U.S. patent application number 12/767646 was filed with the patent office on 2011-07-07 for long lasting fusion peptide inhibitors for hiv infection.
This patent application is currently assigned to CONJUCHEM BIOTECHNOLOGIES, INC.. Invention is credited to Elena Afonina, Dominique P. Bridon, Sandra De Meyer, John W. Erickson, Grant A. Krafft, Jun Liang, Martin Robitaille, Dong Xie.
Application Number | 20110166061 12/767646 |
Document ID | / |
Family ID | 23132509 |
Filed Date | 2011-07-07 |
United States Patent
Application |
20110166061 |
Kind Code |
A1 |
Erickson; John W. ; et
al. |
July 7, 2011 |
LONG LASTING FUSION PEPTIDE INHIBITORS FOR HIV INFECTION
Abstract
The present invention is concerned with This invention relates
to C34 peptide derivatives that are inhibitors of viral infection
and/or exhibit antifusogenic properties. In particular, this
invention relates to C34 derivatives having inhibiting activity
against human immunodeficiency virus (HIV), respiratory syncytial
virus (RSV), human parainfluenza virus (HPV), measles virus (MeV),
and simian immunodeficiency virus (SIV) with long duration of
action for the treatment of the respective viral infections.
Inventors: |
Erickson; John W.;
(Frederick, MD) ; Bridon; Dominique P.; (Quebec,
CA) ; Robitaille; Martin; (Quebec, CA) ;
Krafft; Grant A.; (Glenview, IL) ; Xie; Dong;
(Germantown, MD) ; Afonina; Elena; (Frederick,
MD) ; Liang; Jun; (Boyds, MD) ; De Meyer;
Sandra; (Beerse, BE) |
Assignee: |
CONJUCHEM BIOTECHNOLOGIES,
INC.
Montreal
CA
|
Family ID: |
23132509 |
Appl. No.: |
12/767646 |
Filed: |
April 26, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10478811 |
Dec 10, 2003 |
7741453 |
|
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PCT/CA02/00806 |
May 31, 2002 |
|
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12767646 |
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60294241 |
May 31, 2001 |
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Current U.S.
Class: |
514/3.8 ;
514/21.3; 530/324 |
Current CPC
Class: |
A61P 31/18 20180101;
A61K 38/00 20130101; C12N 2740/16122 20130101; C07K 14/005
20130101; A61P 31/14 20180101; C07K 2319/00 20130101 |
Class at
Publication: |
514/3.8 ;
530/324; 514/21.3 |
International
Class: |
A61K 38/16 20060101
A61K038/16; C07K 14/00 20060101 C07K014/00; A61P 31/18 20060101
A61P031/18 |
Claims
1. A compound of Formulae I-VIII ##STR00003## ##STR00004##
2. A pharmaceutical composition comprising a compound as claimed in
claim 1 in combination with a pharmaceutically acceptable
carrier.
3. A composition as claimed in claim 2 for inhibiting the activity
of HIV, RSV, HPV, MeV or SIV.
4. A method for inhibiting the activity of HIV, RSV, HPV, MeV or
SIV in a subject comprising administering to a subject an effective
amount of a compound as claimed in claim 1, alone or in combination
with a pharmaceutically acceptable carrier.
5. A conjugate comprising a compound as claimed in claim 1
covalently bonded to a blood component.
6. A method for extending the in vivo half-life of a compounds as
claimed in claim 1, the method comprising covalently bonding the
compound to a blood component.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 10/478,811, filed on Dec. 10, 2003, which is a 371 of
PCT/CA02/00806, filed May 31, 2002, which claims benefit of U.S.
Provisional Ser. No. 60/294,241, filed May 31, 2001.
FIELD OF INVENTION
[0002] This invention relates to C34 peptide derivatives that are
inhibitors of viral infection and/or exhibit antifusogenic
properties. In particular, this invention relates to C34
derivatives having inhibiting activity against human
immunodeficiency virus (HIV), respiratory syncytial virus (RSV),
human parainfluenza virus (HPV), measles virus (MeV), and simian
immunodeficiency virus (SIV) with long duration of action for the
treatment of the respective viral infections.
Sequence Listing
[0003] The instant application contains a Sequence Listing which
has been submitted via EFS-Web and is hereby incorporated by
reference in its entirety. Said ASCII copy, created on Dec. 21,
2009, is named C2077700.txt, and is 6,094 bytes in size.
BACKGROUND OF THE INVENTION
[0004] Membrane fusion events, while commonplace in normal cell
biological processes, are also involved in a variety of disease
states, including, for example the entry of enveloped viruses into
cells. Peptides are known to inhibit or otherwise disrupt membrane
fusion-associated events, including, for example, inhibiting
retroviral transmission to uninfected cells.
[0005] HIV is a member of the lentivirus family of retroviruses,
and there are two prevalent types of HIV, HIV-1 and HIV-2, with
various strain of each having been identified. HIV targets CD-4+
cells, and viral entry depends on binding of the HIV protein gp120
to the CD4 glycoprotein and a chemokine receptor on cell surface.
C34 is known to exhibit anti-viral activity against HIV, including
inhibiting CD-4+ cell infection by free virus and/or inhibiting
HIV-induced syncytia formation between infected and uninfected
CD-4+ cells. The inhibition is believed to occur by binding of C34
to the first heptad repeat region in gp41 and thus preventing the
first and second heptad repeat regions from formatting the
fusigenic hairpin structure.
[0006] C34 is known to possess antifusogenic activity, i.e., it has
the ability to inhibit or reduce the level of membrane fusion
events between two or more entities, e.g., virus-cell or cell-cell,
relative to the level of membrane fusion that occurs in the absence
of the peptide. More specifically, WO 00/06599 teaches the use of
C34 to inactivate gp41, and thus, prevent or reduce HIV-1 entry
into cells.
[0007] While many of the anti-viral or anti-fusogenic peptides
described in the art exhibit potent anti-viral and/or
anti-fusogenic activity, C34, like all such peptides, suffers from
short half-life in vivo, primarily due to rapid serum clearance and
peptidase and protease activity. This in turn greatly reduces its
effective anti-viral activity.
[0008] There is therefore a need for a method of prolonging the
half-life of peptides like C34 in vivo without substantially
affecting the anti-fusogenic activity.
SUMMARY OF THE INVENTION
[0009] In accordance with the present invention, there is now
provided C34 peptide derivatives having an extended in vivo
half-life when compared with the corresponding unmodified C34
peptide sequence. More specifically, the present invention is
concerned with compounds of Formulae I-VIII illustrated below,
which are capable of reacting with thiol groups on a blood
component, either in vivo or ex vivo, to form a stable covalent
bond. Formulae I-VIII disclose SEQ ID NOS 2, 3, 4, 5, 6, 7, 8 and
9, respectively, in order of appearance.
##STR00001## ##STR00002##
[0010] Preferred blood components comprise proteins such as
immunoglobulins, including IgG and IgM, serum albumin, ferritin,
steroid binding proteins, transferrin, thyroxin binding protein,
.alpha.-2-macroglobulin etc., serum albumin and IgG being more
preferred, and serum albumin being the most preferred.
[0011] In another aspect of the invention, there is provided a
pharmaceutical composition comprising the derivatives of Formulae
I-VIII in combination with a pharmaceutically acceptable carrier.
Such composition is useful for inhibiting the activity of HIV, RSV,
HPV, MeV or SIV.
[0012] In a further embodiment of the present invention, there is
provided a method for inhibiting the activity of HIV, RSV, HPV, MeV
or SIV. The method comprises administering to a subject, preferably
a mammal, an effective amount of the compounds of Formulae I-VIII
or a conjugate thereof, alone or in combination with a
pharmaceutical carrier.
[0013] In a further aspect of the present invention, there is
provided a conjugate comprising the compounds of Formulae I-VIII
covalently bonded to a blood component.
[0014] In a further aspect of the present invention, there is
provided a method for extending the in vivo half-life of the C34
peptide in a subject, the method comprising covalently bonding the
compounds of Formulae I-VIII to a blood component.
DETAILED DESCRIPTION OF THE INVENTION
[0015] The present invention meets these and other needs and is
directed to C34 peptides derivatives of Formulae I-VIII having
anti-viral activity and/or anti-fusogenic activity. These C34
peptides derivatives provide for an increased stability in vivo and
a reduced susceptibility to peptidase or protease degradation. As a
result, the compounds of Formulae I-VIII minimize the need for more
frequent, or even continual, administration of the peptides. The
present C34 derivatives can be used, e.g., as a prophylactic
against and/or treatment for infection of a number of viruses,
including human immunodeficiency virus (HIV), human respiratory
syncytial virus (RSV), human parainfluenza virus (HPV), measles
virus (MeV) and simian immunodeficiency virus (SIV).
[0016] The modification made to the native C34 peptide sequence
allows it to react with available thiol groups on blood components
to form stable covalent bonds. In one embodiment of the invention,
the blood component comprises a blood protein, including a mobile
blood protein such as albumin, which is most preferred.
[0017] The compounds of Formulae I-VIII inhibit viral infection of
cells, by, for example, inhibiting cell-cell fusion or free virus
infection. The route of infection may involve membrane fusion, as
occurs in the case of enveloped or encapsulated viruses, or some
other fusion event involving viral and cellular structures.
[0018] The blood components to which the present anti-viral C34
derivatives covalently bonds may be either fixed or mobile. Fixed
blood components are non-mobile blood components and include
tissues, membrane receptors, interstitial proteins, fibrin
proteins, collagens, platelets, endothelial cells, epithelial cells
and their associated membrane and membraneous receptors, somatic
body cells, skeletal and smooth muscle cells, neuronal components,
osteocytes and osteoclasts and all body tissues especially those
associated with the circulatory and lymphatic systems. Mobile blood
components are blood components that do not have a fixed situs for
any extended period of time, generally not exceeding 5 minutes, and
more usually one minute. These blood components are not
membrane-associated and are present in the blood for extended
periods of time in a minimum concentration of at least 0.1
.mu.g/ml. Mobile blood components include serum albumin,
transferrin, ferritin and immunoglobulins such as IgM and IgG. The
half-life of mobile blood components is at least about 12
hours.
[0019] Protective groups may be required during the synthesis
process of the present C34 derivative. These protective groups are
conventional in the field of peptide synthesis, and can be
generally described as chemical moieties capable of protecting the
peptide derivative from reacting with other functional groups.
Various protective groups are available commercially, and examples
thereof can be found in U.S. Pat. No. 5,493,007 which is hereby
incorporated by reference. Typical examples of suitable protective
groups include acetyl, fluorenylmethyloxycarbonyl (FMOC),
t-butyloxycarbonyl (BOC), benzyloxy-carbonyl (CBZ), etc. In
addition, Table 1 provides both the three letter and one letter
abbreviations for amino acids.
TABLE-US-00001 TABLE 1 NATURAL AMINO ACIDS AND THEIR ABBREVIATIONS
3-letter 1-letter Name abbreviation abbreviation Alanine Ala A
Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C
Glutamic acid Glu E Glutamine Gln Q Glycine Gly G Histidine His H
Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M
Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T
Tryptophan Trp W Tyrosine Tyr Y Valine Val V
[0020] The present C34 derivatives may be administered in vivo such
that conjugation with blood components occurs in vivo, or they may
be first conjugated to blood components in vitro and the resulting
conjugated derivative administered in vivo.
[0021] The present invention takes advantage of the properties of
existing anti-viral and antifusogenic peptides. The viruses that
may be inhibited by the peptides include, but are not limited to
all strains of viruses listed, e.g., in U.S. Pat. No. 6,013,263 and
U.S. Pat. No. 6,017,536 at Tables V-VII and IX-XIV therein. These
viruses include, e.g., human retroviruses, including HIV-1, HIV-2,
and human T-lymphocyte viruses (HTLV-I and HTLV-II), and non-human
retroviruses, including bovine leukosis virus, feline sarcoma
virus, feline leukemia virus, simian immunodeficiency virus (SIV),
simian sarcoma virus, simian leukemia, and sheep progress pneumonia
virus. Non-retroviral viruses may also be inhibited by the present
C34 derivatives, including human respiratory syncytial virus (RSV),
canine distemper virus, Newcastle Disease virus, human
parainfluenza virus (HPIV), influenza viruses, measles viruses
(MeV), Epstein-Barr viruses, hepatitis B viruses, and simian
Mason-Pfizer viruses. Non-enveloped viruses may also be inhibited
by the present C34 derivatives, and include, but are not limited
to, picornaviruses such as polio viruses, hepatitis A virus,
enteroviruses, echoviruses, coxsackie viruses, papovaviruses such
as papilloma virus, parvoviruses, adenoviruses, and reoviruses.
[0022] The focus of the present invention is to modify the C34
peptide sequence to confer to this peptide improved
bio-availability, extended half-life and better distribution
through selective conjugation of the peptide onto a protein carrier
without substantially modifying the peptide's anti-viral
properties. The carrier of choice (but not limited to) for this
invention would be albumin conjugated through its free thiol.
[0023] The present C34 derivatives are designed to specifically
react with thiol groups on mobile blood proteins. Such reaction is
established by covalent bonding of the peptide modified with a
maleimide link to a thiol group on a mobile blood protein such as
serum albumin or IgG.
[0024] Thiol groups being less abundant in vivo than, for example,
amino groups, the maleimide-modified C34 peptide of the present
invention, will covalently bond to fewer proteins. For example, in
albumin (the most abundant blood protein) there is only a single
thiol group. Thus, a C34-maleimide-albumin conjugate will tend to
comprise approximately a 1:1 molar ratio of C34 peptide to albumin.
In addition to albumin, IgG molecules (class II) also have free
thiols. Since IgG molecules and serum albumin make up the majority
of the soluble protein in blood they also make up the majority of
the free thiol groups in blood that are available to covalently
bond to the C34 peptide derivative.
[0025] Further, even among free thiol-containing blood proteins,
including IgGs, specific labeling with a maleimide leads to the
preferential formation of a C34-maleimide-albumin conjugate due to
the unique characteristics of albumin itself. The single free thiol
group of albumin, highly conserved among species, is located at
amino acid residue 34 (Cys.sup.34). It has been demonstrated
recently that the Cys.sup.34 of albumin has increased reactivity
relative to free thiols on other free thiol-containing proteins.
This is due in part to the very low pK value of 5.5 for the
Cys.sup.34 of albumin. This is much lower than typical pK values
for cysteine residues in general, which are typically about 8. Due
to this low pK, under normal physiological conditions Cys.sup.34 of
albumin is predominantly in the ionized form, which dramatically
increases its reactivity. In addition to the low pK value of
Cys.sup.34, another factor which enhances the reactivity of
Cys.sup.34 is its location, which is in a hydrophobic pocket close
to the surface of one loop of region V of albumin. This location
makes Cys.sup.34 very available to ligands of all kinds, and is an
important factor in Cys.sup.34's biological role as free radical
trap and free thiol scavenger. These properties make Cys.sup.34
highly reactive with maleimide-C34, and the reaction rate
acceleration can be as much as 1000-fold relative to rates of
reaction of maleimide-C34 with other free-thiol containing
proteins.
[0026] Another advantage of C34-maleimide-albumin conjugates is the
reproducibility associated with the 1:1 loading of C34 to albumin
specifically at Cys.sup.34. Other techniques, such as
glutaraldehyde, DCC, EDC and other chemical activations of, e.g,
free amines, lack this selectivity. For example, albumin contains
52 lysine residues, 25-30 of which are located on the surface of
albumin and therefore accessible for conjugation. Activating these
lysine residues, or alternatively modifying C34 to couple through
these lysine residues, results in a heterogenous population of
conjugates. Even if 1:1 molar ratios of C34 to albumin are
employed, the yield will consist of multiple conjugation products,
some containing 0, 1, 2 or more C34 per albumin, and each having
C34 randomly coupled at any one or more of the 25-30 available
lysine sites. Given the numerous possible combinations,
characterization of the exact composition and nature of each
conjugate batch becomes difficult, and batch-to-batch
reproducibility is all but impossible, making such conjugates less
desirable as a therapeutic. Additionally, while it would seem that
conjugation through lysine residues of albumin would at least have
the advantage of delivering more therapeutic agent per albumin
molecule, studies have shown that a 1:1 ratio of therapeutic agent
to albumin is preferred. In an article by Stehle, et al., "The
Loading Rate Determines Tumor Targeting properties of
Methotrexate-Albumin Conjugates in Rats," Anti-Cancer Drugs, Vol.
8, pp. 677-685 (1988), incorporated herein in its entirety, the
authors report that a 1:1 ratio of the anti-cancer methotrexate to
albumin conjugated via glutaraldehyde gave the most promising
results. These conjugates were preferentially taken up by tumor
cells, whereas conjugates bearing 5:1 to 20:1 methotrexate
molecules had altered HPLC profiles and were quickly taken up by
the liver in vivo. It is postulated that at these higher ratios,
conformational changes to albumin diminish its effectiveness as a
therapeutic carrier.
[0027] Through controlled administration of the present C34
derivatives in vivo, one can control the specific labeling of
albumin and IgG in vivo. In typical administrations, 80-90% of the
administered C34 derivatives will label albumin and less than 5%
will label IgG. Trace labeling of free thiols such as glutathione
will also occur. Such specific labeling is preferred for in vivo
use as it permits an accurate calculation of the estimated
half-life of C34.
[0028] In addition to providing controlled specific in vivo
labeling, the present C34 derivatives can provide specific labeling
of serum albumin and IgG ex vivo. Such ex vivo labeling involves
the addition of the C34 derivatives to blood, serum or saline
solution containing serum albumin and/or IgG. Once conjugation has
occurred ex vivo with the C34 derivative, the blood, serum or
saline solution can be readministered to the patient's blood for in
vivo treatment, or lyophilized.
[0029] The present C34 derivatives may be synthesized by standard
methods of solid phase peptide chemistry well known to any one of
ordinary skill in the art. For example, the peptide may be
synthesized by solid phase chemistry techniques following the
procedures described by Steward et al. in Solid Phase Peptide
Synthesis, 2nd Ed., Pierce Chemical Company, Rockford, Ill., (1984)
using a Rainin PTI Symphony synthesizer. Similarly, peptides
fragments may be synthesized and subsequently combined or linked
together to form the C34 peptide sequence (segment
condensation).
[0030] For solid phase peptide synthesis, a summary of the many
techniques may be found in Stewart et al. in "Solid Phase Peptide
Synthesis", W. H. Freeman Co. (San Francisco), 1963 and Meienhofer,
Hormonal Proteins and Peptides, 1973, 2 46. For classical solution
synthesis, see for example Schroder et al. in "The Peptides",
volume 1, Acacemic Press (New York). In general, such method
comprise the sequential addition of one or more amino acids or
suitably protected amino acids to a growing peptide chain on a
polymer. Normally, either the amino or carboxyl group of the first
amino acid is protected by a suitable protecting group. The
protected and/or derivatized amino acid is then either attached to
an inert solid support or utilized in solution by adding the next
amino acid in the sequence having the complimentary (amino or
carboxyl) group suitably protected and under conditions suitable
for forming the amide linkage. The protecting group is then removed
from this newly added amino acid residue and the next amino acid
(suitably protected) is added, and so forth.
[0031] After all the desired amino acids have been linked in the
proper sequence, any remaining protecting groups (and any solid
support) are cleaved sequentially or concurrently to afford the
final peptide. By simple modification of this general procedure, it
is possible to add more than one amino acid at a time to a growing
chain, for example, by coupling (under conditions which do not
racemize chiral centers) a protected tripeptide with a properly
protected dipeptide to form, after deprotection, a
pentapeptide.
[0032] A particularly preferred method of preparing the present C34
derivatives involves solid phase peptide synthesis wherein the
amino acid .alpha.-N-terminal is protected by an acid or base
sensitive group. Such protecting groups should have the properties
of being stable to the conditions of peptide linkage formation
while being readily removable without destruction of the growing
peptide chain or racemization of any of the chiral centers
contained therein. Examples of N-protecting groups and
carboxy-protecting groups are disclosed in Greene, "Protective
Groups In Organic Synthesis," (John Wiley & Sons, New York pp.
152-186 (1981)), which is hereby incorporated by reference.
Examples of N-protecting groups comprise, without limitation,
loweralkanoyl groups such as formyl, acetyl ("Ac"), propionyl,
pivaloyl, t-butylacetyl and the like; other acyl groups include
2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl,
phthalyl, o-nitrophenoxy-acetyl, -chlorobutyryl, benzoyl,
4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl and the like;
sulfonyl groups such as benzenesulfonyl, p-toluenesulfonyl,
o-nitrophenylsulfonyl, 2,2,5,7,8-pentamethylchroman-6-sulfonyl
(pmc), and the like; carbamate forming groups such as
t-amyloxycarbonyl, benzyloxycarbonyl, p-chlorobenzyloxycarbonyl,
p-methoxy-benzyloxycarbonyl, p-nitrobenzyloxycarbonyl,
2-nitrobenzyloxycarbonyl, p-bromo-benzyloxycarbonyl,
3,4-dimethoxybenzyloxycarbonyl, 3,5-dimethoxybenzyloxycarbonyl,
2,4-dimethoxybenzyloxycarbonyl, 4-ethoxybenzyloxycarbonyl,
2-nitro-4,5-dimethoxy-benzyloxycarbonyl,
3,4,5-trimethoxybenzyloxycarbonyl,
1-(p-biphenylyl)-1-methyl-ethoxycarbonyl,
.alpha.,.alpha.-dimethyl-3,5-dimethoxybenzyloxycarbonyl,
benzhydryloxycarbonyl, t-butyloxycarbonyl (boc),
diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxy-carbonyl,
methoxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl,
phenoxy-carbonyl, 4-nitrophenoxycarbonyl,
fluorenyl-9-methoxycarbonyl, isobornyloxycarbonyl,
cyclopentyloxycarbonyl, adamantyloxycarbonyl,
cyclohexyloxycarbonyl, phenylthio-carbonyl and the like; arylalkyl
groups such as benzyl, biphenylisopropyloxycarbonyl,
triphenylmethyl, benzyloxymethyl, 9-fluorenylmethyloxycarbonyl
(Fmoc) and the like and silyl groups such as trimethylsilyl and the
like. Preferred .alpha.-N-protecting group are
o-nitrophenylsulfenyl; 9-fluorenylmethyl oxycarbonyl;
t-butyloxycarbonyl (boc), isobornyloxycarbonyl;
3,5-dimethoxybenzyloxycarbonyl; t-amyloxycarbonyl;
2-cyano-t-butyloxycarbonyl, and the like,
9-fluorenyl-methyloxycarbonyl (Fmoc) being more preferred, while
preferred side chain N-protecting groups comprise
2,2,5,7,8-penta-methylchroman-6-sulfonyl (pmc), nitro,
p-toluenesulfonyl, 4-methoxybenzene-sulfonyl, Cbz, Boc, and
adamantyloxycarbonyl for side chain amino groups like lysine and
arginine; benzyl, o-bromobenzyloxycarbonyl, 2,6-dichlorobenzyl,
isopropyl, t-butyl (t-Bu), cyclohexyl, cyclopenyl and acetyl (Ac)
for tyrosine; t-butyl, benzyl and tetrahydropyranyl for serine;
trityl, benzyl, Cbz, p-toluenesulfonyl and 2,4-dinitrophenyl for
histidine; formyl for tryptophan; benzyl and t-butyl for aspartic
acid and glutamic acid; and triphenylmethyl (trityl) for
cysteine.
[0033] A carboxy-protecting group conventionally refers to a
carboxylic acid protecting ester or amide group. Such carboxy
protecting groups are well known to those skilled in the art,
having been extensively used in the protection of carboxyl groups
in the penicillin and cephalosporin fields as described in U.S.
Pat. No. 3,840,556 and U.S. Pat. No. 3,719,667, the disclosures of
which are hereby incorporated herein by reference. Representative
carboxy protecting groups comprise, without limitation,
C.sub.1-C.sub.8 loweralkyl; arylalkyl such as phenethyl or benzyl
and substituted derivatives thereof such as alkoxybenzyl or
nitrobenzyl groups; arylalkenyl such as phenylethenyl; aryl and
substituted derivatives thereof such as 5-indanyl;
dialkylaminoalkyl such as dimethylaminoethyl; alkanoyloxyalkyl
groups such as acetoxymethyl, butyryloxymethyl, valeryloxymethyl,
isobutyryloxymethyl, isovaleryloxymethyl, 1-(propionyloxy)-1-ethyl,
1-(pivaloyloxyl)-1-ethyl, 1-methyl-1-(propionyloxy)-1-ethyl,
pivaloyloxymethyl, propionyloxymethyl; cycloalkanoyloxyalkyl groups
such as cyclopropylcarbonyloxymethyl, cyclobutylcarbonyloxymethyl,
cyclo-pentylcarbonyloxymethyl, cyclohexylcarbonyloxy-methyl;
aroyloxyalkyl such as benzoyloxymethyl, benzoyloxyethyl;
arylalkylcarbonyloxyalkyl such as benzylcarbonyl-oxymethyl,
2-benzylcarbonyloxyethyl; alkoxycarbonylalkyl or
cycloalkyloxycarbonyl-alkyl such as methoxycarbonylmethyl,
cyclohexyloxycarbonylmethyl, 1-methoxy-carbonyl-1-ethyl;
alkoxycarbonyloxyalkyl or cycloalkyloxycarbonyloxyalkyl such as
methoxycarbonyloxymethyl, t-butyloxycarbonyl-oxymethyl,
1-ethoxycarbonyloxy-1-ethyl, 1-cyclohexyloxycarbonyloxy-1-ethyl;
aryloxy-carbonyloxyalkyl such as 2-(phenoxycarbonyloxy)ethyl,
2-(5-indanyloxycarbonyloxy)-ethyl; alkoxyalkylcarbonyl-oxyalkyl
such as 2-(1-methoxy-2-methylpropan-2-oyloxy)-ethyl;
arylalkyloxycarbonyl-oxyalkyl such as
2-(benzyloxycarbonyloxy)ethyl; arylalkenyloxycarbonyloxyalkyl such
as 2-(3-phenylpropen-2-yloxycarbonyloxy)ethyl;
alkoxycarbonylaminoalkyl such as t-butyloxycarbonylaminomethyl;
alkylaminocarbonyl-aminoalkyl such as
methylamino-carbonylaminomethyl; alkanoylaminoalkyl such as
acetylaminomethyl; heterocyclic-carbonyloxyalkyl such as
4-methylpiperazinyl-carbonyloxymethyl; dialkylamino-carbonylalkyl
such as dimethylaminocarbonylmethyl, diethylaminocarbonylmethyl;
(5-(loweralkyl)-2-oxo-1,3-dioxolen-4-yl)alkyl such as
(5-t-butyl-2-oxo-1,3-dioxolen-4-yl)methyl; and
(5-phenyl-2-oxo-1,3-dioxolen-4-yl)alkyl such as
(5-phenyl-2-oxo-1,3-dioxolen-4-yl)methyl. Representative amide
carboxy protecting groups comprise, without limitation,
aminocarbonyl and loweralkylaminocarbonyl groups. Of the above
carboxy-protecting groups, loweralkyl, cycloalkyl or arylalkyl
ester, for example, methyl ester, ethyl ester, propyl ester,
isopropyl ester, butyl ester, sec-butyl ester, isobutyl ester, amyl
ester, isoamyl ester, octyl ester, cyclohexyl ester, phenylethyl
ester and the like or an alkanoyloxyalkyl, cycloalkanoyloxyalkyl,
aroyloxyalkyl or an arylalkylcarbonyloxyalkyl ester are preferred.
Preferred amide carboxy protecting groups are
loweralkylamino-carbonyl groups.
[0034] In the solid phase peptide synthesis method, the
.alpha.-C-terminal amino acid is attached to a suitable solid
support or resin. Suitable solid supports useful for the above
synthesis are those materials that are inert to the reagents and
reaction conditions of the stepwise condensation-deprotection
reactions, as well as being insoluble in the media used. The
preferred solid support for synthesis of .alpha.-C-terminal carboxy
peptides is 4-hydroxymethylphenoxyacetyl-4'-methylbenzyhydrylamine
resin (HMP resin). The preferred solid support for
.alpha.-C-terminal amide peptides is an Fmoc-protected Ramage
resin, manufactured and sold by Bachem Inc., California.
[0035] At the end of the solid phase synthesis, the peptide is
removed from the resin and deprotected, either in successive
operations or in a single operation. Removal of the peptide and
deprotection can be accomplished conventionally in a single
operation by treating the resin-bound polypeptide with a cleavage
reagent comprising thioanisole, triisopropyl silane, phenol, and
trifluoroacetic acid. In cases wherein the .alpha.-C-terminal of
the peptide is an alkylamide, the resin is cleaved by aminolysis
with an alkylamine. Alternatively, the peptide may be removed by
transesterification, e.g. with methanol, followed by aminolysis or
by direct transamidation. The protected peptide may be purified at
this point or taken to the next step directly. The removal of the
side chain protecting groups is accomplished using the cleavage
mixture described above. The fully deprotected peptide can be
purified by a sequence of chromatographic steps employing any or
all of the following types: ion exchange on a weakly basic resin
(acetate form); hydrophobic adsorption chromatography on
underivatized polystyrene-divinylbenzene (such as Amberlite
XAD.TM.); silica gel adsorption chromatography; ion exchange
chromatography on carboxymethylcellulose; partition chromatography,
e.g. on Sephadex G25.TM., LH20.TM. or countercurrent distribution;
high performance liquid chromatography (HPLC), especially
reverse-phase HPLC on octyl- or phenyl/hexylsilyl-silica bonded
phase column packing. Anyone of ordinary skill in the art will be
able to determine easily what would be the preferred
chromatographic steps or sequences required to obtain acceptable
purification of the C34 peptide.
[0036] Molecular weights of these peptides are determined using
Electrospray mass spectroscopy.
[0037] The present C34 derivatives may be used alone or in
combination to optimize their therapeutic effects. They can be
administered in a physiologically acceptable medium, e.g. deionized
water, phosphate buffered saline (PBS), saline, aqueous ethanol or
other alcohol, plasma, proteinaceous solutions, mannitol, aqueous
glucose, alcohol, vegetable oil, or the like. Other additives which
may be included include buffers, where the media are generally
buffered at a pH in the range of about 5 to 10, where the buffer
will generally range in concentration from about 50 to 250 mM,
salt, where the concentration of salt will generally range from
about 5 to 500 mM, physiologically acceptable stabilizers, and the
like. The compositions may be lyophilized for convenient storage
and transport.
[0038] The C34 derivatives may be administered parenterally, such
as intravascularly (IV), intraarterially (IA), intramuscularly
(IM), subcutaneously (SC), or the like. Administration may in
appropriate situations be by transfusion. In some instances, where
reaction of the functional group is relatively slow, administration
may be oral, nasal, rectal, transdermal or aerosol, where the
nature of the conjugate allows for transfer to the vascular system.
Usually a single injection will be employed although more than one
injection may be used, if desired. The peptide derivative may be
administered by any convenient means, including syringe, trocar,
catheter, or the like. The particular manner of administration will
vary depending upon the amount to be administered, whether a single
bolus or continuous administration, or the like. Preferably, the
administration will be intravascularly, where the site of
introduction is not critical to this invention, preferably at a
site where there is rapid blood flow, e.g., intravenously,
peripheral or central vein. Other routes may find use where the
administration is coupled with slow release techniques or a
protective matrix. The intent is that the C34 derivative be
effectively distributed in the blood, so as to be able to react
with the blood components. The concentration of the conjugate will
vary widely, generally ranging from about 1 pg/ml to 50 mg/ml. The
total administered intravascularly will generally be in the range
of about 0.1 mg/ml to about 10 mg/ml, more usually about 1 mg/ml to
about 5 mg/ml.
[0039] By bonding to long-lived components of the blood, such as
immunoglobulin, serum albumin, red blood cells and platelets, a
number of advantages ensue. The activity of the C34 derivatives is
extended for days to weeks. Only one administration need to be
given during this period of time. Greater specificity can be
achieved, since the active compound will be primarily bound to
large molecules, where it is less likely to be taken up
intracellularly to interfere with other physiological
processes.
[0040] The formation of the covalent bond between the blood
component may occur in vivo or ex vivo. For ex vivo covalent bond
formation, the C34 derivative is added to blood serum or a saline
solution containing purified blood components such as human serum
albumin or IgG, to permit covalent bond formation between the
derivative and the blood component. In a preferred format, the C34
derivative is reacted with human serum albumin in saline solution.
After formation of the conjugate, the latter may be administered to
the subject or lyophilized.
[0041] The blood of the mammalian host may be monitored for the
activity of the C34 peptide and/or presence of the C34 derivatives.
By taking a blood sample from the host at different times, one may
determine whether C34 peptide has become bonded to the long-lived
blood components in sufficient amount to be therapeutically active
and, thereafter, the level of C34 in the blood. If desired, one may
also determine to which of the blood components C34 is covalently
bonded. Monitoring may also take place by using assays of C34
activity, HPLC-MS or antibodies directed to C34.
[0042] The following examples are provided to illustrate preferred
embodiments of the invention and shall by no means be construed as
limiting its scope.
[0043] The present C34 derivatives can be administered to patients
according to the methods described below and other methods known in
the art. Effective therapeutic dosages of the present C34
derivatives may be determined through procedures well known by
those in the art and will take into consideration any concerns over
potential toxicity of C34.
[0044] The present C34 derivative can also be administered
prophylactically to previously uninfected individuals. This can be
advantageous in cases where an individual has been subjected to a
high risk of exposure to a virus, as can occur when individual has
been in contact with an infected individual where there is a high
risk of viral transmission. This can be especially advantageous
where there is known cure for the virus, such as the HIV virus. As
an example, prophylactic administration of a C34 derivative would
be advantageous in a situation where a health care worker has been
exposed to blood from an HIV-infected individual, or in other
situations where an individual engaged in high-risk activities that
potentially expose that individual to the HIV virus.
[0045] The invention having been fully described can be further
appreciated and understood with reference to the following
non-limiting examples.
General
[0046] Unless stated otherwise, the synthesis of each C34
derivative was performed using an automated solid-phase procedure
on a Symphony Peptide Synthesizer with manual intervention during
the generation of the derivative. The synthesis was performed on
Fmoc-protected Ramage amide linker resin, using Fmoc-protected
amino acids. Coupling was achieved by using
O-benzotriazol-1-yl-N,N,N',N'-tetramethyl-uronium
hexafluorophosphate (HBTU) as activator in N,N-dimethylformamide
(DMF) solution and diisopropylethylamine (DIEA) as base. The Fmoc
protective group was removed using 20% piperidine/DMF. When needed,
a Boc-protected amino acid was used at the N-terminus in order to
generate the free N.sub..alpha.-terminus after the peptide is
cleaved from resin. All amino acids used during the synthesis
possess the L-stereochemistry. Glass reaction vessels were used
during the synthesis.
Example 1
Compound of Formula I
[0047] Step 1: The example describes the solid phase peptide
synthesis of the compound on a 100 .mu.mole scale. The following
protected amino acids were sequentially added to resin:
Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Glu(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Lys(Aloc)-OH,
Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ser(tBu)-OH,
Fmoc-Glu(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH,
Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Tyr(tBu)-OH,
Fmoc-Asn(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ile-OH, Fmoc-Glu(tBu)-OH,
Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Trp(Boc)-OH,
Fmoc-Glu(tBu)-OH, Fmoc-Met-OH, Fmoc-Trp(Boc)-OH. They were
dissolved in N,N-dimethylformamide (DMF) and, according to the
sequence, activated using
O-benzotriazol-1-yl-N,N,N',N'-tetramethyl-uronium
hexafluorophosphate (HBTU) and diisopropylethylamine (DIEA).
Removal of the Fmoc protecting group was achieved using a solution
of 20% (V/V) piperidine in N,N-dimethylformamide (DMF) for 20
minutes (step 1). The amino group of the final amino acid was
acetylated using acetic acid activated using
O-benzotriazol-1-yl-N,N,N',N'-tetramethyl-uronium
hexafluorophosphate (HBTU) and diisopropylethylamine (DIEA). Step
2: The selective deprotection of the Lys (Aloc) group was performed
manually and accomplished by treating the resin with a solution of
3 eq of Pd(PPh.sub.3).sub.4 dissolved in 5 mL of
C.sub.6H.sub.6CHCl.sub.3 (1:1): 2.5% NMM (v:v): 5% AcOH (v:v) for 2
h (Step 2). The resin is then washed with CHCl.sub.3 (6.times.5
mL), 20% AcOH in DCM (6.times.5 mL), DCM (6.times.5 mL), and DMF
(6.times.5 mL). Step 3: The synthesis was then re-automated for the
addition of the Fmoc-AEEA-OH and the 3-maleimidopropionic acid
(Step 3). Between every coupling, the resin was washed 3 times with
N,N-dimethylformamide (DMF) and 3 times with isopropanol
(.sup.iPrOH). Step 4: The peptide was cleaved from the resin using
85% TFA/5% triisopropyl-silane (TIPS)/5% thioanisole and 5% phenol,
followed by precipitation by dry-ice cold Et.sub.2O (Step 4).
Example 2
Compound of Formula II
[0048] Step 1: The example describes the solid phase peptide
synthesis of the compound on a 100 mmole scale. The following
protected amino acids were sequentially added to resin:
Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Glu(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH,
Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ser(tBu)-OH,
Fmoc-Glu(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH,
Fmoc-Lys(Aloc)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Tyr(tBu)-OH,
Fmoc-Asn(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ile-OH, Fmoc-Glu(tBu)-OH,
Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Trp(Boc)-OH,
Fmoc-Glu(tBu)-OH, Fmoc-Met-OH, Fmoc-Trp(Boc)-OH. They were
dissolved in N,N-dimethylformamide (DMF) and, according to the
sequence, activated using O-benzotriazol-1-yl-N,N,
N',N'-tetramethyl-uronium hexafluorophosphate (HBTU) and
diisopropylethylamine (DIEA). Removal of the Fmoc protecting group
was achieved using a solution of 20% (V/V) piperidine in
N,N-dimethylformamide (DMF) for 20 minutes (step 1). The amino
group of the final amino acid was acetylated using acetic acid
activated using O-benzotriazol-1-yl-N,N, N',N'-tetramethyl-uronium
hexafluorophosphate (HBTU) and diisopropylethylamine (DIEA). Step
2: The selective deprotection of the Lys (Aloc) group was performed
manually and accomplished by treating the resin with a solution of
3 eq of Pd(PPh.sub.3).sub.4 dissolved in 5 mL of
C.sub.6H.sub.6CHCl.sub.3 (1:1): 2.5% NMM (v:v): 5% AcOH (v:v) for 2
h (Step 2). The resin is then washed with CHCl.sub.3 (6.times.5
mL), 20% AcOH in DCM (6.times.5 mL), DCM (6.times.5 mL), and DMF
(6.times.5 mL). Step 3: The synthesis was then re-automated for the
addition of the Fmoc-AEEA-OH and the 3-maleimidopropionic acid
(Step 3). Between every coupling, the resin was washed 3 times with
N,N-dimethylformamide (DMF) and 3 times with isopropanol
(.sup.iPrOH). Step 4: The peptide was cleaved from the resin using
85% TFA/5% TIS/5% thioanisole and 5% phenol, followed by
precipitation by dry-ice cold Et.sub.2O (Step 4).
Example 3
Compound of Formula III
[0049] Step 1: The example describes the solid phase peptide
synthesis of the compound on a 100 .mu.mole scale. The following
protected amino acids were sequentially added to resin:
Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Glu(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH,
Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ser(tBu)-OH,
Fmoc-Glu(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH,
Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Tyr(tBu)-OH,
Fmoc-Lys(Aloc)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ile-OH, Fmoc-Glu(tBu)-OH,
Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Trp(Boc)-OH,
Fmoc-Glu(tBu)-OH, Fmoc-Met-OH, Fmoc-Trp(Boc)-OH. They were
dissolved in N,N-dimethylformamide (DMF) and, according to the
sequence, activated using
O-benzotriazol-1-yl-N,N,N',N'-tetramethyl-uronium
hexafluorophosphate (HBTU) and diisopropylethylamine (DIEA).
Removal of the Fmoc protecting group was achieved using a solution
of 20% (V/V) piperidine in N,N-dimethylformamide (DMF) for 20
minutes (step 1). The amino group of the final amino acid was
acetylated using acetic acid activated using
O-benzotriazol-1-yl-N,N,N',N'-tetramethyl-uronium
hexafluorophosphate (HBTU) and diisopropylethylamine (DIEA). Step
2: The selective deprotection of the Lys (Aloc) group was performed
manually and accomplished by treating the resin with a solution of
3 eq of Pd(PPh.sub.3).sub.4 dissolved in 5 mL of
C.sub.6H.sub.6CHCl.sub.3 (1:1): 2.5% NMM (v:v): 5% AcOH (v:v) for 2
h (Step 2). The resin is then washed with CHCl.sub.3 (6.times.5
mL), 20% AcOH in DCM (6.times.5 mL), DCM (6.times.5 mL), and DMF
(6.times.5 mL). Step 3: The synthesis was then re-automated for the
addition of the Fmoc-AEEA-OH and the 3-maleimidopropionic acid
(Step 3). Between every coupling, the resin was washed 3 times with
N,N-dimethylformamide (DMF) and 3 times with isopropanol (iPrOH).
Step 4: The peptide was cleaved from the resin using 85% TFA/5%
TIS/5% thioanisole and 5% phenol, followed by precipitation by
dry-ice cold Et.sub.2O (Step 4).
Example 4
Compound of Formula IV
[0050] Step 1: The example describes the solid phase peptide
synthesis of the compound on a 100 .mu.mole scale. The following
protected amino acids were sequentially added to resin:
Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Glu(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH,
Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ser(tBu)-OH,
Fmoc-Glu(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH,
Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Tyr(tBu)-OH,
Fmoc-Asn(Trt)-OH, Fmoc-Lys(Aloc)-OH, Fmoc-Ile-OH, Fmoc-Glu(tBu)-OH,
Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Trp(Boc)-OH,
Fmoc-Glu(tBu)-OH, Fmoc-Met-OH, Fmoc-Trp(Boc)-OH. They were
dissolved in N,N-dimethylformamide (DMF) and, according to the
sequence, activated using
O-benzotriazol-1-yl-N,N,N',N'-tetramethyl-uronium
hexafluorophosphate (HBTU) and diisopropylethylamine (DIEA).
Removal of the Fmoc protecting group was achieved using a solution
of 20% (V/V) piperidine in N,N-dimethylformamide (DMF) for 20
minutes (step 1). The amino group of the final amino acid was
acetylated using acetic acid activated using
O-benzotriazol-1-yl-N,N,N',N'-tetramethyl-uronium
hexafluorophosphate (HBTU) and diisopropyl-ethylamine (DIEA). Step
2: The selective deprotection of the Lys (Aloc) group was performed
manually and accomplished by treating the resin with a solution of
3 eq of Pd(PPh.sub.3).sub.4 dissolved in 5 mL of
C.sub.6H.sub.6CHCl.sub.3 (1:1): 2.5% NMM (v:v): 5% AcOH (v:v) for 2
h (Step 2). The resin is then washed with CHCl.sub.3 (6.times.5
mL), 20% AcOH in DCM (6.times.5 mL), DCM (6.times.5 mL), and DMF
(6.times.5 mL). Step 3: The synthesis was then re-automated for the
addition of the Fmoc-AEEA-OH and the 3-maleimidopropionic acid
(Step 3). Between every coupling, the resin was washed 3 times with
N,N-dimethylformamide (DMF) and 3 times with isopropanol
(.sup.iPrOH). Step 4: The peptide was cleaved from the resin using
85% TFA/5% TIS/5% thioanisole and 5% phenol, followed by
precipitation by dry-ice cold Et.sub.2O (Step 4).
Example 5
Compound of Formula V
[0051] Step 1: The example describes the solid phase peptide
synthesis of the compound on a 100 .mu.mole scale. The following
protected amino acids were sequentially added to resin:
Fmoc-Lys(Aloc)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH,
Fmoc-Gln(Trt)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Asn(Trt)-OH,
Fmoc-Ser(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Gln(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Ser(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Ile-OH,
Fmoc-Leu-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Tyr(tBu)-OH,
Fmoc-Asn(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ile-OH, Fmoc-Glu(tBu)-OH,
Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Trp(Boc)-OH,
Fmoc-Glu(tBu)-OH, Fmoc-Met-OH, Fmoc-Trp(Boc)-OH. They were
dissolved in N,N-dimethylformamide (DMF) and, according to the
sequence, activated using
O-benzotriazol-1-yl-N,N,N',N'-tetramethyl-uronium
hexafluorophosphate (HBTU) and diisopropylethylamine (DIEA).
Removal of the Fmoc protecting group was achieved using a solution
of 20% (V/V) piperidine in N,N-dimethylformamide (DMF) for 20
minutes (step 1). The amino group of the final amino acid was
acetylated using acetic acid activated using
O-benzotriazol-1-yl-N,N, N',N'-tetramethyl-uronium
hexafluorophosphate (HBTU) and diisopropylethyl-amine (DIEA). Step
2: The selective deprotection of the Lys (Aloc) group was performed
manually and accomplished by treating the resin with a solution of
3 eq of Pd(PPh.sub.3).sub.4 dissolved in 5 mL of
C.sub.6H.sub.6CHCl.sub.3 (1:1): 2.5% NMM (v:v): 5% AcOH (v:v) for 2
h (Step 2). The resin is then washed with CHCl.sub.3 (6.times.5
mL), 20% AcOH in DCM (6.times.5 mL), DCM (6.times.5 mL), and DMF
(6.times.5 mL). Step 3: The synthesis was then re-automated for the
addition of the 3-maleimido-propionic acid (Step 3). Between every
coupling, the resin was washed 3 times with N,N-dimethylformamide
(DMF) and 3 times with isopropanol (.sup.iPrOH). Step 4: The
peptide was cleaved from the resin using 85% TFA/5% TIS/5%
thioanisole and 5% phenol, followed by precipitation by dry-ice
cold Et.sub.2O (Step 4).
Example 6
Compound of Formula VI
[0052] Step 1: The example describes the solid phase peptide
synthesis of the compound on a 100 .mu.mole scale. The following
protected amino acids were sequentially added to resin:
Fmoc-Lys(Aloc)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH,
Fmoc-Gln(Trt)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Asn(Trt)-OH,
Fmoc-Ser(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Gln(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Ser(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Ile-OH,
Fmoc-Leu-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Tyr(tBu)-OH,
Fmoc-Asn(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ile-OH, Fmoc-Glu(tBu)-OH,
Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Trp(Boc)-OH,
Fmoc-Glu(tBu)-OH, Fmoc-Met-OH, Fmoc-Trp(Boc)-OH. They were
dissolved in N,N-dimethylformamide (DMF) and, according to the
sequence, activated using
O-benzotriazol-1-yl-N,N,N',N'-tetramethyl-uronium
hexafluorophosphate (HBTU) and diisopropylethylamine (DIEA).
Removal of the Fmoc protecting group was achieved using a solution
of 20% (V/V) piperidine in N,N-dimethylformamide (DMF) for 20
minutes (step 1). The amino group of the final amino acid was
acetylated using acetic acid activated using
O-benzotriazol-1-yl-N,N, N',N'-tetramethyl-uronium
hexafluoro-phosphate (HBTU) and diisopropylethyl-amine (DIEA). Step
2: The selective deprotection of the Lys (Aloc) group was performed
manually and accomplished by treating the resin with a solution of
3 eq of Pd(PPh.sub.3).sub.4 dissolved in 5 mL of
C.sub.6H.sub.6CHCl.sub.3 (1:1): 2.5% NMM (v:v): 5% AcOH (v:v) for 2
h (Step 2). The resin is then washed with CHCl.sub.3 (6.times.5
mL), 20% AcOH in DCM (6.times.5 mL), DCM (6.times.5 mL), and DMF
(6.times.5 mL). Step 3: The synthesis was then re-automated for the
addition of the Fmoc-AEEA-OH and the 3-maleimidopropionic acid
(Step 3). Between every coupling, the resin was washed 3 times with
N,N-dimethylformamide (DMF) and 3 times with isopropanol
(.sup.iPrOH). Step 4: The peptide was cleaved from the resin using
85% TFA/5% TIS/5% thioanisole and 5% phenol, followed by
precipitation by dry-ice cold Et.sub.2O (Step 4).
Example 7
Compound of Formula VII
[0053] Step 1: The example describes the solid phase peptide
synthesis of the compound on a 100 .mu.mole scale. The following
protected amino acids were sequentially added to resin:
Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Glu(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH,
Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ser(tBu)-OH,
Fmoc-Glu(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH,
Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Tyr(tBu)-OH,
Fmoc-Asn(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ile-OH, Fmoc-Glu(tBu)-OH,
Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Trp(Boc)-OH,
Fmoc-Glu(tBu)-OH, Fmoc-Met-OH, Fmoc-Trp(Boc)-OH. They were
dissolved in N,N-dimethylformamide (DMF) and, according to the
sequence, activated using
O-benzotriazol-1-yl-N,N,N',N'-tetramethyl-uronium
hexafluorophosphate (HBTU) and diisopropylethylamine (DIEA).
Removal of the Fmoc protecting group was achieved using a solution
of 20% (V/V) piperidine in N,N-dimethylformamide (DMF) for 20
minutes (step 1). The amino group of the final amino acid was
acetylated using acetic acid activated using
O-benzotriazol-1-yl-N,N,N',N'-tetramethyl-uronium
hexafluorophosphate (HBTU) and diisopropylethylamine (DIEA). Step
2: The synthesis was continued for the addition of the
3-maleimidopropionic acid (Step 2). Between every coupling, the
resin was washed 3 times with N,N-dimethyl-formamide (DMF) and 3
times with isopropanol (.sup.iPrOH). Step 3: The peptide was
cleaved from the resin using 85% TFA/5% TIS/5% thioanisole and 5%
phenol, followed by precipitation by thy-ice cold Et.sub.2O (Step
3).
Example 8
Compound of Formula VIII
[0054] Step 1: The example describes the solid phase peptide
synthesis of the compound on a 100 .mu.mole scale. The following
protected amino acids were sequentially added to resin:
Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Glu(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH,
Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ser(tBu)-OH,
Fmoc-Glu(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH,
Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Tyr(tBu)-OH,
Fmoc-Asn(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ile-OH, Fmoc-Glu(tBu)-OH,
Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Trp(Boc)-OH,
Fmoc-Glu(tBu)-OH, Fmoc-Met-OH, Fmoc-Trp(Boc)-OH. They were
dissolved in N,N-dimethylformamide (DMF) and, according to the
sequence, activated using
O-benzotriazol-1-yl-N,N,N',N'-tetramethyl-uronium
hexafluorophosphate (HBTU) and diisopropylethylamine (DIEA).
Removal of the Fmoc protecting group was achieved using a solution
of 20% (V/V) piperidine in N,N-dimethylformamide (DMF) for 20
minutes (step 1). The amino group of the final amino acid was
acetylated using acetic acid activated using
O-benzotriazol-1-yl-N,N,N',N'-tetramethyl-uronium
hexafluorophosphate (HBTU) and diisopropylethylamine (DIEA). Step
2: The synthesis was continued for the addition of the FMOC-AEEA-OH
and the 3-maleimidopropionic acid (Step 2). Between every coupling,
the resin was washed 3 times with N,N-dimethylformamide (DMF) and 3
times with isopropanol (.sup.iPrOH). Step 3: The peptide was
cleaved from the resin using 85% TFA/5% TIS/5% thioanisole and 5%
phenol, followed by precipitation by dry-ice cold Et.sub.2O (Step
3).
Cellular Anti-HIV Assay (MTT Assay)
[0055] The antiviral activity was determined as described in
Journal of Virological Methods, 1988, 20, 309-321. Briefly, various
concentrations of the test compound were brought into each well of
a flat-bottom microtiter plate. Subsequently, HIV strain (HIV-1
IIIB) and MT-4 cells were added to a final concentration of 200
CCID.sub.50/well and 30,000 cells/well, respectively. In order to
determine the toxicity of the test compound, mock-infected cell
cultures containing an identical compound concentration range, were
incubated in parallel with the HIV-infected cell cultures. After 5
days of incubation (37.degree. C., 5% CO.sub.2), the viability of
the cells was determined by the tetrazolium colorimetric MTT
method. The results of both assays appear in Table 2 below.
TABLE-US-00002 TABLE 2 Antiviral assay Compound Comment IC50
(.mu.M) Native C34 -- 0.0064 Formula I quenched 0.0063 HSA
conjugate 0.1149 Formula II quenched 0.0052 HSA conjugate 0.0200
Formula III quenched 0.0077 HSA conjugate 0.0232 Formula IV
quenched 0.0048 HSA conjugate 0.0207 Formula V quenched 0.0072 HSA
conjugate 0.439 Formula VI quenched 0.0047 HSA conjugate 0.0253
Formula VII quenched 0.3171 HSA conjugate 0.6602 Formula VIII
quenched 0.0015 HSA conjugate 0.0175
[0056] While the invention has been described in connection with
specific embodiments thereof, it will be understood that it is
capable of further modifications, and this application is intended
to cover any variations, uses or adaptations of the invention
following, in general, the principles of the invention, and
including such departures from the present description as come
within known or customary practice within the art to which the
invention pertains, and as may be applied to the essential features
hereinbefore set forth, and as follows in the scope of the appended
claims.
Sequence CWU 1
1
9134PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 1Trp Met Glu Trp Asp Arg Glu Ile Asn Asn Tyr
Thr Ser Leu Ile His1 5 10 15Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln
Glu Lys Asn Glu Gln Glu 20 25 30Leu Leu234PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
2Trp Met Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr Ser Leu Ile His1 5
10 15Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu Xaa Asn Glu Gln
Glu 20 25 30Leu Leu334PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 3Trp Met Glu Trp Asp Arg
Glu Ile Asn Asn Tyr Thr Xaa Leu Ile His1 5 10 15Ser Leu Ile Glu Glu
Ser Gln Asn Gln Gln Glu Lys Asn Glu Gln Glu 20 25 30Leu
Leu434PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 4Trp Met Glu Trp Asp Arg Glu Ile Asn Xaa Tyr
Thr Ser Leu Ile His1 5 10 15Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln
Glu Lys Asn Glu Gln Glu 20 25 30Leu Leu534PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
5Trp Met Glu Trp Asp Arg Glu Ile Xaa Asn Tyr Thr Ser Leu Ile His1 5
10 15Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys Asn Glu Gln
Glu 20 25 30Leu Leu635PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 6Trp Met Glu Trp Asp Arg
Glu Ile Asn Asn Tyr Thr Ser Leu Ile His1 5 10 15Ser Leu Ile Glu Glu
Ser Gln Asn Gln Gln Glu Lys Asn Glu Gln Glu 20 25 30Leu Leu Xaa
35735PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 7Trp Met Glu Trp Asp Arg Glu Ile Asn Asn Tyr
Thr Ser Leu Ile His1 5 10 15Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln
Glu Lys Asn Glu Gln Glu 20 25 30Leu Leu Xaa 35834PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
8Xaa Met Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr Ser Leu Ile His1 5
10 15Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys Asn Glu Gln
Glu 20 25 30Leu Leu934PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 9Xaa Met Glu Trp Asp Arg
Glu Ile Asn Asn Tyr Thr Ser Leu Ile His1 5 10 15Ser Leu Ile Glu Glu
Ser Gln Asn Gln Gln Glu Lys Asn Glu Gln Glu 20 25 30Leu Leu
* * * * *