1 mg) dissolved in methanol (0.2 mL) was
added. The mixture was allowed to stand 2 hours in the dark,
quenched with ethyl maleimide (0.5 mg, Sigma Chemical Co.), and
desalted by gel filtration on a PD-10 column (10 mM potassium
phosphate, 0.1 M sodium chloride, 1 mM EDTA, pH 8.0, elution).
Protein quantification by Coomassie Blue protein assay (Bio-Rad
Laboratories, Calif.; modified Bradford protein assay) showed
quantitative recover of protein at 4.3 mg/mL. UV difference
spectroscopy showed the ratio of hapten to BSA to be 1:1.
EXAMPLE 60
Synthesis of conjugate 2R of
N-maleimidopropionyl-L-glutamyl-(gamma-O.sup.c-saquinavir)-L-alanine
with SATP-modified KLH
[0153] Keyhole limpet hemocyanin (CALBIOCHEM, CN Biosciences, San
Diego, Calif.; slurry in 65% ammonium sulfate) was dialyzed
exhaustively against 50 mM potassium phosphate buffer pH 7.5 (>8
buffer changes; dilution factor more than 10.sup.10) at room
temperature (2-3 buffer changes) then at 4.degree. C. The retentate
was lyophilized almost to dryness, then reconstituted with an
appropriate volume of 50 mM phosphate to give purified KLH at a
relatively high concentration. Unused portions of the purified KLH
were frozen and stored at -20.degree. C. until needed.
[0154] Purified keyhole limpet hemocyanin (20 mg) and
N-succinimidyl S-acetylthiopropionate (SATP, 10 mg, Pierce
Biotechnology, Inc.) were allowed to stand 1 hour in 50 mM
potassium phosphate, 1 mM EDTA, pH 7.5, and desalted by gel
filtration on a PD-10 column (Amersham-Pharmacia) eluting with 50
mM potassium phosphate, 1 mM EDTA, pH 7.5. Derivatized protein (10
mg) was allowed to stand 2 hours in the dark in 50 mM potassium
phosphate, 2.5 mM EDTA, 50 mM hydroxylamine hydrochloride, pH 7.5,
and desalted by gel filtration (50 mM potassium phosphate, 5 mM
EDTA, pH 7.2 elution).
N-maleimidopropionyl-L-glutamyl-(gamma-O.sup.c-saquinavir)-L-alanine
(2P) from Example 28 (6 mg) dissolved in DMSO (1 mL) was added and
the reaction was stirred 16 hours. Ethyl maleimide (0.5 mg) was
added and the reaction was stirred 8 hours. The mixture was
sequentially dialyzed against 30%, 20%, 10% and 0% DMSO in 50 mM
potassium phosphate, pH 7.5 at room temperature, followed by
dialysis against 50 mM potassium phosphate, pH 7.5 at 4.degree. C.
Protein quantification by Coomassie Blue showed quantitative
recovery of protein at 1.6 mg/mL. UV difference spectroscopy showed
up to 25% lysine substitution by hapten.
EXAMPLE 61
Synthesis of
O.sup.c-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-saquinavir
conjugate with BSA (2D)
[0155] Bovine serum albumin (30 mg) and
O.sup.c-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-saquinavir
(2C) from Example 17 (1 mg) were stirred 2 days in 30% DMSO in 50
mM potassium phosphate, pH 7.5 (1.5 mL), at room temperature. The
mixture was sequentially dialyzed against 30%, 20%, 10% and 0% DMSO
in 1 liter 50 mM potassium phosphate, pH7.5, at room temperature,
followed by dialysis against 1 liter 50 mM potassium phosphate, pH
7.5, at 4.degree. C. Protein quantification by Coomassie Blue
showed quantitative recovery of protein at 10.4 mg/mL. UV
difference spectroscopy showed the ratio of hapten to BSA to be
1:1.
EXAMPLE 62
Synthesis of
O.sup.c-[(4'-succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-saquinavir
conjugate with BSA (2G)
[0156]
O.sup.c-[(4'-succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-saquin-
avir BSA conjugate was prepared from bovine serum albumin (30 mg)
and
O.sup.c-[(4'-succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-saquinavir
(2F) from Example 18 (1 mg) following the conditions described in
Example 61. Protein quantification by Coomassie Blue showed
quantitative recovery of protein at 10.4 mg/mL. UV difference
spectroscopy showed the ratio of hapten to BSA to be 1:1.
EXAMPLE 63
Synthesis of
O.sup.c-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-saquinavir
conjugate with KLH (2E)
[0157]
O.sup.c-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-saquinavir
KLH conjugate was prepared from purified keyhole limpet hemocyanin
(30 mg) and
O.sup.c(succinimido-oxycarbonyl-butyryl-aminocaproyl)-saquinavir
(2C) from Example 17 (10 mg) following the general conditions
described in Example 61. Protein quantification by Coomassie Blue
showed quantitative recovery of protein at 10.9 mg/mL. Amine
quantification by trinitrobenzenesulfonic acid (TNBS, Sigma
Chemical Co.) colorimetric assay showed 60% lysine
modification.
EXAMPLE 64
Synthesis of
O.sup.c-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-ritonavir
conjugate with LPH (1E)
[0158]
O.sup.c-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-ritonavir
LPH conjugate was prepared from horseshoe crab hemocyanin (LPH, 30
mg; Sigma Chemical Co.) and
O.sup.c-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-ritonavir
(1C) from Example 15 (7 mg) following the general conditions
described in Example 61. Protein quantification by Coomassie Blue
showed quantitative recovery of protein at 7.9 mg/ml. Amine
quantification by TNBS colorimetric assay showed 26% lysine
modification.
EXAMPLE 65
Synthesis of
O.sup.c-[4'-(succinimido-oxycarbonyl-benzoyl-aminocaproyl]-ritonavir
conjugate with BSA (1F)
[0159]
O.sup.c-[4'-(succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-ritona-
vir BSA conjugate was prepared from bovine serum albumin (30 mg)
and
O.sup.c-[4'-(succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-ritonavir
(1D) from Example 16 (1 mg) following the general conditions
described in Example 61. Protein quantification by Coomassie Blue
showed quantitative recovery of protein at 10.3 mg/mL. TNBS
colorimetric assay showed the ratio of hapten to BSA to be 2:1.
EXAMPLE 66
Synthesis of O.sup.c-(succinimido-oxycarbonyl-butyryl)-ritonavir
conjugate with KLH (1H)
[0160] O.sup.c-(succinimido-oxycarbonyl-butyryl)-ritonavir KLH
conjugate was prepared from purified keyhole limpet hemocyanin (30
mg) and O.sup.c-(succinimido-oxycarbonyl-butyryl)-ritonavir (1G)
from Example 8 (10 mg) following the general conditions described
in Example 61. Protein quantification by Coomassie Blue showed
quantitative recovery of proteins at 11.9 mg/mL. Amine
quantification by TNBS colorimetric assay showed 60% lysine
modification.
EXAMPLE 67
Synthesis of
O.sup.c-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-amprenavir
conjugate with KLH (3E)
[0161]
O.sup.c-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-amprenavir
KLH conjugate was prepared from purified keyhole limpet hemocyanin
(30 mg) and
O.sup.c-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-amprenavir
(3C) from Example 19 (8 mg) following the general conditions
described in Example 61. Protein quantification by Coomassie Blue
showed quantitative recovery of protein at 6.8 mg/mL. Amine
quantification by TNBS colorimetric assay showed 20% lysine
modification.
EXAMPLE 68
Synthesis of
O.sup.c-[(succinimido-oxycarbonyl)-butyryl-aminocaproyl]-indinavir
conjugate with KLH (4G)
[0162]
O.sup.c-[(4'-succinimido-oxycarbonyl-butyryl)-aminocaproyl]-indina-
vir KLH conjugate was prepared from purified keyhole limpet
hemocyanin (30 mg) and
O.sup.c-[(-succinimido-oxycarbonyl-butyryl)-aminocaproyl]-indinav-
ir (4E) from Example 21 (9 mg) following the general conditions
described in Example 61. Protein quantification by Coomassie Blue
showed quantitative recovery of protein at 7.4 mg/mL. Amine
quantification by TNBS colorimetric assay showed 20% lysine
modification.
EXAMPLE 69
Synthesis of
O.sup.c-[4'-(succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-amprenavir
conjugate with BSA (3F)
[0163]
O.sup.c-[4'-(succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-ampren-
avir BSA conjugate was prepared from bovine serum albumin (30 mg)
and
O.sup.c-[(4'-succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-amprenavir
(3D) from Example 20 (1 mg) following the general conditions
described in Example 61. Protein quantification by Coomassie Blue
showed quantitative recovery of protein at 11.5 mg/mL. UV
difference spectroscopy showed the ratio of hapten to BSA to be
2:1.
EXAMPLE 70
Synthesis of
O.sup.c-[(4'-succinimido-oxycarbonyl-benzoyl)-aminocaproyl]-indinavir
conjugate with BSA (4H)
[0164]
O.sup.c-[4'-(succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-indina-
vir BSA conjugate was prepared from bovine serum albumin (30 mg)
and
O.sup.c-[4'-(succinimido-oxycarbonyl-benzoyl)-aminocaproyl]-indinavir
(4F) from Example 22 (1 mg) following the general conditions
described in Example 61. Protein quantification by Coomassie Blue
showed quantitative recovery of protein at 10.8 mg/mL. UV
difference spectroscopy showed the ratio of hapten to BSA to be
2:1.
EXAMPLE 71
Synthesis of
O.sup.c-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-nelfinavir
conjugate with KLH (5F)
[0165]
O.sup.c-[(succinimido-oxycarbonyl-butyryl-aminocaproyl)-nelfinavir
KLH conjugate was prepared from purified keyhole limpet hemocyanin
(30 mg) and
O.sup.c-[(succinimido-oxycarbonyl-butyryl-aminocaproyl)-nelfinavi-
r (5D) from Example 23 (9 mg) following the general conditions
described in Example 61. Protein quantification by Coomassie Blue
showed quantitative recovery of protein at 9.7 mg/mL. Amine
quantification by TNBS colorimetric assay showed 36% lysine
modification.
EXAMPLE 72
Synthesis of
O.sup.c-(4'-[succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-nelfinavir
conjugate with BSA (5G).
[0166]
O.sup.c-[4'-(succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-nelfin-
avir BSA conjugate was prepared from bovine serum albumin (30 mg)
and
O.sup.c-[(4'-succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-nelfinavir
(5E) from Example 24 (1 mg) following the general conditions
described in Example 61. Protein quantification by Coomassie Blue
showed quantitative recovery of protein at 10.9 mg/mL. UV
difference spectroscopy showed the ratio of hapten to BSA to be
2:1.
EXAMPLE 73
Synthesis of
O.sup.ar-(succinimido-oxycarbonyl-propylamino-.sup.co-glycyl-glycyl-propo-
xy)-nelfinavir conjugate with KLH (5L)
[0167]
O.sup.ar-(succinimido-oxycarbonyl-propylamino-.sup.co-glycyl-glycy-
l-propoxy)-nelfinavir KLH conjugate was prepared from keyhole
limpet hemocyanin (30 mg) and
O.sup.ar-(succinimido-oxycarbonyl-propylamino-.sup.co-glycyl-glycyl-propo-
xy)-nelfinavir (5K, 10 mg) of Example 39 following the general
conditions described in Example 61. Protein quantification by
Coomassie Blue showed quantitative recovery of protein at 14.6
mg/mL. Amine quantification by TNBS colorimetric assay showed 57%
lysine modification.
EXAMPLE 74
Synthesis of
O.sup.c-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-lopinavir
conjugate with KLH (6F)
[0168]
O.sup.c-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-lopinavir
KLH conjugate was prepared from keyhole limpet hemocyanin (40 mg)
and
O.sup.c-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-lopinavir
(6C) from Example 25 (16 mg) in 40% dimethyl sulfoxide in 50 mM
potassium phosphate, pH 7.5 (3.4 mL), in a similar manner to
Example 61, followed by sequential dialysis against 40%, 30%, 20%,
10% and 0% DMSO in 50 mM potassium phosphate, pH 7.5 at room
temperature, followed by dialysis against 50 mM potassium
phosphate, pH 7.5 at 4.degree. C. Protein quantification by
Coomassie Blue showed quantitative recovery of protein at 6.9
mg/mL. Amine quantification showed 38% lysine modification.
EXAMPLE 75
Synthesis of
O.sup.c-[4'-(succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-lopinavir
conjugate with BSA (6E)
[0169]
O.sup.c-[4'-(succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-lopina-
vir BSA conjugate was prepared from bovine serum albumin (93 mg)
and
O.sup.c-[4'-(succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-lopinavir
(6D) from Example 26 (3 mg) following the general conditions
described in Example 61. Protein quantification by Coomassie Blue
showed quantitative recovery of protein at 11.1 mg/mL. UV
difference spectroscopy showed the ratio of hapten to BSA to be
2:1.
EXAMPLE 76
Synthesis of
N-(succinimidyl-oxycarbonyl-propylamino-.sup.co-glycyl-glycyl-glutaryl)-a-
mprenavir conjugate with KLH (3I)
[0170]
N-(succinimidyl-oxycarbonyl-propylamino-.sup.co-glycyl-glycyl-glut-
aryl)-amprenavir KLH conjugate was prepared from purified keyhole
limpet hemocyanin (30 mg) and
N-(succinimidyl-oxycarbonyl-propylamino-.sup.co-glycyl-glycyl-glutaryl)-a-
mprenavir (3H) from Example 41 (9 mg) following the general
conditions described in Example 61. Protein quantification by
Coomassie Blue showed quantitative recovery of protein at 8.7
mg/mL. Amine quantification by TNBS colorimetric assay showed 40%
lysine modification.
Development of Antibodies to Protease Inhibitors
EXAMPLE 77
Antibody response to saquinavir KLH immunogen
[0171] Saquinavir-KLH (2E) was used to immunize mice of both the
C57 Black and Swiss Webster strains. The doses and routes of
immunization were the same for both strains of mice. The
immunization schedule is given in Table 1. TABLE-US-00001 TABLE 1
Immunization Schedule Date Dose/Route Adjuvant Day 0 75 .mu.g FP
& IP Complete Freund's Day 25 100 .mu.g FP & IP Incomplete
Freund's Day 53 100 .mu.g IP Incomplete Freund's FP = Foot pad; IP
= Intraperitoneal
[0172] Blood samples were taken from each mouse by retro-orbital
bleeds thirteen days after the last immunization. The blood was
immediately centrifuged and the serum drawn off and stored in a
micro-vial after being diluted 10 times with phosphate buffered
saline with 0.02% thimerosal preservative.
[0173] The next day an ELISA was conducted to establish the titers
of antibody present. The ELISA consisted of microtiter plates
coated with different saquinavir-BSA conjugates, all at 1 .mu.g/mL
in biocarbonate buffer (0.1 M, pH 9.6, 100 .mu.L/well, 4.degree. C.
overnight). After coating the plates were emptied and 200 .mu.L of
post coat solution consisting of Tris buffer, 1% gelatin
hydrolysate, 2% sucrose and 0.17% TWEEN-20 emulsifying agent (ICI
Americas, Inc.) was added. This was incubated at 37.degree. C. for
1 hour to block any uncoated regions of the wells. The sera were
tested by carrying out a pre-dilution to 1000, then serial
dilutions down each column at a 1:3 ratio. The volume of diluted
serum in each well was 100 .mu.l, which was allowed to incubate at
37.degree. C. in a humidified container for 1 hour and 20 minutes.
The plates were then washed with phosphate buffered saline, and 100
.mu.L of goat anti-mouse IgG-HRP (horseradish peroxidase) conjugate
(Zymed, Inc., diluted 1:5000 in PBS) was added to each well. The
plates were again incubated for 2 hours under the same conditions,
then washed again. Development consisted of addition of 100 .mu.L
of K-BLUE SUBSTRATE (Neogen Corporation) to each well, and
incubation at room temperature in the dark for 30 minutes.
Development was stopped by the addition of 100 .mu.L of 1N HCl to
each well. The optical densities of each plate were read with a
microplate reader at 450 nm and captured to a computer.
[0174] The serum titers when examined with respect to
saquinavir-BSA conjugate 2D with the same linker structure and
position as the immunogen were substantially higher than for the
other conjugates, indicating there was some linker recognition in
the polyclonal antibody population. As illustrated in FIG. 14,
titers decreased as the structure and position of the linker
differed from the immunogen. FIG. 14 is a graph of titers of mouse
#333 serum using saquinavir conjugates 2G, 2W, 2D and 2S. (Note:
Preparation of conjugate 2W is described in previously cited
copending application EP 1 207 394 A2 as Example V.) The optical
density at 450 nm read at 30 minutes is plotted on the Y-axis and
serum dilutions are plotted on the X-axis.
[0175] From these analyses, it was clear that the saquinavir-KLH
conjugate was suitable for use in raising polyclonal antibodies and
could also therefore be used for the development of monoclonal
antibodies.
EXAMPLE 78
Development of monoclonal antibodies to saquinavir
[0176] Female Swiss-Webster mice, at least 3 months of age, were
used for immunizations. The KLH immunogen 2E was emulsified in 50%
Complete Freund's Adjuvant, 50% saline, at a final concentration of
0.75 mg/ml. Each mouse was injected twice with 10 .mu.L
subcutaneously in the rear thigh region, and with 90 .mu.l in the
peritoneal space. Twenty five days latter, similar injections were
given in the same routes, using Freund's Incomplete Adjuvant and a
concentration of 1 mg/ml, total volume per mouse was 0.1 ml.
Thirteen days later each mouse was bled retro-orbitally to obtain a
serum sample for analysis. A third immunization was administered 49
days later, identical to the second formulation. The mouse selected
for use in fusion was given a booster immunization thirteen days
later, identical to that of the second and third injections. Four
days later, the mouse was used for cell fusion to develop
monoclonal antibody secreting hybridomas.
[0177] The conjugate 2D featuring the linker homologous to the
immunogen showed the greatest efficacy of binding of serum
antibodies. The binding was directly related to the degree of
homology of the conjugate linker to that of the immunogen. Efficacy
of binding was found to be, from the strongest to the weakest,
2D>2G>2S>2W. Based on the observations made in analyzing
the serum antibodies above, it was decided to devise a strategy for
screening of monoclonal antibodies in the fusion phase of the work
in which the effect of linker homology could be distinguished and
only those clones showing little or no linker preference would be
selected.
[0178] The strategy featured two tactics. First, antibody binding
would be tested using a linker shown by the above analysis to
provide less than maximal binding of the sera antibodies. Second, a
second well coated with the same conjugate, in which 400 ng/mL of
free drug was included, would be employed to estimate the
competitive effect of the drug on binding. The result would allow
the selection of only those monoclonals which competitively bound
the free drug (i.e., without any linker attached).
[0179] The mouse selected for fusion was killed via exsanguination.
The popliteal, inguinal, subclavial and deep inguinal lymph nodes
and spleen were harvested and pooled. The tissues were ground
between two sterile glass slides to release the lymphocytes.
One-half of the resulting lymphocyte suspension was used to fuse
with the F0 myeloma cell line (ATCC CRL 1646), the remaining half
was fused with the P3 myeloma (both myelomas were from ATCC).
[0180] Fusion consisted with adding myeloma cells (1/5 the number
of lymphocytes) to the lymphocytes, washing via centrifugation,
resuspension in serum-free warm Iscove's modified Dulbecco's media
(IMDM), and re-centrifugation. The centrifuge tubes containing the
resulting pellets were gently tapped to loosen the cells, then 1 mL
of warmed PEG/DMSO solution (Sigma Chemicals) was slowly added with
gentle mixing. The cells were kept warm for 1.5 minutes, after
which pre-warmed serum-free IMDM was added at the following rates:
1 ml/min, 2 ml/min, 4 ml/min, 10 ml/min, then the tube was filled
to 50 ml, sealed and incubated for 15 minutes. The cell suspensions
were centrifuged, the supernatant decanted, and IMDM containing 10%
fetal calf serum was added. The cells were centrifuged once again,
and resuspended in complete cloning medium. This consisted of IMDM,
10% FCS, 10% Condimed H1 (Roche Molecular Systems), 4 mM Glutamine,
50 .mu.M 2-mercaptoethanol, 40 .mu.M ethanolamine, pen/strep
antibiotics. The cells were suspended at a density of
4.times.10.sup.5 lymphocytes/ml, distributed 100 .mu.L/well into
sterile 96-well sterile microculture plates and incubated at
37.degree. C. in 5% CO.sub.2 for 24 hours. The next day, 100 .mu.L
of hypoxanthine-methotrexate-thymidine (HMT) selective medium
(cloning medium+1:25 HMT supplement from Sigma Chemicals) was
added. On the 6th day of incubation, approximately 150 .mu.L of
media was drawn from each well using a sterile 8-place manifold
connected to a light vacuum source. One hundred fifty microliters
of hypoxanthine-thymidine (HT) media was then added. This consists
of cloning medium+1:50 HT supplement (Sigma Chemicals). The plates
were returned to the incubator and inspected daily for signs of
growth. When growth was judged sufficient, wells were screened for
antibody production via ELISA.
[0181] Microplates were coated with 100 .mu.L saquinavir-BSA
conjugate at 1 .mu.g/mL in 0.1 M carbonate buffer, pH 9.5 for 1
hour at 37.degree. C. (humidified). The plates were then emptied
and filled with a post-coat solution. The plates were incubated for
an additional 1 hour at 37.degree. C. (humidified) after which they
were washed with phosphate-buffered saline containing 0.1% TWEEN
20. The plates were then filled with a 2% sucrose solution in 0.15
M Tris, pH 7.2-7.4 briefly, then emptied and allowed to air dry at
room temperature. When dried, the plates were packed in zip-lock
bags containing several desiccant pillows, sealed and stored at
4.degree. C. until use.
[0182] When the growing clones were judged ready for testing, 25
.mu.L of supernatant from the wells were taken and transferred to
96-well flexible plates. Culture medium was added to each well to
provide a 1:10 dilution of the media sample. Two saquinavir-BSA
coated wells were used for each culture well tested. One well
received 50 .mu.L of PBS buffer, the other received 50 .mu.L of PBS
containing a saquinavir drug at a concentration of 800 ng/ml. Fifty
microliters of the diluted sample were transferred to each of two
of the coated wells above. The plates were incubated covered for 1
hour at 37.degree. C., then washed with PBS-TWEEN. The wells were
then filled with 100 .mu.L of goat anti-mouse IgG-HRP conjugate
(Zymed Labs) diluted 1:5,000 in PBS-TWEEN and the plates
re-incubated for 1 hour. The plates were then washed again, and 100
.mu.L of K-BLUE SUBSTRATE (Neogen Corp) were added to each well.
This was allowed to develop for 5-15 minutes, the reaction being
stopped by the addition of 100 .mu.L of 1 N HCl. Color was read via
a microplate reader at 450 nm and collected by computer for
analysis. Criteria for selection were binding to the saquinavir-BSA
conjugate and significant inhibition of binding in the second well
due to the free drug. TABLE-US-00002 TABLE 2 Representative portion
of the screening of the plates Culture well OD in absence of free
drug OD in presence of free drug 1 H12 3.568 0.504 37F5 0.738 0.358
2B11 3.942 3.649 19D5 1.152 0.225 24D11 3.305 1.342
[0183] Subsequent to the selection of a clone from the fusion
culture plates, the cells were subjected to stringent cloning via
limiting dilution. Subclones growing from those wells in which
single cells had been verified by microscopy were then re-tested by
the above method. Stability of antibody expression was judged on
the number of wells showing antibody, the level of binding and the
presence of any wells showing growth but little or no antibody. If
any of the latter were found, a well showing high antibody
secretion was then used to repeat stringent subcloning. This was
repeated as necessary to obtain 100% of the subclones secreting
equivalent quantities of antibody. Cells from selected wells were
then expanded in culture, and used to prepare preliminary cell
banks. The supernatant from those cultures was then subjected to
specificity analysis.
[0184] The antibody containing culture supernatants from the
expansion cultures were subjected to specificity analysis by the
following procedure. First, the titer appropriate for analysis was
determined by dilution analysis. A dilution of antibody providing
for approximately 50% of maximal binding was selected for
proceeding to the next step. Second, binding to the saquinavir-BSA
conjugate was examined at the above antibody dilution, in the
presence of varying amounts of six HIV protease inhibitor drugs.
The data was subjected to analysis by non-linear regression curve
fitting to a 4-parameter logistic function. That parameter which
describes the concentration of the free drug which corresponds to
50% of the binding in the absence of free drug is termed the
ED.sub.50 for that drug. The specificity of the antibody can thus
be described by comparing the ED.sub.50 of the cognate drug,
saquinavir, or saq ED.sub.50 with the other values for other drugs
fitted from those data according to the following equation (using
nelfinavir data for this example): % .times. .times. cross .times.
- .times. reactivity = saq .times. .times. ED 50 nel .times.
.times. ED 50 100. ##EQU1## The four parameter logistic function
used is ODx = OD .times. .times. max ( 1 + ( ED 50 X ) S - OD
.times. .times. min ##EQU2## where S is the curvature parameter,
ODmax is the optical density with 0 drug concentration. ODmin is
the optical density of the background of the instrument, and ODx is
the optical density observed at drug concentration X is moles/liter
(M/l).
[0185] By this analysis, the cross-reactivites of two
anti-saquinavir antibodies are given in Table 3. Murine hybridomas
SAQ 10.2.1 and SAQ 14.1.1 were deposited with the American Type
Culture Collection (ATCC) on Jan. 18, 2002 and assigned ATCC No.
PTA-3973 and ATCC No. PTA-3974, respectively. TABLE-US-00003 TABLE
3 Specificity of saquinavir 10.2.1 and 14.1.1 antibodies Clone
Saquinavir Nelfinavir Indinavir Amprenavir Ritonavir Lopinavir
14.1.1 % Cross Rx 100 0.003 0.100 0.053 0.134 0.075 ED.sub.50 (M/l)
4.9 .times. 10.sup.-8 1.72 .times. 10.sup.-3 4.9 .times. 10.sup.-5
9.35 10.sup.-5 3.66 10.sup.-5 6.52 10.sup.-5 10.2.1 % Cross Rx 100
0 0 0 0 0 ED.sub.50 (M/l) 1.7E.sup.-8 <1E.sup.-4 <1E.sup.-4
<1E.sup.-4 <1E.sup.-4 <1E.sup.-4
EXAMPLE 79
Development of monoclonal antibodies to nelfinavir
[0186] The procedures used for the development of monoclonals to
nelfinavir were similar to those used for saquinavir. Female Balb/c
mice 8 weeks of age, were immunized with 100 .mu.g of conjugate 5F
emulsified in Complete Freund's Adjuvant via intraperitoneal
injection. Twenty one days later, another immunization of the same
dose followed in Incomplete Freund's adjuvant. Four further
injections were carried out, using the same dosage and alternating
with Ribi adjuvant, at approximately 21 day intervals. All
adjuvants were from the Signal Chemical Co.
[0187] Four days following the last injection, a mouse was killed
by exsanguination and cervical dislocation. Spleen cells were taken
and fused to the F0 myeloma line by the same procedure as for
saquinavir. Culturing and feeding were also the same.
[0188] Screening of growing hybridomas was as for saquinavir, with
the exception that nelfinavir-BSA (5G) and free nelfinavir were
substituted for the saquinavir-BSA and free saquinavir,
respectively. Table 4 presents a portion of the screening data thus
obtained. TABLE-US-00004 TABLE 4 Development of nelfinavir clones
OD w/o OD with Culture well free nelfinavir free nelfinavir 9 D4
4.200 1.812 56 A9 3.906 0.469 12 G3 3.948 2.482 46 B12 3.946 1.869
12 A6 3.955 0.456 40 E7 3.820 0.271
[0189] Further processing to assure stability was by the same
methods as for saquinavir monoclonal antibodies. Specificity
analysis was using the same panel of drugs, with competitive
binding by nelfinavir taken as 100%. Table 5 shows the
specificities of subclones of the lines shown in Table 4.
TABLE-US-00005 TABLE 5 Specificities of selected stabilized
subclones of nelfinavir clones Clone Saquinavir Nelfinavir
Indinavir Amprenavir Ritonavir Lopinavir 5.4.1 % Cross Rx 0 100 0 0
0 0 ED.sub.50 (M/l) >4 .times. 10.sup.-4 1.1 .times. 10.sup.-8
>4 .times. 10.sup.-4 >4 .times. 10.sup.-4 >4 .times.
10.sup.-4 >4 .times. 10.sup.-4 15.3.1 % Cross Rx 0 100 0 0 0 0
ED.sub.50 (M/l) >4 .times. 10.sup.-4 2.4 .times. 10.sup.-8 >4
.times. 10.sup.-4 >4 .times. 10.sup.-4 >4 .times. 10.sup.-4
>4 .times. 10.sup.-4 21.4 % Cross Rx 0.041 100 0.033 0.018 0
0.07 ED.sub.50 (M/l) 1.3 .times. 10.sup.-8 .sup. 5.3 .times.
10.sup.-10 1.6 .times. 10.sup.-6 2.9 .times. 10.sup.-6 >4
.times. 10.sup.-4 7.7 .times. 10.sup.-8
[0190] Murine hybridoma NEL 5.4.1 was deposited with the American
Type Culture Collection (ATCC) on Jun. 25, 2002 and assigned ATCC
No. PTA-4475.
EXAMPLE 80
Development of monoclonal antibodies to indinavir
[0191] 12-week old female Babl/c mice were given a primary
intraperitoneal immunization with 100 .mu.g indinavir KLH conjugate
4G together with the adjuvant CFA (complete Freund's adjuvant).
This was followed by three further intraperitoneal immunizations
after 6 weeks at monthly intervals. In this case each mouse was
administered with 100 .mu.g indinavir KLH conjugate 4G together
with IFA (incomplete Freund's adjuvant). Subsequently the last
immunizations were carried out intravenously with 100 .mu.g
indinavir KLH conjugate 4G in PBS buffer on the second day and on
the last day before fusion.
[0192] The spleen cells of the mice immunized as described above
were fused with myeloma cells according to Galfre, Methods in
Enzymology, Vol. 73, 3 (1981). Approximately 1.times.10.sup.8
spleen cells of the immunized mouse were mixed with
2.times.10.sup.7 myeloma cells (P3X63-Ag8-653, ATCC CRL 1580) and
centrifuged (10 minutes at 300 G and room temperature). The cells
were then washed once with RPMI 1640 medium without fetal calf
serum (FCS) and again centrifuged at 400 G in a 50 mL conical tube.
Subsequently 1 mL PEG (polyethylene glycol, molecular weight 4000,
Merck, Darmstadt) was added, and it was mixed by gentle shaking.
After 1 minute in a water bath at 37.degree. C., 5 mL RPMI 1640
without FCS were added dropwise, mixed, made up to 30 mL with
medium (RPMI 1640) and subsequently centrifuged. The sedimented
cells were taken up in RPMI 1640 medium containing 10% FCS and
plated in hypoxanthine-azaserine selection medium (100 mmol/l
hypoxanthine, 1 .mu.g/mL azaserine in RPMI 1640+10% FCS).
Interleukin 6 from mouse (Roche Diagnostics GmbH, Catalog No. 1 444
581, 50 U/ml) was added to the medium as a growth factor.
[0193] After approximately 11 days the primary cultures were tested
for specific antibody synthesis. Primary cultures which exhibited a
positive reaction with indinavir and no cross-reaction with
saquinavir, nelfinavir, ritonavir and amprenavir, were cloned in
96-well cell culture plates by means of a cell sorter.
[0194] The deposited cell lines/clones listed in Table 6 were
obtained in this manner. Murine hybridomas <INDIN>M 1.003.12
and <INDIN>M 1.158.8 were deposited with the Deutsche
Sammlung von Mikroorganismen and Zellkulturen GmbH (DSMZ) on Jun.
18, 2002 and assigned DSM No. ACC2547 and DSM No. ACC2546,
respectively. TABLE-US-00006 TABLE 6 Immunoglobulin subclass Clone
IgG subclass 1.158.8 IgG1 kappa 1.003.12 IgG2a kappa
[0195] For determination of the specificity of the antibodies in
the culture supernatant of the hybridoma cells, microtiter plates
coated with recombinant streptavidin (MicroCoat Co. Penzberg,
Catalog No. 148051001) were coated with 500 ng/mL of indinavir
biotin conjugate 4I (100 .mu.L per well diluted in PBS/1.0% CROTEIN
C/0.1% TWEEN 20; incubation overnight at 4.degree. C.) and
subsequently washed 3 times with 0.9% NaCl/0.1% TWEEN 20. (CROTEIN
C is a trademark of Croda Colloids, Ltd. for hydrolyzed collagen
protein.)
[0196] Free streptavidin binding sites were then blocked by
incubation with 100 .mu.g/mL of biotin (1 hour; ambient temperature
while shaking) and subsequently washed 3 times with 0.9% NaCl/0.1%
TWEEN 20.
[0197] Next, 50 .mu.L of the analyte to be tested for
cross-reaction was added to a coated well in a concentration series
of 0-25 .mu.g/mL (diluted in PBS plus 1.0% CROTEIN C, 0.1% TWEEN
20) and together with 50 .mu.L of the antibody solution (culture
supernatant) to be examined and incubated for 1 hour at room
temperature while shaking. After washing 3 times with 0.9% sodium
chloride/0.1% TWEEN 20, 100 .mu.L of a horseradish
peroxidase-labeled Fab fragment of a polyclonal antibody from the
sheep against mouse Fc (pab<mouse Fc gamma>S-Fab-POD, Roche;
25 mU/ml) was added to each well to detect bound antibody from the
sample, incubated for 1 hour at room temperature while shaking and
subsequently washed 3 times with 0.9% sodium chloride/0.1% TWEEN
20.
[0198] Finally 100 .mu.L/well ABTS solution (Roche Diagnostics
GmbH, cat. no. 1684302) was added and the absorbance at 405/492 nm
was measured after 30 minutes at room temperature in a SLT Spectra
Image microplate reader from TECAN.
[0199] Using the test system described above, it was shown that the
monoclonal antibodies <INDIN> M 1.158.8 and <INDIN> M
1.003.12 exhibited less than 10% cross-reactivity with indinavir,
nelfinavir, ritonavir, saquinavir, and amprenavir. FIG. 17 shows
graphs of the cross-reaction of mab <INDIN> M 1.158.8 and mab
<INDIN> M 1.003.12 with indinavir, nelfinavir, ritonavir,
saquinavir and amprenavir.
EXAMPLE 81
Development of monoclonal antibodies to amprenavir
[0200] Monoclonal antibodies to amprenavir were developed using the
procedures for immunization, fusion, culture, and cloning as
described above in Example 80.
O.sup.c-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-amprenavir
conjugate with KLH (3E) was used as the immunogen.
[0201] ELISA screening was accomplished using the amprenavir-biotin
conjugate 3J. Murine hybridoma <AMPREN> M 1.1.52 was
deposited with the DSMZ on Sep. 16, 2003 and assigned DSM No. ACC
2612.
[0202] Specificity was determined as described above in Example 80.
It was shown that the monoclonal antibody =21 AMPREN> M 1.1.52
exhibited less than 10% cross-reactivity with indinavir,
nelfinavir, ritonavir, saquinavir, and lopinavir. FIG. 20 shows a
graph of the cross-reaction of mab <AMPREN> M 1.1.52 with
indinavir, nelfinavir, ritonavir, saquinavir and lopinavir.
EXAMPLE 82
Development of monoclonal antibodies to lopinavir
[0203] Monoclonal antibodies to lopinavir were developed using the
procedures for immunization, fusion, culture, and cloning as
described above in Example 80.
O.sup.c-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-lopinavir
conjugate with KLH (6F) was used as the immunogen.
[0204] ELISA screening was accomplished using the lopinavir-biotin
conjugate 6G. Murine hybridoma <LOPIN> M 1.1.85 was deposited
with the DSMZ on Sep. 16, 2003 and assigned DSM No. ACC 2611.
[0205] Specificity was determined as described above in Example 80.
It was shown that the monoclonal antibody <LOPIN> M 1.1.85
exhibited less than 10% cross-reactivity with indinavir,
nelfinavir, ritonavir, saquinavir, and amprenavir. FIG. 21 shows a
graph of the cross-reaction of mab <LOPIN> M 1.1.85 with
indinavir, nelfinavir, ritonavir, saquinavir and amprenavir.
EXAMPLE 83
Development of monoclonal antibodies to ritonavir
[0206] Monoclonal antibodies to ritonavir were developed using the
procedures for immunization, fusion, culture, and cloning as
described above in Example 80.
O.sup.c-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-ritonavir
conjugate with LPH (1E) was used as the immunogen.
[0207] ELISA screening was accomplished using the ritonavir-biotin
conjugate 1D. Murine hybridoma <RITON> M 1.5.44 was deposited
with the DSMZ on Sep. 16, 2003 and assigned DSM No. ACC 2613.
[0208] Specificity was determined as described above in Example 80.
It was shown that the monoclonal antibody <RITON> M 1.5.44
exhibited less than 10% cross-reactivity with indinavir,
nelfinavir, lopinavir, saquinavir, and amprenavir. FIG. 22 shows a
graph of the cross-reaction of mab <RITON> M 1.5.44 with
indinavir, nelfinavir, lopinavir, saquinavir and amprenavir.
EXAMPLE 84
Synthesis of O.sup.c-(N-FMOC-aminocaproyl)-atazanavir (7A)
[0209] O.sup.c-(N-FMOC-aminocaproyl)-atazanavir (7A) was prepared
by stirring atazanavir (7, 0.20 g), FMOC-aminocaproic acid (0.010
g, 1 eg), DCC (0.059 g, 1 eg), and DMAP (0.038 g, 1 eq) in dry
methylene chloride (40 mL) in a similar manner to Example 1, except
that after stirring overnight at room temperature, an additional
0.5 eq of FMOC-aminocaproic acid and 0.5 eq of DCC were added, and
stirring continued for a further 3 days. Work-up and purification
in a similar manner to that given in Example 1 gave the product 7A
(210 mg; 71%) as a white solid. M+H 1040.5
EXAMPLE 85
Synthesis of O.sup.c-(aminocaproyl)-atazanavir (7B)
[0210] O.sup.c-(aminocaproyl)-atazanvir (7B) was prepared from
O.sup.c-(N-FMOC-aminocaproyl)-atazanavir (7A) of Example 84 (0.092
g) following the conditions described in Example 9, except that two
silica gel chromatography purifications were performed (first
column using 40% methanol in ethyl acetate (EtOAc), second column
using 20% methanol in EtOAc) to give the product 7B as a solid
(0.070 g, 97%). M+H 818.4.
[0211] In another run, 7B was isolated as the trifluoroacetic acid
(TFA) salt after purification by preparative RP-HPLC (C18, gradient
of 5% to 100% of 0.1% TFA-acetonitrile in 0.1% TFA-water).
EXAMPLE 86
Synthesis of
O.sup.c-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-atazanavir
(7C)
[0212] O.sup.c-(aminocaproyl)-atazanavir (7B) as the TFA salt
(0.070 g), triethylamine (22 .mu.L), and succinimido-oxycarbonyl
butyryl chloride (0.0195 g) were stirred for 3 hours in dry THF at
about 0.degree. C. (ice-water bath). The reaction was evaporated to
dryness, redissolved in 15% THF in ethyl acetate, and purified by
silica gel chromatography (elution with 30% THF in EtOAc, column
pre-washed with several column volumes of 15% THF in EtOAc).
Fractions containing product were combined, evaporated, redissolved
in dry methylene chloride (CH.sub.2C.sub.12) and re-evaporated
(repeated several times) to yield
O.sup.c-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-atazanavir
(7C) as a solid (24 mg, 31%). M+H 1029.4
EXAMPLE 87
Synthesis of
O.sup.c-[4'-(succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-atazanavir
(7D)
[0213] A solution of O.sup.c-(aminocaproyl)-atazanavir (7B, 0.054
g) in 2 mL of dry DMF was added slowly to a stirring, cooled
solution (ice-water bath) of disuccinimidyl terephthalate (0.0228
g) in 4.5 mL of dry DMF. After brief stirring, triethylamine (50
.mu.L) was added and the reaction stirred overnight. Analysis by
HPLC indicated essential completion of the reaction. Solvent was
removed on a rotovap under high vacuum (at less than 25.degree.
C.), the residue redissolved in acetonitrile-water and purified by
preparative RP-HPLC (C18, gradient of 5% to 100% of 0.1%
TFA-acetonitrile in 0.1% TFA-water) to give, from the main peak
after evaporation of acetonitrile, freezing and lyophilization, the
product
O.sup.c-[4'-(succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-atazanavir
(7D), assigned as the trifluoracetic acid salt, in two cuts (0.036
g and 0.007 g, combined 0.043 g, 55%). M+H 1063.5 (free base)
EXAMPLE 88
Synthesis of
O.sup.c-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-atazanavir
conjugate with KLH (7E)
[0214]
O.sup.c-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-atazanavir
KLH conjugate was prepared from purified keyhole limpet hemocyanin
(60 mg) and
O.sup.c-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-atazanavir
(7C) from Example 86 (17 mg) following the general conditions
described in Example 61, except that the reaction was performed in
40% DMSO. Protein quantification of the retentate by Coomassie Blue
Protein Assay showed 10.8 mg/mL, 92% protein recovery (KLH
standard/control). Amine quantification by TNBS colorimetric assay
showed 56% lysine modification.
EXAMPLE 89
Synthesis of
O.sup.c-[4'-(succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-atazanvir
conjugate with BSA (7F)
[0215]
O.sup.c-[4'-(succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-atazan-
avir BSA conjugate was prepared from bovine serum albumin (100 mg)
and
O.sup.c-[4'-(succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-atazanavir
(7D) as the TFA salt, from Example 87 (3 mg) following the general
conditions described in Example 61, except that the reaction was
performed in 40% DMSO. Protein quantification by Coomassie Blue
protein assay showed quantitative recovery of protein at 10.0 mg/mL
(BSA standard/control). UV difference spectroscopy showed the ratio
of hapten to BSA to be 1:1.7.
* * * * *