U.S. patent application number 16/986863 was filed with the patent office on 2020-11-26 for compounds for inhibiting ns3 and compositions containing the inhibited protein.
This patent application is currently assigned to King Abdulaziz University. The applicant listed for this patent is King Abdulaziz University. Invention is credited to Hani Zakariah Asfour, Faida Hassan Bamane, Moustafa E. EL-ARABY, Mahmoud Abdelkhalek El-Faky, Maan Talaat Khayat, Abdelsattar Mansour Omar, Sameh Hamdy Abdelmageed Soror.
Application Number | 20200368204 16/986863 |
Document ID | / |
Family ID | 1000005207278 |
Filed Date | 2020-11-26 |
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United States Patent
Application |
20200368204 |
Kind Code |
A1 |
EL-ARABY; Moustafa E. ; et
al. |
November 26, 2020 |
COMPOUNDS FOR INHIBITING NS3 AND COMPOSITIONS CONTAINING THE
INHIBITED PROTEIN
Abstract
Imidazole-based compounds as hepatitis C virus (HCV) inhibitors.
The compounds have an imidazole core that is disubstituted via
amide links. Also described are a pharmaceutical composition
incorporating the imidazole-based compound, a method of preparing
these compounds, and a method for using the pharmaceutical
composition in the treatment of HCV infection.
Inventors: |
EL-ARABY; Moustafa E.;
(Jeddah, SA) ; Omar; Abdelsattar Mansour; (Jeddah,
SA) ; El-Faky; Mahmoud Abdelkhalek; (Jeddah, SA)
; Soror; Sameh Hamdy Abdelmageed; (Jeddah, SA) ;
Khayat; Maan Talaat; (Jeddah, SA) ; Asfour; Hani
Zakariah; (Jeddah, SA) ; Bamane; Faida Hassan;
(Jeddah, SA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
King Abdulaziz University |
Jeddah |
|
SA |
|
|
Assignee: |
King Abdulaziz University
Jeddah
SA
|
Family ID: |
1000005207278 |
Appl. No.: |
16/986863 |
Filed: |
August 6, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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16384472 |
Apr 15, 2019 |
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16986863 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 31/14 20180101;
A61K 31/4164 20130101 |
International
Class: |
A61K 31/4164 20060101
A61K031/4164; A61P 31/14 20060101 A61P031/14 |
Claims
1: A composition, comprising: non-structural protein 3 (NS3), and a
compound of formula (I) ##STR00016## a salt thereof, a solvate
thereof, or a mixture thereof, wherein R.sub.1 is n-pentyl; and m
is 4, wherein the compound of formula (I) is bonded to an active
site of the NS3 to inhibit protease activity of the NS3.
2-12. (canceled)
13: The composition of claim 1, further comprising: a
pharmaceutically acceptable carrier and/or excipient.
14. (canceled)
15: The composition of claim 13, wherein the pharmaceutically
acceptable carrier and/or excipient is at least one selected from
the group consisting of a buffer, an inorganic salt, a fatty acid,
a vegetable oil, a synthetic fatty ester, a surfactant, and a
polymer.
16: The composition of claim 13, further comprising an antiviral
agent.
17-20. (canceled)
21: The composition of claim 13, further comprising a body fluid.
Description
STATEMENT OF FUNDING ACKNOWLEDGEMENT
[0001] This project was funded by the National Plan for Science,
Technology and Innovation (MAARIFAH), King Abdulaziz City for
Science and Technology, the Kingdom of Saudi Arabia under award
number 12-BIO3193-03. The project was also supported by the Science
and Technology Unit of King Abdulaziz University.
BACKGROUND OF THE INVENTION
Technical Field
[0002] The present disclosure relates to a family of disubstituted
imidazole compounds. A pharmaceutical composition involving the
compounds, a method of preparing the compounds, and a method of
treating or inhibiting hepatitis C infection via inhibiting the
functioning of the NS3 protease using the compounds are
disclosed.
Description of the Related Art
[0003] The "background" description provided herein is for the
purpose of generally presenting the context of the disclosure. Work
of the presently named inventors, to the extent it is described in
this background section, as well as aspects of the description
which may not otherwise qualify as prior art at the time of filing,
are neither expressly or impliedly admitted as prior art against
the present invention.
[0004] Hepatitis C is a life-threatening viral infection that is
wide spread in the world [Organization, W. H., Global hepatitis
report 2017. 2017]. Initially, the virus infects liver cells but
remains asymptomatic for an extended period [Dickson, R. C.,
Clinical manifestations of hepatitis C. Clinics in liver disease
1997, 1 (3), 569-85]. This is an epidemiological challenge because
patients diagnosed with hepatitis C come from two pools;
recently-infected and later-stage asymptomatic. The initial
symptoms of hepatitis C such as fever, fatigue, nausea, and liver
tenderness can be misleading because they are tolerated by most
patients. After several years of infection, the virus activates,
replicates, and causes complications that start with liver
scarring, fibrosis followed by cirrhosis and eventually liver
failure and carcinoma [Friedrich, M. J., Third millennium
challenge: hepatitis C. Jama 1999, 282 (3), 221-2]. The hepatitis C
virus (HCV) is usually transmitted via the blood of an infected
person [Bocket, L.; Chevaliez, S.; Talbodec, N.; Sobaszek, A.;
Pawlotsky, J. M.; Yazdanpanah, Y., Occupational transmission of
hepatitis C virus resulting from use of the same supermarket meat
slicer. Clinical microbiology and infection: the official
publication of the European Society of Clinical Microbiology and
Infectious Diseases 2011, 17 (2), 238-41]. Awareness of hepatitis C
health problems and forceful efforts to combat the spread on HCV in
healthcare settings have led to a significant decline in new
infection cases [Organization, W. H., Global hepatitis report 2017.
2017; and Hatzakis, A. et al. The present and future disease burden
of hepatitis C virus (HCV) infections with today's treatment
paradigm--volume 2. J Viral Hepat 2015, 22 Suppl 1, 26-45].
However, the death toll from hepatitis C complications remains as
high as 400,000 annually due to the above described unique clinical
vs. epidemic profile of hepatitis C. An estimated 70 million people
globally are infected with HCV, constituting a major health problem
[Collaborators, P. O. H., Global prevalence and genotype
distribution of hepatitis C virus infection in 2015: a modelling
study. Lancet Gastroenterol Hepatol 2017, 2 (3), 161-176]. In 2013,
the death rate from hepatitis C complications surpassed that of
HIV. In 2015, hepatitis C-related deaths exceeded those of
tuberculosis and malaria combined [Stanaway, J. D. et al. The
global burden of viral hepatitis from 1990 to 2013: findings from
the Global Burden of Disease Study 2013. Lancet (London, England)
2016, 388 (10049), 1081-1088].
[0005] Up until 2013, interferon (or pegylated interferon) and
ribavirin were the most effective available therapies, however the
long-term efficacies were as low as 3% [North, C. S.; Hong, B. A.;
Adewuyi, S. A.; Pollio, D. E.; Jain, M. K.; Devereaux, R.; Quartey,
N. A.; Ashitey, S.; Lee, W. M.; Lisker-Melman, M., Hepatitis C
treatment and SVR: the gap between clinical trials and real-world
treatment aspirations. General hospital psychiatry 2013, 35 (2),
122-8]. Identification of the hepatitis C genome and proteome in
early 1990s prompted research efforts that led to development of
direct antiviral agents (DAA) in 2011 including bocepreivir and
telaprevir. The DAAs revolutionized the treatment of HCV
infection.sup.9-11 and the current situation of the treatment looks
promising. Nonetheless, new therapies will likely be needed to
address emerging resistance of this rapidly mutating virus
[Bartenschlager, R.; Baumert, T. F.; Bukh, J.; Houghton, M.; Lemon,
S. M.; Lindenbach, B. D.; Lohmann, V.; Moradpour, D.; Pietschmann,
T.; Rice, C. M.; Thimme, R.; Wakita, T., Critical challenges and
emerging opportunities in hepatitis C virus research in an era of
potent antiviral therapy: Considerations for scientists and funding
agencies. Virus research 2018, 248, 53-62].
[0006] The HCV genome is a positive-sense, single-stranded RNA
virus belonging to Flaviviridae [Murray, C. L.; Jones, C. T.; Rice,
C. M., Architects of assembly: roles of Flaviviridae non-structural
proteins in virion morphogenesis. Nature reviews microbiology 2008,
6 (9), 699-708]. It is composed of about 9,400 nucleotides with
highly conserved 5' and 3' terminal regions. The untranslated 5'
terminus is followed by a single open reading frame that encodes a
polyprotein of 3010 to 3303 amino acids. The virus genome
translates mainly to structural and non-structural proteins. The
non-structural (NS) proteins including NS3, NS4A, NS4B, NS5A and
NS5B comprise proteins that are important for maturation and
replication of the virus.
[0007] NS3 dually functions as protease (N-terminal domain) and RNA
helicase (C-terminal domain). When conformed as protease, NS3
catalyzes the processing of the viral proteome to functional
proteins by cleaving NS3-NS4A, NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B
junctions. NS5A is a non-enzymatic protein that is important for
viral replication however the exact mechanism is poorly understood.
NS5B is well characterized to be the viral RNA polymerase. NS4A is
a small (54 amino acids) and versatile multi-functioning peptide.
It acts as the activating cofactor of NS3 protease (N-terminal) and
NS3 helicase (C-terminal) [Ishido, S.; Fujita, T.; Hotta, H.,
Complex formation of NS5B with NS3 and NS4A proteins of hepatitis C
virus. Biochemical and biophysical research communications 1998,
244 (1), 35-40; and Kim, D. W.; Gwack, Y.; Han, J. H.; Choe, J.,
C-terminal domain of the hepatitis C virus NS3 protein contains an
RNA helicase activity. Biochemical and biophysical research
communications 1995, 215 (1), 160-166]. It is also important for
the integration of NS3 to the host cell endoplasmic reticulum
[Wolk, B.; Sansonno, D.; Krausslich, H.-G.; Dammacco, F.; Rice, C.
M.; Blum, H. E.; Moradpour, D., Subcellular localization,
stability, and trans-cleavage competence of the hepatitis C virus
NS3-NS4A complex expressed in tetracycline-regulated cell lines.
Journal of virology 2000, 74 (5), 2293-2304] and neutralization of
the immune response of the host cell towards the viral invasion
[Li, K.; Foy, E.; Ferreon, J. C.; Nakamura, M.; Ferreon, A. C.;
Ikeda, M.; Ray, S. C.; Gale, M.; Lemon, S. M., Immune evasion by
hepatitis C virus NS3/4A protease-mediated cleavage of the
Toll-like receptor 3 adaptor protein TR1F. Proceedings of the
National Academy of Sciences 2005, 102 (8), 2992-2997; and Meylan,
E.; Curran, J.; Hofmann, K.; Moradpour, D.; Binder, M.;
Bartenschlager, R.; Tschopp, J., Cardif is an adaptor protein in
the RIG-I antiviral pathway and is targeted by hepatitis C virus.
Nature 2005, 437 (7062), 1167]. The current approved DAAs elevated
the patient sustained cure to 95% in less than two months of use
[Kardashian, A. A.; Pockros, P. J., Novel emerging treatments for
hepatitis C infection: a fast-moving pipeline. Therapeutic advances
in gastroenterology 2017, 10 (2), 277-282]. Drugs available for
prescription are categorized to three classes: NS3/4A inhibitors,
e.g. telaprevir, boceprovir, simeprevir, asunaprevir, paritaprevir,
grazopreivir, and voxilaprevir (binding at the substrate site),
NS5A inhibitors, e.g. daclatasvir, ledipasvir, ombitasvir,
elbasvir, and velpatasvir, and NS5B inhibitors, e.g. soforbuvir,
and dasabuvir [Feld, J. J., Direct-Acting Antivirals for Hepatitis
C Virus (HCV): The Progress Continues. Current drug targets 2017,
18 (7), 851-862].
[0008] The use of DAA combinations with high rates of sustained
viral recovery (SVR) became routine in therapy regimens [Aghemo,
A.; Piroth, L.; Bhagani, S., What do clinicians need to watch for
with direct-acting antiviral therapy? Journal of the International
AIDS Society 2018, 21 Suppl 2, e25076]. For instance, a combination
of voxilaprevir (NS3/4A inhibitor), velpatasvir (NS5A inhibitor),
and sofosbuvir (NS5B inhibitor) that covers all HCV genotypes 1-6
has been prescribed to patients suffering relapse subsequent to
failure of DAA monotherapy [Chahine, E. B.; Kelley, D.;
Childs-Kean, L. M., Sofosbuvir/Velpatasvir/Voxilaprevir: A
Pan-Genotypic Direct-Acting Antiviral Combination for Hepatitis C.
The Annals of pharmacotherapy 2018, 52 (4), 352-363].
[0009] As outlined in the World Health Organization Global
Hepatitis Report, there are several remaining challenges that must
be met to eradicate HCV by 2030 [Organization, W. H., Global
hepatitis report 2017. 2017]: 1) access to the treatment in
economically-challenged areas must be increased [Chhatwal, J.;
Chen, Q.; Aggarwal, R., Estimation of Hepatitis C Disease Burden
and Budget Impact of Treatment Using Health Economic Modeling.
Infect Dis Clin North Am 2018, 32 (2), 461-480]; 2) emerging drug
resistance must be addressed [Cuypers, L.; Libin, P.; Schrooten,
Y.; Theys, K.; Di Maio, V. C.; Cento, V.; Lunar, M. M.; Nevens, F.;
Poljak, M.; Ceccherini-Silberstein, F., Exploring resistance
pathways for first-generation NS3/4A protease inhibitors boceprevir
and telaprevir using Bayesian network learning. Infection, Genetics
and Evolution 2017, 53, 15-23]; 3) efficacy in broader sectors of
HCV patients, such as elder and impaired kidney or liver patients,
must be increased [Hellard, M. E.; Chou, R.; Easterbrook, P., WHO
guidelines on testing for hepatitis B and C-meeting targets for
testing. BioMed Central: 2017]. Thus, there is a clear need to
identify new approaches to inhibit viral protein targets and treat
HCV infection. The current disclosure introduces a new class of
compounds that interfere with NS4A in its binding with NS3 protease
and deactivate the enzyme [Joyce, M.; Williams, M.; Hindsgaul, O.;
Tyrrell, D. Inhibitors of hepatitis C virus protease, U.S. patent
application Ser. No. 10/319,402].
[0010] In view of the forgoing, one objective of the present
disclosure is to provide therapeutic di-substituted imidazole-based
compounds and a process of synthesizing these compounds. A further
objective of the present disclosure is to provide a pharmaceutical
composition comprising the imidazole-based compound and a method of
treating or preventing HCV infection.
BRIEF SUMMARY OF THE INVENTION
[0011] According to a first aspect, the present disclosure relates
to a compound of formula (I)
##STR00001##
or a salt thereof, a solvate thereof, a tautomer thereof, a
stereoisomer thereof, or a mixture thereof, wherein (i) R.sub.1 is
selected from the group consisting of an optionally substituted
alkyl, an optionally substituted cycloalkyl, an optionally
substituted arylalkyl, and an optionally substituted aryl, and (ii)
m is an integer in a range of 2-8.
[0012] In one embodiment, R.sub.1 is an optionally substituted
C.sub.4-10 alkyl.
[0013] In one embodiment, R.sub.1 is a C.sub.4-C.sub.10 alkyl
optionally substituted with at least one substituent selected from
the group consisting of an alkyloxy, a cycloalkyloxy, an aryloxy,
an amine, and an amide.
[0014] In one embodiment, R.sub.1 is a linear C.sub.4-10 alkyl.
[0015] In one embodiment, R.sub.1 is at least one selected from the
group consisting of n-pentyl, n-hexyl, n-heptyl,
2-(cyclohexyloxy)ethyl, 3-(cyclohexyloxy)propyl, 2-phenoxyethyl,
3-phenoxypropyl, 4-isopropoxybutyl, 6-methylheptyl, and
3-methylpentyl.
[0016] In one embodiment, R.sub.1 is n-pentyl
[0017] In one embodiment, R.sub.1 is n-hexyl.
[0018] In one embodiment, m is 4.
[0019] In one embodiment, the compound of formula (I) is
##STR00002##
[0020] According to a second aspect, the present disclosure relates
to a compound of formula (II)
##STR00003##
or a salt thereof, a solvate thereof, a tautomer thereof, a
stereoisomer thereof, or a mixture thereof, wherein (i) R.sub.2 is
selected from the group consisting of an optionally substituted
amide, an optionally substituted aryl, and an optionally
substituted heteroaryl, R.sub.3 is selected from the group
consisting of an optionally substituted alkyl, an optionally
substituted cycloalkyl, an optionally substituted arylalkyl, an
optionally substituted alkenyl, and an optionally substituted aryl,
and (iii) n is an integer in a range of 1-4.
[0021] In one embodiment, R.sub.2 is selected from the group
consisting of an unsubstituted amide (--C(O)NH.sub.2),
N-methylamide (--C(O)NHCH.sub.3), a pyridyl, and a furyl.
[0022] In one embodiment, R.sub.3 is selected from the group
consisting of n-pentyl, n-hexyl, n-heptyl, 2-(cyclohexyloxy)ethyl,
3-(cyclohexyloxy)propyl, 2-phenoxyethyl, 3-phenoxypropyl,
4-isopropoxybutyl, 6-methylheptyl, 3-methylpentyl,
2-acetamidoethyl, and 3,7-dimethylocta-2,6-dien-1-yl.
[0023] According to a third aspect, the present disclosure relates
to a pharmaceutical composition involving the compound of formula
(I) of the first aspect and a pharmaceutically acceptable carrier
and/or excipient.
[0024] In one embodiment, the pharmaceutical composition contains
0.5-500 .mu.M of the compound of formula (I) relative to a total
volume of the composition.
[0025] In one embodiment, the pharmaceutically acceptable carrier
and/or excipient is at least one selected from the group consisting
of a buffer, an inorganic salt, a fatty acid, a vegetable oil, a
synthetic fatty ester, a surfactant, and a polymer.
[0026] In one embodiment, the pharmaceutical composition further
includes an antiviral agent.
[0027] In one embodiment, the compound of formula (I) is
##STR00004##
[0028] According to a fourth aspect, the present disclosure relates
to a method of preventing or treating hepatitis C virus (HCV)
infection. The method involves administering the pharmaceutical
composition of the third aspect to a subject in need of
therapy.
[0029] In one embodiment, 0.1-100 mg/kg of the compound of formula
(I) is administered per body weight of the subject.
[0030] In one embodiment, the compound of formula (I) binds to an
activation site of hepatitis C virus (HCV) serine protease.
[0031] The foregoing paragraphs have been provided by way of
general introduction, and are not intended to limit the scope of
the following claims. The described embodiments, together with
further advantages, will be best understood by reference to the
following detailed description taken in conjunction with the
accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] A more complete appreciation of the disclosure and many of
the attendant advantages thereof will be readily obtained as the
same becomes better understood by reference to the following
detailed description when considered in connection with the
accompanying drawings, wherein:
[0033] FIG. 1 is a schematic diagram illustrating the design of
imidazole scaffolds based on the planar region of NS4A.
[0034] FIG. 2 is a synthesis scheme for the preparation of starting
materials and compounds of formulae (I) and (II).
[0035] FIG. 3 shows a list of MOC compounds of the present
disclosure.
[0036] FIG. 4 shows the purity of NS3 using SDS-Page
electrophoresis method.
[0037] FIG. 5 shows a competition assay using fluorescence
anisotropy that determines ability of compounds of formula (I)
replacing FITC-NS4A in binding with NS3.
[0038] FIG. 6 is an enzyme inhibition assay that shows the effect
of compound MOC-23 on NS3 activity.
DETAILED DESCRIPTION OF THE EMBODIMENTS
[0039] Embodiments of the present disclosure will now be described
more fully hereinafter with reference to the accompanying drawings,
in which some, but not all embodiments of the disclosure are
shown.
[0040] As used herein, the words "a" and "an" and the like carry
the meaning of "one or more". Within the description of this
disclosure, where a numerical limit or range is stated, the
endpoints are included unless stated otherwise. Also, all values
and subranges within a numerical limit or range are specifically
included as if explicitly written out.
[0041] As used herein, the terms "complex", "compound", and
"product" are used interchangeably, and are intended to refer to a
chemical entity, whether in the solid, liquid or gaseous phase, and
whether in a crude mixture or purified and isolated.
[0042] As used herein, the term "solvate" refers to a physical
association of a compound of this disclosure with one or more
solvent molecules, whether organic or inorganic. This physical
association includes hydrogen bonding. In certain instances, the
solvate will be capable of isolation, for example when one or more
solvent molecules are incorporated in the crystal lattice of the
crystalline solid. The solvent molecules in the solvate may be
present in a regular arrangement and/or a non-ordered arrangement.
The solvate may comprise either a stoichiometric or
nonstoichiometric amount of the solvent molecules. Solvate
encompasses both solution phase and isolable solvates. Exemplary
solvents include, but are not limited to, water, methanol, ethanol,
n-propanol, isopropanol, n-butanol, isobutanol, tert-butanol, ethyl
acetate and other lower alkanols, glycerine, acetone,
dichloromethane (DCM), dimethyl sulfoxide (DMSO), dimethyl acetate
(DMA), dimethylformamide (DMF), isopropyl ether, acetonitrile,
toluene, N-methylpyrrolidone (NMP), tetrahydrofuran (THF),
tetrahydropyran, other cyclic mono-, di- and tri-ethers,
polyalkylene glycols (e.g. polyethylene glycol, polypropylene
glycol, propylene glycol), and mixtures thereof in suitable
proportions. Exemplary solvates include, but are not limited to,
hydrates, ethanolates, methanolates, isopropanolates and mixtures
thereof. Methods of solvation are generally known to those of
ordinary skill in the art.
[0043] As used herein, the term "tautomer" refers to constitutional
isomers of organic compounds that readily convert by
tautomerization or tautomerism. The interconversion commonly
results in the formal migration of a hydrogen atom or proton,
accompanied by a switch of a single bond and adjacent double bond.
Tautomerism is a special case of structural isomerism, and because
of the rapid interconversion, tautomers are generally considered to
be the same chemical compound. In solutions in which
tautomerization is possible, a chemical equilibrium of the
tautomers will be reached. The exact ratio of the tautomers depends
on several factors including, but not limited to, temperature,
solvent and pH. Exemplary common tautomeric pairs include, but are
not limited to, ketone and enol, enamine and imine, ketene and
ynol, nitroso and oxime, amide and imidic acid, lactam and lactim
(an amide and imidic tautomerism in heterocyclic rings), and
open-chain and cyclic forms of an acetal or hemiacetal (e.g., in
reducing sugars).
[0044] As used herein, the term "stereoisomer" refers to isomeric
molecules that have the same molecular formula and sequence of
bonded atoms (i.e. constitution), but differ in the
three-dimensional orientations of their atoms in space. This
contrasts with structural isomers, which share the same molecular
formula, but the bond connection of their order differs. By
definition, molecules that are stereoisomers of each other
represent the same structural isomer. Enantiomers are two
stereoisomers that are related to each other by reflection, they
are non-superimposable mirror images. Every stereogenic center in
one has the opposite configuration in the other. Two compounds that
are enantiomers of each other have the same physical properties,
except for the direction in which they rotate polarized light and
how they interact with different optical isomers of other
compounds. Diastereomers are stereoisomers not related through a
reflection operation, they are not mirror images of each other.
These include meso compounds, cis- and trans- (E- and Z-) isomers,
and non-enantiomeric optical isomers. Diastereomers seldom have the
same physical properties. In terms of the present disclosure,
stereoisomers may refer to enantiomers, diastereomers, or both.
[0045] Conformers, rotamers, or conformational isomerism refers to
a form of isomerism that describes the phenomenon of molecules with
the same structural formula but with different shapes due to
rotations around one or more bonds. Different conformations can
have different energies, can usually interconvert, and are very
rarely isolatable. There are some molecules that can be isolated in
several conformations. Atropisomers are stereoisomers resulting
from hindered rotation about single bonds where the steric strain
barrier to rotation is high enough to allow for the isolation of
the conformers. In terms of the present disclosure, stereoisomers
may refer to conformers, atropisomers, or both.
[0046] In terms of the present disclosure, stereoisomers of the
double bonds, ring systems, stereogenic centers, and the like can
all be present in the compounds, and all such stable isomers are
contemplated in the present disclosure. Cis- and trans- (or E- and
Z-) stereoisomers of the compounds of the present disclosure
wherein rotation around the double bond is restricted, keeping the
substituents fixed relative to each other, are described and may be
isolated as a mixture of isomers or as separated isomeric forms. S-
and R- (or L- and D-) stereoisomers of the compounds of the present
disclosure are described and may be isolated as a mixture of
isomers or as separated isomeric forms. All processes or methods
used to prepare compounds of the present disclosure and
intermediates made therein are considered to be part of the present
disclosure. When stereoisomeric products are prepared, they may be
separated by conventional methods, for example, by chromatography,
fractional crystallization, or use of a chiral agent.
[0047] As used herein, the term "substituted" refers to at least
one hydrogen atom that is replaced with a non-hydrogen group,
provided that normal valencies are maintained and that the
substitution results in a stable compound. When a substituent is
noted as "optionally substituted", the substituents are selected
from halo, hydroxyl, alkoxy, oxo, alkanoyl, aryloxy, alkanoyloxy,
amino, alkylamino, arylamino, arylalkylamino, disubstituted amines
(e.g. in which the two amino substituents are selected from the
exemplary group including, but not limited to, alkyl, aryl or
arylalkyl), alkanoylamino, aroylamino, aralkanoylamino, substituted
alkanoylamino, substituted arylamino, substituted aralkanoylamino,
thiol, alkylthio, arylthio, arylalkylthio, alkylthiono, arylthiono,
aryalkylthiono, alkyl sulfonyl, arylsulfonyl, arylalkylsulfonyl,
sulfonamide (e.g. --SO.sub.2NH.sub.2), substituted sulfonamide,
nitro, cyano, carboxy, unsubstituted amide (i.e. --CONH.sub.2),
substituted amide (e.g. --CONHalkyl, --CONHaryl, --CONHarylalkyl or
cases where there are two substituents on one nitrogen from alkyl,
aryl, or alkylalkyl), alkoxycarbonyl, aryl, substituted aryl,
guanidine, heterocyclyl (e.g. indolyl, imidazoyl, furyl, thienyl,
thiazolyl, pyrrolidyl, pyridyl, pyrimidiyl, pyrrolidinyl,
piperidinyl, morpholinyl, piperazinyl, homopiperazinyl and the
like), substituted heterocyclyl and mixtures thereof. The
substituents may themselves be optionally substituted, and may be
either unprotected, or protected as necessary, as known to those
skilled in the art, for example, as taught in Greene, et al.,
"Protective Groups in Organic Synthesis", John Wiley and Sons,
Second Edition, 1991, hereby incorporated by reference in its
entirety.
[0048] As used herein, the term "alkyl" unless otherwise specified
refers to both branched and straight chain saturated aliphatic
primary, secondary, and/or tertiary hydrocarbons of typically
C.sub.1 to C.sub.21, for example C.sub.1, C.sub.2, C.sub.3,
C.sub.4, C.sub.5, C.sub.6, C.sub.7, C.sub.8, C.sub.9, C.sub.10,
C.sub.11, C.sub.12, C.sub.13, C.sub.14, and specifically includes,
but is not limited to, methyl, trifluoromethyl, ethyl, propyl,
isopropyl, cyclopropyl, butyl, isobutyl, t-butyl, pentyl,
cyclopentyl, isopentyl, neopentyl, hexyl, isohexyl, cyclohexyl,
cyclohexylmethyl, 3-methylpentyl, 2,2-dimethylbutyl,
2,3-dimethylbutyl, 2-ethylhexyl, heptyl, octyl, nonyl,
3,7-dimethyloctyl, decyl, undecyl, dodecyl, tridecyl,
2-propylheptyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl,
octadecyl, nonadecyl, and eicosyl.
[0049] The term "cycloalkyl" refers to cyclized alkyl groups.
Exemplary cycloalkyl groups include, but are not limited to,
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl, and
adamantyl. Branched cycloalkyl groups such as exemplary
1-methylcyclopropyl and 2-methylcyclopropyl groups are included in
the definition of cycloalkyl as used in the present disclosure.
[0050] The term "alkenyl" refers to a straight, branched, or cyclic
hydrocarbon fragment containing at least one C.dbd.C double bond.
Exemplary alkenyl groups include, without limitation, 1-propenyl,
2-propenyl (or "allyl"), 1-butenyl, 2-butenyl, 3-butenyl,
1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl,
2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-heptenyl, 2-heptenyl,
3-heptenyl, 4-heptenyl, 5-heptenyl, 6-heptenyl, 1-octenyl,
2-octenyl, 3-octenyl, 4-octenyl, 5-octenyl, 6-octenyl, 7-octenyl,
1-nonenyl, 2-nonenyl, 3-nonenyl, 4-nonenyl, 5-nonenyl, 6-nonenyl,
7-nonenyl, 8-nonenyl, 1-decenyl, 2-decenyl, 3-decenyl, 4-decenyl,
5-decenyl, 6-decenyl, 7-decenyl, 8-decenyl, and 9-decenyl.
[0051] The term "arylalkyl", as used herein, refers to a straight
or branched chain alkyl moiety having 1 to 8 carbon atoms that is
substituted by an aryl group as defined herein, and includes, but
is not limited to, benzyl, phenethyl, 2-methylbenzyl,
3-methylbenzyl, 4-methylbenzyl, 2,4-dimethylbenzyl,
2-(4-ethylphenyl)ethyl, 3-(3-propylphenyl)propyl, and the like.
[0052] The term "aryl", as used herein, and unless otherwise
specified, refers to phenyl, biphenyl, naphthyl, anthracenyl, and
the like.
[0053] The term "heteroaryl" refers to an aryl group where at least
one carbon atom is replaced with a heteroatom (e.g. nitrogen,
oxygen, sulfur) and can be indolyl, furyl, imidazolyl, triazolyl,
triazinyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl,
pyrazolyl, pyrrolyl, pyrazinyl, tetrazolyl, pyridyl (or its
N-oxide), thienyl, pyrimidinyl (or its N-oxide), 1H-indolyl,
isoquinolyl (or its N-oxide), or quinolyl (or its N-oxide), for
example.
[0054] The terms "alkoxy" and "alkyloxy" refer to a straight or
branched alkyl group attached to an oxygen atom. Exemplary alkyloxy
groups include, but are not limited to, methoxy, ethoxy, propoxy,
isopropoxy, butoxy, isobutoxy, secondary butoxy, tertiary butoxy,
pentoxy, isopentoxy, hexyloxy, heptyloxy, octyloxy, nonyloxy, and
decyloxy.
[0055] The terms "cycloalkoxy" and `cycloalkyloxy" as used herein
means a cycloalkyl group linked to an oxygen atom, such as, for
example, cyclobutyloxy or cyclopropyloxy.
[0056] The term "aryloxy" refers to an aryl group bonded to an
oxygen atom. Exemplary aryloxy groups include, but are not limited
to, phenoxy, 4-methylphenoxy, and naphthaloxy.
[0057] The term "amide", as used herein, and unless otherwise
specified, refers to an amide (--C(O)NR.sub.cR.sub.d) that is
unsustituted (--C(O)NH.sub.2), monosubstituted (where R.sub.c is a
hydrogen), or disubstituted where R.sub.c and R.sub.d are
independently an optionally substituted alkyl, an optionally
substituted cycloalkyl, an optionally substituted arylalkyl, or an
optionally substituted aryl.
[0058] As used herein, the term "amine" includes unsubstituted
amine (--NH.sub.2), monosubstituted amine (--NHR.sub.a), as well as
disubstituted amine (--NR.sub.aR.sub.b), wherein R.sub.a and
R.sub.b are independently an optionally substituted alkyl, an
optionally substituted cycloalkyl, an optionally substituted
arylalkyl, or an optionally substituted aryl.
[0059] The present disclosure is intended to include all isotopes
of atoms occurring in the present compounds. Isotopes include those
atoms having the same atomic number but different mass numbers. By
way of general example, and without limitation, isotopes of
hydrogen include deuterium and tritium, isotopes of carbon include
.sup.13C and .sup.14C, isotopes of nitrogen include .sup.14N and
.sup.15N, and isotopes of oxygen include .sup.16O, .sup.17O and
.sup.18O. Isotopically labeled compounds of the disclosure can
generally be prepared by conventional techniques known to those
skilled in the art or by processes and methods analogous to those
described herein, using an appropriate isotopically labeled reagent
in place of the non-labeled reagent otherwise employed.
[0060] According to a first aspect, the present disclosure relates
to a compound of formula (I)
##STR00005##
or a salt thereof, a solvate thereof, a tautomer thereof, a
stereoisomer thereof, or a mixture thereof.
[0061] R.sub.1 is selected from the group consisting of an
optionally substituted alkyl, an optionally substituted cycloalkyl,
an optionally substituted arylalkyl, and an optionally substituted
aryl.
[0062] In one or more embodiments, R.sub.1 is an optionally
substituted alkyl, which may be linear or branched.
[0063] In one or more embodiments, R.sub.1 is a substituted alkyl,
preferably a substituted C.sub.3-10 alkyl, preferably a substituted
C.sub.4-9 alkyl, preferably a substituted C.sub.5-8 alkyl,
preferably a substituted C.sub.6-7 alkyl. The carbon counts
described herein refers to a number of carbon atoms of the alkyl
group of R.sub.1 which excludes the carbon atoms of optionally
present substituents. The alkyl of R.sub.1 may be substituted with
at least one substituent selected from the group consisting of an
alkyloxy, a cycloalkyloxy, and an aryloxy, for example, methoxy,
ethoxy, isopropoxy, cyclopentyloxy, cyclohexyloxy, and phenoxy.
Alternatively, the alkyl of R.sub.1 may be substituted with an
amine and/or an amide. The alkyl of R.sub.1 may be preferably
substituted with an amide group such as acetamide, propanamide, and
isobutyramide. In a preferred embodiment, the alkyl of R.sub.1 is
substituted with acetamide.
[0064] In an alternative embodiment, R.sub.1 is an unsubstituted
alkyl, preferably a linear alkyl, preferably a linear C.sub.3-10
alkyl, preferably a linear C.sub.4-9 alkyl, preferably a linear
C.sub.5-8 alkyl, preferably a linear C.sub.6-7 alkyl. Exemplary
linear alkyls include, but are not limited to n-propyl, n-butyl,
n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl.
Alternatively, R.sub.1 is a branched alkyl, such as 6-methylheptyl,
3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 2-ethylhexyl,
3,7-dimethyloctyl, and 2-propylheptyl. In a preferred embodiment,
R.sub.1 is 6-methylheptyl, or 3-methylpentyl.
[0065] In one or more embodiments, R.sub.1 is at least one selected
from the group consisting of n-pentyl, n-hexyl, n-heptyl,
2-(cyclohexyloxy)ethyl, 3-(cyclohexyloxy)propyl, 2-phenoxyethyl,
3-phenoxypropyl, 4-isopropoxybutyl, 6-methylheptyl, 3-methylpentyl
and 2-acetamidoethyl. In a preferred embodiment, R.sub.1 is
n-pentyl. In another preferred embodiment, R.sub.1 is n-hexyl. In a
most preferred embodiment, R.sub.1 is n-pentyl.
[0066] As used herein, the value of m denotes an alkyl chain of
--CH.sub.2-- groups connected between guanidino and amide groups of
the compound of formula (I). In one or more embodiments, m is an
integer in a range of 2-9, preferably 3-8, preferably 4-7,
preferably 5-6. Most preferably, m is 4.
[0067] In some embodiments, the compound of formula (I) is one or
more of the following structures:
##STR00006##
[0068] In a most preferred embodiment, the compound of formula (I)
is
##STR00007##
[0069] According to a second aspect, the present disclosure relates
to a compound of formula (II)
##STR00008##
or a salt thereof, a solvate thereof, a tautomer thereof, a
stereoisomer thereof, or a mixture thereof.
[0070] R.sub.2 is selected from the group consisting of an
optionally substituted amide, an optionally substituted aryl, and
an optionally substituted heteroaryl.
[0071] In one or more embodiments, R.sub.2 is a monosubstituted
alkylamide, preferably a monosubstituted C.sub.2-5 alkylamide,
preferably a monosubstituted C.sub.3-4 alkylamide, such as
N-methylamide (--C(O)NHCH.sub.3), N-ethylamide
(--C(O)NHC.sub.2H.sub.5), and N-(n-propyl)amide
(--C(O)NHC.sub.3H.sub.7). Most preferably, R.sub.2 is
N-methylamide. In another embodiment, R.sub.2 is an unsubstituted
amide (--C(O)NH.sub.2).
[0072] Alternatively, R.sub.2 is an optionally substituted
heteroaryl. In a preferred embodiment, R.sub.2 is an optionally
substituted pyridyl, or an optionally substituted furyl. Most
preferably, R.sub.2 is a pyridyl, or a furyl.
[0073] As used herein, the value of n denotes an alkyl chain of
--CH.sub.2-- groups connected between --R.sub.2 and amide groups of
the compound of formula (II). In one or more embodiments, n is an
integer in a range of 1-4, or 2-3. In a preferred embodiment, n is
1.
[0074] R.sub.3 is selected from the group consisting of an
optionally substituted alkyl, an optionally substituted cycloalkyl,
an optionally substituted arylalkyl, an optionally substituted
alkenyl, and an optionally substituted aryl.
[0075] In one embodiment, R.sub.2 is a monosubstituted alkylamide,
and R.sub.3 is a substituted alkyl, preferably a substituted
C.sub.3-10 alkyl, preferably a substituted C.sub.4-9 alkyl,
preferably a substituted C.sub.5-8 alkyl, preferably a substituted
C.sub.6-7 alkyl. The alkyl of R.sub.3 may be substituted with at
least one substituent selected from the group consisting of an
alkyl, an alkyloxy, a cycloalkyloxy, and an aryloxy, for example,
methoxy, ethoxy, isopropoxy, cyclopentyloxy, cyclohexyloxy, and
phenoxy. Alternatively, R.sub.2 is a monosubstituted alkylamide,
and R.sub.3 is a linear (i.e. unsubstituted) alkyl, preferably a
linear C.sub.3-10 alkyl, preferably a linear C.sub.4-9 alkyl,
preferably a linear C.sub.5-8 alkyl, preferably a linear C.sub.6-7
alkyl. Exemplary linear alkyls include, but are not limited to
n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl,
n-decyl. In a preferred embodiment, the linear alkyl is n-pentyl,
n-hexyl, or n-heptyl. In another embodiment, R.sub.2 is an
unsubstituted amide (--C(O)NH.sub.2), and R.sub.3 is a substituted
alkyl, preferably a substituted C.sub.3-10 alkyl, preferably a
substituted C.sub.4-9 alkyl, preferably a substituted C.sub.5-8
alkyl, preferably a substituted C.sub.6-7 alkyl, most preferably
6-methylheptyl, or 3-methylpentyl. Alternatively, R.sub.2 is a
monosubstituted alkylamide, and R.sub.3 is an optionally
substituted alkenyl, preferably R.sub.3 is a substituted C.sub.5-16
alkenyl, preferably a substituted C.sub.6-14 alkenyl, preferably a
substituted C.sub.7-12 alkenyl, preferably a substituted C.sub.8-10
alkenyl. Most preferably, R.sub.3 is
3,7-dimethylocta-2,6-dien-1-yl.
[0076] In another embodiment, R.sub.2 is a heteraryl, and R.sub.3
is a substituted alkyl, preferably a substituted C.sub.3-10 alkyl,
preferably a substituted C.sub.4-9 alkyl, preferably a substituted
C.sub.5-8 alkyl, preferably a substituted C.sub.6-7 alkyl. The
alkyl of R.sub.3 may be substituted with an amide group. Exemplary
amide groups include, but are not limited to, acetamide,
propanamide, and isobutyramide. In a preferred embodiment, the
alkyl of R.sub.3 is substituted with acetamide. Alternatively,
R.sub.2 is a heteraryl, and R.sub.3 is a linear (i.e.
unsubstituted) alkyl, preferably a linear C.sub.3-10 alkyl,
preferably a linear C.sub.4-9 alkyl, preferably a linear C.sub.5-8
alkyl, preferably a linear C.sub.6-7 alkyl. Exemplary linear alkyls
include, but are not limited to n-propyl, n-butyl, n-pentyl,
n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl. In a preferred
embodiment, the linear alkyl is n-hexyl.
[0077] In at least one embodiment, R.sub.3 is selected from the
group consisting of n-pentyl, hexyl n-heptyl,
2-(cyclohexyloxy)ethyl, 3-(cyclohexyloxy)propyl, 2-phenoxyethyl,
3-phenoxypropyl, 4-isopropoxybutyl, 6-methylheptyl, 3-methylpentyl,
2-acetamidoethyl, and 3,7-dimethylocta-2,6-dien-1-yl.
[0078] In some embodiments, the compound of formula (II) has one of
the following structures:
##STR00009##
[0079] The compounds of the present disclosure may be prepared by
methods known to those of ordinary skills in the art. The following
methods set forth below are provided for illustrative purposes and
are not intended to limit the scope of the disclosure. It will be
recognized that it may be preferred or necessary to prepare such a
compound in which a functional group is protected using a
conventional protecting group then to remove the protecting group
to provide a compound of the present disclosure.
[0080] The compounds of formulae (I) and (II) may, for example, be
synthesized according to a process illustrated in FIG. 2. Briefly,
a 2,4-disubstituted imidazole (i.e.
2-(ethoxycarbonyl)-1H-imidazole-4-carboxylic acid (MOC-02)) may be
formed via (i) a cyclization reaction between an oxime (i.e.
(Z)-2-amino-2-(hydroxyimino) acetate) and an alkyne (i.e.
tert-butyl propiolate) thereby forming a disubstituted imidazole
tert-butyl ester (i.e. 5-(tert-butyl) 2-ethyl
1H-imidazole-2,5-dicarboxylate (MOC-01)), and (ii) deprotection of
tert-butyl ester group of MOC-01 thereby forming MOC-02 [Duguay,
G.; Guemas, J.-P.; Meslin, J.-C.; Pradere, J.-P.; Reliquet, F.;
Reliquet, A.; Tea-Gokou, C.; Quiniou, H.; Rabiller, C.,
Heteroatomic chains and their products of cyclisation. IV.
t-butyl-2-phthalimido-2-(3,6-dihydro-1,3-2H-thiazine-2-yliden)-acetates
substituted in position 5 by a functional group. Journal of
Heterocyclic Chemistry 1980, 17 (4), 767-770; and Vuilhorgne, M.;
Malpart, J.; Mutti, S.; Mignani, S., Preparative route to
2-ethoxycarbonylimidazole-4-phosphonate and
diethylimidazole-2,4-dicarboxylate. Journal of Heterocyclic
Chemistry 2003, 40 (1), 159-162, each incorporated herein by
reference in their entirety]. Alternatively, MOC-02 may be
available from commercial vendors including, without limitation,
Accel Pharmtech, Asta Tech, Ambeed, Alichem, BLD Pharmatech, and
FCH group.
[0081] In one embodiment, the guanidino side chain of compound of
formula (I) may be incorporated via amidation reaction between the
carboxylic group of MOC-02 and an amine of formula (III)
##STR00010##
wherein m is as previously specified, and R.sub.p is a protecting
group intended to avoid formation of undesired bonds during the
amidation. The details concerning the use of protecting groups in
accordance with the present invention are known to those of
ordinary skills in the art. Protecting groups that can be used are
listed, for example, in Greene, et al., "Protective Groups in
Organic Synthesis", Wiley-Interscience, 1999, hereby incorporated
by reference in its entirety. Exemplary protecting groups include,
but are not limited to, acyl groups such as formyl,
trifluoroacetyl, phthalyl, and p-toluenesulfonyl, aromatic
carbamate groups such as benzyloxycarbonyl (Cbz or Z), and
substituted bensyloxycarbonyls, and 9-fluorenylmethyloxycarbonyl
(Fmoc), aliphatic carbamate groups such as tert-butyloxycarbonyl
(Boc), ethoxycarbonyl, diisopropylmethoxycarbonyl, and allyloxy
carbonyl, cyclic alkyl carbamate groups such as
cyclopentyloxycarbonyl and adamantyloxycarbonyl, alkyl groups such
as triphenylmethyl and benzyl, trialkylsilyl such as
trimethylsilyl, and thiol containing groups such as
phenylthiocarbonyl, and dithiasuccinoyl. In a preferred embodiment,
R.sub.p of the amine of formula (III) is Boc. After the first
amidation reaction, a guanidino compound of formula (IV) may be
obtained
##STR00011##
The protecting group R.sub.p may be cleaved after the amidation
reaction using deprotection protocols generally known to those of
ordinary skill in the art. For example, when the Boc group is used,
the methods of choice may be trifluoroacetic acid, neat or in
dichloromethane, or HCl in dioxane or in ethyl acetate.
[0082] In another embodiment, the R.sub.2 amide side chain of the
compound of formula (II) may be incorporated via a first amidation
reaction between the carboxylic group of MOC-02 and an amine of
formula (V):
##STR00012##
wherein R.sub.2 and n are as previously specified. After the first
amidation reaction, a compound of formula (VI) may be obtained
##STR00013##
[0083] Generally, the first amidation reaction may be performed in
a solvent such as tetrahydrofuran, benzene, xylene,
dimethylformamide, ethyl acetate, diethyl ether, acetonitrile,
dimethyl sulfoxide, methylene chloride, chloroform, nitrobenzene,
isopropanol, and mixtures thereof. Preferably, tetrahydrofuran is
used as the solvent. In a preferred embodiment, a molar ratio of
the amine of formula (III) or (V) to the disubstituted imidazole
(i.e. MOC-02) is in a range of 1:1 to 3:1, preferably 1.05:1 to
2:1, preferably 1.1:1 to 1.5:1, or about 1.2:1.
[0084] In preferred embodiments, a base such as trimethylamine,
trimethylamine, diisopropylethylamine (DIPEA), triisopropylamine,
dimethylaminopropylamine, N-methylmorpholine, N-methylpyrrolidine,
4-dimethylaminopyridine (DMAP), and
1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) is present in the first
amidation reaction. Preferably, the base is DIPEA. In a preferred
embodiment, a molar ratio of the base to the disubstituted
imidazole (i.e. MOC-02) is in a range of 1:1 to 5:1, preferably
12:1 to 3:1, preferably 1.5:1 to 2:1, or about 1.75:1.
[0085] In a related embodiment, amide bond formation coupling
reagents may be used to facilitate and/or accelerate the first
amidation reaction. Examples of such coupling reagents include,
without limitation, carbodiimides such as
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC or EDCI),
N,N-dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide
(DTC), 1H-benzotriazole derivatives such as
1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium
3-oxid hexafluorophosphate (HATU),
O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HBTU),
O-benzotriazol-1-yl-1,1,3,3-tetramethyluronium tetrafluoroborate
(TBTU), and
N-[(5-chloro-3-oxido-1H-benzotriazol-1-yl)-4-morpholinylmethylene]-N-meth-
ylmethanaminium hexafluorophosphate (HDMC), and carbonyldiimidazole
(CDI).
[0086] In another related embodiment, an additive that minimizes
racemization during the amidation reaction may be used in
combination of the aforementioned coupling reagent. Exemplary
additives include, but are not limited to,
1-hydroxy-7-azabenzotriazole (HOAt), hydroxybenzotriazole (HOBt),
6-chloro-1-hydroxybenzotriazole (6-ClHOBt), ethyl
1-hydroxy-1H-1,2,3-triazole-4-carboxylate (HOCt),
1-hydroxy-2-pyridinone (HOPy), ethyl
2-cyano-2-(hydroxyimino)acetate (Oxyma),
N-hydroxy-5-norbornene2,3-dicarboximide (HONB),
N-hydroxysuccinimide (HOSu),
3-hydroxy-4-oxo-3,4-dihydro1,2,3-benzotriazine (HODhbt), and
copper(I) iodide. Preferably, the additive is HOBt.
[0087] In a most preferred embodiment, the coupling reagent is EDCI
and the additive is HOBt. A molar ratio of the coupling reagent
(e.g. EDCI) to the disubstituted imidazole (i.e. MOC-02) is in a
range of 1:1 to 3:1, preferably 1.2:1 to 2:1, or about 1.5:1. A
molar ratio of the coupling reagent (e.g. EDCI) to the additive
(e.g. HOBt) is in a range of 1:2 to 2:1, preferably 1:1.5 to 1.5:1,
or about 1:1.
[0088] In a preferred embodiment, the base, the coupling reagent,
and the additive are each introduced to the first amidation
reaction mixture in a two-stage or multi-stage fashion. For
example, a first portion of the base, the coupling reagent, or the
additive which is 50-75%, 55-70%, or 57-67% of a total mole of the
base, the coupling reagent, or the additive used herein may be
added to the reaction mixture and allowed to react for 1-5 hours,
2-4 hours, or about 3 hours, and subsequently a second portion of
the base, the coupling reagent, or the additive which is 25-50%,
30-45%, or 33-43% of a total mole of the base, the coupling
reagent, or the additive used herein may be added to the same
reaction mixture. Alternatively, the base, the coupling reagent,
and the additive may be introduced to the reaction mixture in one
batch. In one or more embodiments, the first amidation reaction
mixture is reacted at a temperature of 20-80.degree. C., preferably
30-70.degree. C., preferably 40-60.degree. C. under agitation. The
first amidation reaction may be conducted in inert gas (e.g.
nitrogen, argon, helium). Also, in some embodiments, the reaction
may not be conducted in inert gas, but in a vacuum.
[0089] Methods of agitation include, without limitation, using an
agitator, a vortexer, a rotary shaker, a magnetic stirrer, a
centrifugal mixer, or an overhead stirrer. In another embodiment,
the mixture and/or the reaction mixture is left to stand (i.e. not
stirred). Alternatively, the mixture and/or the reaction mixture is
sonicated in an ultrasonic bath or with an ultrasonic probe.
[0090] The compound of formula (I) may be produced via a second
amidation reaction. In one embodiment, the amidation reaction
involves (i) adding a base to the compound of formula (IV) to form
a carboxylate salt, and (ii) reacting the carboxylate salt with a
proper amine of formula (VII)
H.sub.2N--R.sub.1 (VII),
thereby forming the compound of formula (I), wherein R.sub.1 is as
previously specified.
[0091] The compound of formula (II) may be synthesized similarly
via a second amidation reaction between the compound of formula
(VI) and a proper amine of formula (VIII)
H.sub.2N--R.sub.3 (VIII),
wherein R.sub.3 is as previously described.
[0092] The second amidation reaction may be performed in a solvent
such as tetrahydrofuran, benzene, xylene, dimethylformamide, ethyl
acetate, diethyl ether, acetonitrile, dimethyl sulfoxide, methylene
chloride, chloroform, nitrobenzene, isopropanol, and mixtures
thereof. Preferably, tetrahydrofuran is used as the solvent for the
second amidation. In a preferred embodiment, a molar ratio of the
amine of formula (VII) to the compound of formula (IV) is in a
range of 0.9:1 to 2:1, preferably 1:1 to 1.5:1, or about 1.2:1.
[0093] Exemplary bases used herein for the second amidation
include, without limitation, inorganic bases such as lithium
hydroxide, sodium hydroxide, potassium hydroxide, rubidium
hydroxide, and cesium hydroxide, and organic bases such as
trimethylamine, trimethylamine, diisopropylethylamine (DIPEA),
triisopropylamine, dimethylaminopropylamine, N-methylmorpholine,
N-methylpyrrolidine, 4-dimethylaminopyridine (DMAP),
1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), and mixtures thereof.
Preferably, an inorganic base is used herein for the second
amidation. More preferably, lithium hydroxide is used as the base.
In one embodiment, a molar ratio of the base to the compound of
formula (IV) is in a range of 1:1 to 6:1, preferably 2:1 to 5:1,
more preferably 3:1 to 4:1.
[0094] A coupling reagent may be added to the reaction mixture of
the carboxylate salt and the amine of formula (VII) to facilitate
the second amidation reaction. Preferably, the coupling reagent is
a phosphorus-based coupling reagent. Exemplary phosphorus-based
coupling reagents include, but are not limited to, propylphosphonic
anhydride (T3P.RTM.), diphenylphosphorazidate (DPPA),
diethyl-2-(3-oxo-2,3-dihydro-1,2-benzisosulfonazolyl)phosphonate
(DEBP), N,N'-bismorpholinophosphonic chloride (BMPCl),
1-oxo-chlorophospholane (CptCl),
3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one diethyl
tartarate (DEPBT), phosphoric acid
3,5-dioxo-10-oxa-4-azatricyclo[5.2.1.0]dec-8-en-4-yl ester diphenyl
ester (ENDPP), norborn-5-ene-2,3-dicarboximidodiphenylphosphate
(NDPP), and bis(2-oxooxazolidin-3-yl)phosphinic chloride (BopCl).
In a preferred embodiment, T3P.RTM. is used herein as the coupling
reagent. Alternatively, the coupling reagents and additives
described previously for the first amidation reaction may be used
in addition to, or in lieu of the phosphorus-based coupling
reagent.
[0095] In one embodiment, a molar ratio of the phosphorus-based
coupling reagent to the amine of formula (VII) is in a range of 4:1
to 20:1, preferably 6:1 to 16:1, preferably 8:1 to 12:1, or about
10:1. Preferably, the phosphorus-based coupling reagent may be
introduced to the second amidation reaction in a two-stage or
multi-stage fashion. Alternatively, the phosphorus-based coupling
reagent may be introduced to the reaction mixture in one batch. In
one or more embodiments, the second amidation reaction is performed
at a temperature of 4-40.degree. C., preferably 10-30.degree. C.,
preferably 15-25.degree. C. under agitation. The second amidation
reaction may be conducted in inert gas (e.g. nitrogen, argon,
helium). Also, in some embodiments, the reaction may not be
conducted in inert gas, but in a vacuum.
[0096] The compound of formulae (I) and (II) may be isolated and
purified by methods known to those of ordinary skill in the art,
such as crystallization, filtration through a celite containing
cartridge, evaporating the reaction mixture to dryness, aqueous
work-up, extraction with organic solvents, distillation, column
chromatography, and high pressure liquid chromatography (HPLC) on
normal phase or reversed phase. Preferred methods include column
chromatography and recrystallization.
[0097] Drug research and development efforts made by global
research institutions and pharmaceutical industries have been
focused on other HCV targets rather than NS4A. NS4A binding pocket
remained an under-explored target for discovering new antiviral
agents, which may possibly overcome emerging resistance against
available drugs. Structurally, NS4A is a small peptide with 54
amino acids, which are inclined of forming proper assembly with
other viral proteins. For instance, the hydrophobic N-terminal
initiates a sandwich-filling binding that brings the N-terminal
(A.sub.0 and A.sub.1 sheets) of the NS3 to rearrange and conform
properly. This process is critical for the enzyme's catalytic site
to attain a suitable shape that accommodates the peptide substrate
[De Francesco, R.; Tomei, L.; Altamura, S.; Summa, V.; Migliaccio,
G., Approaching a new era for hepatitis C virus therapy: inhibitors
of the NS3-4A serine protease and the NS5B RNA-dependent RNA
polymerase. Antiviral Res 2003, 58 (1), 1-16; Ishido, S.; Fujita,
T.; Hotta, H., Complex formation of NS5B with NS3 and NS4A proteins
of hepatitis C virus. Biochemical and biophysical research
communications 1998, 244 (1), 35-40; Failla, C.; Tomei, L.; De
Francesco, R., Both NS3 and NS4A are required for proteolytic
processing of hepatitis C virus nonstructural proteins. Journal of
virology 1994, 68 (6), 3753-3760; and Hamad, H. A.; Thurston, J.;
Teague, T.; Ackad, E.; Yousef, M. S., The NS4A cofactor dependent
enhancement of HCV NS3 protease activity correlates with a 4D
geometrical measure of the catalytic triad region. PloS one 2016,
11 (12), e0168002]. It was found that the central part of NS4A
(Gly21-Leu34) with only 14 amino acids is required for the
activation of NS3 protease. Shimizu et al. observed inhibition of
the enzyme via mutating the basic Arg-28 residue to a neutral
glutamine (Gln) [Shimizu, Y.; Yamaji, K.; Masuho, Y.; Yokota, T.;
Inoue, H.; Sudo, K.; Satoh, S.; Shimotohno, K., Identification of
the sequence on NS4A required for enhanced cleavage of the NS5A/5B
site by hepatitis C virus NS3 protease. Journal of virology 1996,
70 (1), 127-132]. Other works that discussed the NS4A site
inhibitors are either presumptive [De Francesco, R.; Pessi, A.;
Steinkuhler, C., Mechanisms of hepatitis C virus NS3 proteinase
inhibitors. Journal of viral hepatitis 1999, 6, 23-30; and Kim, J.;
Morgenstern, K.; Lin, C.; Fox, T.; Dwyer, M.; Landro, J.; Chambers,
S.; Markland, W.; Lepre, C.; O'malley, E., Crystal structure of the
hepatitis C virus NS3 protease domain complexed with a synthetic
NS4A cofactor peptide. Cell 1996, 87 (2), 343-355] or virtual (i.e.
computational modeling) [Hamad, H. A.; Thurston, J.; Teague, T.;
Ackad, E.; Yousef, M. S., The NS4A cofactor dependent enhancement
of HCV NS3 protease activity correlates with a 4D geometrical
measure of the catalytic triad region. PloS one 2016, 11 (12),
e0168002]. As a result, non-peptide competitors of NS4A central
part may be viable therapeutic options in DAA class of HCV
therapeutics.
[0098] After careful inspection of 3D-visualization of crystal
structures of NS4A in bound form, it was found that it forms a
.beta.-sheet that is mostly extended except a turn featuring a
nearly planar area composed of an eclipsed cis bond and four trans
bonds with the backbone that span through Val-26 to Arg-28. This
turn is facilitated by the presence of the Gly-27 due to its small
size (no side chains) (see FIG. 1). Accordingly, the compounds of
formulae (I) and (II) disclosed herein are proposed via a De novo
structure-based design by deriving the planar area into imidazole
nucleus. Similar imidazole cores are found in several
commercialized drugs such as nitronidazole (antimicrobial),
azathioprine (immunosuppressive), nilotinib (anticancer), and many
others [Zhang, L.; Peng, X. M.; Damu, G. L.; Geng, R. X.; Zhou, C.
H., Comprehensive review in current developments of imidazole-based
medicinal chemistry. Med Res Rev 2014, 34 (2), 340-437].
[0099] According to a third aspect, the present disclosure relates
to a pharmaceutical composition involving the compound of formula
(I) of the first aspect and a pharmaceutically acceptable carrier
and/or excipient.
[0100] In one or more embodiments, the compound of formula (I)
is
##STR00014##
In certain embodiments, the compound of formula (II) is present in
addition to, or in lieu of the compound of formula (I) in the
pharmaceutical composition.
[0101] As used herein, a "composition" or a "pharmaceutical
composition" refers to a mixture of the active ingredient with
other chemical components, such as pharmaceutically acceptable
carriers and excipients. One purpose of a composition is to
facilitate administration of the compound disclosed herein in any
of its embodiments to a subject. Pharmaceutical compositions of the
present disclosure may be manufactured by processes well known in
the art, e.g., by means of conventional mixing, dissolving,
granulating, dragee-making, levigating, emulsifying, encapsulating,
entrapping or lyophilizing processes. Depending on the intended
mode of administration (oral, parenteral, or topical), the
composition can be in the form of solid, semi-solid or liquid
dosage forms, such as tablets, suppositories, pills, capsules,
powders, liquids, or suspensions, preferably in unit dosage form
suitable for single administration of a precise dosage.
[0102] The term "active ingredient", as used herein, refers to an
ingredient in the composition that is biologically active, for
example, the compound represented by formula (I), the compound
represented by formula (II), a salt thereof, a solvate thereof, a
tautomer thereof, a stereoisomer thereof, or any mixtures thereof.
In some embodiments, other active ingredients in addition to the
compound of the current disclosure may be incorporated into a
pharmaceutical composition.
[0103] In one or more embodiments, the pharmaceutical composition
disclosed herein further includes an antiviral agent that is
structurally distinct from the compounds of formulae (I) and (II).
In one embodiment, the antiviral agent used herein does not have
anti-HCV activity. Preferably, the antiviral agent has anti-HCV
activity. As used herein, the term "anti-HCV activity" means the
agent is effective to inhibit the function of at least one target
selected from the group consisting of HCV metalloprotease, HCV
serine protease, HCV polymerase, HCV helicase, HCV NS4B protein,
HCV entry, HCV assembly, HCV egress, HCV NS5A protein, IMPDH and a
nucleoside analog for the treatment of an HCV infection. In one
embodiment, the antiviral agent having anti-HCV activity is
effective to inhibit the function of a target in the HCV life cycle
other than the HCV serine protease.
[0104] Exemplary antiviral agents with anti-HCV activity include,
but are not limited to, Imiqimod, ribavirin, amantadine,
rimantadine, interferon such as interferon alpha 2B, pegylated
interferon alpha, consensus interferon, interferon alpha 2A, and
lymphoblastiod interferon tau, interleukins such as interleukin 2,
interleukin 6, and interleukin 12, other inhibitors of HCV NS3
protease, inhibitors of other targets in the HCV life cycle such as
helicase, polymerase, metalloprotease, or internal ribosome entry
site; or combinations thereof, and the like. Other antiviral agents
such as oseltamivir (Tamiflu), zanamivir, peramivir (Rapivab.RTM.)
may be further combined into the pharmaceutical composition for the
prevention or treatment of secondary infection. Alternatively, the
antiviral agent may act as an additional therapy, and the compounds
of formula (I) and/or (II) may be administered with the antiviral
agent in combination therapy, either jointly or separately.
[0105] In one or more embodiments, the pharmaceutical composition
comprises up to 0.01%, up to 0.1%, up to 1%, up to 5%, or up to 10%
by weight of the pharmaceutically acceptable carrier and/or
excipient relative to a total weight of the pharmaceutical
composition. In one or more embodiments, the pharmaceutical
composition comprises at least 0.01 wt %, at least 0.05 wt %, at
least 0.1 wt %, at least 0.5 wt %, at least 5 wt %, at least 10 wt
%, at least 15 wt %, at least 20 wt %, at least 25 wt %, at least
30 wt %, at least 35 wt %, at least 40 wt %, at least 45 wt %, at
least 50 wt %, at least 55 wt %, at least 60 wt %, at least 65 wt
%, at least 70 wt %, at least 75 wt %, at least 80 wt %, at least
85 wt %, at least 90 wt %, at least 95 wt %, at least 99 wt %, or
at least 99.9 wt % of the compound of formula (1) relative to a
total weight of the pharmaceutical composition. The pharmaceutical
composition may contain 0.5-500 .mu.M of the compound of formula
(I) relative to a total volume of the composition, preferably 1-400
.mu.M, preferably 10-300 .mu.M, preferably 20-200 .mu.M of the
compound of formula (I) relative to the total volume of the
composition. In some embodiments, the composition comprises up to
0.1 wt %, 1 wt %, 5 wt %, or 10 wt % of a pharmaceutically
acceptable salt of the compound of formula (I). In some
embodiments, the composition comprises up to 0.1 wt %, 1 wt %, 5 wt
%, or 10 wt % of a pharmaceutically acceptable solvate of the
compound of formula (I). Preferably, the composition may further
comprise pharmaceutically acceptable binders, such as sucrose,
lactose, xylitol, and pharmaceutically acceptable excipients such
as calcium carbonate, calcium phosphate, and dimethyl sulfoxide
(DMSO).
[0106] As used herein, a "pharmaceutically acceptable carrier"
refers to a carrier or diluent that does not cause significant
irritation to an organism, does not abrogate the biological
activity and properties of the administered active ingredient,
and/or does not interact in a deleterious manner with the other
components of the composition in which it is contained. The term
"carrier" encompasses any excipient, binder, diluent, filler, salt,
buffer, solubilizer, lipid, stabilizer, or other material well
known in the art for use in pharmaceutical formulations. The choice
of a carrier for use in a composition will depend upon the intended
route of administration for the composition. The preparation of
pharmaceutically acceptable carriers and formulations containing
these materials is described in, e.g. Remington's Pharmaceutical
Sciences, 21st Edition, ed. University of the Sciences in
Philadelphia, Lippincott, Williams & Wilkins, Philadelphia Pa.,
2005, which is incorporated herein by reference in its entirety).
Examples of physiologically acceptable carriers include
antioxidants including ascorbic acid; low molecular weight (less
than about 10 residues) polypeptides; proteins, such as serum
albumin, gelatin, or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, arginine or lysine; monosaccharides, disaccharides, and
other carbohydrates including glucose, mannose, or dextrins;
chelating agents such as EDTA; sugar alcohols such as mannitol or
sorbitol; salt-forming counterions such as sodium; and/or nonionic
surfactants such as TWEEN.RTM. (ICI, Inc.; Bridgewater, N.J.),
polyethylene glycol (PEG), and PLURONICS.TM. (BASF; Florham Park,
N.J.). An "excipient" refers to an inert substance added to a
composition to further facilitate administration of a compound.
Examples, without limitation, of excipients include calcium
carbonate, calcium phosphate, various sugars and types of starch,
cellulose derivatives, gelatin, vegetable oils, and polyethylene
glycols.
[0107] In one or more embodiments, the pharmaceutically acceptable
carrier and/or excipient is at least one selected from the group
consisting of a buffer, an inorganic salt, a fatty acid, a
vegetable oil, a synthetic fatty ester, a surfactant, and a
polymer.
[0108] Exemplary buffers include, but are not limited to, phosphate
buffers, citrate buffer, acetate buffers, borate buffers, carbonate
buffers, bicarbonate buffers, and buffers with other organic acids
and salts.
[0109] Exemplary inorganic salts include, but are not limited to,
calcium carbonate, calcium phosphate, disodium hydrogen phosphate,
potassium hydrogen phosphate, sodium chloride, zinc oxide, zinc
sulfate, and magnesium trisilicate.
[0110] Exemplary fatty acids include, but are not limited to, an
omega-3 fatty acid (e.g., linolenic acid, docosahexaenoic acid,
eicosapentaenoic acid) and an omega-6 fatty acid (e.g., linoleic
acid, eicosadienoic acid, arachidonic acid). Other fatty acids,
such as oleic acid, palmitoleic acid, palmitic acid, stearic acid,
and myristic acid, may be included.
[0111] Exemplary vegetable oils include, but are not limited to,
avocado oil, olive oil, palm oil, coconut oil, rapeseed oil,
soybean oil, corn oil, sunflower oil, cottonseed oil, and peanut
oil, grape seed oil, hazelnut oil, linseed oil, rice bran oil,
safflower oil, sesame oil, brazil nut oil, carapa oil, passion
fruit oil, and cocoa butter.
[0112] Exemplary synthetic fatty esters include, without
limitation, methyl, ethyl, isopropyl and butyl esters of fatty
acids (e.g., isopropyl palmitate, glyceryl stearate, ethyl oleate,
isopropyl myristate, isopropyl isostearate, diisopropyl sebacate,
ethyl stearate, di-n-butyl adipate, dipropylene glycol
pelargonate), C.sub.12-C.sub.16 fatty alcohol lactates (e.g., cetyl
lactate and lauryl lactate), propylene dipelargonate, 2-ethylhexyl
isononoate, 2-ethylhexyl stearate, isopropyl lanolate, 2-ethylhexyl
salicylate, cetyl myristate, oleyl myristate, oleyl stearate, oleyl
oleate, hexyl laurate, isohexyl laurate, propylene glycol fatty
ester, and polyoxyethylene sorbitan fatty ester. As used herein,
the term "propylene glycol fatty ester" refers to a monoether or
diester, or mixtures thereof, formed between propylene glycol or
polypropylene glycol and a fatty acid. The term "polyoxyethylene
sorbitan fatty ester" denotes oleate esters of sorbitol and its
anhydrides, typically copolymerized with ethylene oxide.
[0113] Surfactants may act as detergents, wetting agents,
emulsifiers, foaming agents, and dispersants. Surfactants that may
be present in the compositions of the present disclosure include
zwitterionic (amphoteric) surfactants, e.g., phosphatidylcholine,
and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate
(CHAPS), anionic surfactants, e.g., sodium lauryl sulfate, sodium
octane sulfonate, sodium decane sulfonate, and sodium dodecane
sulfonate, non-ionic surfactants, e.g., sorbitan monolaurate,
sorbitan monopalmitate, sorbitan trioleate, polysorbates such as
polysorbate 20 (Tween 20), polysorbate 60 (Tween 60), and
polysorbate 80 (Tween 80), cationic surfactants, e.g.,
decyltrimethylammonium bromide, dodecyltrimethylammonium bromide,
tetradecyltrimethylammonium bromide, tetradecyltrimethyl-ammonium
chloride, and dodecylammonium chloride, and combinations
thereof.
[0114] Exemplary polymers include, without limitation,
polylactides, polyglycolides, polycaprolactones, polyanhydrides,
polyurethanes, polyesteramides, polyorthoesters, polydioxanones,
polyacetals, polyketals, polycarbonates, polyorthocarbonates,
polyphosphazenes, polyhydroxybutyrates, polyhydroxyvalerates,
polyalkylene oxalates, polyalkylene succinates, poly(malic acid),
poly(maleic anhydride), a polyvinyl alcohols, and copolymers,
terpolymers, or combinations or mixtures therein. The
copolymer/terpolymer may be a random copolymer/terpolymer, or a
block copolymer/terpolymer.
[0115] Depending on the route of administration e.g. oral,
parental, or topical, the composition may be in the form of solid
dosage form such as tablets, caplets, capsules, powders, and
granules, semi-solid dosage form such as ointments, creams,
lotions, gels, pastes, and suppositories, liquid dosage forms such
as solutions, and dispersions, inhalation dosage form such as
aerosols, and spray, or transdermal dosage form such as
patches.
[0116] Solid dosage forms for oral administration can include
capsules, tablets, pills, powders, and granules. In such solid
dosage forms, the active ingredient is ordinarily combined with one
or more adjuvants appropriate to the indicated route of
administration. If administered per os, the active ingredient can
be admixed with lactose, sucrose, starch powder, cellulose esters
of alkanoic acids, cellulose alkyl esters, talc, stearic acid,
magnesium stearate, magnesium oxide, sodium and calcium salts of
phosphoric and sulfuric acids, gelatin, acacia gum, sodium
alginate, polyvinylpyrrolidone, and/or polyvinyl alcohol, and then
tableted or encapsulated for convenient administration. Such
capsules or tablets can contain a controlled-release formulation as
can be provided in a dispersion of active compound in
hydroxypropylmethyl cellulose. In the case of capsules, tablets,
and pills, the dosage forms can also comprise buffering ingredients
such as sodium citrate, magnesium or calcium carbonate or
bicarbonate. Tablets and pills can additionally be prepared with
enteric coatings.
[0117] Liquid dosage forms for oral administration can include
pharmaceutically acceptable emulsions, solutions, suspensions,
syrups, and elixirs containing inert diluents commonly used in the
art, such as water. Such compositions can also comprise adjuvants,
such as wetting ingredients, emulsifying and suspending
ingredients, and sweetening, flavouring, and perfuming
ingredients.
[0118] For therapeutic purposes, formulations for parenteral
administration can be in the form of aqueous or non-aqueous
isotonic sterile injection solutions or suspensions. The term
"parenteral", as used herein, includes intravenous, intravesical,
intraperitoneal, subcutaneous, intramuscular, intralesional,
intracranial, intrapulmonal, intracardial, intrasternal, and
sublingual injections, or infusion techniques. These solutions and
suspensions can be prepared from sterile powders or granules having
one or more of the carriers or diluents mentioned for use in the
formulations for oral administration. The active ingredient can be
dissolved in water, polyethylene glycol, propylene glycol, ethanol,
corn oil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol,
sodium chloride, and/or various buffers. Other adjuvants and modes
of administration are well and widely known in the pharmaceutical
art.
[0119] Injectable preparations, for example, sterile injectable
aqueous or oleaginous suspensions can be formulated according to
the known art using suitable dispersing or wetting ingredients and
suspending ingredients. The sterile injectable preparation can also
be a sterile injectable solution or suspension in a non-toxic
parenterally acceptable diluent or solvent, for example, as a
solution in 1,3-butanediol. Among the acceptable vehicles and
solvents that can be employed are water, Ringer's solution, and
isotonic sodium chloride solution. In addition, sterile, fixed oils
are conventionally employed as a solvent or suspending medium. For
this purpose any bland fixed oil can be employed including
synthetic mono- or di-glycerides. In addition, fatty acids, such as
oleic acid, find use in the preparation of injectables. Dimethyl
acetamide, surfactants including ionic and non-ionic detergents,
polyethylene glycols can be used. Mixtures of solvents and wetting
ingredients such as those discussed above are also useful.
[0120] Suppositories for rectal administration can be prepared by
mixing the active ingredient with a suitable non-irritating
excipient, such as cocoa butter, synthetic mono-, di-, or
triglycerides, fatty acids, and polyethylene glycols that are solid
at ordinary temperatures but liquid at the rectal temperature and
will therefore melt in the rectum and release the drug.
[0121] Topical administration may involve the use of transdermal
administration such as transdermal patches or iontophoresis
devices. Formulation of drugs is discussed in, for example, Hoover,
J. E. Remington's pharmaceutical sciences, Mack Publishing Co.,
Easton, Pa., 1975; and Liberman, H. A.; Lachman, L Eds.
Pharmaceutical dosage forms, Marcel Decker, New York, N.Y., 1980,
which are incorporated herein by reference in their entirety.
[0122] In other embodiments, the pharmaceutical composition having
the compound of formula (I), the salt thereof, the solvate thereof,
the tautomer thereof, the stereoisomer thereof, or the mixture
thereof has different release rates categorized as immediate
release and controlled- or sustained-release.
[0123] As used herein, immediate release refers to the release of
an active ingredient substantially immediately upon administration.
In another embodiment, immediate release occurs when there is
dissolution of an active ingredient within 1-20 minutes after
administration. Dissolution can be of all or less than all (e.g.
about 70%, about 75%, about 80%, about 85%, about 90%, about 91%,
about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,
about 98%, about 99%, about 99.5%, 99.9%, or 99.99%) of the active
ingredient. In another embodiment, immediate release results in
complete or less than complete dissolution within about 1 hour
following administration. Dissolution can be in a subject's stomach
and/or intestine. In one embodiment, immediate release results in
dissolution of an active ingredient within 1-20 minutes after
entering the stomach. For example, dissolution of 100% of an active
ingredient can occur in the prescribed time. In another embodiment,
immediate release results in complete or less than complete
dissolution within about 1 hour following rectal administration. In
some embodiments, immediate release is through inhalation, such
that dissolution occurs in a subject's lungs.
[0124] Controlled-release, or sustained-release, refers to a
release of an active ingredient from a composition or dosage form
in which the active ingredient is released over an extended period
of time. In one embodiment, controlled-release results in
dissolution of an active ingredient within 20-180 minutes after
entering the stomach. In another embodiment, controlled-release
occurs when there is dissolution of an active ingredient within
20-180 minutes after being swallowed. In another embodiment,
controlled-release occurs when there is dissolution of an active
ingredient within 20-180 minutes after entering the intestine. In
another embodiment, controlled-release results in substantially
complete dissolution after at least 1 hour following
administration. In another embodiment, controlled-release results
in substantially complete dissolution after at least 1 hour
following oral administration. In another embodiment,
controlled-release results in substantially complete dissolution
after at least 1 hour following rectal administration. In one
embodiment, the pharmaceutical composition described herein is not
a controlled-release composition.
[0125] According to a fourth aspect, the present disclosure relates
to a method of preventing or treating hepatitis C virus (HCV)
infection. The method involves administering the pharmaceutical
composition of the third aspect to a subject in need of
therapy.
[0126] As used herein, the term "preventing" in the context of the
administration of a therapy to a subject in need thereof refers to
preventing a disease, disorder or condition from occurring in a
subject which may be predisposed to the disease, disorder and/or
condition but has not yet been diagnosed as having it. The term
"treating" refers to (i) inhibiting the disease, disorder or
condition, i.e., arresting its development; and (ii) relieving the
disease, disorder or condition, i.e., causing regression of the
disease, disorder and/or condition. Treatment is preferably
commenced at the time of infection or post infection with HCV. It
is recommended that the treatment continues until the virus is no
longer present or active. For protecting a non-infected subject
from future infection, the treatment continues for as long as there
is a potential exposure to the virus.
[0127] The term "subject" and "patient" are used interchangeably.
As used herein, they refer to any subject for whom or which
therapy, including with the compositions according to the present
disclosure is desired. In most embodiments, the subject is a
mammal, including but not limited to a human, a non-human primate
such as a chimpanzee, a domestic livestock such as a cattle, a
horse, a swine, a pet animal such as a dog, a cat, and a rabbit,
and a laboratory subject such as a rodent, e.g. a rat, a mouse, and
a guinea pig. In preferred embodiments, the subject is a human.
[0128] The terms "administer", "administering", "administration",
and the like, as used herein, refer to the methods that may be used
to enable delivery of the active ingredient and/or the composition
to the desired site of biological action. Routes or modes of
administration are as set forth herein. These methods include, but
are not limited to, oral routes, intraduodenal routes, parenteral
injection (including intravenous, subcutaneous, intraperitoneal,
intramuscular, intravascular, or infusion), topical and rectal
administration. Those of ordinary skill in the art are familiar
with administration techniques that can be employed with the
compounds and methods described herein. In a preferred embodiment,
the active ingredient and/or the pharmaceutical composition
described herein are administered orally.
[0129] In one or more embodiments, the pharmaceutical composition
administered comprises the compound of formula (I), or a salt
thereof, a solvate thereof, a tautomer thereof, a stereoisomer
thereof, or a mixture thereof. In a preferred embodiment, the
pharmaceutical composition administered comprises a compound which
is
##STR00015##
[0130] The dosage amount and treatment duration are dependent on
factors, such as bioavailability of a drug, administration mode,
toxicity of a drug, gender, age, lifestyle, body weight, the use of
other drugs and dietary supplements, the disease stage, tolerance
and resistance of the body to the administered drug, etc., and then
determined and adjusted accordingly. The terms "effective amount",
"therapeutically effective amount", or "pharmaceutically effective
amount" refer to that amount of the active ingredient being
administered which will relieve to some extent one or more of the
symptoms of the disease being treated. The result can be a
reduction and/or alleviation of the signs, symptoms, or causes of a
disease, or any other desired alteration of a biological system. An
appropriate "effective amount" may differ from one individual to
another. An appropriate "effective amount" in any individual case
may be determined using techniques, such as a dose escalation
study. In one or more embodiments, an effective amount of the
compound of formula (I) in a range of 0.1-100 mg/kg, preferably
0.5-50 mg/kg, more preferably 1-25 mg/kg is administered per body
weight of the subject. However, in certain embodiments, the
effective amount of the compound of formula (1) is less than 0.1
mg/kg or greater than 100 mg/kg. In general, the compound is most
desirably administered at a concentration level that will generally
afford antivirally effective results without causing any harmful or
deleterious side effects.
[0131] A treatment method may comprise administering a
pharmaceutical composition containing the compound of formula (I)
of the current disclosure in any of its embodiments as a single
dose or multiple individual divided doses. In some embodiments, the
interval of time between the administration of the composition and
the administration of one or more additional therapies (e.g.
interferon, ribavirin) may be about 1-5 minutes, 1-30 minutes, 30
minutes to 60 minutes, 1 hour, 1-2 hours, 2-6 hours, 2-12 hours,
12-24 hours, 1-2 days, 2 days, 3 days, 4 days, 5 days, 6 days, 7
days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks,
8 weeks, 9 weeks, 10 weeks, 15 weeks, 20 weeks, 26 weeks, 52 weeks,
11-15 weeks, 15-20 weeks, 20-30 weeks, 30-40 weeks, 40-50 weeks, 1
month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months,
8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2
years, or any period of time in between. Preferably, the
pharmaceutical compositions disclosed herein will be administered
from about 1 to about 5 times per day or alternatively, as a
continuous infusion for at least 2 days, at least 5 days, at least
6 days, or at least 7 days. Such administration can be used as a
chronic or acute therapy. In certain embodiments, the composition
and one or more additional therapies are administered less than 1
day, less than 1 week, less than 2 weeks, less than 3 weeks, less
than 4 weeks, less than 1 month, less than 2 months, less than 3
months, less than 6 months, less than 1 year, less than 2 years, or
less than 5 years apart.
[0132] The pharmaceutical composition may be administered in vivo
to the subject to inhibit HCV NS3 protease or to treat or prevent
HCV virus infection. In one or more embodiments, the compound of
formula (I) of the current disclosure in any of its embodiments
binds to an activation site of hepatitis C virus (HCV) serine
protease. As used herein, the "serine protease" is referred to as
NS3 protease. The compound of formula (I) may preferably bind to
the activation site of HCV NS3 protease such as but not limited to
linear peptides, cyclic peptides, macrolactons, macrolactams, and
peptidomimetics. In a preferred embodiment, the compound of formula
(I) of the current disclosure in any of its embodiments binds to
NS3/4A serine protease.
[0133] As used herein, "bind" or "binding" refers to a process
involving an active ingredient (e.g. the compound of formula (1))
reversibly or irreversibly binds to HCV N S3 protease or variant
thereof at the activation site. Binding may involve the formation
of bonds which may be covalent or non-covalent. Non-covalent bonds
may be e.g. hydrogen bonds, ionic bonds, or hydrophobic
interactions. The active ingredient (e.g. the compound of formula
(I)) is expected to interfere and inhibit the interactions leading
to the formation of the active form of the NS3 protease. Binding
can be quantitated in accordance with methods well-known in the art
and described herein below.
[0134] In certain embodiments, a suitable assay is used to
characterize the potential binding ability of the active ingredient
(e.g. compound of formula (I)) to bind to the activation site. This
may involve directly testing the active ingredient's ability to
bind, and/or determining whether the active ingredient has an
influence on the binding of the NS4A to HCV NS3 protease or
variants thereof. To evaluate binding properties of binding
compounds, assays may be used. Exemplary assay methods include, but
not limited to, calorimetric techniques, surface plasmon resonance
(SPR), and spectroscopic methods such as NMR, fluorescence, and
UV-vis spectroscopies.
[0135] Calorimetric methods include but not limited to isothermal
titration calorimetry and differential scanning calorimetry. SPR is
the resonant oscillation of conduction electrons at the interface
between negative and positive permittivity material stimulated by
incident light. The method involves immobilizing one molecule of a
binding pair on the sensor chip surface ("ligand", in Biacore
parlance) and injecting a series of concentrations of its partner
("analyte") across the surface. Changes in the index of refraction
at the surface where the binding interaction occurs are detected by
the hardware and recorded as RU (resonance units) in the control
software. Curves are generated from the RU trace and are evaluated
by fitting algorithms which compare the raw data to well-defined
binding models. These fits allow determination of a variety of
thermodynamic constants, including the apparent affinity of the
binding interaction. SPR main advantage is that it does not require
labeling the protein or the binding compound.
[0136] The kinetics of enzymatic-catalyzed reactions is a useful
tool not only to determine the inhibition constants (Ki) for an
inhibitor but also the site at which an inhibitor binds to the
enzyme. An inhibitor that binds exclusively to the catalytic active
site displays a competitive inhibition pattern with the substrate.
In contrast, an inhibitor that binds to a different site from that
of the substrate displays an uncompetitive inhibition pattern with
the substrate. If the inhibitor binds to both an active site and a
different site from that of the active site, it would display a
non-competitive pattern. Thus, the active ingredient (e.g. the
compound of formula (I)) would be competitive with the activation
peptide and uncompetitive with the substrate.
[0137] NMR methods and optical spectroscopic methods such as
fluorescence (e.g. fluorescence anisotropy (FA)), UV-vis, and
circular dichroism (CD) are well-known assays utilized in measuring
the kinetics of the interaction between an active ingredient and a
protein. In a preferred embodiment, fluorescence anisotropy is
used.
[0138] The fluorescence method is suitable for high throughput
screening method amenable to automation in a laboratory
environment. Since HCV NS3 contains two tryptophan residues and
four tyrosine residues, the binding of an inhibitor (e.g. the
compound of formula (I), NS4A) to the activation site may be
accompanied by significant change in the intrinsic fluorescence of
the protein, and hence the enzyme inhibition may be evaluated (see
FIG. 6). A peptide inhibitor (e.g. NS4A) may be labeled with a
fluorescent probe and the binding of the labeled peptide to the
enzyme is accompanied by fluorescent change (see Example 12, 13,
FIG. 6). Another fluorescence assay method for determining the
binding constant of the compound of formula (I) disclosed herein is
a competitive displacement assay (see Examples 11, 13, FIG. 5).
[0139] NMR methods may be used to observe the binding of an active
ingredient (e.g. the compound of formula (1)) to the activation
site of HCV NS3 protease. In its simple form, the observation of
broadening of an NMR signal as a function of concentration would
allow the determination of binding constants.
[0140] The examples below are intended to further illustrate
protocols for preparing, characterizing the compound of formulae
(I) and (II), and uses thereof, and are not intended to limit the
scope of the claims.
Example 1
Synthesis of Screened Compounds
[0141] The compounds were synthesized using straightforward
chemistry [Duguay, G.; Guemas, J.-P.; Meslin, J.-C.; Pradere,
J.-P.; Reliquet, F.; Reliquet, A.; Tea-Gokou, C.; Quiniou, H.;
Rabiller, C., Heteroatomic chains and their products of
cyclisation. IV.
t-butyl-2-phthalimido-2-(3,6-dihydro-1,3-2H-thiazine-2-yliden)-acetates
substituted in position 5 by a functional group. Journal of
Heterocyclic Chemistry 1980, 17 (4), 767-770; and Vuilhorgne, M.;
Malpart, J.; Mutti, S.; Mignani, S., Preparative route to
2-ethoxycarbonylimidazole-4-phosphonate and
diethylimidazole-2,4-dicarboxylate. Journal of Heterocyclic
Chemistry 2003, 40 (1), 159-162, each incorporated herein by
reference in their entirety] as illustrated in the synthesis scheme
(FIG. 2). All final compounds are new and their structures were
confirmed by spectral analyses (e.g. .sup.1H NMR, .sup.13C NMR, and
LC/MS).
Example 2
Chemical Synthesis: General
[0142] Solvents and reagents were purchased from Sigma-Aldrich
(USA), VWR (USA) or Alfa Aesar (UK). When needed, solvents were
dried according procedures described in literature. Unless stated
otherwise, reactions were performed under inert atmosphere of
nitrogen. Melting points (m.p.) were determined in open capillary
tubes using Electrothermal apparatus (Stuart, UK) and were
uncorrected. NMR spectra were recorded on Bruker DPX-300 MHz
(Bruker, Switzerland). HPLC-Mass Spectrometry was performed on
Agilent 1100/ZQ MSD including diod-array UV detector.
Example 3
Synthesis of 5-(tert-butyl) 2-ethyl 1H-imidazole-2,5-dicarboxylate
(MOC-01)
[0143] Water (0.6 mL/mmol) was added dropwise over a period of 2 h
to a stirred mixture of ethyl cyanoformate (1.0 equiv, 3 g, 30.3
mmol), hydroxylamine hydrochloride (1.5 equiv, 3.16 g, 45.45 mmol),
and sodium carbonate (0.77 equiv, 2.47 g, 23.331 mmol) in ethanol
(1 mL/mmol) at r.t. Upon completion, the reaction was quenched and
the solvent was removed under vacuum. The resulting residue was
extracted with DCM, and the combined organic layers were washed
with brine, dried over MgSO.sub.4, filtered, and concentrated to
afford a white solid. The intermediate (Z)-2-amino-2-(hydroxyimino)
acetate was recrystallized from chloroform and n-heptane to afford
white crystals (1.88 g, 47%). This intermediate (1.0 equiv, 132 mg,
1 mmol) was added to a solution of tBu-propiolate (1.0 equiv, 126
mg, 1 mmol), Et.sub.3N (1.0 equiv, 101 mg, 1 mmol) and toluene (5.5
mL/mmol). The resulting solution was MW irradiated at 120.degree.
C. for 3 min (300 W) and then for an additional 10 min. The solvent
was evaporated and the final product was purified via preparative
HPLC. .sup.1H NMR (300 MHz, DMSO-d.sub.6) d.sub.H ppm 13.72 (br.,
1H), 7.87 (s, 1H), 4.34 (q, J=7.18 Hz, 2H), 1.51 (s, 9H), 1.32 (t,
J=6.99 Hz, 3H); .sup.13C NMR (214 MHz, CDCl.sub.3) d.sub.c 169.7,
136.5, 129.5, 129.1, 128.9, 127.8, 123.8, 29.7; IR (FT-IR,
cm.sup.-1): 3025.37, 2916.82, 2843.61, 1695.04, 1619.31, 1594.07,
1571.35, 1505.72, 1445.14, 1399.70, 1359.31, 1270.96, 1205.32;
LC-MS (ESI), RT=1.41 min, m/z 241.3 [M+H].sup.+.
Example 4
Synthesis of 2-(ethoxycarbonyl)-1H-imidazole-4-carboxylic Acid
(MOC-02)
[0144] A solution of 5-tert-butyl 2-ethyl
1H-imidazole-2,5-dicarboxylate (1 equiv, 4.299 g, 17.6 mmol) and
CF.sub.3COOH (1.2 mL/mmol) in DCM (0.84 mL/mmol) was stirred for 12
h at r.t. The solvent was evaporated under vacuum and the remaining
residue was treated with MeOH (4 mL/mmol) and filtered to give a
yellow solid (2.841 g, 86.29%). .sup.1H NMR (300 MHz, DMSO-d.sub.6)
d.sub.H ppm 13.72 (br. s, 1H); 12.59 (br. s, 1H), 7.91 (s, 1H),
4.33 (q, J=6.97 Hz, 2H), 1.32 (t, J=7.06 Hz, 3H); LC-MS (ESI),
RT=1.55 min, m/z 185.3 [M+H].sup.+.
Example 5
General Procedure for the Synthesis of ethyl
4-(N-substitutedcarbamoyl)-1H-imidazole-2-carboxylate (MOC-03 to
MOC-6)
[0145] Under inert atmosphere, the carboxylic acid MOC-02 (1.0
equiv., 5.21 mmol) was dissolved in dry THF (5 mL/mmol) then EDCI
(1.0 equiv, 0.998 g, 5.21 mmol), HOBt (1.0 equiv, 0.704 mg, 5.21
mmol), DIPEA (1.5 equiv, 1.01 g, 7.8 mmol), and the amine (1.2
equiv, 6.252 mmol) were added orderly. The mixture was stirred at
r.t. for 1 h then heated to 60.degree. C. After 3 h, EDCI (0.5
equiv, 0.5 g, 2.6 mmol), HOBt (0.5 equiv, 0.352 g, 2.6 mmol), DIPEA
(0.75 equiv, 0.505 g, 3.9 mmol) were added to the mixture. After
completion, the mixture was purified by column chromatography using
50-100% EtOAc in cyclohexane.
Ethyl
4-((2-(methylamino)-2-oxoethyl)carbamoyl)-1H-imidazole-2-carboxylate
(MOC-03)
[0146] Yield was 77%. .sup.1H NMR (300 MHz, DMSO-d.sub.6) d.sub.H
ppm 8.19 (br. s., 1H), 7.81 (s, 2H), 4.35 (q, J=7.16 Hz, 2H), 3.81
(d, J=5.65 Hz, 2H), 2.59 (d, J=4.52 Hz, 3H), 1.33 (t, J=7.16 Hz,
3H); LC-MS (ESI), RT=1.57 min, m/z 255.3 [M+H].sup.+.
Synthesis of (((4-aminobutyl)amino)(carboxyamino)methylene)carbamic
Acid ditert-butyl Ester (Amine Reactant for MOC-04)
[0147] To a solution of 1,4-diaminobutane (2.0 equiv, 303.24 mg,
3.44 mmol) in THE (1.3 mL/mmol), a solution of
1,3-bis(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea (1.0 equiv,
500 mg, 1.72 mmol) in THF (1.7 mL/mmol) was added within 0.5 h. The
solution was stirred at room temperature for 1 h. After completion,
the solvent was removed under vacuum and the product was purified
by column chromatography on silica gel using a mixture of DCM/MeOH
to give (((4-aminobutyl)amino)(carboxyamino)methylene)carbamic acid
ditert-butyl ester (250 mg, 52.88%); .sup.1H NMR (300 MHz,
CDCl.sub.3) d.sub.H ppm 11.51 (br. s., 1H), 8.36 (br. s., 1H),
3.40-3.52 (m, 2H), 2.75 (t, J=6.69 Hz, 2H), 1.51-1.71 (m, 4H), 1.52
(s, 9H), 1.51 (s, 9H); LC-MS (ESI), RT=2.44 min, m/z 331.8
[M+H].sup.+.
Ethyl
(E)-4-((4-(2,3-bis(tert-butoxycarbonyl)guanidino)butyl)carbamoyl)-1H-
-imidazole-2-carboxylate (MOC-04)
[0148] Yield was 36%. The compound was used as the starting
material for the next step without further characterization.
Ethyl 5-((pyridin-2-ylmethyl)carbamoyl)-1H-imidazole-2-carboxylate
(MOC-05)
[0149] Yield was 94%. .sup.1H NMR (300 MHz, DMSO-d.sub.6) d.sub.H
ppm 13.24 (br. s. 1H), 8.69 (t, J=5.65 Hz, 1H), 8.51 (d, J=4.14 Hz,
1H), 8.41 (s, 1H), 7.81 (s, 1H), 7.74 (t, J=7.06 Hz, 1H), 7.19-7.35
(m, 2H), 4.53 (d, J=6.03 Hz, 2H), 4.34 (q, J=7.16 Hz, 2H), 1.32 (t,
J=7.06 Hz, 3H); LC-MS (ESI), RT=1.14 min, m/z 275.3
[M+H].sup.+.
Ethyl 5-((furan-2-ylmethyl)carbamoyl)-1H-imidazole-2-carboxylate
(MOC-06)
[0150] Yield was 76%. .sup.1H NMR (300 MHz, DMSO-d.sub.6) d.sub.H
ppm 13.76 (br. s., 1H), 8.53 (br. s., 1H), 7.82 (s, 1H), 7.50-7.58
(m, 1H), 6.22 (d, J=3.02 Hz, 1H), 6.38 (dd, J=3.02, 1.89 Hz, 1H),
6.22 (d, J=3.02 Hz, 1H), 4.41 (d, J=6.04 Hz, 2H), 4.34 (q, J=7.18
Hz, 2H), 1.32 (t, J=6.99 Hz, 3H).
Example 6
General Procedure for the Synthesis of MOC-07 to MOC-10
[0151] The ethyl esters (1.0 equiv) was dissolved in THF/water 4:1
(4 mL/mmol) then LiOH (3.0 equiv) was added. The mixture was
stirred at r.t. until completion. Afterwards, the solvent was
evaporated under vacuum. The remaining residue was re-suspended in
EtOH/toluene and evaporated. No further purification or
characterization was performed and the salts were used for the next
step.
Example 7
General Procedure for the Synthesis of MOC-11 to MOC-36
[0152] Under inert atmosphere, the appropriate lithium salt
imidazole-carboxylic acid derivative of MOC-7 to MOC-10 (1.0
equiv.) was dissolved in dry THF (3 mL/mmol) then propanephosphonic
acid anhydride (T.sub.3P, 50% solution in THF, 6.0 equiv.) and the
requisite amine derivative (1.2 equiv.) were added. The mixture was
stirred at r.t. for 72 h. After 24 h, 6.0 equiv. of T.sub.3P (50%
solution in THF) was added to the mixture. Upon reaction
completion, the solvent was evaporated and the residue was purified
on a silica gel column with 0-20% MeOH in CDCl.sub.3, and then the
crude product was collected and purified by prep HPLC.
N.sup.5-(2-(methylamino)-2-oxoethyl)-N.sup.2-(n-pentyl)-1H-imidazole-2,5-d-
icarboxamide (MOC-11)
[0153] .sup.1H NMR (300 MHz, METHANOL-d.sub.4) d.sub.H ppm 7.76 (s,
1H), 4.04 (s, 1H), 3.74 (t, J=6.42 Hz, 2H), 3.38-3.51 (m, 1H), 2.78
(s, 2H), 1.80-2.00 (m, 2H), 1.53-1.80 (m, 2H), 1.20-1.53 (m, 3H),
0.86-1.07 (m, 3H); .sup.13C NMR (75 MHz, METHANOL-d.sub.4) d.sub.C
ppm 170.8, 48.5, 48.2, 47.9, 47.6, 47.4, 47.1, 46.8, 41.8, 38.9,
29.0, 28.9, 25.0, 22.1, 13.0; LC-MS (ESI), RT=1.46 min, m/z 296.4
[M+H].sup.+.
N.sup.2-(n-hexyl)-N.sup.5-(2-(methylamino)-2-oxoethyl)-1H-imidazole-2,5-di-
carboxamide (MOC-12)
[0154] .sup.1H NMR (300 MHz, METHANOL-d.sub.4) d.sub.H ppm 7.76 (s,
1H), 4.04 (s, 1H), 3.39 (t, J=7.18 Hz, 1H), 2.78 (s, 2H), 1.56-1.91
(m, 3H), 1.38 (br. s., 5H), 1.04 (t, J=6.42 Hz, 2H), 0.77-0.98 (m,
3H); .sup.13C NMR (75 MHz, METHANOL-d.sub.4) d.sub.C ppm 48.5,
48.2, 47.9, 47.6, 47.4, 47.1, 46.8, 41.8, 39.0, 31.3, 29.2, 26.3,
25.0, 22.3, 13.0; LC-MS (ESI), RT=1.39 min, m/z 310.3
[M+H].sup.+.
N.sup.2-(n-heptyl)-N.sup.5-(2-(methylamino)-2-oxoethyl)-1H-imidazole-2,5-d-
icarboxamide (MO-13)
[0155] .sup.1H NMR (300 MHz, METHANOL-d.sub.4) d.sub.H ppm 7.68 (s,
1H), 3.95 (s, 2H), 2.69 (s, 3H), 1.45-1.73 (m, 2H), 1.25 (s, 5H),
1.29 (s, 4H), 0.63-1.01 (m, 4H); .sup.13C NMR (75 MHz,
METHANOL-d.sub.4) d.sub.C ppm 48.5, 48.2, 47.9, 47.6, 47.4, 47.1,
46.8, 38.9, 31.6, 29.3, 28.8, 26.6, 25.0, 22.3, 13.0; LC-MS (ESI),
RT=2.59 min, m/z 324.4 [M+H].sup.+.
N.sup.2-(2-(cyclohexyloxy)ethyl)-N.sup.5-(2-(methylamino)-2-oxoethyl)-1H-i-
midazole-2,5-dicarboxamide (MOC-14)
[0156] .sup.1H NMR (300 MHz, METHANOL-d.sub.4) d.sub.H ppm 7.77 (s,
1H), 3.84-4.24 (m, 2H), 3.66 (t, J=5.48 Hz, 2H), 3.56 (t, J=5.29
Hz, 2H), 2.77 (s, 3H), 1.93 (br. s., 2H), 1.62-1.84 (m, 4H),
1.45-1.62 (m, 1H), 1.16-1.41 (m, 6H), 0.98-1.12 (m, 1H); .sup.13C
NMR (75 MHz, METHANOL-d.sub.4) d.sub.C ppm 122.2, 77.7, 65.8, 48.5,
48.2, 47.9, 47.6, 47.4, 47.1, 46.8, 41.8, 39.3, 31.9, 25.5, 25.0,
23.7; LC-MS (ESI), RT=1.60 min, m/z 352.3 [M+H].sup.+.
N.sup.2-(3-(cyclohexyloxy)propyl)-N.sup.5-(2-(methylamino)-2-oxoethyl)-1H--
imidazole-2,5-dicarboxamide (MOC-15)
[0157] .sup.1H NMR (300 MHz, METHANOL-d.sub.4) d.sub.H ppm 7.76 (s,
1H), 3.86-4.19 (m, 2H), 3.41-3.70 (m, 4H), 2.78 (s, 3H), 1.83-2.03
(m, 4H), 1.44-1.83 (m, 5H), 1.17-1.44 (m, 5H), 0.97-1.17 (m, 1H);
.sup.13C NMR (75 MHz, METHANOL-d.sub.4) d.sub.C ppm 77.6, 65.5,
48.5, 48.2, 47.9, 47.6, 47.4, 47.1, 46.8, 41.7, 36.9, 31.9, 29.5,
25.6, 25.0, 23.7; LC-MS (ESI), RT=1.59 min, m/z 366.4
[M+H].sup.+.
N.sup.5-(2-(methylamino)-2-oxoethyl)-N.sup.2-(2-phenoxyethyl)-1H-imidazole-
-2,5-dicarboxamide (MOC-16)
[0158] .sup.1H NMR (300 MHz, METHANOL-d.sub.4) d.sub.H ppm
7.63-7.83 (m, 1H), 7.13-7.43 (m, 2H), 6.79-7.13 (m, 3H), 4.11-4.30
(m, 2H), 4.03 (s, 2H), 3.80 (t, J=5.29 Hz, 2H), 2.77 (s, 3H), 1.42
(t, =6.99 Hz, 1H); .sup.13C NMR (75 MHz, METHANOL-d.sub.4) d.sub.C
ppm 129.1, 114.3, 48.5, 48.2, 47.9, 47.6, 47.3, 47.1, 46.8, 24.9;
LC-MS (ESI), RT=1.40 min, m/z 346.3 [M+H].sup.+.
N.sup.5-(2-(methylamino)-2-oxoethyl)-N.sup.2-(3-phenoxypropyl)-1H-imidazol-
e-2,5-dicarboxamide (MOC-17)
[0159] .sup.1H NMR (300 MHz, METHANOL-d.sub.4) d.sub.H ppm
7.65-7.88 (m, 1H), 7.16-7.37 (m, 2H), 6.79-7.05 (m, 3H), 3.91-4.22
(m, 4H), 3.61 (t, J=6.80 Hz, 2H), 2.77 (s, 3H), 2.11 (quin, J=6.33
Hz, 2H), 1.43 (t, J=6.99 Hz, 1H); .sup.13C NMR (75 MHz,
METHANOL-d.sub.4) d.sub.C ppm 129.1, 120.4, 114.2, 65.3, 48.5,
48.2, 47.9, 47.6, 47.4, 47.1, 46.8, 41.8, 36.4, 29.0, 25.0; LC-MS
(ESI), RT=1.39 min, m/z 360.4 [M+H].sup.+.
N.sup.2-(4-isopropoxybutyl)-N.sup.5-(2-(methylamino)-2-oxoethyl)-1H-imidaz-
ole-2,5-dicarboxamide (MOC-18)
[0160] .sup.1H NMR (300 MHz, METHANOL-d.sub.4) d.sub.H ppm 7.76 (s,
1H), 3.91-4.25 (m, 2H), 3.56-3.78 (m, 1H), 3.35-3.56 (m, 4H), 2.78
(s, 3H), 1.55-1.87 (m, 8H), 1.11-1.26 (m, 7H), 1.05 (t, J=6.61 Hz,
3H); .sup.13C NMR (75 MHz, METHANOL-d.sub.4) d.sub.C ppm 67.4,
48.5, 48.2, 47.9, 47.6, 47.4, 47.1, 46.8, 38.7, 27.1, 26.1, 25.0,
21.0; LC-MS (ESI), RT=1.79 min, m/z 340.4 [M+H].sup.+.
N.sup.5-(2-(methylamino)-2-oxoethyl)-N.sup.2-(6-methylheptyl)-1H-imidazole-
-2,5-dicarboxamide (MOC-19)
[0161] .sup.1H NMR (300 MHz, METHANOL-d.sub.4) d.sub.H ppm 7.77 (s,
1H), 4.04 (s, 2H), 2.78 (s, 3H), 1.63 (br. s., 1H), 1.25-1.55 (m,
9H), 0.76-1.11 (m, 7H); .sup.13C NMR (75 MHz, METHANOL-d.sub.4)
d.sub.C ppm 48.5, 48.2, 47.9, 47.6, 47.4, 47.1, 46.8, 42.1, 39.5,
30.6, 28.6, 25.0, 23.8, 22.7, 13.0, 9.8; LC-MS (ESI), RT=2.66 min,
m/z 338.4 [M+H].sup.+.
(E)-N.sup.2-(3,7-dimethylocta-2,6-dien-1-yl)-N.sup.5-(2-(methylamino)-2-ox-
oethyl)-1H-imidazole-2,5-dicarboxamide (MOC-20)
[0162] .sup.1H NMR (300 MHz, METHANOL-d.sub.4) d.sub.H ppm 7.76 (s,
1H), 5.31 (t, J=6.42 Hz, 1H), 5.12 (t, 0.1=6.23 Hz, 1H), 3.86-4.17
(m, 4H), 2.78 (s, 3H), 1.94-2.24 (m, 4H), 1.76 (s, 3H), 1.62 (s,
3H), 1.67 (s, 3H); .sup.13C NMR (75 MHz, METHANOL-d.sub.4) d.sub.C
ppm 123.7, 119.9, 48.5, 48.2, 47.9, 47.6, 47.4, 47.1, 46.8, 41.8,
39.3, 36.7, 26.1, 25.0, 24.5, 16.4, 15.0; LC-MS (ESI), RT=1.60 min,
m/z 362.4 [M+H].sup.+.
N.sup.5-(2-(methylamino)-2-oxoethyl)-N.sup.2-(3-methylpentyl)-1H-imidazole-
-2,5-dicarboxamide (MOC-21)
[0163] .sup.1H NMR (300 MHz, METHANOL-d.sub.4) d.sub.H ppm 7.76 (s,
1H), 4.04 (s, 2H), 3.38-3.56 (m, 2H), 2.78 (s, 3H), 1.59-1.84 (m,
2H), 1.34-1.59 (m, 2H), 1.16-1.34 (m, 1H), 0.76-1.16 (m, 7H);
.sup.13C NMR (75 MHz, METHANOL-d.sub.4) d.sub.C ppm 170.8, 122.1,
48.5, 48.2, 47.9, 47.6, 47.4, 47.1, 46.8, 41.8, 37.1, 35.9, 32.1,
29.1, 25.0, 18.0, 10.2; LC-MS (ESI), RT=1.39 min, m/z 310.4
[M+H].sup.+.
N.sup.2-(2-acetamidoethyl)-N.sup.5-(2-(methylamino)-2-oxoethyl)-1H-imidazo-
le-2,5-dicarboxamide (MOC-22)
[0164] .sup.1H NMR (300 MHz, METHANOL-d.sub.4) d.sub.H ppm 7.76 (s,
1H), 4.04 (s, 1H), 3.35-3.65 (m, 3H), 2.78 (s, 2H), 1.88-2.07 (m,
3H), 1.66 (d, J=8.67 Hz, 2H), 1.04 (t, J=6.03 Hz, 1H); .sup.13C NMR
(75 MHz, METHANOL-d.sub.4) & ppm 48.5, 48.2, 47.9, 47.6, 47.4,
47.1, 46.8, 41.8, 39.1, 38.7, 25.0, 21.2; LC-MS (ESI), RT=2.82 min,
m/z 311.33 [M+H].sup.+.
N.sup.5-(4-guanidinobutyl)-N.sup.2-(n-pentyl)-1H-imidazole-2,5-dicarboxami-
de (MOC-23)
[0165] .sup.1H NMR (300 MHz, METHANOL-d.sub.4) d.sub.H ppm 7.83 (s,
1H), 3.36-3.60 (m, 5H), 3.25 (br. s., 3H), 1.69 (br. s., 7H),
1.17-1.50 (m, 6H), 0.95 (br. s., 4H); .sup.13C NMR (75 MHz,
METHANOL-d.sub.4) d.sub.C ppm 48.5, 48.3, 48.0, 47.7, 47.4, 47.1,
46.8, 40.8, 39.3, 28.9, 28.7, 25.9, 22.1, 13.0; LC-MS (ESI),
RT=1.90 min, m/z 338.4 [M+H].sup.+.
N.sup.5-(4-guanidinobutyl)-N.sup.2-(n-hexyl)-1H-imidazole-2,5-dicarboxamid-
e (MOC-24)
[0166] .sup.1H NMR (300 MHz, METHANOL-d.sub.4) d.sub.H ppm 7.81
(br. s., 1H), 3.39-3.57 (m, 4H), 3.07-3.27 (m, 3H), 1.61-1.84 (m,
8H), 1.55 (t, J=7.16 Hz, 1H), 1.18-1.48 (m, 9H), 0.93 (br. s., 4H);
.sup.13C NMR (75 MHz, METHANOL-d.sub.4) d.sub.C ppm 48.5, 48.2,
48.0, 47.7, 47.4, 47.1, 46.8, 40.8, 39.2, 31.3, 26.4, 25.9, 22.3,
13.0; LC-MS (EST), RT=1.78 min, m/z 352.3 [M+H].sup.+.
N5-(4-guanidinobutyl)-N2-(n-heptyl)-1H-imidazole-2,5-dicarboxamide
(MOC-25)
[0167] .sup.1H NMR (300 MHz, METHANOL-d.sub.4) d.sub.H ppm 7.90 (s,
1H), 3.35-3.57 (m, 5H), 3.25 (br. s., 2H), 1.60-1.86 (m, 7H), 1.34
(s, 6H), 1.39 (s, 4H), 0.79-1.03 (m, 4H); .sup.13C NMR (75 MHz,
METHANOL-d.sub.4) & ppm 48.6, 48.3, 48.0, 47.7, 47.4, 47.1,
46.9, 40.8, 39.6, 38.5, 31.6, 28.9, 28.7, 26.7, 26.3, 25.9, 22.3,
13.1; LC-MS (EST), RT=1.78 min, m/z 366.4 [M+H].sup.+.
N.sup.2-(2-(cyclohexyloxy)ethyl)-N.sup.5-(4-guanidinobutyl)-1H-imidazole-2-
,5-dicarboxamide (MOC-26)
[0168] .sup.1H NMR (300 MHz, METHANOL-d.sub.4) d.sub.H ppm 7.76
(br. s., 1H), 3.50-3.75 (m, 7H), 3.44 (br. s., 3H), 3.25 (br. s.,
3H), 1.81-2.09 (m, 5H), 1.63-1.81 (m, 9H), 1.45-1.63 (m, 2H),
1.11-1.44 (m, 7H); .sup.13C NMR (75 MHz, METHANOL-d.sub.4) d.sub.C
ppm 77.5, 65.6, 48.5, 48.2, 47.9, 47.6, 47.4, 47.1, 46.8, 40.7,
37.0, 31.9, 25.8, 25.6, 23.7; LC-MS (ESI), RT=2.46 min, m/z 213.2
[M+H].sup.+.
N.sup.2-(3-(cyclohexyloxy)propyl)-N.sup.5-(4-guanidinobutyl)-1H-imidazole--
2,5-dicarboxamide (MOC-27)
[0169] .sup.1H NMR (300 MHz, METHANOL-d.sub.4) d.sub.H ppm 7.73 (s,
1H), 3.38-3.76 (m, 7H), 3.13-3.30 (m, 3H), 1.79-2.07 (m, 5H),
1.63-1.79 (m, 7H), 1.56 (d, J=7.54 Hz, 1H), 1.12-1.42 (m, 5H);
.sup.13C NMR (75 MHz, METHANOL-d.sub.4) d.sub.C ppm 77.5, 65.6,
48.5, 48.2, 47.9, 47.6, 47.4, 47.1, 46.8, 40.7, 37.0, 31.9, 25.8,
25.6, 23.7; LC-MS (ESI), RT=1.98 min, m/z 408.3 [M+H].sup.+.
N.sup.5-(4-guanidinobutyl)-N.sup.2-(2-phenoxyethyl)-1H-imidazole-2,5-dicar-
boxamide (MOC-28)
[0170] .sup.1H NMR (300 MHz, METHANOL-d.sub.4) d.sub.H ppm 8.01 (s,
1H), 7.28 (t, J=7.91 Hz, 2H), 6.82-7.10 (m, 3H), 4.19 (t, J=5.09
Hz, 2H), 3.84 (t, J=5.09 Hz, 2H), 3.44 (br. s., 2H), 3.25 (br. s.,
2H), 1.69 (br. s., 5H); .sup.13C NMR (75 MHz, METHANOL-d.sub.4)
d.sub.C ppm 129.2, 114.2, 48.5, 48.2, 47.9, 47.6, 47.4, 47.1, 46.8;
LC-MS (ESI), RT=1.86 min, m/z 388.4 [M+H].sup.+.
N.sup.5-(4-guanidinobutyl)-N.sup.2-(3-phenoxypropyl)-1H-imidazole-2,5-dica-
rboxamide (MOC-29)
[0171] .sup.1H NMR (300 MHz, METHANOL-d.sub.4) d.sub.H ppm 7.73 (s,
1H), 7.13-7.38 (m, 2H), 6.80-7.05 (m, 3H), 4.10 (t, J=5.93 Hz, 2H),
3.61 (t, J=6.78 Hz, 2H), 3.44 (br. s., 2H), 3.14-3.30 (m, 2H), 2.11
(quin, J=6.36 Hz, 2H), 1.52-1.84 (m, 5H); .sup.13C NMR (75 MHz,
METHANOL-d.sub.4) d.sub.C ppm 129.1, 120.4, 114.2, 65.3, 48.5,
48.2, 47.9, 47.6, 47.4, 47.1, 46.8, 40.8, 37.9, 36.4, 29.0, 26.6,
25.8; LC-MS (ESI), RT=1.51 min, m/z 402.3 [M+H].sup.+.
N.sup.5-(4-guanidinobutyl)-N.sup.2-(4-isopropoxybutyl)-1H-imidazole-2,5-di-
carboxamide (MOC-30)
[0172] .sup.1H NMR (300 MHz, METHANOL-d.sub.4) d.sub.H ppm 7.73 (s,
1H), 3.35-3.74 (m, 8H), 3.14-3.28 (m, 2H), 1.69 (br. s., 9H), 1.16
(d, J=6.22 Hz, 6H); .sup.13C NMR (75 MHz, METHANOL-d.sub.4) d.sub.C
ppm 71.5, 67.4, 48.5, 48.2, 47.9, 47.6, 47.4, 47.1, 46.8, 40.8,
38.7, 37.9, 27.1, 26.6, 26.1, 25.8, 21.0; LC-MS (ESI), RT=2.08 min,
m/z 382.3 [M+H].sup.+.
N.sup.5-(4-guanidinobutyl)-N.sup.2-(6-methylheptyl)-1H-imidazole-2,5-dicar-
boxamide (MOC-31)
[0173] .sup.1H NMR (300 MHz, METHANOL-d.sub.4) d.sub.H ppm 7.74 (s,
1H), 3.38-3.57 (m, 2H), 3.14-3.30 (m, 2H), 1.54-1.83 (m, 6H),
1.22-1.54 (m, 9H), 0.81-1.09 (m, 7H); .sup.13C NMR (75 MHz,
METHANOL-d.sub.4) d.sub.C ppm 48.5, 48.2, 47.9, 47.6, 47.3, 47.1,
46.8, 40.8, 39.5, 30.6, 28.6, 26.7, 25.8, 23.8, 22.7, 13.0, 9.8;
LC-MS (ESI), RT=1.19 min, m/z 380.3 [M+H].sup.+.
N.sup.5-(4-guanidinobutyl)-N.sup.2-(3-methylpentyl)-1H-imidazole-2,5-dicar-
boxamide (MOC-32)
[0174] .sup.1H NMR (300 MHz, METHANOL-d.sub.4) d.sub.H ppm 7.73 (s,
1H), 3.35-3.58 (m, 4H), 3.14-3.30 (m, 2H), 1.69 (br. s., 6H),
1.35-1.59 (m, 3H), 1.24 (td, J=7.06, 13.75 Hz, 1H), 0.77-1.09 (m,
6H); .sup.13C NMR (75 MHz, METHANOL-d.sub.4) d.sub.C ppm 48.5,
48.2, 47.9, 47.6, 47.4, 47.1, 46.8, 40.8, 37.1, 35.9, 32.1, 29.1,
26.6, 25.8, 18.0, 10.2; LC-MS (ESI), RT=1.79 min, in/z 352.3
[M+H].sup.+.
N.sup.2-(n-hexyl)-N.sup.5-(pyridin-2-ylmethyl)-1H-imidazole-2,5-dicarboxam-
ide (MOC-33)
[0175] .sup.1H NMR (300 MHz, METHANOL-d.sub.4) d.sub.H ppm 8.52 (d,
J=4.53 Hz, 1H), 7.70-7.93 (m, 2H), 7.45 (d, J=7.93 Hz, 1H),
7.19-7.40 (m, 1H), 4.71 (s, 2H), 3.39 (t, J=6.99 Hz, 2H), 1.51-1.76
(m, 2H), 1.37 (br. s., 6H), 0.93 (t, J=6.42 Hz, 3H); .sup.13C NMR
(75 MHz, METHANOL-d.sub.4) d.sub.C ppm 158.6, 157.8, 148.5, 137.5,
122.5, 121.9, 121.6, 48.5, 48.2, 47.9, 47.7, 47.4, 47.1, 46.8,
43.7, 39.0, 31.3, 29.2, 26.3, 22.3; LC-MS (ESI), RT=2.74 min, m/z
330.3 [M+H].sup.+.
N.sup.2-(2-acetamidoethyl)-N.sup.5-(pyridin-2-ylmethyl)-1H-imidazole-2,5-d-
icarboxamide (MOC-34)
[0176] .sup.1H NMR (300 MHz, METHANOL-d.sub.4) d.sub.H ppm 8.65 (d,
J=4.91 Hz, 1H), 8.08-8.32 (m, 1H), 7.69-7.93 (m, 2H), 7.54-7.69 (m,
1H), 3.47 (dd, J=5.85, 19.83 Hz, 4H), 1.97 (s, 3H), 0.92-1.19 (m,
1H); .sup.13C NMR (75 MHz, METHANOL-d.sub.4) d.sub.C ppm 142.1,
124.0, 123.6, 48.5, 48.2, 47.9, 47.7, 47.4, 47.1, 46.8, 39.3, 38.7,
21.2; LC-MS (ESI), RT=3.18 min, m/z 331.3 [M+H].sup.+.
N.sup.5-(furan-2-ylmethyl)-N.sup.2-(n-hexyl)-1H-imidazole-2,5-dicarboxamid-
e (MOC-35)
[0177] .sup.1H NMR (300 MHz, METHANOL-d.sub.4) d.sub.H ppm 7.75 (s,
1H), 7.45 (s, 1H), 6.18-6.55 (m, 2H), 4.57 (s, 2H), 3.36-3.42 (m,
2H), 1.61 (d, J=7.18 Hz, 2H), 1.37 (br. s., 7H), 0.76-1.06 (m, 3H);
.sup.13C NMR (75 MHz, METHANOL-d.sub.4) d.sub.C ppm 158.6, 151.7,
142.0, 121.8, 110.0, 106.8, 48.5, 48.2, 47.9, 47.7, 47.4, 47.1,
46.8, 38.9, 35.4, 31.3, 29.2, 26.3, 22.3, 13.0; LC-MS (ESI),
RT=2.43 min, m/z 319. 3 [M+H].sup.+.
N.sup.2-(2-acetamidoethyl)-N.sup.5-(furan-2-ylmethyl)-1H-imidazole-2,5-dic-
arboxamide (MOC-36)
[0178] .sup.1H NMR (300 MHz, METHANOL-d.sub.4) d.sub.H ppm 7.75 (s,
1H), 7.45 (s, 1H), 6.32 (s, 1H), 6.37 (s, 1H), 4.57 (s, 2H),
3.35-3.65 (m, 5H), 1.96 (s, 3H); .sup.13C NMR (75 MHz,
METHANOL-d.sub.4) d.sub.C ppm 172.6, 159.0, 151.8, 142.0, 110.0,
106.8, 48.5, 48.2, 47.9, 47.7, 47.4, 47.1, 46.8, 39.0, 38.7, 35.4,
21.2; LC-MS (ESI), RT=1.71 min, m/z 319.13 [M+H].sup.+.
Example 8
Biological Screening: General
[0179] All reagents used in the biological screenings were
purchased from Sigma-Aldrich (UK) of molecular biology grade unless
stated otherwise.
Example 9
NS3 Protein
(i) NS3 Constructs
[0180] A synthetic gene coding for the HCV NS3 domain of genotype
4a, the most abundant HCV in Saudi Arabia and Egypt [Massariol,
M.-J.; Zhao, S.; Marquis, M.; Thibeault, D.; White, P. W., Protease
and helicase activities of hepatitis C virus genotype 4, 5, and 6
NS3-NS4A proteins. Biochemical and Biophysical Research
Communications 2010, 391 (1), 692-697], was synthesized by
GenScript (Hong Kong), the nucleotide sequence was optimized for E.
coli codon usage. The synthetic gene was cloned as NdeI-BamHI
fragment into the expression vector pET-3a Novagen.RTM.. The
obtained construct was sequenced to confirm that we had the right
clone and the gene was in the correct frame.
(ii) NS3 Protein Information
Accession GU085486.1
[0181] HCV genotype 4a (The most common genotype in Saudi Arabia)
[Bawazir, A.; AlGusheri, F.; Jradi, H.; AlBalwi, M.; Abdel-Gader,
A.-G., Hepatitis C virus genotypes in Saudi Arabia: a future
prediction and laboratory profile. Virology journal 2017, 14 (1),
208-208] NS3 from 4 to 182 aa (L/E, F/E, I/Q, V/E, L/Q, C/S) NS4A
632 to 685 aa (i/n) G svvivgrvnl sgdtayaqqt rgeestqets qtgrdtnenc
gevqvlstat qsflgtavng vmwtvyhgag sktisgpkgp vnqmytnvdq dlvgwpsppg
vksltpctcg asdlylvtrh advvpvrrrg dtrgallspr pistlkgssg gpllcpmgha
aglfraayst rgvakavdfv pveslett mrsp NS4A/NS3 Fusion protein
expression in pET-28a NS3 protease domain 1-181 aa+N-terminal T7
tag and C-terminal His tag M ASMTGGQQMG apitayaqqt rglfstivts
ltgrdtnenc gevqvlstat qsflgtavng vmwtvyhgag sktisgpkgp vnqmytnvdq
dlvgwpsppg vksltpctcg asdlylvtrh advvpvrrrg dtrgallspr pistlkgssg
gpllcpmgha aglfraavct rgvakavdfv pveslettmr sGSHHHHHH Expression in
pET-3a (iii) Protein eNS4Axpression
[0182] The sequence of NS3 domain for genotype 4a, was expressed in
E. coli Rosette (DE3) pLysS according to standard protocol [Kim,
J.; Morgenstern, K.; Lin, C.; Fox, T.; Dwyer, M.; Landro, J.;
Chambers, S.; Markland, W.; Lepre, C.; O'malley, E., Crystal
structure of the hepatitis C virus NS3 protease domain complexed
with a synthetic NS4A cofactor peptide. Cell 1996, 87 (2),
343-355]. Therefore, a synthetic gene for NS3 domain was subcloned
in the expression vector pET-3a. In the process, a 100 mL of
bacterial culture in Luria Broth medium was grown overnight at
37.degree. C. and used for inoculation of 10 L LB in a 14-liter
fermenter flask (New Brunswick Scientific Co., CT, USA). The media
was supplemented with 50 .mu.g/mL ampicillin. The culture grew
until the OD.sub.600 reached 0.5-0.6, then it was cooled to
25.degree. C. and 1 mM IPTG was added. Expression was followed
overnight, and then cells were harvested.
(iv) Protein Purification
[0183] The produced protein was purified using equilibrated Ni-NTA
beads and the poly-histidine tag was not removed. In the process,
cells were re-suspended (1 g/5 mL) in buffer (50 mM HEPES, 0.3 M
NaCl, 10% glycerol, 2 mM .beta.-mercaptoethanol, pH 8). Lysozymes
were added (1 mg/mL) followed by protease inhibitor cocktail tablet
and the suspension was sonicated. Cell lysate was centrifuged to
collect the clear supernatant that contained the desired NS3
protein. The protein was purified using pre-equilibrated Ni-NTA
beads (Qiagen, USA). Beads were washed with buffer (50 mM HEPES,
0.3 M NaCl, 10% glycerol, 2 mM .beta.-mercaptoethanol, 20 mM
imidazole, pH 8) and eluted with another buffer (50 mM HEPES, 0.3 M
NaCl, 10% glycerol, 2 mM .beta.-mercaptoethanol, 350 mM imidazole,
pH 8). Fractions were collected and concentrated using Amicon
Ultra-4 3000 MWCO centrifugal device (Millipore, Germany). Protein
purity after Ni-affinity purification step was not less than 70%.
The purity, as estimated by SDS-PAGE, was sufficient to perform all
investigations of this study and the protein was stable for several
hours at test conditions (see FIG. 4) [Massariol, M.-J.; Zhao, S.;
Marquis, M.; Thibeault, D.; White, P. W., Protease and helicase
activities of hepatitis C virus genotype 4, 5, and 6 NS3-NS4A
proteins. Biochemical and Biophysical Research Communications 2010,
391 (1), 692-697]. The concentration of NS3 in the final
concentrate was measured using Nanodrop.TM. nanoscale
spectrophotometer.
[0184] When needed, further purification of the protein was
accomplished on Superdex 75 16/90 column (GE Healthcare, USA)
equilibrated in 20 mM HEPES, 10 mM DDT, 200 mM NaCl, pH 7.6 run at
rate of 1 mL/min followed by SDS-PAGE for purity estimation.
Example 10
NS4A
[0185] The cofactor NS4A and the fluorescent fluorescein
isothiocyanate NS4A (FITC-NS4A) were purchased from GenScript (Hong
Kong). NS4A structure was identical to that of HCV genotype 4a with
two lysine residues added at both the N- and C-termini. Thus the
structure of NS4A used in this study was
LL-G.sub.21SVVIVGRIVLSG.sub.33-LL.
Example 11
Binding and Competition Assay
[0186] Binding buffer (20 mM HEPES, 10 mM DTT, 200 mM NaCl, pH
7.6), a mixture of NS3 (1.8 .mu.M) and FITC-NS4A (0.1 .mu.M) at the
calculated affinity constant concentration were placed in a 96-well
plate as binding test solution. A serial dilution of MOC compounds
(dissolved in 1.5% DMSO in binding buffer) (1/2 dilution starting
from 150 .mu.M to 0.292 .mu.M) was mixed gently with the binding
text solution for 60 minutes in the dark. The dissociation constant
was calculated according to the recommended equation embedded in
GraphPad Prism v8.
Example 12
Enzyme Inhibition Assay
[0187] The assay was performed using SensoLyte-520.RTM. HCV
protease assay kit fluorometric* (Anaspec, Fremont, Calif., USA)
according to a modified procedure to suit the purpose of
determination of allosteric inhibition. NS3 (4.0 .mu.M) was mixed
with MOC-23 (6.3 .mu.M) for 15 minutes. Afterwards,
5-FAM/QXL.TM.520 fluorescence resonance energy transfer (FRET)
peptide was added as instructed by the assay kit manual. The
sequence of this FRET peptide (5-FAM-SLGRKIQIQ) is derived from the
cleavage site of NS4A/NS4B. In the FRET peptide, the fluorescence
of 5-FAM is quenched by QXL.TM.520. Upon cleavage into two separate
fragments by HCV NS3/4A protease, the fluorescence of 5-FAM is
recovered, and can be monitored at 490 nm/520 nm
(excitation/emission). Controls included buffer, compound,
compound+NS3, NS3+NS4A and FRET peptide separately. All test wells
and controls were repeated in triplicates at the same 96-well
plate.
Example 13
Biological Screening Results
[0188] The binding affinity test was performed using fluorescence
anisotropy technique. In this test, maximum fluorescence was first
determined at optimal ratio of NS3 (1.8 .mu.M) and FITC-NS4A (0.1
.mu.M). Compounds that compete with NS4A should be able to decrease
the fluorescence of this mixture because they prevent the binding
to occur and it should be concentration-dependent. Results showed
some compounds suppressed the fluorescence emitted upon binding of
NS4A with NS3. However, we found that MOC-23 showed highest
affinity and more reproducible results than other compounds (see
FIG. 5). It could effectively compete with FITC-NS4A at
dissociation constant K.sub.d=6.303.+-.0.3 .mu.M). The
reproducibility was checked using another batch of the express NS3
protein following the same procedure and the value was
calculated=7.815.+-.1.014 .mu.M. Compound MOC-24 showed binding
affinity at similar level but results were less reproducible and
standard error was higher. Therefore, compound MOC-23 was tested
for its ability to inhibit the NS3 enzyme. MOC-23 at its binding
affinity concentration (6.3 .mu.M) was able to inhibit NS3 and
abolished any proteolytic activity of the enzyme (see FIG. 6).
* * * * *