[0001] The present invention relates to macrocyclic compounds that are useful as inhibitors
of the hepatitis C virus (HCV) NS3 protease, their synthesis, and their use for treating
or preventing HCV infection.
BACKGROUND OF THE INVENTION
[0002] Hepatitis C virus (HCV) infection is a major health problem that leads to chronic
liver disease, such as cirrhosis and hepatocellular carcinoma, in a substantial number
of infected individuals, estimated to be 2-15% of the world's population. There are
an estimated 3.9 million infected people in the United States alone, according to
the U.S. Center for Disease Control, roughly five times the number of people infected
with the human immunodeficiency virus (HIV). According to the World Health Organization,
there are more than 170 million infected individuals worldwide, with at least 3 to
4 million people being infected each year. Once infected, about 20% of people clear
the virus, but the rest harbor HCV the rest of their lives. Ten to twenty percent
of chronically infected individuals eventually develop liver-destroying cirrhosis
or cancer. The viral disease is transmitted parenterally by contaminated blood and
blood products, contaminated needles, or sexually and vertically from infected mothers
or carrier mothers to their off-spring.
[0003] Current treatments for HCV infection, which are restricted to immunotherapy with
recombinant interferon-α alone or in combination with the nucleoside analog ribavirin,
are of limited clinical benefit. Moreover, there is no established vaccine for HCV.
Consequently, there is an urgent need for improved therapeutic agents that effectively
combat chronic HCV infection. The current state of the art in the treatment of HCV
infection has been discussed in the following references:
B. Dymock, et al., "Novel approaches to the treatment of hepatitis C virus infection,"
Antiviral Chemistry & Chemotherapy, 11: 79-96 (2000);
H. Rosen, et al., "Hepatitis C virus: current understanding and prospects for future
therapies," Molecular Medicine Today, 5: 393-399 (1999);
D. Moradpour, et al., "Current and evolving therapies for hepatitis C," European J.
Gastroenterol. Hepatol., 11: 1189-1202 (1999);
R. Bartenschlager, "Candidate Targets for Hepatitis C Virus-Specific Antiviral Therapy,"
Intervirology, 40: 378-393 (1997);
G.M. Lauer and B.D. Walker, "Hepatitis C Virus Infection," N. Engl. J. Med., 345:
41-52 (2001);
B.W. Dymock, "Emerging therapies for hepatitis C virus infection," Emerging Drugs,
6: 13-42 (2001); and
C. Crabb, "Hard-Won Advances Spark Excitement about Hepatitis C," Science: 506-507
(2001).
[0004] Several virally-encoded enzymes are putative targets for therapeutic intervention,
including a metalloprotease (NS2-3), a serine protease (NS3), a helicase (NS3), and
an RNA-dependent RNA polymerase (NSSB). The NS3 protease is located in the N-terminal
domain of the NS3 protein, and is considered a prime drug target since it is responsible
for an intramolecular cleavage at the NS3/4A site and for downstream intermolecular
processing at the NS4A/4B, NS4B/5A and NS5A/5B junctions. Previous research has identified
classes of peptides, such as hexapeptides as well as tripeptides discussed in U.S.
patent applications
US2005/0020503,
US2004/0229818, and
US2004/00229776, showing degrees of activity in inhibiting the NS3 protease. The international application
WO 03/064455 shows macrocyclic peptides which are useful as inhibitors of the the HCV NS3 protease.
The aim of the present invention is to provide further compounds which exhibit activity
against the HCV NS3 protease.
SUMMARY OF THE INVENTION
[0005] The present invention relates to novel macrocyclic compounds of formula (
I) and/or pharmaceutically acceptable salts or hydrates thereof. These compounds are
useful in the inhibition of HCV (hepatitis C virus) NS3 (non-structural 3) protease,
the prevention or treatment of one or more of the symptoms of HCV infection, either
as compounds or their pharmaceutically acceptable salts or hydrates (when appropriate),
or as pharmaceutical composition ingredients, whether or not in combination with other
HCV antivirals, anti-infectives, immunomodulators, antibiotics or vaccines. More particularly,
the present invention relates to a compound of formula (
I) and/or a pharmaceutically acceptable salt or hydrate thereof:

wherein:
p and q are both 1;
R1 is CONR10SO2R6;
R2 is C1-C6 alkyl or C2-C6 alkenyl;, wherein said alkyl or alkenyl is optionally substituted with 1 to 3 halo;
R3 is C1-C8 alkyl or C3-C8 cycloalkyl;
R5 is H;
R6 is C3-C6 cycloalkyl;
Y is C(=O);
ZisO;
M is C1-C12 alkylene or C1-C12 alkenylene; and
each R10 is independently H or C1-C6 alkyl.
[0006] The present invention also includes pharmaceutical compositions containing a compound
of the present invention and methods of preparing such pharmaceutical compositions.
The present invention further relates to methods of treating or preventing one or
more symptoms of HCV infection.
[0007] Other embodiments, aspects and features of the present invention are either further
described in or will be apparent from the ensuing description, examples and appended
claims.
DETAILED DESCRIPTION OF THE INVENTION
[0008] The present invention includes compounds of formula
I above, and pharmaceutically acceptable salts and/or hydrates thereof. These compounds
and their pharmaceutically acceptable salts and/or hydrates are HCV protease inhibitors
(e.g., HCV NS3 protease inhibitors). The present invention also includes compounds
of formulae
II-a and
III-a wherein all variables are as defined for formula
I.

[0009] A first embodiment of the present invention is a compound of formula
I, II-a or
III-a, or a pharmaceutically acceptable salt or hydrate thereof, wherein R
1 is CONHSO
2R
6, and all other variables are as originally defined (i.e., as defined in the Summary
of the Invention). In a first aspect of the first embodiment, R
1 is CONHSO
2R
6 wherein R
6 is C
3-C
5 cycloalkyl; and all other variables are as defined in the first embodiment. In a
feature of the first aspect of the first embodiment, R
1 is CONHSO
2R
6 wherein R
6 is cyclopropyl; and all other variables are as defined in the first embodiment.
[0010] A second embodiment of the present invention is a compound of formula
I, II-a or
III-a, or a pharmaceutically acceptable salt or hydrate thereof, wherein R
2 is C
1-C
6 alkyl or C
2-C
6 alkenyl; and all other variables are as originally defined or as defined in any one
of the preceding embodiments. In a first aspect of the second embodiment, R
2 is C
1-C
4 alkyl or C
2-C
4 alkenyl; and all other variables are as originally defined or as defined in any one
of the preceding embodiments. In a second aspect of the second embodiment, R
2 is C
2-C
4 alkenyl; and all other variables are as originally defined or as defined in any one
of the preceding embodiments. In a feature of the second aspect of the second embodiment,
R
2 is vinyl; and all other variables are as defined in the third embodiment or as defined
in any one of the preceding embodiments. In a third aspect of the second embodiment,
R
2 is C
1-C
4 alkyl; and all other variables are as originally defmed or as defined in any one
of the preceding embodiments. In a feature of the third aspect of the third embodiment,
R
2 is ethyl; and all other variables are as defined in the third embodiment or as defined
in any one of the preceding embodiments.
[0011] A third embodiment of the present invention is a compound of formula
I,
II-a or
III-a, or a pharmaceutically acceptable salt or hydrate thereof, wherein R
3 is C
3-C
8 cycloalkyl or C
1-C
8 alkyl; and all other variables are as originally defmed or as defined in any one
of the preceding embodiments. In a first aspect of the third embodiment, R
3 is C
5-C
7 cycloalkyl or C
1-C
8 alkyl; and all other variables are as defined in the third embodiment or as defmed
in any one of the preceding embodiments. In a second aspect of the third embodiment,
R
3 is C
5-C
6 cycloalkyl or C
1-C
8 alkyl; and all other variables are as defined in the third embodiment or as defmed
in any one of the preceding embodiments. In a third aspect of the third embodiment,
R
3 is propyl or butyl; and all other variables are as defined in the third embodiment
or as defined in any one of the preceding embodiments. In a feature of the third aspect
of the third embodiment, R
3 is i-propyl, n-butyl or t-butyl; and all other variables are as defined in the third
embodiment or as defined in any one of the preceding embodiments. In a fourth aspect
of the third embodiment, R
3 is cyclopentyl or cyclohexyl; and all other variables are as defined in the third
embodiment or as defined in any one of the preceding embodiments.
[0012] A fourth embodiment of the present invention is a compound of formula
I, II-a or
III-a, or a pharmaceutically acceptable salt or hydrate thereof, wherein M is C
1-C
10 alkylene or C
2-C
10 alkenylene (including linear and branched chain alkylene or alkenylene); and all
other variables are as originally defined or as defined in any one of the preceding
embodiments. In a first aspect of the fourth embodiment, M is C
1-C
8 alkylene or C
2-C
8 alkenylene (including linear and branched chain alkylene or alkenylene); and all
other variables are as originally defmed or as defmed in any one of the preceding
embodiments. In a second aspect of the fourth embodiment, M is C
4 alkylene or C
4 alkenylene (including linear and branched chain alkylene or alkenylene); and all
other variables are as defined in the fourth embodiment or as defined in any one of
the preceding embodiments. In a third aspect of the fourth embodiment, M is C
5 alkylene or C
5 alkenylene (including linear and branched chain alkylene or alkenylene); and all
other variables are as defined in the fourth embodiment or as defined in any one of
the preceding embodiments. In a fourth aspect of the fourth embodiment, M is C
6 alkylene or C
6 alkenylene (including linear and branched chain alkylene or alkenylene); and all
other variables are as defined in the fourth embodiment or as defined in any one of
the preceding embodiments. In a fifth aspect of the fourth embodiment, M is C
7 alkylene or C
7 alkenylene (including linear and branched chain alkylene or alkenylene); and all
other variables are as defined in the fourth embodiment or as defined in any one of
the preceding embodiments. In a sixth aspect of the fourth embodiment, M is C
8 alkylene or C
8 alkenylene (including linear and branched chain alkylene or alkenylene); and all
other variables are as defined in the fourth embodiment or as defined in any one of
the preceding embodiments. In a seventh aspect of the fourth embodiment, M is C
9 alkylene or C
9 alkenylene (including linear and branched chain alkylene or alkenylene); and all
other variables are as defined in the fourth embodiment or as defined in any one of
the preceding embodiments. In an eighth aspect of the fourth embodiment, M is C
10 alkylene or C
10 alkenylene (including linear and branched chain alkylene or alkenylene); and all
other variables are as defined in the fourth embodiment or as defmed in any one of
the preceding embodiments. In an nineth aspect of the fourth embodiment, M is selected
from the following; and all other variables are as defined in the fourth embodiment
or as defined in any one of the preceding embodiments.

III-225
[0014] Other embodiments of the present invention relate to the following:
- (a) A pharmaceutical composition comprising an effective amount of a compound of formula
I, II-a, or III-a and a pharmaceutically acceptable carrier.
- (b) The pharmaceutical composition of (a), further comprising a second therapeutic
agent selected from the group consisting of a HCV antiviral agent, an immunomodulator,
and an anti-infective agent.
- (c) The pharmaceutical composition of (b), wherein the HCV antiviral agent is an antiviral
selected from the group consisting of a HCV protease inhibitor and a HCV NS5B polymerase
inhibitor.
- (d) A pharmaceutical combination which is (i) a compound of formula I, II-a, or III-a and (ii) a second therapeutic agent selected from the group consisting of a HCV antiviral
agent, an immunomodulator, and an anti-infective agent; wherein the compound of formula
I, II-a, or III-a and the second therapeutic agent are each employed in an amount that renders the
combination effective for inhibiting HCV NS3 protease, or for treating or preventing
infection by HCV.
- (e) The combination of (d), wherein the HCV antiviral agent is an antiviral selected
from the group consisting of a HCV protease inhibitor and a HCV NS5B polymerase inhibitor.
- (f) A method of inhibiting HCV NS3 protease in a subject in need thereof which comprises
administering to the subject an effective amount of a compound of formula I, II-a, or III-a.
- (g) A method of preventing or treating infection by HCV in a subject in need thereof
which comprises administering to the subject an effective amount of a compound of
formula I, II-a, or III-a.
- (h) The method of (g), wherein the compound of formula I, II-a, or III-a is administered in combination with an effective amount of at least one second therapeutic
agent selected from the group consisting of a HCV antiviral agent, an immunomodulator,
and an anti-infective agent.
- (i) The method of (h), wherein the HCV antiviral agent is an antiviral selected from
the group consisting of a HCV protease inhibitor and a HCV NS5B polymerase inhibitor.
- (j) A method of inhibiting HCV NS3 protease in a subject in need thereof which comprises
administering to the subject the pharmaceutical composition of (a), (b), or (c) or
the combination of (d) or (e).
- (k) A method of preventing or treating infection by HCV in a subject in need thereof
which comprises administering to the subject the pharmaceutical composition of (a),
(b), or (c) or the combination of (d) or (e).
[0015] The present invention also includes a compound of the present invention (i) for use
in, (ii) for use as a medicament for, or (iii) for use in the preparation of a medicament
for: (a) inhibiting HCV NS3 protease, or (b) preventing or treating infection by HCV.
In these uses, the compounds of the present invention can optionally be employed in
combination with one or more second therapeutic agents selected from HCV antiviral
agents, anti-infective agents, and immunomodulators.
[0016] Additional embodiments of the invention relate to the pharmaceutical compositions,
combinations and methods set forth in (a)-(k) above and the uses set forth in the
preceding paragraph, wherein the compound of the present invention employed therein
is a compound of one of the embodiments, aspects, classes, sub-classes, or features
of the compounds described above. In all of these embodiments, the compound may optionally
be used in the form of a pharmaceutically acceptable salt or hydrate as appropriate.
[0017] As used herein, the term "alkyl" refers to any linear or branched chain alkyl group
having a number of carbon atoms in the specified range. Thus, for example, "C
1-
6 alkyl" (or "C
1-C
6 alkyl") refers to all of the hexyl alkyl and pentyl alkyl isomers as well as n-,
iso-, sec- and t-butyl, n- and isopropyl, ethyl and methyl. As another example, "C
1-4 alkyl" refers to n-, iso-, sec- and t-butyl, n- and isopropyl, ethyl and methyl.
[0018] The term "haloalkyl" refers to an alkyl group wherein a hydrogen has been replaced
by a halogen. The term "alkoxy" refers to an "alkyl-O-" group.
[0019] The term "alkylene" refers to any linear or branched chain alkylene group having
a number of carbon atoms in the specified range. Thus, for example, "-C
1-6 alkylene-" refers to any of the C
1 to C
6 linear or branched alkylenes. A class of alkylenes of particular interest with respect
to the invention is -(CH
2)
1-6-, and sub-classes of particular interest include -(CH
2)
1-4-
, -(CH
2)
1-3-, -(CH
2)
1-2-, and -CH
2-. Also of interest is the alkylene -CH(CH
3)-.
[0020] The term "alkenylene" refers to any linear or branched chain divalent alkenylene
group having a number of carbon atoms in the specified range.
[0021] The terms "cycloalkyl" refers to any cyclic ring of an alkane or alkene having a
number of carbon atoms in the specified range. Thus, for example, "C
3-8 cycloalkyl" (or "C
3-C
8 cycloalkyl") refers to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,
and cyclooctyl. The term "cycloalkoxy" refers to a "cycloalkyl-O-" group.
[0022] The term "halogen" (or "halo") refers to fluorine, chlorine, bromine and iodine (alternatively
referred to as fluoro, chloro, bromo, and iodo).
[0023] Unless expressly stated to the contrary, all ranges cited herein are inclusive. For
example, a heteroaryl ring described as containing from "1 to 3 heteroatoms" means
the ring can contain 1, 2, or 3 heteroatoms. It is also to be understood that any
range cited herein includes within its scope all of the sub-ranges within that range.
The oxidized forms of the heteroatoms N and S are also included within the scope of
the present invention.
[0024] When any variable (e.g., R
10) occurs more than one time in any constituent or in formula
I, II-a, or
III-a or in any other formula depicting and describing compounds of the invention, its
definition on each occurrence is independent of its definition at every other occurrence.
Also, combinations of substituents and/or variables are permissible only if such combinations
result in stable compounds.
[0025] Unless expressly stated to the contrary, substitution by a named substituent is permitted
on any atom in a ring (e.g., aryl, a heteroaromatic ring, or a saturated heterocyclic
ring) provided such ring substitution is chemically allowed and results in a stable
compound. A "stable" compound is a compound which can be prepared and isolated and
whose structure and properties remain or can be caused to remain essentially unchanged
for a period of time sufficient to allow use of the compound for the purposes described
herein (e.g., therapeutic or prophylactic administration to a subject).
[0026] As a result of the selection of substituents and substituent patterns, certain of
the compounds of the present invention can have asymmetric centers and can occur as
mixtures of stereoisomers, or as individual diastereomers, or enantiomers. All isomeric
forms of these compounds, whether isolated or in mixtures, are within the scope of
the present invention.
[0027] As would be recognized by one of ordinary skill in the art, certain of the compounds
of the present invention can exist as tautomers. For the purposes of the present invention
a reference to a compound of formula
I, II-a, or
III-a is a reference to the compound per se, or to any one of its tautomers per se, or
to mixtures of two or more tautomers.
[0028] The compounds of the present inventions are useful in the inhibition of HCV protease
(e.g., HCV NS3 protease) and the prevention or treatment of infection by HCV. For
example, the compounds of this invention are useful in treating infection by HCV after
suspected past exposure to HCV by such means as blood transfusion, exchange of body
fluids, bites, accidental needle stick, or exposure to patient blood during surgery.
[0029] The compounds of this invention are useful in the preparation and execution of screening
assays for antiviral compounds. For example, the compounds of this invention are useful
for isolating enzyme mutants, which are excellent screening tools for more powerful
antiviral compounds. Furthermore, the compounds of this invention are useful in establishing
or determining the binding site of other antivirals to HCV protease, e.g., by competitive
inhibition. Thus the compounds of this invention are commercial products to be sold
for these purposes.
[0030] The compounds of the present invention may be administered in the form of pharmaceutically
acceptable salts. The term "pharmaceutically acceptable salt" refers to a salt which
possesses the effectiveness of the parent compound and which is not biologically or
otherwise undesirable (e.g., is neither toxic nor otherwise deleterious to the recipient
thereof). Suitable salts include acid addition salts which may, for example, be formed
by mixing a solution of the compound of the present invention with a solution of a
pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, acetic
acid, trifluoroacetic acid, or benzoic acid. Many of the compounds of the invention
carry an acidic moiety, in which case suitable pharmaceutically acceptable salts thereof
can include alkali metal salts (e.g., sodium or potassium salts), alkaline earth metal
salts (e.g., calcium or magnesium salts), and salts formed with suitable organic ligands
such as quaternary ammonium salts. Also, in the case of an acid (-COOH) or alcohol
group being present, pharmaceutically acceptable esters can be employed to modify
the solubility or hydrolysis characteristics of the compound.
[0031] The term "administration" and variants thereof (e.g., "administering" a compound)
in reference to a compound of the invention mean providing the compound or a prodrug
of the compound to the individual in need of treatment. When a compound of the invention
or a prodrug thereof is provided in combination with one or more other active agents
(e.g., antiviral agents useful for treating HCV infection), "administration" and its
variants are each understood to include concurrent and sequential provision of the
compound or salt (or hydrate) and other agents.
[0032] As used herein, the term "composition" is intended to encompass a product comprising
the specified ingredients, as well as any product which results, directly or indirectly,
from combining the specified ingredients.
[0033] By "pharmaceutically acceptable" is meant that the ingredients of the pharmaceutical
composition must be compatible with each other and not deleterious to the recipient
thereof.
[0034] The term "subject" (alternatively referred to herein as "patient") as used herein
refers to an animal, preferably a mammal, most preferably a human, who has been the
object of treatment, observation or experiment.
[0035] The term "effective amount" as used herein means that amount of active compound or
pharmaceutical agent that elicits the biological or medicinal response in a tissue,
system, animal or human that is being sought by a researcher, veterinarian, medical
doctor or other clinician. In one embodiment, the effective amount is a "therapeutically
effective amount" for the alleviation of the symptoms of the disease or condition
being treated. In another embodiment, the effective amount is a "prophylactically
effective amount" for prophylaxis of the symptoms of the disease or condition being
prevented. The term also includes herein the amount of active compound sufficient
to inhibit HCV NS3 protease and thereby elicit the response being sought (i.e., an
"inhibition effective amount"). When the active compound (i.e., active ingredient)
is administered as the salt, references to the amount of active ingredient are to
the free acid or free base form of the compound.
[0036] For the purpose of inhibiting HCV NS3 protease and preventing or treating HCV infection,
the compounds of the present invention, optionally in the form of a salt or a hydrate,
can be administered by any means that produces contact of the active agent with the
agent's site of action. They can be administered by any conventional means available
for use in conjunction with pharmaceuticals, either as individual therapeutic agents
or in a combination of therapeutic agents. They can be administered alone, but typically
are administered with a pharmaceutical carrier selected on the basis of the chosen
route of administration and standard pharmaceutical practice. The compounds of the
invention can, for example, be administered orally, parenterally (including subcutaneous
injections, intravenous, intramuscular, intrasternal injection or infusion techniques),
by inhalation spray, or rectally, in the form of a unit dosage of a pharmaceutical
composition containing an effective amount of the compound and conventional non-toxic
pharmaceutically-acceptable carriers, adjuvants and vehicles. Liquid preparations
suitable for oral administration (e.g., suspensions, syrups, elixirs and the like)
can be prepared according to techniques known in the art and can employ any of the
usual media such as water, glycols, oils, alcohols and the like. Solid preparations
suitable for oral administration (e.g., powders, pills, capsules and tablets) can
be prepared according to techniques known in the art and can employ such solid excipients
as starches, sugars, kaolin, lubricants, binders, disintegrating agents and the like.
Parenteral compositions can be prepared according to techniques known in the art and
typically employ sterile water as a carrier and optionally other ingredients, such
as a solubility aid. Injectable solutions can be prepared according to methods known
in the art wherein the carrier comprises a saline solution, a glucose solution or
a solution containing a mixture of saline and glucose. Further description of methods
suitable for use in preparing pharmaceutical compositions of the present invention
and of ingredients suitable for use in said compositions is provided in
Remington's Pharmaceutical Sciences, 18th edition, edited by A. R. Gennaro, Mack
Publishing Co., 1990.
[0037] The compounds of this invention can be administered orally in a dosage range of 0.001
to 1000 mg/kg of mammal (e.g., human) body weight per day in a single dose or in divided
doses. One preferred dosage range is 0.01 to 500 mg/kg body weight per day orally
in a single dose or in divided doses. Another preferred dosage range is 0.1 to 100
mg/kg body weight per day orally in single or divided doses. For oral administration,
the compositions can be provided in the form of tablets or capsules containing 1.0
to 500 milligrams of the active ingredient, particularly 1, 5, 10, 15, 20, 25, 50,
75, 100, 150, 200, 250, 300, 400, and 500 milligrams of the active ingredient for
the symptomatic adjustment of the dosage to the patient to be treated. The specific
dose level and frequency of dosage for any particular patient may be varied and will
depend upon a variety of factors including the activity of the specific compound employed,
the metabolic stability and length of action of that compound, the age, body weight,
general health, sex, diet, mode and time of administration, rate of excretion, drug
combination, the severity of the particular condition, and the host undergoing therapy.
[0038] As noted above, the present invention also relates to a method of inhibiting HCV
NS3 protease, inhibiting HCV replication, or preventing or treating HCV infection
with a compound of the present invention in combination with one or more therapeutic
agents and a pharmaceutical composition comprising a compound of the present invention
and one or more therapeutic agents selected from the group consisting of a HCV antiviral
agent, an immunomodulator, and an anti-infective agent. Such therapeutic agents active
against HCV include, but are not limited to, ribavirin, levovirin, viramidine, thymosin
alpha-1, R7025 (an enhanced interferon (Roche)), interferon-β, interferon-α, pegylated
interferon-α (peginterferon-α), a combination of interferon-α and ribavirin, a combination
of peginterferon-α and ribavirin, a combination of interferon-α and levovirin, and
a combination of peginterferon-α and levovirin. Interferon-α includes, but is not
limited to, recombinant interferon-α2a (such as Roferon interferon available from
Hoffmann-LaRoche, Nutley, NJ), pegylated interferon-α2a (Pegasys
™), interferon-α2b (such as Intron-A interferon available from Schering Corp., Kenilworth,
NJ), pegylated interferon-α2b (PegIntron
™), a recombinant consensus interferon (such as interferon alphacon-1), albuferon (interferon-α
bound to human serum albumin (Human Genome Sciences)), and a purified interferon-α
product. Amgen's recombinant consensus interferon has the brand name Infergen®. Levovirin
is the L-enantiomer of ribavirin which has shown immunomodulatory activity similar
to ribavirin. Viramidine represents an analog of ribavirin disclosed in
WO 01/60379 (assigned to ICN Pharmaceuticals). In accordance with the method of the present invention,
the individual components of the combination can be administered separately at different
times during the course of therapy or concurrently in divided or single combination
forms.
[0039] For the treatment of HCV infection, the compounds of the present invention may also
be administered in combination with an agent that is an inhibitor of HCV NS3 serine
protease. HCV NS3 serine protease is an essential viral enzyme and has been described
to be an excellent target for inhibition of HCV replication. Both substrate and non-substrate
based inhibitors of HCV NS3 protease inhibitors are disclosed in
WO 98/22496,
WO 98/46630,
WO 99/07733,
WO 99/07734,
WO 99/38888,
WO 99/50230,
WO 99/64442,
WO 00/09543,
WO 00/59929,
GB-2337262,
WO 02/48116,
WO 02/48172, and
U.S. Patent No. 6,323,180.
[0040] Ribavirin, levovirin, and viramidine may exert their anti-HCV effects by modulating
intracellular pools of guanine nucleotides via inhibition of the intracellular enzyme
inosine monophosphate dehydrogenase (IMPDH). IMPDH is the rate-limiting enzyme on
the biosynthetic route in
de novo guanine nucleotide biosynthesis. Ribavirin is readily phosphorylated intracellularly
and the monophosphate derivative is an inhibitor of IMPDH. Thus, inhibition of IMPDH
represents another useful target for the discovery of inhibitors of HCV replication.
Therefore, the compounds of the present invention may also be administered in combination
with an inhibitor of IMPDH, such as VX-497, which is disclosed in
WO 97/41211 and
WO 01/00622 (assigned to Vertex); another IMPDH inhibitor, such as that disclosed in
WO 00/25780 (assigned to Bristol-Myers Squibb); or mycophenolate mofetil [see
A.C. Allison and E.M. Eugui, Agents Action, 44 (Suppl.): 165 (1993)].
[0041] For the treatment of HCV infection, the compounds of the present invention may also
be administered in combination with the antiviral agent amantadine (1-aminoadamantane)
[for a comprehensive description of this agent, see
J. Kirschbaum, Anal. Profiles Drug Subs. 12: 1-36 (1983)].
[0042] For the treatment of HCV infection, the compounds of the present invention may also
be administered in combination with the antiviral agent polymerase inhibitor R7128
(Roche).
[0043] The compounds of the present invention may also be combined for the treatment of
HCV infection with antiviral 2'-C-branched ribonucleosides disclosed in
R. E. Harry-O'kuru, et al., J. Org. Chem., 62: 1754-1759 (1997);
M. S. Wolfe, et al., Tetrahedron Lett., 36: 7611-7614 (1995);
U.S. Paten No. 3,480,613 (Nov. 25, 1969); International Publication Number
WO 01/90121 (29 November 2001); International Publication Number
WO 01/92282 (6 December 2001); and International Publication Number
WO 02/32920 (25 April 2002); and International Publication Number
WO 04/002999 (8 January 2004); and International Publication Number
WO 04/003000 (8 January 2004); and International Publication Number
WO 04/002422 (8 January 2004). Such 2'-
C-branched ribonucleosides include, but are not limited to, 2'-
C-methyl-cytidine, 2'-
C-methyl-uridine, 2'-
C-methyl-adenosine, 2'-
C-methyl-guanosine, and 9-(2-
C-methyl-β-D-ribofuranosyl)-2,6-diatninopurine, and the corresponding amino acid ester
of the ribose C-2', C-3', and C-5' hydroxyls and the corresponding optionally substituted
cyclic 1,3-propanediol esters of the 5'-phosphate derivatives.
[0044] The compounds of the present invention may also be combined for the treatment of
HCV infection with other nucleosides having anti-HCV properties, such as those disclosed
in
WO 02/51425 (4 July 2002), assigned to Mitsubishi Pharma Corp.;
WO 01/79246,
WO 02/32920,
WO 02/48165 (20 June 2002), and
WO2005003147 (13 Jan. 2005)(including R1656, (2'
R)-2'-deoxy-2'-fluoro-2'-
C-methylcytidine, shown as compounds
3-6 on page 77) assigned to Pharmasset, Ltd.;
WO 01/68663 (20 September 2001), assigned to ICN Pharmaceuticals;
WO 99/43691 (2 Sept. 1999);
WO 02/18404 (7 March 2002),
US2005/0038240 (Feb. 17, 2005) and
WO2006021341 (2 March 2006), including 4'-azido nucleosides such as R1626, 4'-azidocytidine, assigned to Hoffmann-LaRoche;
U.S. 2002/0019363 (14 Feb. 2002);
WO 02/100415 (19 Dec. 2002);
WO 03/026589 (3 Apr. 2003);
WO 03/026675 (3 Apr. 2003);
WO 03/093290 (13 Nov. 2003);:
US 2003/0236216 (25 Dec. 2003);
US 2004/0006007 (8 Jan. 2004);
WO 04/011478 (5 Feb. 2004);
WO 04/013300 (12 Feb. 2004);
US 2004/0063658 (1 Apr. 2004); and
WO 04/028481 (8 Apr. 2004).
[0045] For the treatment of HCV infection, the compounds of the present invention may also
be administered in combination with an agent that is an inhibitor of HCV NS5B polymerase.
Such HCV NS5B polymerase inhibitors that may be used as combination therapy include,
but are not limited to, those disclosed in
WO 02/057287,
US 6,777,395,
WO 02/057425,
US 2004/0067901,
WO 03/068244,
WO 2004/000858,
WO 04/003138 and
WO 2004/007512. Other such HCV polymerase inhibitors include, but are not limited to, valopicitabine
(NM-283; Idenix) and 2'-F-2'-beta-methylcytidine (see also
WO 2005/003147, assigned to Pharmasset, Ltd.).
[0046] In one embodiment, nucleoside HCV NS5B polymerase inhibitors that are used in combination
with the present HCV NS3 protease inhibitors are selected from the following compounds:
4-amino-7-(2-C-methyl-β-D-arabinofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine; 4-amino-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine;
4-methylamino-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine; 4-dimethylamino-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine;
4-cyclopropylamino-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine; 4-amino-7-(2-C-vinyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine;
4-amino-7-(2-C-hydroxymethyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine; 4-amino-7-(2-C-fluoromethyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine;
4-amino-5-methyl-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine; 4-amino-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxylic
acid; 4-amino-5-bromo-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine;
4-amino-5-chloro-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine; 4-amino-5-fluoro-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine;
2,4-diamino-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine; 2-amino-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine;
2-amino-4-cyclopropylamino-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine;
2-amino-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidin-4(3H)-one; 4-amino-7-(2-C-ethyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine;
4-amino-7-(2-C,2-O-dimethyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine; 7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidin-4(3H)-one;
2-amino-5-methyl-7-(2-C, 2-O-dimethyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidin-4(3H)-one;
4-amino-7-(3-deoxy-2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine; 4-amino-7-(3-deoxy-2-C-methyl-β-D-arabinofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine;
4-amino-2-fluoro-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine; 4-amino-7-(3-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine;
4-amino-7-(3-C-methyl-β-D-xylofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine; 4-amino-7-(2,4-di-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine;
4-amino-7-(3-deoxy-3-fluoro-2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2 ,3-d]pyrimidine;
and the corresponding 5'-triphosphates; or a pharmaceutically acceptable salt thereof.
[0048] In one embodiment, non-nucleoside HCV NS5B polymerase inhibitors that are used in
combination with the present HCV NS3 protease inhibitors are selected from the following
compounds: 14-cyclohexyl-6-[2-(dimethylamino)ethyl]-7-oxo-5,6,7,8-tetrahydroindolo[2,1-a][2,5]benzodiazocine-11-carboxylic
acid; 14-cyclohexyl-6-(2-morpholin-4-ylethyl)-5,6,7,8-tetralrydroindolo[2,1-a][2,5]benzodiazocine-11-carboxylic
acid; 14-cyclohexyl-6-[2-(dimethylamino)ethyl]-3-methoxy-5,6,7,8-tetrahydroindolo[2,1-
a][2,5]benzodiazocine-11-carboxylic acid; 14-cyclohexyl-3-methoxy-6-methyl-5,6,7,8-tetrahydroindolo[2,1-
a][2,5]benzodiazoeine-11-carboxlic acid; methyl ({[(14-cyclohexyl-3-methoxy-6-methyl-5,6,7,8-tetrahydroindolo[2,1-a][2,5]benzodiazocin-11-yl)carbonyl]amino}sulfonyl)acetate;
({[(14-cyclohexyl-3-methoxy-6-methyl-5,6,7,8-tetrahydroindolo[2,1-a][2,5]benzodiazocin-11-yl)carbonyl]amino}sulfonyl)acetic
acid; 14-cyclohexyl-
N-[(dimethylamino)sulfonyl]-3-methoxy-6-methyl-5,6,7,8-tetrahydroindolo[2,1-
a][2,5]benzodiazocine-11-carboxamide; 3-chloro-14-cyclohexyl-6-[2-(dimethylamino)ethyl]-7-oxo-5,6,7,8-tetrahydroindolo[2,1-a][2,5]benzodiazocine
11-carboxylic acid;
N'-(11-carboxy-14-cyclohexyl-7,8-dihydro-6
H-indolo[1,2-e][1,5]benzoxazocin-7-yl)-
N,
N-dimethylethane-1,2-diaminium bis(trifluoroacetate); 14-cyclohexyl-7,8-dihydro-6H-indolo[1,2-e][1,5]benzoxazocine-11-carboxylic
acid; 14-cyclohexyl-6-methyl-7-oxo-5,6,7,8-tetrahydroindolo[2,1-
a][2,5]benzodiazocine-11-carboxylic acid; 14-cyclohexyl-3-methoxy-6-methyl-7-oxo-5,6,7,8-tetrahydroindolo[2,1-a][2,5]benzodiazocine-11-carboxylic
acid; 14-cyclohexyl-6-[2-(dimethylamino)ethyl]-3-methoxy-7-oxo-5,6,7,8-tetrahydroindolo[2,1-a][2,5]benzodiazocine-11-carboxylic
acid; 14-cyclohexyl-6-[3-(dimethylamino)propyl]-7-oxo-5,6,7,8-tetrahydroindolo[2,1-a][2,5]benzodiazocine-11-carboxylic
acid; 14-cyclohexyl-7-oxo-6-(2-piperidin-1-ylethyl)-5,6,7,8-tetrahydroindolo[2,1-a][2,5]benzodiazocine-11-carboxylic
acid; 14-cyclohexyl-6-(2-morpholin-4-ylethyl)-7-oxo-5,6,7,8-tetrahydroindolo[2,1-a][2,5]benzodiazocine-11-carboxylic
acid; 14-cyclohexyl-6-[2-(diethylamino)ethyl]-7-oxo-5,6,7,8-tetrahydroindolo[2,1-
a][2,5]benzodiazocine-11-carboxylic acid; 14-cyclohexyl-6-(1-methylpiperidin-4-yl)-7-oxo-5,6,7,8-tetrahydroindolo[2,1-
a][2,5]benzodiazocine-11-carboxylic acid; 14-cyclohexyl-N-[(dimethylamino)sulfonyl]-7-oxo-6-(2-piperidin-1-ylethyl)-5,6,7,8-tetrahydroindolo[2,1-a][2,5]benzodiazocine-11-carboxamide;
14-cyclohexyl-6-[2-(dimethylamino)ethyl]-
N-[(dimethylamino)sulfonyl]-7-oxo-5,6,7,8-tetrahydroindolo[2,1-
a][2,5]benzodiazocine-11-carboxamide; 14-cyclopentyl-6-[2-(dimethylamino)ethyl]-7-oxo-5,6,7,8-tetrahydroindolo[2,1-
a][2,5]benzodiazocine-11-carboxylic acid; 14-cyclohexyl-5,6,7,8-tetrahydroindolo[2,1-a][2,5]benzodiazocine-11-carboxylic
acid; 6-allyl-14-cyclohexyl-3-methoxy-5,6,7,8-tetrahydroindolo[2,1-a][2,5]benzodiazocine-11-carboxylic
acid; 14-cyclopentyl-6-[2-(dimethylamino)ethyl]-5,6,7,8-tetrahydroindolo[2,1-
a][2,5]benzodiazocine-11-carboxylic acid; 14-cyclohexyl-6-[2-(dimethylamino)ethyl]-5,6,7,8-tetrahydroiridolo[2,1-
a][2,5]benzodiazocine-11-carboxylic acid; 13-cyclohexyl-5-methyl-4,5,6,7-tetrahydrofuro[3',2':6,7][1,4]diazocino[1,8-
a]indole-10-carboxylic acid; 15-cyclohexyl-6-[2-(dimethylamino)ethyl]-7-oxo-6,7,8,9-tetrahydro-5
H-indolo[2,1-
a][2,6]benzodiazonine-12-carbox-ylic acid; 15-cyclohexyl-8-oxo-6,7,8,9-tetrahydro-5
H-indolo[2,1-
a][2,5]benzodiazonine-12-carboxylic acid; 13-cylohexyl-6-oxo-6,7-dihydro-5
H-indolo[1,2-
d][1,4]benzodiazepine-10-carboxylic acid; and pharmaceutically acceptable salts thereof.
[0049] The above tetracyclic indole-based HCV NS5B polymerase inhibitors may be obtained
following methods A-E as outlined below, wherein different variables may be selected
in accordance with the specific tetracyclic indole compound to be prepared:
Method A
[0050]

2-Bromoindole intermediate (prepared as described in published International patent
application
WO2004087714) was functionalized on the indole nitrogen to introduce pre-cursor functionality
W'/X' to either or both of the elements W/X of the tether. Pd-mediated cross-coupling
methodology (eg, Suzuki, Stille
etc) then brought in the C2 aromatic bearing pre-cursor functionality Z'/Y' to either
or both of the elements Z/Y of the tether. Functional group manipulation followed
by ring closure afforded the tetracyclic system. Ester deprotection then yielded the
target indole carboxylic acids, with the C2 aromatic tethered to the indole nitrogen.
Method B
[0051]

Following tether assembly out to the appropriate 2-haloaromatic, Pd-mediated ring
closure afforded the fused tetracyclic system. Ester deprotection then yielded the
target indole carboxylic acids, with the C2 aromatic tethered to the indole nitrogen.
Method C
[0052]

The C2 aromatic was introduced at the outset
via Pd-mediated cross-coupling methodology (Suzuki, Stille
etc). The tether was then built up, with cyclisation onto the indole nitrogen finally
closing the ring. Ester deprotection then yielded the target indole carboxylic acids,
with the C2 aromatic tethered to the indole nitrogen.
Method D
[0053]

Fused tetracyclic intermediates arising from Methods A-C underwent manipulation of
the functionality in the tether prior to ester deprotection to yield the target C2-tethered
indole carboxylic acids.
Method E
[0055] The HCV NS3 protease inhibitory activity of the present compounds may be tested using
assays known in the art. One such assay is HCV NS3 protease time-resolved fluorescence
(TRF) assay as described in Example 56. Other examples of such assays are described
in e.g., International patent publication
WO2005/046712. Compounds useful as HCV NS3 protease inhibitors would have a Ki less than 50 µM,
more preferably less than 10 µM, and even more preferably less than 100 nM.
[0056] The present invention also includes processes for making compounds of formula
I, II-a, or
III-a. The compounds of the present invention can be readily prepared according to the following
reaction schemes and examples, or modifications thereof, using readily available starting
materials, reagents and conventional synthesis procedures. In these reactions, it
is also possible to make use of variants which are themselves known to those of ordinary
skill in this art, but are not mentioned in greater detail. Furthermore, other methods
for preparing compounds of the invention will be readily apparent to the person of
ordinary skill in the art in light of the following reaction schemes and examples.
Unless otherwise indicated, all variables are as defined above. The following reaction
schemes and examples serve only to illustrate the invention and its practice. The
examples are not to be construed as limitations on the scope or spirit of the invention.
General Description of Synthesis:
[0057] The compounds of the present invention may be synthesized as outlined in the general
Schemes 1 and 2.

[0058] Scheme 1 (n=0-9) outlines the synthesis of a representative molecule. An appropriately
protected 4-hydroxyproline derivative (for example, a carbamate protected nitrogen
and an ester protected acid can be reacted with carbonyldiimidazole or equivalent
reagent and then reacted with an appropriately substituted isoindoline or tetrahydroisoquinoline.
The alkenyl functionality may be introduced at this or a later stage by palladium
catalyzed reaction of a halide substituent such as chloride, bromide and iodide, or
other functionality such as a triflate with an organometallic reagent such as a vinyl
or allyltrialkyltin. Alternatively, the alkenyl functionality may be introduced prior
to the reaction with protected prolinol.
[0059] Scheme 2 describes the synthesis of the olefin containing amino acid portion. An
amino acid (either commercially available or may be prepared readily using known methods
in the art) in which the acid functionality is protected as an ester (for example,
R=methyl) can be converted to amides A by coupling an olefinic carboxylic acid utilizing
a wide range of peptide coupling agents known to those skilled in the art such as
DCC, EDC, BOP, TBTU, etc. Preparation of the sulfonamides B can be accomplished by
reaction with the appropriate sulfonyl chloride in an organic solvent (e.g., THF)
with an amine base as scavenger. Urea derivatives C may be prepared by reacting the
aminoester with a reagent such as carbonyldiimidazole, to form an intermediate isocyanate
(
Catalano et al., WO 03/062192) followed by addition of a second olefin containing amine. Alternatively, phosgene,
diphosgene or triphosgene may be used in place of carbonyldiimidazole. Cyanoguanidine
derivatives D can be prepared by reaction of the amino acid ester with diphenyl C-cyanocarbonimidate
in an organic solvent, followed by addition of a second olefm containing amine. Carbamate
derivatives E may be prepared by reacting an olefm containing alcohol with carbonyldiimidazole
(or phosgene, triphosgene or diphosgene) in an organic solvent, followed by addition
of the amino ester.

[0062] To complete the synthesis of the compounds of this invention, the amino acid derivative
can be coupled to the proline derivative via a wide range of peptide coupling reagents
such as DCC, EDC, BOP, TBTU etc (see Scheme 1). Macrocyclization is then achieved
by an olefin metathesis using a range of catalysts that have been described in the
literature for this purpose. At this stage the olefmic bond produced in the ring closing
metathesis may be optionally hydrogenated to give a saturated linkage or functionalized
in alternative ways such as cyclopropanation. The proline ester is then hydrolyzed
under basic conditions and coupled with the cyclopropylamino acid ester (the appropriate
alkenyl or alkylcyclopropane portion of the molecule can be prepared as described
previously (Llinas-Brunet et al.,
US 6,323,180) and subjected to an additional basic hydrolysis step to provide the final compounds.
The proline ester can also be hydrolyzed and directly coupled to an appropriately
functionalized cyclopropylamino acid acyl sulfonamide (which can be prepared according
to
Wang X.A. et al. WO2003/099274) to provide the final compounds.
[0063] Olefin metathesis catalysts include the following Ruthenium based species:
F: Miller et al J. Am. Chem. Soc 1996, 118, 9606;
G: Kingsbury et al J.Am. Chem. Soc 1999, 121, 791;
H: Scholl et al Org. Lett. 1999, 1, 953;
Hoveyda et al US2002/0107138;
K: Furstner et al. J. Org. Chem 1999, 64, 8275. The utility of these catalysts in ring closing metathesis is well known in the literature
(e.g.
Trnka and Grubbs, Acc. Chem. Res. 2001, 34, 18).

List of Abbreviations
[0064]
BOP |
Benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate |
DCC |
Dicyclohexylcarbodiimide CH3CN Acetonitrile |
DBU |
1,8-Diazabicyclo[5.4.0]undec-7-ene |
DCE |
Dichloroethane |
DCM |
Dichloromethane |
DMAP |
4-Dimethylamino pyridine |
DIPEA |
Diisoproylethylamine |
DMF |
Dimethylformamide |
DMSO |
Dimethyl sulfoxide |
EDC |
N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide |
Et3N |
Triethylamine |
Et2O |
Diethyl ether |
EtOAc |
Ethyl acetate |
EtOH |
Ethanol |
HATU |
O-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate |
HBr |
Hydrobromic acid |
|
|
HCl |
Hydrochloric acid |
HOAc |
Acetic acid |
HOAt |
1-Hydroxy-7-azabenzotriazole |
LiOH |
Lithium hydroxide |
MeOH |
Methanol |
MgSO4 |
Magnesium Sulfate |
NaHCO3 |
Sodium bicarbonate |
Na2SO4 |
Sodium sulfate |
NaOH |
Sodium hydroxide |
NH4Cl |
Ammonium chloride |
NH4OH |
Ammonium hydroxide |
Pd/C |
Palladium on carbon |
Pd(PPh3)4 |
tetrakis(triphenylphosphine)palladium (0) |
PhMe |
Toluene |
PPh3 |
Triphenylphosphine |
RT |
room temperature |
THF |
Tetrahydofuran |
TBTU |
O-Benzotriazol-1-yl-N,N,N',N'-tetramethyluronium tetrafluoroborate |
EXAMPLE 1
(5R,7S,10S)-10-Butyl-N-((1R,2S)-1-{[(cyclopropylsulfonyl)amino]carbonyl}-2-vinylcyclopropyl)-3,9,12-trioxo-1,6,7,9,10,11,12,14,15,16-decahydro-5H-2,22:5,8-dimethano-4,13,2,8,11-benzodioxatrtazacycloicosine-7-carboxamide
(III-1)
[0065]

Step 1: 4-Chloroisoindoline
[0066]

[0067] A mixture of 3-chlorophthalic acid anhydride (9 g, 49.2 mmol) and formamide (100
mL) was heated to 125 °C and stirred for 3 h. Water (300 mL) was then added and the
mixture was cooled to room temperature. The mixture was filtered and the resulting
white solid was washed with water and dried to give 4-chloro-1H-isoindole-1,3(2H)-dione
(7.7 g, 86% yield).
[0068] To solid 4-chloro-1H-isoindole-1,3(2H)-dione (4.0 g, 22.0 mmol) was added borane-THF
complex (1 M/THF, 88.1 mL, 88.1 mmol) dropwise with stirring. When the addition was
complete, the reaction mixture was heated to reflux (80 °C) and stirred for 6 h. The
reaction mixture was then cooled to 0°C, methanol (2.8 mL, 88.1 mmol) was carefully
added dropwise and the reaction mixture was warmed to room temperature. HCl (6 N)
was added until the mixture was acidic and then the mixture was concentrated. The
crude product was dissolved in 1 M HCl and extracted twice with ethyl ether and twice
with dichloromethane. The pH of the aqueous layer was adjusted to pH = 11 with solid
NaOH and extracted three times with ethyl acetate. The combined ethyl acetate extracts
were dried over Na
2SO
4, filtered and concentrated to give 4-chloroisoindoline (1.8 g, 53% yield). LRMS (ESI)
m/
z 154 [(M+H)
+; calcd for C
8H
9ClN: 154].
Step 2: 1-tert-Butyl 2-methyl (2S,4R)-4-{[(4-chloro-1,3-dihydro-2H-isoindol-2-yl)carbonyl]oxy} pyrrolidine-1,2-dicarboxylate
[0069]

[0070] To a solution of
N-Boc proline methyl ester (2.87 g, 11.7 mmol) in DMF (15 mL) at 0°C was added carbonyldiimidazole
(1.9 g, 11.7 mmol). The reaction was warmed to room temperature and stirred for 30
min. A solution of 4-chloroisoindoline (1.8 g, 11.7 mmol) in DMF (10 mL) was then
added and the reaction mixture was heated to 50°C and stirred for 2 h. The reaction
mixture was poured onto ethyl ether and 0.5 M HCl and the layers were separated. The
organic layer was washed with water, dried over Na
2SO
4, filtered and concentrated. The crude product was purified on silica gel (gradient
elution 10% to 90% ethyl acetate in hexanes) to give 1-
tert-butyl 2-methyl (2S,4R)-4-{[(4-chloro-1,3-dihydro-2H-isoindol-2-yl)carbonyl]oxy}pyrrolidine-1,2-dicarboxylate
(3.3 g, 66% yield). LRMS (ESI)
m/
z 325 [(M+H-Boc)
+; calcd for C
15H
18ClN
2O
4: 325].
Step 3: 1-tert-Butyl 2-methyl (2S,4R)-4-{[(4-vinyl-1,3-dihydro-2H-isoindol-2-yl)carbonyl]oxy} pyrrolidine-1,2-dicarboxylate
[0071]

[0072] A solution of 1-
tert-butyl 2-methyl (2S,4R)-4-{[(4-vinyl-1,3-dihydro-2H-isoindol-2-yl)carbonyl]oxy}pyrrolidine-1,2-dicarboxylate
(40 mg, 0.09 mmol), vinyl tributylystannane (36 mg, 0.11 mmol) and cesium fluoride
(31 mg, 0.21 mmol) in dioxane (0.5 mL) was degassed with N
2 for 15 min. Bis(tributylphospine)palladium(0) (2 mg, 0.005 mmol) was then added and
the reaction vessel was sealed and heated to 100 °C for 18h. After cooling, the reaction
mixture was concentrated and purifed by silica gel chromatography (10% to 90% ethyl
acetate in hexanes) to give 1-
tert-butyl 2-methyl (2S,4R)-4-{[(4-vinyl-1,3-dihydro-2H-isoindol-2-yl)carbonyl]oay}pyrrolidine-1,2-dicarboxylate
(10 mg, 25 % yield). LRMS (ESI)
m/
z 317 [(M+H-Boc)
+; calcd for C
17H
21N
2O
4: 317].
Step 4: Methyl N-[(pent-4-enyloxy)carbonyl]-L-norleucyl-(4R)-{[(4-vinyl-1,3-dihydro-2H-isoindol-2-yl)carbonyl]oxy}-L-prolinate
[0073]

[0074] To a flask containing 1-
tert-butyl 2-methyl (2S,4R)-4-{[(4-vinyl-1,3-dihydro-2H-isoindol-2-yl)carbonyl]oxy}pyrrolidine-1,2-dicarboxylate
(60 mg, 0.14 mmol) was added a 4 M solution of HCl in dioxane (2 mL). After 1 h, LC-MS
analysis indicated complete consumption of the starting material and formation of
the desired Boc product. The volatile components were then removed in vacuo, and the
crude material was taken up in DMF (2 mL).
[0075] To this mixture was added
N-[(pent4-en-1-yloxy)carbonyl]-L-norleucine (41 mg, 0.17 mmol) (prepared according
to the procedure below), DIPEA (0.076 mL, 0.43 mmol), EDC (54 mg, 0.28 mmol) and HOAt
(44 mg, 0.28 mmol). After stirring at r.t. for 30 min, complete consumption of the
amine was evidenced via LC-MS. The reaction mixture was then worked-up with 0.5 N
HCl and EtOAc. The organic layer was washed with brine and dried over MgSO
4. The solvent was then removed in vacuo and the crude product was purified on silica
(10-90 % EtOAc/hexanes) to yield 60 mg (79% yield) of methyl
N-[(pent-4-enyloxy)carbonyl]-L-norleucyl-(4R)-4-{[(4-vinyl-1,3-dihydro-2H-isoindol-2-yl)carbonyl]oxy}-L-prolinate.
LRMS (ESI)
m/
z 542 [(M+H)
+; calcd for C
29H
40N
3O
7: 542].
Step 5: Methyl (5R,7S,10S)-10-butyl-3,9,12-trioxo-1,6,7,9,10,11,12,14,15,16-decahydro-5H-2,22:5,8-dimethano-4,13,2,8,11-benzodioxatriazacycloicosine-7-carboxylate
[0076]

[0077] A solution of methyl
N-[(pent-4-enyloxy)carbonyl]-L-norleucyl-(4R)-4-{[(4-vinyl-1,3-dihydro-2H-isoindol-2-yl)carbonyl]oxy}-L-prolinate
(60 mg, 0.11 mmol) in DCE (20 mL) was degassed with N
2 for 15 min. The Zhan ruthenium metathesis catalyst RC-301 (Zhan Catalyst I (depicted
as J on page 43), RC-301, Zannan Pharma Ltd.) (7 mg, 0.01 mmol) was then added. The
solution was then heated to 100 °C for 1h. At this time, LC-MS and TLC analysis indicated
complete consumption of the starting material and formation of nearly a single product
which had the desired mass. The solvent was then removed in vacuo, and the crude product
was purified on silica (5-70% EtOAc/hexane) to yield 45 mg (79% yield) of methyl (5R,7S,10S)-10-butyl-3,9,12-trioxo-1,6,7,9,10,11,12,14,15,16-decahydro-5H-2,22:5,8-dimethano-4,13,2,8,11-benzodioxatriazacycloicosine-7-carboxylate.
LRMS (ESI)
m/
z 514 [(M+H)
+; calcd for C
27H
36N
3O
7: 514].
Step 6: (5R,7S,10S)-10-Butyl-N-((1R,2S)-1-{[(cyclopropylsulfonyl)amino]carbonyl}-2-vinylcyclopropyl)-3,9,12-trioxo-1,6,7,9,10,11,12,14,15,16-decahydro-5H-2,22:5,8-dimethano-4,13,2,8,11-benzodioxatriazacyloicosine-7-carboxamide
[0078] To a solution of methyl (5R,7S,10S)-10-butyl-3,9,12-trioxo-1,6,7,9,10,11,12,14,15,16-decahydro-5H-2,22:5,8-dimethano-4,13,2,8,11-benzodioxatriazacycloicosine-7-carboxylate
(45 mg, 0.09 mmol) in THF (2 mL), MeOH (0.5 mL), and water (1 mL) was added LiOH (21
mg, 0.87 mmol). The reaction mixture was heated to 40 °C and stirred for 1 h, at which
time complete consumption of the methyl ester starting material was observed by LC-MS.
The mixture was then worked-up with 0.5 N HCl and EtOAc. The organic layer was then
dried over K
2CO
3, and solvent was removed in vacuo. The crude product was taken up in DMF (1 mL).
[0079] To the above solution was added (1R,2S)-1-{[(cyclopropylsulfonyl)amino]carbonyl}-2-vinylcyclopropanaminium
chloride (
Llinas-Brunet et al US03/15755 and
Wang et al WO 03/099274) (32 mg, 0.12 mmol), TBTU (51 mg, 0.16 mmol) and DIPEA (0.071 mL, 0.40 mmol) and
the reaction mixture was stirred at room temperature for 2h. The reaction mixture
was directly purified by reverse phase HPLC to give (5R,7S,10S)-10-butyl-
N-((1R,2S)-1-{[(cyclopropylsulfonyl)amino]carbonyl}-2-vinylcyclopropyl)-3,9,12-trioxo-1,6,7,9,10,11,12,14,15,16-decahydro-5H-2,22:5,8-dimethano-4,13,2,8,11-benzodioxatriazacycloicosine-7-carboxamide
(27 mg, 47% yield).
1H NMR (500 MHz, ppm, CDCl
3) δ 10.01 (s, 1 H), 7.27 (m, 2 H), 7.12 (d, 1 H), 7.04 (s, 1 H), 6.40 (d,
J = 16.1 Hz, 1 H), 6.08 (m, 1 H), 5.76 (m, 1 H), 5.44 (s, 1 H), 5.36 (d, 1 H), 5.25
(d, 1 H), 5.14 (d, 1 H), 4.80-4.68 (m, 3 H), 4.59 (d, 1 H), 4.44 (m, 2 H), 4.38 (m,
1 H), 4.28 (m, 1 H), 3.95 (m, 1 H), 3.77 (dd, 1 H), 2.94 (m, 1 H), 2.43 (m, 2 H),
2.29 (d, 2 H), 2.06 (m, 2 H), 1.94 (m, 1 H), 1.78 (m, 4 H), 1.45 (m, 1 H), 1.38-1.06
(m, 5 H), 1.04 (d, 2 H), 0.92 (t, 3 H) ppm. LRMS (ESI)
m/
z 712 [(M+H)
+; calcd for C
35H
46N
5O
9S: 712].
EXAMPLE 2
(5R,7S10S)-10-tert-Butyl-N-((1R,2S)-1-{[(cyclopropylsulfonyl)aminolcarbonyl}-2-vinylcyclopropyl)-3,9,12-trioxo-1,6,7,9,10,11,12,14,15,16-decahydro-5H-2,22:5S-dimethano-4,13,2,8,11-benzodioxatriazacycloicosine-7-carboxamide (II-2)
[0080]

[0081] EXAMPLE 2 was prepared according to the procedure used for EXAMPLE 1 except that
3-methyl-
N-[(pent-4-enyloxy)carbonyl]-L-valine (prepared according to the procedure below) was
used in place of
N-[(pent-4-en-1-yloxy)carbonyl]-L-norleucine in Step 4.
1H NMR (500 MHz, ppm, CDCl
3) δ 9.90 (s , 1 H), 7.28 (m, 2 H), 7.13 (m, 2 H), 6.31 (d,
J = 15.9 Hz, 1 H), 6.04 (m, 1 H), 5.74 (m, 1 H), 5.45 (m, 2 H), 5.27 (d, 1 H), 5.16
(d, 1 H), 4.77-4.66 (m, 3 H), 4.55 (d, 1 H), 4.48 (t, 1 H), 4.41-4.35 (m, 2 H), 4.27
(m, 1 H), 3.93 (m, 1 H), 3.74 (dd, 1 H), 2.93 (m, 1 H), 2.45 (d, 2 H), 2.32 (m, 2
H), 2.10-1.95 (m, 2 H), 1.74 (m, 1 H), 1.47 (m, 1 H), 1.37 (m, 2 H), 1.07 (s, 9 H)
ppm. LRMS (ESI)
m/
z 712 [(M+H)
+; calcd for C
35H
46N
5O
9S: 712].
EXAMPLE 3
(5R,7S,10S)-10-tert-Butyl-N-((1R,2S)-1-{[(cyclopropylsulfonyl)amino]carbonyl}-2-vinylcyclopropyl)-15,15-dimethyl-3,9,12-trioxo-1,6,7,9,10,11,12,14,15,16-decahydro-5H-2,22:5,8-dimethano-4,13,2,8,11
benzodioxatriazacycloicosine-7-carboxamide (III-8)
[0082]

Step 1: 1-Bromo-2 3-bis(bromomethyl)benzene
[0083]

[0084] A suspension of 3-bromo-o-xylene (196 g, 1.06 mol),
N-bromosuccinimide (377 g, 2.15 mol) and benzoyl peroxide (0.26 g, 1.0 mmol) in carbon
tetrachloride (1800 mL) was heated to reflux under nitrogen for 15 h. The contents
of the reaction flask were cooled, filtered, and the filtrate evaporated. Distilled
crude material under high vacuum. Major fractions distilled between 88 °C and 152°C.
Recovered 108 g pure material. Recovered 182 g slightly crude material which could
be used in the following reaction.
1H NMR (CDCl
3) δ (ppm) 7.56 (d,
J = 8.0 Hz, 1 H), 7.31 (d,
J = 8.0 Hz, 1 H), 7.26 (s, 1 H), 7.16 (t,
J = 8.0 Hz, 1 H), 4.84 (s, 2 H), 4.64 (s, 2 H).
Step 2: 2-Benzyl-4-bromoisoindoline
[0085]

[0086] Postassium bicarbonate (204 g, 2.04 mol) was suspended in acetonitrile (12 L) and
the mixture was heated to 80 °C. Solutions of 1-bromo-2,3-bis(bromomethyl)benzene
(280 g, 0.82 mol in 500 mL acetonitrile) and benzylamine (87.5 g, 0.82 mol in 500
mL acetonitrile) were added concurrently via addition funnels over 1 h. The reaction
mixture was stirred at 77 °C for 16h. The contents of the reaction flask were cooled,
filtered and the solvent removed by evaporation. The reaction was partitioned between
1M K
2CO
3 and EtOAc. The organics were washed with brine, dried with anhydrous Na
2SO
4, filtered, and evaporated. Flash column chromatography (gradient elution: heptane
to 10% EtOAc in heptane) gave after evaporation the title compound as a pale oil.
1H NMR (CDCl
3) δ (ppm) 7.41-7.39 (m, 2 H), 7.37-7.34 (m, 2 H), 7.32-7.27 (m, 2 H), 7.10-7.03 (m,
2 H), 4.02 (s, 2 H), 3.97 (s, 2 H), 3.91 (s, 2 H). LRMS (ESI)
mlz 289 [(M+H)
+; calcd for C
15H
15BrN: 289].
[0087] Converted to HCl salt in HCl/MeOH. Added MTBE and filtered solid to give 118 g of
product as the HCl salt.
Step 3: 2-Benzyl-4-vinylisoindoline
[0088]

[0089] A solution of 2-benzyl-4-bromoisoindoline (16.7 g, 58.0 mmol) and tributyl(vinyl)tin
(20.3 mL, 69.6 mmol) in toluene (400 mL) was degassed by bubbling nitrogen gas through
the solution for 0.25h. Tetrakis(triphenylphosphine)palladium (0) (1.30 g, 1.16 mmol)
was added and the resulting solution heated in a 100°C oil bath under nitrogen for
24h. The contents of the reaction flask were cooled, evaporated and subjected to flash
column chromatography eluting with hexane/ethyl acetate 95/5 to give after evaporation
the title compound as a pale oil that turned pink on standing. LRMS (ESI)
m/
z 236 [(M+H)
+; calcd for C
17H
18N: 236].
Step 4: 4-Vinylisoindoline
[0090]

[0091] A solution of 2-benzyl-4-vinylisoindoline (58 mmol) in 1,2-dichloroethane (150 mL)
was placed in a 1L round bottom flask under nitrogen. To this was attached an addition
funnel containing a solution of 1-chloroethyl chloroformate (7.5.1 mL, 69.6 mmol)
in 1,2-dichloroethane. The reaction flask was cooled in an ice bath and the contents
of the addition funnel were added dropwise over 20 min keeping the internal reaction
temperature <5°C. After the addition was complete the reaction flask was allowed to
warm to room temperature then heated to reflux for 45 min. The contents of the reaction
flask were cooled to room temperature then the solvent removed by evaporation. Methanol
(200 mL) was added and the contents of the reaction flask were heated to reflux for
30 min. The reaction flask was cooled and the solvent removed by evaporation. Water
(200 mL) was added and the resulting mixture washed with ethyl acetate (2 × 250 mL).
The aqueous layer was made basic with 2N sodium hydroxide then extracted with methylene
chloride (4 × 250 mL). The combined organic extracts were dried with anhydrous sodium
sulfate, filtered and the filtrate evaporated. The remaining residue was subjected
to flash column chromatography eluting with methylene chloride/methanol/ammonium hydroxide
97/3/0.3 to 95/5/0.5. Evaporation of fractions gave the title compound as a brown
oil, 6.00g (41.4 mmol, 71% yield for two steps). LRMS (ESI)
mlz 146 [(M+H)
+; calcd for C
10H
12N: 146].
Step 5: 1-tert-Butyl 2-methyl (2S,4R)-4-{[(4-vinyl-1,3-dihydro-2H-isoindol-2-yl)carbonyl]oxy}pyrrolidine-1,2-dicarboxylate
[0092]

[0093] A solution of 1-
tert-butyl 2-methyl (2
S,4
R)-4-hydroxypyrrolidine-1,2-dicarboxylate (10.1 g, 41.4 mmol) in DMF (90 mL) under
nitrogen was cooled to 0 °C. Solid 1,1'-carbonyldiimidazole (6.70 g, 41.4 mmol) was
added to the reaction. The contents of the reaction flask were wanned to room temperature
and after 2h a solution of 4-vinylisoindoline (6.00 g, 41.4 mmol) in DMF (10 mL) was
added. The reaction was heated in a 60 °C oil bath for 2h then cooled and poured into
water and 5% potassium bisulfate. The resulting mixture was extracted with ethyl acetate
(4 × 250 mL). Combined organics were washed with brine, dried with anhydrous sodium
sulfate, filtered and evaporated. Flash column chromatography eluting with hexane/ethyl
acetate 70/30 gave the title compound as a white foam, 13.9 g (33.4 mmol, 81% yield).
LRMS (ESI)
m/
z 417 [(M+H)
+; calcd for C
227H
29N
2O
6: 417].
Step 6: (3R,5S)-5-(Methoxycarbonyl)pyrrolidin-3-yl 4-vinyl-1,3-dihydro-2Hisoindole-2H-carboxylate Hydrochloride
[0094]

[0095] A solution of 1-
tert-Butyl 2-methyl (2
S,4
R)-4-{[(4-vinyl-1,3-dihydro-2
H-isoindol-2-yl)carbonyl]oxy}pyrrolidine-1,2-dicarboaylate (13.9 g, 33.4 mmol) in ethyl
acetate (700 mL) was cooled in an ice bath the saturated with hydrogen chloride gas.
The reaction flask was sealed and allowed to warm to room temperature. After 3.5h
the solvent was removed by evaporation to give the title compound as a gray solid,
11.2 g, 95% yield).
1H NMR (500 MHz, ppm, CD
3OD) δ 7.47-7.45 (m, 1 H), 7.32-7.31 (m, 1 H), 7.26-7.21 (m, 1 H), 6.79-6.73 (m, 1
H), 5.79 - 5.73 (m, 1 H), 5.46 (s, 1 H), 5.41 - 5.38 (m, 1 H), 4.80 - 4.72 (m, 4 H),
3.91 (s, 3 H), 3.74 - 3.63 (m, 2 H), 2.77 - 2.71(m, 1 H), 2.51-2.46 (m, 1 H). LRMS
(ESI)
m/
z 317 [(M+H)
+; calcd for C
17H
21N
2O
4: 317].
Step 7: Methyl N-{[(2,2-dimethylpent-4-enyl)oxy]carbonyl}-3-methyl-L-valyl-(4R)-4-{[(4-vinyl-1,3-dihydro-2H-isoindol-2-yl)carbonyl]oxy}-L-prolinate
[0096]

[0097] To a solution of (3
R,5
S)-5-(methoxycarbonyl)pyrrolidin-3-yl 4-vinyl-1,3-dihydro-2
H-isoindole-2-carboxylate hydrochloride (2.00 g, 5.67 mmol) and
N-{[(2,2-dimethylpent-4-enyl)oxy]carbonyl}-3-methyl-L-valine (1.54 g, 5.67 mmol) in
DMF (100 mL) was added EDC (1.41 g, 7.37 mmol), HOBt (1.00 g, 7.37 mmol) and DIPEA
(3.16 mL, 22.8 mmol). The reaction mixture was stirred at RT for 18 h and then diluted
with ethyl acetate and aqueous NaHCO
3. The layers were separated and the organic layer was washed with water and brine,
dried over Na
2SO
4, filtered and concentrated. The crude residue was purified on silica gel (gradient
elution 5% to 50% ethyl acetate in hexanes) to give methyl
N-{[(2,2-dimethylpent-4-enyl)oxy]carbonyl}-3-methyl-L-valyl-(4R)-4-{[(4-vinyl-1,3-dihydro-2H-isoindol-2-yl)carbonyl]oxy}-L-prolinate
(2.75 g, 85% yield) as a white foam. LRMS (ESI)
m/
z 570 [(M+H)
+; calcd for C
31H
44N
3O
7: 570].
Step 8: Methyl (5R,7S,10S)-10-tert-butyl-15,15-dimethyl-3,9,12-trioxo-1,6,7,9,10,11,12,14,15,16-decahydro-5H-2,22:5,8-dimethano-4,13,2,8,11-benzodioxatriazacycloicosine-7-carboxylate
[0098]

[0099] A solution of methyl
N-{[(2,2-dimethylpent-4-enyl)oxy]carbonyl}-3-methyl-L-valyl-(4R)-4-{[(4-vinyl-1,3-dihydro-2H-isoindol-2-yl)carbonyl]oxy}-L-prolinate
(2.46 g, 4.32 mmol) in anhydrous dichloromethane (450 mL) was purged with nitrogen
for 15 min. A solution of bis(tricylohexylphosphine)-3-phenyl-1H-indene-1-ylideneruthenium
dichloride (Neolyst M1 catalyst purchased from Strem) (0.40 g, 0.43 mmol) in degassed,
anhydrous dichloromethane (50 mL) was then added dropwise over 30 min. The reaction
mixture was stirred at RT, during which time 0.2 g portions of the catalyst were added
approximately every 8-12h. Reaction progress was monitored by HPLC until the reaction
was complete at 48h. The residue was purified by flash chromatography on silica gel,
eluting with 10-70% EtOAc/Hexarie, to give methyl (5R,7S,10S)-10-
tert-butyl-15,15-dimethyl-3,9,12-trioxo-1,6,7,9,10,11,12,14,15,16-decahydro-5H-2,22:5,8-dimethano-4,13,2,8,11-benzodioxatriazacycloicosine-7-carboxylate
(1.85 g, 76% yield). LRMS (ESI)
m/
z 542 [(M+H)
+; calcd for C
29H
40N
3O
7: 542].
Step 9: (5R,7S,10S)-10-tert-Butyl-15,15-dimethyl-3,9,12-trioxo-1,6,7,9,10,11,12,14,15,16-decahydro-5H-2,22:5,8-dimethano-4,13,2,8,11-benzodioxatriazacycloicosine-7-carboxylic
acid
[0100]

[0101] To a solution of methyl (5R,7S,10S)-10-
tert-butyl-15,15-dimethyl-3,9,12-trioxo-1,6,7,9,10,11,12,14,15,16-decahydro-5H-2,22:5,8-dimethano-4,13,2,8,11-benzodioxatriazacycloicosine-7-carboxylate
(0.9 g, 1.67 mmol) in THF:H2O (2:1, 45 mL) was added LiOH (0.40, 16.7 mmol). The reaction
mixture was heated to 40 °C and stirred for 1 h. The reaction mixture was diluted
with aqueous HCl, and extracted with EtOAc. The combined EtOAc layer was washed with
water, brine, dried over Na
2SO
4, filtered and concentrated. The product was used with no further purification. LRMS
(ESI)
m/
z 528 [(M+H)
+; calcd for C
28H
38N
3O
7: 528].
Step 10: (5R,7S,10S)-10-tert-Butyl-N-(1R,2S)-1-{[(cyclopropylsulfonyl)amino]carbonyl}-2-vinylcyclopropyl)-15,15-dimethyl-3,9,12-trioxo-1,6,7,9,10,11,12,14,15,16-decahydro-5H-2,22:5.8-dimethano-4,13,2,8,11-benzodioxatriazacycloicosine-7-carboxamide
[0102] A solution of (5R,7S,10S)-10-
tert-butyl-15,15-dimethyl-3,9,12-trioxo-1,6,7,9,10,11,12,14,15,16-decahydro-5H-2,22:5,8-dimethano-4,13,2,8,11-benzodioxatriazacycloicosine-7-carboxylic
acid (100 mg, 0.19 mmol), (1
R,2
S)-1-{[(cylopropylsulfonyl)amino]carbonyl}-2-vinylcyclopropanaminium chloride (
Llinas-Brunet et al US03/15755 and
Wang et al WO 03/099274) (76 mg, 0.28 mmol), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium phosphorushexafluoride
(HATU, 108 mg, 0.28 mmol), DIPEA (0.073 mL, 0.42 mmol) and 4-dimethylaminopyridine
(2 mg) in dichloromethane (5 mL) was stirred at 40 °C for 1 h. The reaction solution
was diluted with aqueous saturated NaHCO
3, and extracted with EtOAc. The combined EtOAc layer was washed with water, brine,
dried over Na
2SO
4, filtered and concentrated. The residue was purified by flash chromatography eluting
with 3% MeOH/CH
2Cl
2, to give (5R,7S,10S)-10-
tert-butyl-
N-((1R,2S)-1-{[(cyclopropylsulfonyl)amino]carbonyl}-2-vinylcyclopropyl)-15,15-dimethyl-3,9,12-trioxo-1,6,7,9,10,11,12,14,15,16-decahydro-5H-2,22:5,8-dimethano-4,13,2,8,11-benzodioxatriazacycloicosine-7-carboxamide
(80 mg, 57% yield).
1H NMR (400 MHz, ppm, CDCl
3) δ 7.48 (s, 1 H), 7.23 (s, 1 H), 7.12 (d, 1 H), 6.23 (d,
J = 15.9 Hz, 1 H), 5.94 (m, 1 H), 5.76 (m, 1 H), 5.50 (m, 2 H), 5.43 (s, 1 H), 5.24
(d,
J = 16.6 Hz, 1 H), 5.11 (d, 1 H), 4.70 (s, 2 H), 4.61 (d, 1 H), 4.48 (m, 3 H), 4.35
(d, 1 H), 4.14 (d, 1 H), 3.74 (d, 1 H), 3.34 (d, 1 H), 2.89 (m, 1 H), 2.43 (dd, 2
H), 2.06 (m, 1 H), 1.93 (m, 1 H), 1.89 (dd, 1 H), 1.43 (d, 1 H), 1.25 (m, 3 H), 1.09
(s, 3 H), 1.06 (s, 9 H), 0.86 (s, 3 H). LRMS (ESI)
m/
z 740 [(M+H)
+; calcd for C
37H
50N
5O
9S: 740].
EXAMPLE 4
(5R,7S,10S)-10-tert-Butyl-N-((1R,2S)-1-{[(cylopropylsulfonyl)amino]carbonyl}-2-vinylcypropyl)-3,9,12-trioxo-6,7,9,10,11,12,14,15,16,17-decahydro-1H,5H-2,23:5,8-dimethano-4,13,2,8,11-benzodioxatriazacyclohenicosine-7-carboxamide
(III-12)
[0103]

[0104] The title compound was prepared according to the procedure used for EXAMPLE 3 except
that 3-methyl-
N-[(hex-5-enyloxy)carbonyl]-L-valine (prepared according to the procedure below) was
used in place of
N-{[(2,2-dimethylpent-4-enyl)oxy]carbonyl}-3-methyl-L-valine in Step 7.
1H NMR (500 MHz, ppm, CD
3OD) δ 9.13 (s, 1 H), 7.26 (t, 1 H), 7.23 (d, 1 H), 7.16 (d, 1 H), 6.39 (d,
J = 16.4 Hz, 1 H), 6.08 (m, 1H), 5.76 (m, 1 H), 5.38 (s, 1 H), 5.29 (d, 1 H), 5.12
(d, 1 H), 4.79 (d, 1 H), 4.73 - 4.63 (m, 4 H), 4.41 (s, 1 H), 4.37 (q, 1 H), 4.24
(d, 1 H), 3.96 (dd, 1 H), 3.77 (quin, 1 H), 2.94 (m, 1 H), 2.51 (q, 1 H), 2.29 - 2.13
(m, 4 H), 1.87 (dd, 1 H), 1.68 (m, 2 H), 1.53 (quin, 2 H), 1.44 (dd, 1 H), 1.25 (m,
2 H), 1.05 (s, 9 H). LRMS (ESI)
m/
z 726 [(M+H)
+; calcd for C
36H
48N
SO
9S: 726].
EXAMPLE 5
(5R,7S,10S)-10-Butyl-N-((1R,2S)-1-{[(cyclopropylsulfonyl)amino]carbonyl}-2-vinylcyclopropyl)-3,9,12-trioxo-6,7,9,10,11,12,14,15,16,17-decahydro-1H,5H-2,23:5,8-dimethano-4,13,2,8,11-benzodioxatriazacyclohenicosine-7-carboxamide
(III-133)
[0105]

[0106] The title compound was prepared according to the procedure used for EXAMPLE 3 except
that 3-methyl-
N-[(hex-5-enyloxy)carbonyl]-L-norleucine (prepared according to the procedure below)
was used in place of
N-{[(2,2-dimethylpent-4-enyl)oxy]carbonyl}-3-methyl-L-valine in Step 7.
1H NMR (500 MHz, ppm, CD
3OD) δ 7.24 (t, 1 H), 7.23 (d, 1 H), 7.15 (d, 1 H), 6.91 (d, 1 H), 6.37 (d,
J = 16.1 Hz, 1 H), 6.07 (m, 1H), 5.75 (m, 1 H), 5.39 (s, 1 H), 5.29 (d, 1 H), 5.12
(d, 1 H), 4.77 (d, 1 H), 4.66 (m, 3 H), 4.57 (m, 1 H), 4.47 (q, 1 H), 4.39 (q, 1 H),
4.27 (d, 1 H), 3.90 (dd, 1 H), 3.77 (quin, 1 H), 2.96 (m, 1 H), 2.49 (q, 1 H), 2.29
(m, 1 H), 2.22 (m, 3 H), 1.88 (dd, 1 H), 1.75 (m, 2 H), 1.64 (m, 2 H), 1.52 (m, 2
H), 1.39 (m, 5 H), 1.27 (m, 1 H), 1.18 (m, 1 H), 1.09 (m, 2 H), 0.94 (t, 3 H). LRMS
(ESI)
m/
z 726 [(M+H)
+; calcd for C
36H
48N
5O
9S: 726].
EXAMPLE 6
(5R,7S,10S)-10-Butyl-N-((1R,2S)-1-{[(cyclopropylsulfonyl)amino]carbonyl}-2-vinylcylopropyl)-3,9,12-trioxo-1,6,7,9,10,11,12,14,15,16,17,18-dodecahydro-5H-2,24:5,8-dimethano-4,13,2,8,11-benzodioxatriazacyclodocosine-7-carboxamide
(III-198)
[0107]

[0108] The title compound was prepared according to the procedure used for EXAMPLE 3 except
that
N-[(hept-6-en-1-yloxy)carbonyl]-L-norleucine (prepared according to the procedure below)
was used in place of
N-{[(2,2-dimethylpent-4-enyl)oxy]carbonyl}-3-methyl-L-valine in Step 7.
1H NMR (500 MHz, ppm, CD
3OD) δ 9.26 (s, 1 H), 7.39 (d, 1 H), 7.24 (t, 1 H), 7.15 (d, 1 H), 6.30 (d,
J = 15.9 Hz, 1 H), 6.20 (m, 1H), 5.75 (m, 1 H), 5.53 (s, 1 H), 5.31 (d, 1 H), 5.12
(d, 1 H), 4.70 (m, 4 H), 4.43 (dd, 1 H), 4.34 (m, 2 H), 4.27 (q, 1 H), 3.91 (dd, 1
H), 3.79 (quin, 1 H), 3.31 (m, 1 H), 2.97 (m, 1 H), 2.31 (m, 1 H), 2.22 (m, 3 H),
1.89 (dd, 1 H), 1.74 (m, 2 H), 1.66 (m, 1 H), 1.56 (m, 3 H), 1.38 (m, 8 H), 1.19 (m,
1 H), 1.09 (m, 2 H), 0.94 (t, 3 H). LRMS (ESI)
m/
z 740 [(M+H)
+; calcd for C
37H
50N
5O
9S: 740].
EXAMPLE 7
(5R,7S,10S)-10-tert-Butyl-N-((1R,2S)-1-{[(cyclopropylsulfonyl)amino]carbonyl}-2-vinylcyclopropyl)-15,15-dimethyl-3,9,12-tioxo-6,7,9,10,11,12,14,15,16,17-decahydro-1H,5H-2,23:5,8-dimethano-4,13,2,8,11-benzodioxatriazacylohenicosine-7-carboxamide
(III-199)
[0109]

[0110] The title compound was prepared according to the procedure used for EXAMPLE 3 except
that
N-{[(2,2-dimethylhex-5-enyl)oxy]carbonyl}-3-methyl-L-valine (prepared according to the
procedure below) was used in place of
N-{[(2,2-dimethylpent-4-enyl)oxy]carbonyl}-3-methyl-L-valine in Step 7.
1H NMR (500 MHz, ppm, CD
3OD) δ 9.17 (s, 1 H), 7.27 (t,
J = 7.5 Hz, 1 H), 7.21 (t,
J = 7.5 Hz, 2 H), 7.16 (d,
J = 7.5 Hz, 1 H), 6.38 (d,
J = 16 Hz, 1 H), 6.03 (m, 1 H), 5.79 (m, 1 H), 5.32 (m, 2 H), 5.13 (m, 1 H), 4.82-4.77
(m, 1 H), 4.73-4.61 (m, 4 H), 4.48 (s, 1 H), 4.39 (m, 1 H), 4.19 (d,
J = 12 Hz, 1 H), 3.96 (m, 1 H), 2.96 (m, 1 H), 2.59-2.55 (m, 1 H), 2.35-2.12 (m, 4
H), 1.89 (m, 1 H), 1.49-1.23 (m, 6 H), 1.51-0.98 (m, 14 H), 0.95-0.85 (m, 4 H). LRMS
(ESI)
m/
z 754 [(M+H)
+; calcd for C
38H
52N
5O
9S: 754].
EXAMPLE 8
5R,75,10S)-10-tert-Butyl-N-((1R,2R)-1-{[(cylopropylsulfonyl)amino]carbonyl}-2-ethylcylopropyl)-3,9,12-trioxo-1,6,7,9,10,11,12,14,15,16,17,18-dodecahydro-5H-2,22:5,8-dimethano-4,13,2,8,11-benzodioxatriazacycloicosine-7-carboxamide
(III-200)
[0111]

[0112] A solution of EXAMPLE 2 (0.32 mg, 0.45 mmol) and palladium on carbon (10% wt., 0.03
g) in EtOAc (10 mL) was vigorously stirred under a hydrogen balloon for 1 h. The reaction
mixture was filtered and concentrated. The residue was purified by reverse-phase HPLC
(DeltaPak C18 column), running 40-65% CH
3CN in water (with NH
4OAc 1 g/L). The fractions were concentrated, diluted with aqueous saturated NaHCO
3 (20 mL) and extracted with CH
2Cl
2 (3 x 70 mL). The combined CH
2Cl
2 layers were washed with water (50 mL), dried over Na
2SO
4, filtered and concentrated to give (5R,7S,10S)-10-
tert-butyl-
N-((1R,2R)-1-{[(cyclopropylsulfonyl)amino]carbonyl}-2-ethylcyclopropyl)-3,9,12-trioxo-1,6,7,9,10,11,12,14,15,16,17,18-dodecahydro-5H-2,22:5,8-dimethano-4,13,2,8,11-benzodioxatriazacycloicosine-7-carboxamide
(0.31 g, 97% yield).
1H NMR (CD
3OD ppm) δ 7.23 (t, 1 H), 7.14 (d, 1 H), 7.10 (d, 1 H), 7.02 (d, 1 H), 5.52 (s, 1H),
4.74 - 4.60 (m, 4 H), 4.48 - 4.30 (m, 4 H), 3.88 (d, 1 H), 3.75 (s, 1H), 2.99 (m,
1 H), 2.62 (m, 1 H), 2.41 (m, 2 H), 2.14 (m, 1H), 1.79 (m, 1 H), 1.65 - 1.51 (m, 6
H), 1.47 - 1.19 (m, 5 H), 1.07 (s, 9 H), 0.99 (t, 3 H). LRMS (ESI)
m/
z 716 [(M+H)
+; calcd for C
35H
50N
5O
9S: 716].
EXAMPLE 9
(5R,7S,10S)-10-tert-Butyl-N-((1R,2R)-1-{[(cyclopropylsulfonyl)amino]carbonyl}-2-ethylcyclopropyl)-3,9,12-trioxo-6,7,9,10,11,12,14,15,16,17,18,19-dodecahydro-1H,5H-2,23:5,8-dimethano-4,13,2,8,11-benzodioxatriazacyclohenicosine-7-carboxamide
(III-201)
[0113]

[0114] The title compound was prepared from EXAMPLE 4 using the procedure described for
EXAMPLE 8.
1H NMR (500 MHz, ppm, CD
3OD) δ 7.23 (t, 1 H), 7.14 (d, 1 H), 7.10 (d, 1 H), 7.02 (d, 1 H), 5.36 (s, 1 H), 4.71
(m, 3 H), 4.64 (t, 1 H), 4.56 (m, 1 H), 4.40 (m, 2 H), 4.24 (d, 1 H), 3.96 (dd, 1
H), 3.72 (quin, 1 H), 2.98 (m, 1 H), 2.58 (m, 1 H), 2.49 (m, 2 H), 2.15 (t, 1 H),
1.69 -1.19 (m, 15 H), 1.09 (m, 1 H), 1.06 (s, 9 H), 0.98 (t, 3 H). LRMS (ESI)
m/
z 730 [(M+H)
+; calcd for C
36H
52N
5O
9S: 730].
EXAMPLE 10
(5R,7S,10S)-10-Butyl-N-((1R,2R)-1-{[(cylopropylsulfonyl)amino]carbonyl}-2-ethylcydopropyl)-3,9,12-trioxo-6,7,9,10,11,12,14,15,16,17,18,19-dodecahydro-1H,5H-2,23:5,8-dimethano-4,13,2,8,11-benzodioxatriazacyclohenicosine-7-carboxamide
(III-202)
[0115]

[0116] The title compound was prepared from EXAMPLE 5 using the procedure described for
EXAMPLE 8.
1H NMR (500 MHz, ppm, CD
3OD δ 7.23 (t, 1 H), 7.14 (d, 1 H), 7.09 (d, 1 H), 6.99 (d, 1 H), 5.39 (s, 1 H), 4.76
- 4.61 (m, 4 H), 4.43 (m, 3 H), 4.29 (d, 1 H), 3.92 (dd, 1 H), 3.69 (quin, 1 H), 2.99
(m, 1 H), 2.57 (m, 1 H), 2.51 (m, 2 H), 2.19 (tt, 1 H), 1.77 (m, 1 H), 1.70 -1.30
(m, 20 H), 1.17 (m, 2 H), 1.10 (m, 2 H), 0.99 (t, 3 H), 0.95 (t, 3 H). LRMS (ESI)
m/
z 730 [(M+H)
+; calcd for C
36H
5zN
5O
9S: 730].
EXAMPLE 11
(5R,7S,10S)-10-Butyl-N-((1R,2R)-1-{[(cylopropylsulfonyl)amino]carbonyl}-2-ethylcylopropyl)-3,9,12-trioxo-1,6,7,9,10,11,12,14,15,16,17,18,19,20-tetradecihydro-5H-2,24:5,8-dimethano-4,13,2,8,11-benzodioxatriazacyclodocosine-7-carboxamide
(III-203)
[0117]

[0118] The title compound was prepared from EXAMPLE 6 using the procedure described for
EXAMPLE 8.
1H NMR (500 MHz, ppm, CD
3OD) δ 7.2 (t, 1 H), 7.15 (d, 1 H), 7.11 (d, 1 H), 5.55 (s, 1 H), 4.70 (m, 4 H), 4.49
(m, 1 H), 4.38 (t, 1 H), 4.29 (m, 2 H), 3.94 (dd, 1 H), 3.73 (quin, 1 H), 3.00 (m,
1 H), 2.63 (quin, 1 H), 2.51 (m, 1 H), 2.38 (m, 1 H), 2.20 (tt, 1 H), 1.76 (quin,
1 H), 1.68 - 1.07 (m, 24 H), 1.00 (t, 3 H), 0.95 (t, 3 H). LRMS (ESI)
m/
z 744 [(M+H)
+; calcd for C
37H
54N
5O
9S: 744].
EXAMPLE 12
(5R,7S,10S)-10-tert-Butyl-N-((1R,2R)-1-{[(cyclopropylsulfonyl)amino]carbonyl}-2-ethylcyclopropyl)-15,15-dimethyl-3,9,12-trioxo-1,6,7,9,10,11,12,14,15,16,17,18-dodecahydro-5H-2,22:5,8-dimethano-4,13,2,8,11-benzodioxatriazacycloicosine-7-carboxamide
(III-204)
[0119]

[0120] The title compound was prepared from EXAMPLE 3 using the procedure described for
EXAMPLE 8.
1H NMR (400 MHz, ppm, CD
3OD) δ 9.06 (s, 1 H), 7.22 (dd, 1 H), 7.13 (d, 1 H), 7.07 (d, 1 H), 5.51 (s, 1 H),
4.72 (d, 2 H), 4.68 (d, 2 H), 4.44 (d, 2 H), 4.28 (m, 2 H), 3.87 (dd, 1 H), 3.28 (m,
1 H), 2.98 (d, 1 H), 2.85 (m, 3 H), 2.52 (m, 1 H), 2.43 (m, 2 H), 2.15 (m, 1 H), 1.15-1.17
(m, 3 H), 1.41 (m, 2 H), 1.30 (m, 1 H), 1.21 (m, 4 H), 1.08 (m, 1 H), 1.06 (s, 3 H),
1.05 (s, 9 H), 0.98 (t, 3 H), 0.81 (s, 3 H). LRMS (ESI)
m/
z 744 [(M+H)
+; calcd for C
37H
54N
5O
9S: 744].
EXAMPLE 13
(5R,7S,10S)-10-tert-Butyl-N-((1R,2R)-1-{[(cyclopropylsulfonyl)amino]carbonyl}-2-ethylcydopropyl)-15,15-dimethyl-3,9,12-trioxo-6,7,9,10,11,12,14,15,16,17,18,19-dodecahydro-1H,5H-2,23:5,8-dimethano-4,13,2,8,11-benzodioxatriazacyclohenicosine-7-carboxamide
(III-205)
[0121]

[0122] The title compound was prepared from EXAMPLE 7 using the procedure described for
EXAMPLE 8.
1H NMR (500 MHz, ppm, CD
3OD δ 9.09 (s, 1 H), 7.24 (t,
J = 7.5 Hz, 1 H), 7.15 (d,
J = 7.5 Hz, 1 H), 7.10 (d,
J = 7.5 Hz, 1 H), 5.53 (s, 1 H), 4.75 - 4.59 (m, 4 H), 4.44 - 4.37 (m, 3 H), 4.20 (d,
J = 12 Hz, 1 H), 3.95 - 3.91 (m, 1 H), 3.31 (m, 2 H), 2.99 - 2.96 (m, 1 H), 2.62 -
2.46 (m, 3 H), 2.17 - 2.13 (m, 1 H), 1.67 - 1.50 (m, 6 H), 1.37 - 1.18 (m, 7 H), 1.15
- 0.96 (m, 16 H), 0.80 (s, 3 H). LRMS (ESI)
m/
z 758 [(M+H)
+; calcd for C
38H
56N
5O
9S: 758].
Alternative Preparation:
Step 1: 1-Bromo-2,3-bis(bromomethyl)benzene
[0123]

[0124] To a suspension of 3-bromo-o-xylene (999 g, 5.40 mol) in chlorobenzene (9 L) at RT
was added
N-bromosuccinimide (1620 g, 9.1 mol) and benzoyl peroxide (2.6 g, 10.8 mmol). The reaction
mixture was heated to 80 °C and stirred under nitrogen for 18 h. The reaction mixture
was cooled to 70 °C and an additional portion ofNBS (302 g, 1.7 mol) was added. The
reaction mixture was heated to 80 °C and stirred under nitrogen for 22 h. The reaction
mixture was cooled to RT, diluted with heptane (6 L) and filtered. The filter cake
was washed with heptane (4 L) and the combined filtrates were evaporated. The crude
product was dissolved in heptane (2 L) and chloroform (200 mL) and filtered through
basic alumina (500 g). The alumina pad was washed with heptane (4 L) and the combined
filtrates were evaporated to give 1-bromo-2,3-bis(bromomethyl)benzene (1760 g, crude
weight) which was used without further purification.
1H NMR (CDCl
3) δ (ppm) 7.56 (d,
J = 8.0 Hz, 1 H), 7.31 (d,
J = 8.0 Hz, 1 H), 7.26 (s, 1 H), 7.16 (t,
J = 8.0 Hz, 1 H), 4.84 (s, 2 H), 4.64 (s, 2 H).
Step 2: 2-Benzyl-4-bromoisoindoline hydrochloride
[0125]

[0126] Potassium bicarbonate (657 g, 6.56 mol) was suspended in MeCN (17 L) and the mixture
was heated to 80 °C. Solutions of crude 1-bromo-2,3-bis(bromomethyl)benzene (900 g,
2.63 mol in 1 L MeCN) and benzylamine (281 g, 2.63 mol in 1 L MeCN) were added concurrently
via addition funnels over 2 h. The reaction mixture was stirred at 77 °C for 2 h and
then cooled to RT and stirred for 16 h. The contents of the reaction flask were cooled,
filtered and the solvent removed by evaporation. The reaction was partitioned between
water (6 L) and EtOAc (2 L). The pH was adjusted to >9 by the addition of 1M K
2CO
3, the layers were separated and the aqueous phase extracted with an additional portion
of EtOAc (2 L). The combined organics were washed with brine, dried with anhydrous
Na
2SO
4, filtered, and evaporated. The crude oil was diluted with EtOH (300 mL) and cooled
to 0 °C. Methanolic HCl was added until the mixture was acidic, followed by MTBE (700
mL) and the mixture sonicated, then stirred for 15 h. MTBE (1 L) was added and the
mixture was filtered and washed with 20% EtOH in MTBE followed by MTBE. The solid
was air dried to give 2-benzyl-4-bromoisoindoline hydrochloride (211g). An additional
portion of product (86 g) was isolated by concentration of the mother liquors. LRMS
(ESI)
m/
z 289 [(M+H)
+; calcd for C
15H
15BrN: 289].
Step 3: 4-Bromoisoindoline
[0127]

[0128] To a solution of 2-benzyl-4-bromoisoindoline hydrochloride (11 g, 30.96 mmol) in
200 mL EtOAc was added 1M NaOH (100 mL) and the mixture stirred for 30 min. The organic
layer was separated, washed with brine, dried over anhydrous Na
2SO
4 and solvent evaporated to an oil which was azeotroped once with toluene (50 mL).
The oil was dissolved in chlorobenzene (50 mL) and 4A molecular sieves (5 g) added
to the stirred solution. After 10 min, 1-chloroethylchloroformate (5.6 mL, 51 mmol)
was added dropwise over 5 min. The reaction mixture was then heated to 90 °C for 2
h, cooled to room temperature and filtered. The solids were washed with chlorobenzene
(5 mL) and methanol (40 mL). The filtrate was heated to 70 °C for 1 h., allowed to
cool and stirred at room temperature overnight. The solids were filtered, washed with
chlorobenzene (2 mL) and hexane and dried to give 6.84 g of title compound. LRMS (ESI)
m/
z 198.1 [(M+H)
+; calcd for C
8H
9BrN: 198.0].
Step 4: 1-t-Butyl 2-methyl (2S,4R)-4-{[(4-bromo-1,3-dihydro-2H-isoindol-2-yl)carbonyl]oxy}pyrrolidine-1,2-dicarboxylate
[0129]

[0130] To a solution of (2S,4R)-BOC-4-hydroxyproline methyl ester (126.3 g, 515 mmol) in
DMF (960 mL) at 0 °C was added N,N'-carbonyldiimidazole (83.51 g, 515 mmol). The reaction
mixture was stirred at room temperature for 3 h. 4-Bromoisoindoline hydrochloride
(120 g, 515 mmol) and diisopropylethylamine (96.3 mL, 540 mmol) were added and the
reaction mixture heated to 50 °C for 6 h then allowed to cool to room temperature
and stirred overnight. The reaction mixture was partitioned between EtOAc (3 L) and
10% aqueous KHSO
4 (6 L), the aqueous re-extracted with EtOAc (2 L) and the combined organic phases
washed with 10% aqueous NaHCO
3, brine, dried over Na
2SO
4 and solvent evaporated to a foam (239 g). LRMS (ESI)
m/
z 471.0 [(M+H)
+; calcd for C
20H
26BrN
2O
6: 471.1].
Step 5: 1-t-Butyl 2-methyl (2S,4R)-4-{[(4-vinyl-1,3-dihydro-2H-isoindol-2-yl)carbonyl]oxy}pyrrolidine-1,2-dicarboxylate
[0131]

[0132] To a solution of 1-t-butyl 2-methyl (2S,4R)-4-{[(4-bromo-1,3-dihydro-2H-isoindol-2-yl)carbonyl]oxy}pyrrolidine-1,2-dicarboxylate
(10.0 g, 21.3 mmol) in ethanol (200 mL) was added potassium vinyltrifluoroborate (4.28
g, 32 mmol) and triethylamine (4.5 mL, 32 mmol) followed by dichloro[1,1-bis(diphenylphosphino)ferrocene]palladium
(II) chloride dichloromethane adduct (175 mg, 0.21 mmol). The reaction mixture was
heated to reflux for 6 h, cooled to room temperature, diluted with 10% aqueous KHSO
4 and the ethanol removed by evaporation in vacuo. The aqueous residue was extracted
with EtOAc and the organic phase washed with brine, dried over Na
2SO
4, solvent evaporated and crude product purified by chromatography on silica eluting
with 40-60% EtOAc/ hexane to give, after evaporation, the title compound (8.18 g).
LRMS (ESI)
mlz 417.2 [(M+H)
+; calcd for C
22H
29N
2O
6: 417.2].
Step 6: (3R,5S)-5-(Methoxycarbonyl)pyrrolidin-3-14-vinyl-1,3-dihydro-2H-isoindole-2-carboxylate hydrochloride
[0133]

[0134] A mixture of 1-t-butyl 2-methyl (2S,4R)-4-{[(4-vinyl-1,3-dihydro-2H-isoindol-2-yl)carbonyl]oxy}pyrrolidine-1,2-dicarboxylate
(18.0 g, 43.2 mmol) and HCl/dioxane (4 M) (43.2 mL, 173 mmol) was stirred at RT for
2h. The reaction mixture was concentrated to remove the dioxane followed by concentration
from Et
2O to give (3
R,5
S)-5-(methoxycarbonyl)pyrrolidin-3-yl 4-vinyl-1,3-dihydro-2
H-isoindole-2-carboxylate hydrochloride as an off-white solid (15 g) which was used
without further purifcation. LRMS (ESI)
m/
z 317 [(M+H)
+; calcd for C
17H
21N
2O
4: 317].
Step 7: Methyl N-{[(2,2-dimethylhex-5-en-1-yl)oxy]carbonyl}-3-methyl-L-valvl-(4R)-4-{[(4-vinyl-1,3-dihydro-2H-isoindol-2-yl)carbonyl]oxy}-L-prolinate
[0135]

[0136] To a solution of (3
R,5
S)-5-(methoxycarbonyl)pyrrolidin-3-yl 4-vinyl-1,3-dihydro-2
H-isoindole-2-carboxylate hydrochloride (5.0 g, 14.2 mmol) and
N-{[(2,2-dimethylhex-5-enyl)oxy]carbonyl}-3-methyl-L-valine (4.0 g, 14.2 mmol) in DMF
(20 ml) at RT was added DIPEA (2.5 mL, 14.2 mmol), EDC (5.5 g, 28.4 mmol), and HOAt
(1.9 g, 14.2 mmol). After 18 h the reaction mixture was poured into Et
2O, and extracted with 1 N HCl. The aqueous layer was extracted with EtOAc, and the
combined organic layers were washed with 1 N HCl, water, NaHCO
3 and brine. The organic layer was dried over MgSO
4 and the solvent was removed in vacuo. The crude product was purified on silica (30%
EtOAc in hexanes) to yield 4.2 g of the title compound as a thick oil. LRMS (ESI)
mlz 584.4 [(M+H)
+; calcd for C
32H
46N
3O
7: 584.3].
Step 8: Methyl (5R,7S,10S,18E)-10-tert-butyl-15,15-dimethyl-3,9,12-trioxo-6,7,9,10,11,12,14,15,16,17-decahydro-1H,5H-2,23:5,8-dimethano-4,13,2,8,11-benzodioxatriazacyclohenicosine-7-carboxylate
[0137]

[0138] To a solution of methyl
N-{[(2,2-dimethylhex-5-en-1-yl)oxy]carbonyl}-3-methyl-L-valyl-(4
R)-4-{[(4-vinyl-1,3-dihydro-2
H-isoindol-2-yl)carbonyl]oxy}-L-prolinate (4.7 g, 8.05 mmol) in degassed (nitrogen
bubbling for 30 min) DCM (1410 mL) was added Zhan 1B catalyst (Zhan catalyst 1B, RC-303,
Zannan Pharma Ltd.) (0.591 g, 0.805 mmol). The mixture was then stirred at RT under
an N
2 atmosphere. After 19 h, the reaction was complete and DMSO (57 µL, 0.805 mmol) was
added. The mixture was stirred for 2 h and the mixture was concentrated in vacuo to
∼70 mL. The crude product was then directly purified on silica (gradient elution,
0-50% EtOAc in hexanes) to yield 4.4 g of the title compound as an oil. LRMS (ESI)
m/
z 556.3 [(M+H)
+; calcd for C
30H
42N
3O
7: 556.3].
Step 9: Methyl (5R,7S,10S)-10-tert-butyl-15,15-dimethyl-3,9,12-trioxo-6,7,9,10,11,12,14,15,16,17,18, 19-dodecahydro-1H,5H-2,23:5,8-dimethano-4,13,2,8,11-benzodioxatriazacyclohenicosine-7-carboxylate
[0139]

[0140] To a solution of methyl (5
R,7
S,10
S,18
E)-10-
tert-butyl-15,15-dimethyl-3,9,12-trioxo-6,7,9,10,11,12,14,15,16,17-decahydro-1
H,5
H-2,23:5,8-dimethano-4,13,2,8,11-benzodioxatriazacyclohenicosine-7-carboxylate (4.4
g, 7.92 mmol) in EtOAc (79 mL) was added Pd/C (0.421 g, 0.396 mmol). A H
2 balloon was then placed on the reaction flask. The flask was evacuated quickly and
filled with H
2. After 17 h, the reaction was complete as determined by LC-MS. The Pd/C was filtered
through glass wool, and the crude product was purified on silica (gradient elution,
0-60% EtOAc in hexanes) to yield 4.01 g of the title compound as a white powder. LRMS
(ESI)
m/
z 558.4 [(M+H)
+; calcd for C
30H
44N
3O
7: 558.3].
Step 10: (5R,7S,10S)-10-tert-Butyl-15,15-dimethyl-3,9,12-trioxo-6,7,9,10,11,12,14,15,16,17,18,19-dodecahydro-1H,5H-2,23:5,8-dimethano-4,13,2,8,11-benzodioxatriazacyclohenicosine-7-carboxylic acid
[0141]

[0142] To a solution of methyl (5
R,7
S,10
S)-10-
tert-butyl-15,15-dimethyl-3,9,12-trioxo-6,7,9,10,11,12,14,15,16,17,18,19-dodecahydro-1
H,5
H-2,23:5,8-dimethano-4,13,2,8,11-benzodioxatriazacyclohenicosine-7-carboxylate (5.76
g, 10.33 mmol) in THF (41.3 mL), MeOH (41.3 mL), and water (20.7 mL) at RT was added
LiOH (4.33 g, 103 mmol). After full conversion (45 min), as judged by LC-MS, the reaction
was worked up by partitioning between Et
2O and 1N HCl. The aqueous layer was then extracted with EtOAc. The combined organic
layers were dried over MgSO
4 and the solvent was removed in vacuo to yield 5.53 g of the title compound, which
was used without further purification. LRMS (ESI)
m/
z 544.4 [(M+H)
+; calcd for C
29H
42N
3O
7: 544.3].
Step 11: (5R,7S,10S)-10-tert-Butyl-N-((1R,2R)-1-{[(cylopropylsulfonyl)amino]carbonyl}-2-ethylcyclopropyl)-15,15-dimethyl-3,9,12-trioxo-6,7,9,10,11,12,14,15,16,17,18,19-dodecahydro-1H,5H-2,23:5,8-dimethano-4,13,2,8,11-benzodioxatriazacyclohenicosine-7-carboxamide
(III-205)
[0143] To a solution of (5
R,7
S,10
S)-10-
tert-Butyl-15,15-dimethyl-3,9,12-trioxo-6,7,9,10,11,12, 14,15,16,17,18,19-dodecahydro-1
H,5
H-2,23:5,8-dimethano-4,13,2,8,11-benzodioxatriazacyclohenicosine-7-carboxylic acid
(5.53 g, 10.17 mmol) and (1
R,2
R)-1-amino-
N-(cyclopropylsulfonyl)-2-ethylcyclopropanecarboxamide hydrochloride (3.28 g, 12.21
mmol) in DMF (50.9 mL) was added DIPEA (7.11 ml, 40.7 mmol) and HATU (5.03 g, 13.22
mmol). After full conversion (1h), the reaction mixture was partitioned between EtOAc
and 1N HCl. The organic layer was washed with brine three times, dried over MgSO
4, and the solvent was removed in vacuo. The crude material was then purified on silica
(gradient elution, 20-80% EtOAc in hexanes) to yield 5.8 g of the title compound as
a white powder.
EXAMPLE 14
(5R,7S,10S)-10-tert-Butyl-N-((1R,2S)-1-{[(cylopropylsulfonyl)amino]carbonyl}-2-vinylcylopropyl)-3,9,12-trioxo-1,6,7,9,10,11,12,14,15,16,17,18-dodecahydro-5H-2,22:5,8-dimethano-4,13,2,8,11-benzodioxatriazacycloicosine-7-carboxamide
(III-5)
[0144]

Step 1: Methyl (5R,7S,10S)-10-tert-butyl-3,9,12-trioxo-1,6,7,9,10,11,12,14,15,16-decahydro-5H-2,22:5,8-dimethano-4,13,2,8,11-benzodioxatriazacyloicosine-7-carboxylate
[0145]

[0146] Methyl (5R,7S,10S)-10
-tert-butyl
-3,9,12-trioxo-1,6,7,9,10,11,12,14,15,16-decahydro-5H-2,22:5,8-dimethano-4,13,2,8,11-benzodioxatriazacyloicosine-7-carboxylate
was prepared according to the procedure used for methyl (5R,7S,10S)-10-
tert-butyl-15,15-dimethyl-3,9,12-trioxo-1,6,7,9,10,11,12,14,15,16-decahydro-5H-2,22:5,8-dimethano-4,13,2,8,11-benzodioxatriazacycloicosine-7-carboxylate
(EXAMPLE 3, Step 8) except that 3-methyl-
N-[(pent-4-enyloxy)carbonyl]-L-valine (prepared according to the procedure below) was
used in place of
N-{[(2,2-dimethylpent-4-enyl)oxy]carbonyl}-3-methyl-L-valine in Step 7. LRMS (ESI)
m/
z 514 [(M+H)
+; calcd for C
27H
36N
3O
7: 514].
Step 2: Methyl (5R,75,10S)-10-tert-butyl-3,9,12-trioxo-1,6,7,9,10,11,12,14,15,16,17,18-dodecahydro-5H-2,22:5,8-dimethano-4,13,2,8,11-benzodioxatriazacycloicosine-7-carboxylate
[0147]

[0148] To a solution of methyl (5R,7S,10S)-10-
tert-butyl-3,9,12-trioxo-1,6,7,9,10,11,12,14,15,16,17,18-dodecahydro-5H-2,22:5,8-dimethano-4,13,2,8,11-benzodioxatriazacycloicosine-7-carboxylate
(0.10 g, 0.20 mmol) in ethyl acetate (7 mL) was added 10% palladium on carbon (0.01
g). The reaction mixture was stirred under a balloon of hydrogen for 5 h at room temperature.
Contents of the reaction flask were filtered through celite and the filtrate evaporated.
The crude product was used with no further purification (0.09g, 90% yield). LRMS (ESI)
m/
z 516 [(M+H)
+; calcd for C
27H
38N
3O
7: 516].
Step 3: (5R,7S,10S)-10-tert-Butyl-N-((1R,2S)-1-{[(cyclopropylsulfonyl)amino]carbonyl}-2-vinylcylopropyl)-3,9,12-trioxo-1,6,7,9,10,11,12,14,15,16,17,18-dodecahydro-5H-2,22:5,8-dimethano-4,13,2,8,11-benzodioxatriazapcycloicosine-7-carboxamide
[0149] To a solution of methyl (5R,7S,10S)-10-
tert-butyl-3,9,12-trioxo-1,6,7,9,10,11,12,14,15,16,17,18-dodecahydro-5H-2,22:5,8-dimethano-4,13,2,8,11-benzodioxatriazacycloicosine-7-carboxylate
(90 mg, 0.18 mmol) in THF (2 mL) and MeOH (0.5 mL), was added LiOH (1N 1.75 mL, 1.75
mmol). The reaction mixture was heated to 40 °C and stirred for 1 h, at which time
complete consumption of the methyl ester starting material was observed by LC-MS.
The mixture was then worked-up with 0.5 N HCl and EtOAc. The organic layer was then
dried over K
2CO
3, and solvent was removed in vacuo. The crude product was taken up in DMF (1 mL).
[0150] To the above solution was added (1R,2S)-1-{[(cyclopropylsulfonyl)amino]carbonyl}-2-vinylcyclopropanaminium
chloride (51 mg, 0.19 mmol), TBTU (77 mg, 0.24 mmol) and DIPEA (0.07 mL, 0.40 mmol)
and the reaction mixture was stirred at room temperature for 2h. The reaction mixture
was directly purified by reverse phase HPLC to give (5R,7S,10S)-10-
tert-butyl-
N-((1R,2S)-1-{[(cyclopropylsulfonyl)amino]carbonyl}-2-vinylcyclopropyl)-3,9,12-trioxo-1,6,7,9,10,11,12,14,15,16,17,18-dodecahydro-5H-2,22:5,8-dimethano-4,13,2,8,11-benzodioxatriazacycloicosine-7-carboxamide
(34 mg, 28% yield).
1H NMR (500 MHz, ppm, CD
3OD) δ 9.14 (s, 1 H), 7.23 (t, 1 H), 7.13 (d, 1 H), 7.10 (d, 1 H), 5.75 (quin, 1H),
5.53 (s, 1 H), 5.29 (d, 1 H), 5.12 (d, 1H), 4.75 - 4.59 (m, 5 H), 4.42 (m, 2 H), 4.34
(s, 1 H), 4.30 (d, 1 H), 3.88 (dd, 1 H), 3.75 (m, 1 H), 3.60 (q, 2 H), 2.95 (m, 1
H), 2.63 (m, 1 H), 2.41 (m, 2 H), 2.26 - 2.12 (m, 2H), 1.88 (dd, 1 H), 1.79 (m, 1
H), 1.56 (m, 3 H), 1.41 (m, 3 H), 1.25 (m, 2 H), 1.17 (t, 2 H), 1.06 (s, 9 H). LRMS
(ESI)
m/
z 714 [(M+H)
+; calcd for C
35H
48N
5O
9S: 714].
EXAMPLE 15
(5R,7S,10S)-10-tert-Butyl-N-((1R,2S)-1-{[(cyclopropylsulfonyl)amino]carbonyl}-2-vinylcyclopropyl)-15,15-dimethyl-3,9,12-trioxo-1,6,7,9,10,11,12,14,15,16,17,18-dodecahydro-5H-2,22:5,8-dimethano-4,13,2,8,11-benzodioxatriazacycloicosine-7-carboxamide
(III-206)
[0151]

[0152] The title compound was prepared according to the procedure used for EXAMPLE 14 (using
steps 2 and 3) except that methyl (5R,7S,10S)-10-
tert-butyl-15,15-dimethyl-3,9,12-trioxo-1,6,7,9,10,11,12,14,15,16-decahydro-5H-2,22:5,8-dimethano-4,13,2,8,11-benzodioxatriazacycloicosine-7-carboxylate
(EXAMPLE 3, Step 1) was used in place of methyl (5R,7S,10S)-10-
tert-butyl-3,9,12-trioxo-1,6,7,9,10,11,12,14,15,16-decahydro-5H-2,22:5,8-dimethano-4,13,2,8,11-benzodioxatriazacycloicosine-7-carboxylate
in Step 2.
1H NMR (400 MHz, ppm, CDCl
3) δ 9.91 (s, 1 H), 7.22 (t, 1 H), 7.09 (d, 2 H), 7.05 (d, 1 H), 5.77 (m, 1 H), 5.60
(s, 1 H), 5.45 (d, 1 H), 5.29 (s, 1 H), 5.15 (d, 1 H), 4.72 (q, 2 H), 4.40-4.55 (m,
4 H), 4.30 (d, 1 H), 4.25 (d, 1 H), 3.78 (dd, 1 H), 3.26 (d, 1 H), 2.91 (m, 1 H),
2.50 (m, 3 H), 2.39 (m, 3 H), 2.11 (m, 1 H), 1.98 (m, 2 H), 1.51 (m, 2 H), 1.38 (m,
4 H), 1.18(m, 1 H), 1.04 (s, 9 H), 1.01 (t, 3 H), 0.79 (s, 3 H). LRMS (ESI)
m/
z 742 [(M+H)
+; calcd for C
37H
52N
5O
9S: 742].
EXAMPLE 16
(5R,7S,10S)-10-tert-Butyl-N-((1R,2R)-1-{[(cyclopropylsulfonyl)amino]carbonyl}-2-ethylcyclopropyl)-3,9,12-trioxo-1,6,7,9,10,11,12,14,15,16-decahydro-5H-2,22:5,8-dimethano-4,13,2,8,11-benzodioxatriazacycloicosine-7-carboxamide
(III-16)
[0153]

[0154] To a solution of methyl (5R,7S,10S)-10-
tert-butyl-3,9,12-trioxo-1,6,7,9,10,11,12,14,15,16-decahydro-5H-2,22:5,8-dimethano-4,13,2,8,11-benzodioxatriazacycloicosine-7-carboxylate
(EXAMPLE 14, Step 1) (60 mg, 0.12 mmol) in THF (1 mL) and MeOH (0.5 mL) was added
LiOH (1N 1.17 mL, 1.17 mmol). The reaction mixture was heated to 40 °C and stirred
for 1 h, at which time complete consumption of the methyl ester starting material
was observed by LC-MS. The mixture was then worked-up with 0.5 N HCl and EtOAc. The
organic layer was then dried over K
2CO
3, and solvent was removed in vacuo. The crude product was taken up in DMF (1 mL).
[0155] To the above solution was added (1R,2R)-1-{[(cyclopropylsulfonyl)amino]carbonyl}-2-ethylcyclopropanaminium
chloride (32 mg, 0.12 mmol), TBTU (48 mg, 0.15 mmol) and DIPEA (0.044 mL, 0.25 mmol)
and the reaction mixture was stirred at room temperature for 2h. The reaction mixture
was directly purified by reverse phase HPLC to give (5R,7S,10S)-10-
tert-butyl-
N-((1R,2R)-1-{[(cyclopropylsulfonyl)amino]carbonyl}-2-ethylcyclopropyl)-3,9,12-trioxo-1,6,7,9,10,11,12,14,15,16-decahydro-5H-2,22:5,8-dimethano-4,13,2,8,11-benzodioxatriazacyloicosine-7-carboxamide
(55 mg, 67% yield).
1H NMR (500 MHz, ppm, CD
3OD δ 7.33 (d, 1 H), 7.26 (t, 1 H), 7.16 (d, 1 H), 6.39 (d,
J = 15.7 Hz, 1 H), 6.13 (m, 1H), 5.37 (s, 1 H), 4.69 (m, 4 H), 4.47 - 4.28 (m, 4 H),
3.89 (m, 1 H), 3.83 (d, 1 H), 2.98 (m, 1 H), 2.40 (m, 2 H), 2.31 (m, 1 H), 2.11 (t,
1 H), 1.99 (s, 1 H), 1.73 (s, 1 H), 1.60 (m, 2 H), 1.52 (m, 1 H), 1.29 - 1.15 (m,
3 H), 1.08 (s, 9 H), 0.98 (t, 3 H). LRMS (ESI)
m/
z 714 [(M+H)
+; calcd for C
35H
48N
5O
9S: 714].
EXAMPLE 17
(SR,7S,10S)-10-tert-Butyl-N-((1R,2R)-1-{[(cyclopropylsulfonyl)amino]carbonyl}-2-ethylcyclopropyl)-3,9,12-trioxo-6,7,9,10,11,12,14,15,16,17-decahydro-1H,5H-2,23:5,8-dimethano-4,13,2,8,11-benzodioxatriazacyclohenicosine-7-carboxamide
(III-207)
[0156]

Step 1: Methyl (5R,7S,10S)-10-tert-butyl-3,9,12-trioxo-6,7,9,10,11,12,14,15,16,17-decahydro-1H,5H-2,23:5,8-dimethano-4,13,2,8,11-benzodioxatriazacyclohenicosine-7-carboxylate
[0157]

[0158] Methyl (5R,7S,10S)-10-
tert-butyl-3,9,12-trioxo-6,7,9,10,11,12,14,15,16,17-decahydro-1H,5H-2,23:5,8-dimethano-4,13,2,8,11-benzodioxatriazacyclohenicosine-7-carboxylate
was prepared according to the procedure used for methyl (5R,7S,10S)-10-
tert-butyl-15,15-dimethyl-3,9,12-trioxo-1,6,7,9,10,11,12,14,15,16-decahydro-5H-2,22:5,8-dimethano-4,13,2,8,11-benzodioxatriazacycloicosine-7-carboxylate
(EXAMPLE 3, Step 8) except that 3-methyl-
N-[(hex-5-enyloxy)carbonyl]-L-valine (prepared according to the procedure below) was
used in place of
N-{[(2,2-dimethylpent-4-enyl)oxy]carbonyl}-3-methyl-L-valine in Step 7. LRMS (ESI)
m/
z 528 [(M+H)
+; calcd for C
28H
38N
3O
7: 528].
Step 2: (5R,7S,10S)-10-tert-Butyl-N-((1R,2R)-1-{[(cyclopropylsulfonyl)amino]carbonyl}-2-ethylcyclopropyl)-3,9,12-trioxo-6,7,9,10,11,12,14,15,16,17-decahydro-1H,5H-2,23:5,8-dimethano-4,13,2,8,11-benzodioxatriazacyclohenicosine-7-carboxamide
[0159] EXAMPLE 17 was prepared according to the procedure used for EXAMPLE 16 except using
methyl (5R,7S,10S)-10-
tert-butyl-3,9,12-trioxo-6,7,9,10,11,12,14,15,16,17-decahydro-1H,5H-2,23:5,8-dimethano-4,13,2,8,11-benzodioxatriazacyclohenicosine-7-carboxylate
in place of methyl-(5R,7S,10S)-10-
tert-butyl-3,9,12-trioxo-1,6,7,9,10,11,12,14,15,16-decahydro-5H-2,22:5,8-dimethano-4,13,2,8,11-benzodioxatriazacyloicosine-7-carboxylate
(EXAMPLE 14, Step 1).
1H NMR (500 MHz, ppm, CD
3OD) δ 9.06 (s, 1 H), 7.27 (t, 1 H), 7.24 (d, 1 H), 7.18 (d, 1 H), 6.40 (d,
J = 16.4 Hz, 1 H), 6.11 (m, 1H), 5.39 (t, 1 H), 4.80 (d, 1 H), 4.69 (m, 4 H), 4.42
(s, 1 H), 4.25 (d, 1 H), 3.97 (dd, 1 H), 3.79 (quin, 1 H), 2.98 (m, 1 H), 2.50 (q,
1 H), 2.78 (m, 2 H), 2.15 (m, 1 H), 1.77 - 1.54 (m, 8 H), 1.32 - 1.19 (m, 4 H), 1.11
(m, 1 H), 1.07 (s, 9 H), 0.98 (t, 3 H). LRMS (ESI)
m/
z 728 [(M+H)
+; calcd for C
36H
5ON
5O
9S: 728].
EXAMPLE 18
(5R,7S,10S)-10-tert-Buiyl-N-((1R,2R)-1-{[(cyclopropylsulfonyl)amino]carbopyl}-2-ethylclyclopropyl)-15,15-dimethyl-3,9,12-trioxo-6,7,9,10,11,12,14,15,16,17-decahydro-1H,5H-2,23:5,8-dimethano-4,13,2,8,11-benzodioxatriazacyclohenicosine-7-carboxamide
(III-208)
[0160]

Step 1: Methyl (5R,7S,10S)-10-tert-butyl-15,15-dimethyl-3,9,12-trioxo-6,7,9,10,11,12,14,15,16,17-decahydro-1H,5H-2,23:5,8-dimethano-4,13,2,8,11-benzodioxatriazacyclohenicosine-7-carboxylate
[0161]

[0162] Methyl (5R,7S,10S)-10-
tert-butyl-15,15-dimethyl-3,9,12-trioxo-6,7,9,10,11,12,14,15,16,17-decahydro-1H,5H-2,23:5,8-dimethano-4,13,2,8,11-benzodioxatriazacyclohenicosine-7-carboxylate
was prepared according to the procedure used for methyl (5R,7S,10S)-10-
tert-butyl-15,15-dimethyl-3,9,12-trioxo-1,6,7,9,10,11,12,14,15,16-decahydro-5H-2,22:5,8-dimethano-4,13,2,8,11-benzodioxatriazacycloicosine-7-carboxylate
(EXAMPLE 3, Step 8) except that
N-{[(2,2-dimethylhex-5-enyl)oxy]carbonyl}-3-methyl-L-valine (prepared according to
the procedure below) was used in place of
N-{[(2,2-dimethylpent-4-enyl)oxy]carbonyl}-3-methyl-L-valine in Step 7. LRMS (ESI)
m/
z 556 [(M+H)
+; calcd for C
30H
42N
3O
7: 556]. Step 2: (5R,7S,10S)-10-
tert-Butyl-
N-((1R,2R)-1-{[(cyclopropylsulfonyl)amino]carbonyl}-2-ethylcylopropyl)-15,15-dimethyl-3,9,12-trioxo-6,7,9,10,11,12,14,15,16,17-decahydro-1H,5H-2,23:5,8-dimethano-4,13,2,8,11-benzodioxatriazacyclohenicosine-7-carboxamide
[0163] EXAMPLE 18 was prepared according to the procedure used for EXAMPLE 16 except using
methyl (5R,7S,10S)-10-
tert-butyl-15,15-dimethyl-3,9,12-trioxo-6,7,9,10,11,12,14,15,16,17-decahydro-1H,5H-2,23:5,8-dimethano-4,13,2,8,11-benzodioxatriazacyclohenicosine-7-carboxylate
in place of methyl-(5R,75,10S)-10-
tert-butyl-3,9,12-trioxo-1,6,7,9,10,11,12,14,15,16-decahydro-5H-2,22:5,8-dimethano-4,13,2,8,11-benzodioxatriazacycloicosine-7-carboxylate
(EXAMPLE 14, Step 1).
1H NMR (500 MHz, ppm, CD
3OD) δ 10.05 (s, 1 H), 7.24 (m, 2 H), 7.17 (d, 1 H), 7.11 (d, 1 H), 6.61 (s, 1 H),
6.28 (d,
J = 16.4 Hz, 1 H), 5.95 (m, 1 H), 5.58 (m, 1 H), 5.31 (s, 1 H), 4.71 (m, 2 H), 4.55
(m, 2 H), 4.46 (d, 2 H), 4.29 (dd, 1 H), 4.17 (d, 1 H), 3.89 (d, 1 H), 3.32 (d, 1
H), 2.92 (m, 1 H), 2.59 (m, 1 H), 2.21-2.30 (m, 2 H), 2.08 (m, 1 H), 1.60-1.78 (m,
6 H), 1.22-1.31 (m, 5 H), 1.06 (s, 9 H), 1.04 (t, 3 H), 0.093 (t, 3 H), 0.87 (s, 3
H). LRMS (ESI)
m/
z 756 [(M+H)
+; calcd for C
38H
54N
5O
9S: 756].
Preparation of N-[(Pent-4-eN-yloxy)carbonyl]-L-norleucine:
[0164]

[0165] To a solution of 1-penten-4-ol (0.95 g, 11.0 mmol) in DMF (15 mL) at 0°C was added
carbonyldiimidazole (1.79 g, 11.0 mmol). The reaction mixture was warmed to room temperature
and stirred for 30 min. L-norleucine methyl ester hydrochloride (2.0 g, 11.0 mmol)
was then added, the reaction mixture was heated to 50 °C and stirred for 15 min. Upon
cooling, the reaction mixture was diluted with ethyl ether and washed twice with water.
The organic layer was dried over sodium sulfate, filtered and concentrated. The crude
product was purified by silica gel chromatography (gradient elution 10 to 90% ethyl
acetate in hexanes) to afford 2.1 g (74% yield) methyl
N-[(pent-4-en-1-yloxy)carbonyl]-L-norleucinate as a clear oil.
[0166] To a stirred solution of methyl
N-[(pent-4-enyloxy)carbonyl]-L-norleucinate (8.50g, 33.03 mmol) in THF (20 mL) was
added 1N NaOH (20 mL). This reaction solution was stirred at room temperature for
3 h, then acidified to pH 3 with 1N HCl and extracted with (3 x 250 mL) EtOAc. The
combined EtOAc layer was washed with 50 mL water, 50 mL brine, dried over sodium sulfate,
filtered and concentrated to give 7.09 g (88% yield) of the title product as clear
oil. LRMS (ESI)
m/
z 244 [(M+H)
+; calcd for C
12H
22NO
4: 244].
Preparation of 3-Methyl-N-[(pent-4-enyloxy)carbonyl]-L-valine:
[0167]

[0168] A solution of 4-pentenol (7.22 g, 83.8 mmol) and triphosgene (11.3 g, 38.1 mmol)
in dioxane (160 mL) was cooled to 0 °C followed by a dropwise addition of DIPEA (9.85
g, 76.2 mL). The white suspension was stirred vigorously for 1 h at 25 °C, then cooled
to 0 °C. A 1 N solution of NaOH (76.2 mL) and t-butylglycine (10.0 g, 76.2 mmol) were
added. The resulting suspension was warmed to 25 °C and stirred for 18 h. Approximately
half of the dioxane was removed
in vacuo, the solution was poured into 1 N NaOH (100 mL) and washed with dichloromethane (3
x 150 mL). The aqueous layer was acidified with 6 N HCl and the desired product was
extracted with dichloromethane (3 x 150 mL). The combined organics were dried over
MgSO
4 and concentrated to give 13.7 g (73.9% yield) of 3-methyl-
N-[(pent-4-enyloxy)carbonyl]-L-valine as a colorless oil. LRMS (ESI)
m/
z 244 [(M+M
+; calcd for C
12H
22NO
4: 244].
Preparation of N-[(Hex-5-en-1-yloxy)carbonyl]-L-norleucine:
[0169]

[0170] N-[(Hex-5-en-1-yloxy)carbonyl]-L-norleucine was prepared according to the procedure
for
N-[(pent-4-en-1-yloxy)carbonyl]-L-norleucine by using 5-hexenol instead of 4-pentenol.
LRMS (ESI)
m/
z 258 [(M+H)
+; calcd for C
13H
24NO
4: 258].
Preparation of 3-Methyl-N-[(hex-5-enyloxy)carbonyl]-L-valine:
[0171]

[0172] 3-Methyl-
N-[(hex-5-enyloxy)carbonyl]-L-valine was prepared according to the procedure for 3-methyl-
N-[(pent-4-enyloxy)carbonyl]-L-valine by using 5-hexenol instead of 4-pentenol. LRMS
(ESI)
m/
z 258 [(M+H)
+; calcd for C
13H
24NO
4: 258].
Preparation of N-[Hept-6-en-1-yloxy)carbonyl]-L-norleucine:
[0173]

[0174] N-[(Hept-6-en-1-yloxy)carbonyl]-L-norleucine was prepared according to the procedure
for
N-[(pent-4-en-1-yloxy)carbonyl]-L-norleucine by using 6-heptenol instead of 4-pentenol.
LRMS (ESI)
m/
z 272 [(M+H)
+; calcd for C
14H
26NO
4: 272].
Preparation of N-{[(2,2-Dimethylpent-4-enyl)oxy]carbonyl}-3-methyl-L-valine:
[0175]

Step 1: 2,2-Dimethylpent-4-en-1-ol
[0176]

[0177] A solution of 2,2-dimethyl 4-pentenoic acid (6.0 g, 46.8 mmol) in anhydrous THF was
cooled in an ice bath to 0 °C. A slow stream of 1M lithium aluminum hydride in THF
(56.2 mL, 56.2 mmol) was added and the reaction was allowed to warm to 25°C. The reaction
mixture was stirred for 1h before pouring into 1N HCl and diethyl ether. The organic
layer was separated, dried over MgSO
4 and concentrated to provide 2,2-dimethylpent-4-en-1-ol as a clear oil (4.7 g, 87.9%
yield).
Step 2: N-{[(2,2-Dimethylpent-4-enyl)oxy]carbonyl}-3-methyl-L-valine
[0178] DIPEA (2.48 g, 19.2 mmol) was added dropwise to a 0 °C solution of 2,2-dimethylpent-4-en-1-ol
(2.24 g, 19.6 mmol) and triphosgene (2.56 g, 8.64 mmol) in 60 mL dioxane. The resulting
white suspension was stirred for 5 min at 0 °C, then allowed to warm to 25 °C over
1 h. The suspension was cooled to 0 °C with an ice bath, followed by addition of 1
N NaOH (19.2 mL) and
L-tert-butylglycine (2.52 g, 19.2 mmol). The reaction mixture was warmed to 25 °C and stirred
for 72 h. The dioxane was removed
in vacuo and the reaction mixture was basified to pH 12 with I N NaOH. The aqueous layer was
extracted with dichloromethane (3x 150 mL), then acidified to pH∼1 with 6 N HCl. The
aqueous layer was extracted with dichloromethane (3 x 150 mL). The combined organic
layers were dried over MgSO
4 and concentrated to give
N-{[(2,2-dimethylpent-4-enyl)oxy]carbonyl}-3-methyl-L-valine as a white powder (4.26
g, 827% yield). LRMS (ESI)
mlz 272 [(M+H)
+; calcd for C
14Hz
6NO
4: 272].
Preparation of N-{[(2,2-Dimethylhex-5-enyl)oxy]carbonyl}-3-methyl-L-valine:
[0179]

Step 1: Ethyl 2,2-dimethylhex-5-enoate
[0180]

[0181] To a stirred solution of diisopropylamine (13.38 mL, 94.70 mmol) in anhydrous THF
(50 mL), at -70°C and under nitrogen, was slowly added 2.5 M n-BuLi in ether (36.50
mL, 91.25 mmol). Stirred for 15 minutes, to this reaction solution was then added
dropwise ethyl isobutyrate (11.51 mL, 86.09 mmol) in THF (50 mL), stirred for 20 minutes
before added dropwise 4-bromo-1-butene (9.79 mL, 96.42 mmol) in HMPA (20 mL). The
reaction solution was then stirred to -50°C in 5 hours, quenched with 1M HCl (10 mL)
and water (100 mL), then extracted with (3 x 125 mL) ether. The combined ether layer
was washed with water (4 x 70 mL), aqueous saturated NaHCO
3 (2 x 70 mL), dried over Na
2SO
4, filtered and concentrated. The crude product was flash chromatographed on 120 g
silica gel 60, eluting with 1 - 20% EtOAc / Hexane to give the title product as clear
oil (11.01g, 75% yield). LRMS (ESI)
mlz 171 [(M+H)
+; calcd for C
10H
19O
2: 171].
Step 2: 2,2-Dimethylhex-5-en-1-ol
[0182]

[0183] To a stirred solution of 1M LAH in ether (142.14 mL, 142.14 mmol), at 0°C and under
nitrogen, was added dropwise ethyl 2,2-dimethylhex-5-enoate (11.00 g, 64.61 mmol)
dissolved in 100 mL anhydrous ether over 1 hour. This reaction solution was stirred
at 22°C for 20 hours, then quenched with water (3 mL), 1M NaOH (11 mL) and water (9
mL), dried over Na
2SO
4, filtered and concentrated to give the title product (7.22 g, 87.09%).
1H NMR (500 MHz, CDCl
3) δ 5.85-5.77 (m, 1 H); 5.01 (d, 1 H); 4.93 (d, 1 H); 3.33 (d, 2 H); 2.03 (m, 2 H);
1.34 (m, 2 H); 0.89 (m, 6 H) ppm.
Step 3: N-{[(2,2-Dimethylhex-5-enyl)oxy]carbonyl}-3-methyl-L-valine
[0184] To a stirred solution of 2,2-dimethylhex-5-en-1-ol (10.75 g, 83.85 mmol) in anhydrous
1,4-dioxane (100 mL), at 0°C and under nitrogen, was added triphosgene (13.69 g, 46.12
mmol) and then DIPEA (14.61 mL, 83.85 mmol) cautiously. This reaction solution was
stirred at 22°C for 1 hour, cooled to 0 °C and added slowly 1N NaOH (83.85 mL, 83.85
mmol) and L-
tert-leucine (11.00 g, 83.85 mmol), then stirred at 22°C for 20 hours. The reaction solution
was basified to pH 10 with 1N NaOH, washed with CH
2Cl
2 (3x 100 mL), acidified to pH 5. with 1N HCl and extracted with CH
2Cl
2 (3 x 150 mL). The combined CH
2Cl
2 layer was washed with water (100 mL), dried over Na
2SO
4, filtered and concentrated to give the title product (20.26 g, 84.66%).
1H NMR (500 MHz, CDCl
3) δ 5.85-5.77 (m, 1 H); 5.24 (d, 1 H); 5.01 (d, 1 H); 4.93 (d, 1 H); 4.20 (d, 1 H);
3.86 (d, 1 H); 3.79 (d, 1 H); 2.01 (m, 2 H); 1.36 (m, 2 H); 1.04 (s, 9 H); 0.92 (m,
6 H) ppm. LRMS (ESI)
m/
z 286 [(M+H)
+; calcd for C
15H
28NO
4: 286].
Preparation of (1R,2R-1-{[(cyclopropylsulfonyl)amino]carbonyl}-2-ethylcyclopropanaminium
chloride:
[0185]

[0186] A mixture of (1
R,2
S)-1-{[(cyclopropylsulfonyl)amino]carbonyl}-2-vinylcyclopropanaminium chloride (
Llinas-Brunet et al US03/15755 and
Wang et al WO 03/099274) (0.05 g, 0.187 mmol) and palladium on carbon (10% wt., 0.01g) in EtOAc (5 mL) was
vigorously stirred under hydrogen atmosphere provided by a hydrogen balloon for 1
hour. The reaction mixture was filtered and concentrated to give (1
R,2
R)-1-{[(cyclopropylsulfonyl)amino]carbonyl}-2-ethylcyclopropanaminium chloride (0.045
g, 89% yield).
EXAMPLE 19
(5R,7S,10S)-10-tert-Butyl-N-((1R,2S)-1-{[(cylopropylsulfonyl)amino]carbonyl}-2-vinylcylopropyl)-15,15-dimethyl-3,9,12-trioxo-6,7,9,10,11,12,14,15,16,17,18,19-dodecahydro-1H,5H-2,23:5.8-dimethano-4,13,2,8,11-benzodioxatriazacyclohenicosine-7-carboxamide (III-210)
[0187]

[0188] EXAMPLE 19 was prepared from (5
R,7
S,10
S)-10-
tert-butyl-15,15-dimethyl-3,9,12-trioxo-6,7,9,10,11,12,14,15,16,17,18,19-dodecahydro-1
H,5
H-2,23:5,8-dimethano-4,13,2,8,11-benzodioxatriazacyclohenicosine-7-carboxylic acid
(EXAMPLE 13 Alternative Preparation, Step 4) using the procedure for EXAMPLE 3, Step
10.
1H NMR (500 MHz, CD
3OD, ppm) δ 7.25-7.09 (m, 3 H), 5.82-5.74 (m, 1 H), 5.35-5.29 (m, 2 H), 5.15-5.12 (m,
1 H), 4.75-4.59 (m, 3 H), 4.45-4.38 (m, 2 H), 4.21-4.12 (m, 1 H), 4.13-4.09 (m, 1
H), 3.95-3.92 (m, 1 H), 2.98-2.94 (m, 1 H), 2.62-2.54 (m, 1 H), 2.49-2.46 (m, 2 H),
2.25-2.21 (m, 1 H), 2.19-2.13 (m, 1 H), 1.90-1.88 (m, 1 H), 1.52 (m, 2 H), 1.48-1.45
(m, 1 H), 1.40-1.18 (m, 6 H), 1.15-1.00 (m, 14 H), and 0.81 (m, 4 H). LRMS (ESI)
m/
z 756.4 [(M+H)
+; calcd for C
38H
53N
5O
9S: 755.9].
EXAMPLE 20
(5R,7S,10S,18E)-10-Cyclohexyl-N-((1R,2S)-1-{[(cyclopropylsulfonyl)amino]carbonyl}-2-vinylcyclo propyl)-15,15-dimethyl-3,9,12-trioxo-6,7,9,10,11,12,14,15,16,17-decahydro-1H,5H-2,23:5,8-dimethano- 4,13,2,8,11-benzodioxatriazacyclohenicosine-7-carboxamide (III-225)
[0189]

[0190] EXAMPLE 20 was prepared using the procedures from EXAMPLE 13 Alternate Preparation,
Steps 1, 2, 4 and 5 using (2
S)-cyclohexyl({[(2,2-dimethylhex-5-en-1-yl)oxy]carbonyl} amino)acetic acid in Step
1 and (1R,2S)-1-{[(cyclopropylsulfonyl)amino]carbonyl}-2-vinylcyclo-propanaminium
chloride in Step 5.
1H NMR (500 MHz, CD
3OD, ppm) δ 7.26 (m, 1 H), 7.20 (t,
J = 7.5 Hz, 1 H), 7.15 (d,
J = 9.5 Hz, 1 H), 6.38 (d,
J = 9.5 Hz, 1 H), 5.99-6.02 (m, 1 H), 5.74-5.80 (m, 1 H), 5.29-5.34 (m, 2 H), 5.11-5.14
(m, 1 H), 4.79-4.81 (m, 2 H), 4.64-4.72 (m, 3 H), 4.56 (d,
J = 11.5 Hz, 1 H), 4.36-4.40 (m, 2 H), 4.18 (d,
J = 11.5 Hz, 1 H), 4.10 (d,
J = 5.5 Hz, 0.5 H), 3.91-3.94 (dd,
J = 11.5, 3.5 Hz, 1 H), 3.34 (d,
J = 11.0 Hz, 1 H), 2.95-2.97 (m, 1 H), 2.52-2.56 (m, 1 H), 2.16-2.35 (m, 5 H), 1.65-1.82
(m, 8 H), and 0.85-1.43 (m, 17 H). LRMS (ESI)
m/
z 780.4 [(M+H)
+; calcd for C
40H
53N
5O
9S: 780.9].
EXAMPLE 21
(5R,7S,10S)-10-Cyclohexyl-N-((1R,2R)-1-{[(cyclopropylsulfonyl)amino]carbonyl}-2-ethylcylopropyl)-15,15-dimethyl-3,9,12-trioxo-6,7,9,10,11,12,14,15,16,17,18,19-dodecahydro-1H,5H-2,23:5,8-dimethano-4,13,2,8,11-benzodioxatriazacyclohenicosine-7-carboxamide (III-226)
[0191]

[0192] EXAMPLE 21 was prepared from EXAMPLE 20 using the procedure described for EXAMPLE
8.
1H NMR (500 MHz, CDCl
3, ppm) δ 10.13 (s, 1 H), 7.22 (t,
J = 7.5 Hz, 1 H), 7.10 (d,
J = 7.5 Hz, 1 H), 7.05 (d,
J = 7.5 Hz, 1 H), 6.73 (s, 1 H), 5.40 (d,
J = 9.5 Hz, 1 H), 5.36 (m, 1 H), 4.67-4.76 (m, 2 H), 4.55 (d,
J = 15.5 Hz, 1 H), 4.44 (d,
J = 14.5 Hz, 1 H), 4.41 (d,
J = 11.0 Hz, 1 H), 4.29-4.39 (m, 2 H), 4.16 (d,
J = 11.0 Hz, 1 H), 3.82-3.85 (dd,
J = 11,5, 3.5 Hz, 1 H), 3.25 (d,
J = 11.0 Hz, 1 H), 2.95 (m, 1 H), 2.51-2.59 (m, 2 H), 2.36-2.44 (m, 2 H), 1.73-1.76
(m, 5 H), and 0.79 (br s, 2 H). LRMS (ESI)
m/
z 784.4 [(M+H)
+; calcd for C
40H
57N
5O
9S: 784.4].
Alternative preparation of (1R,2R)-1-amino-N-(cyclopropylsulfonyl)-2-ethylcyclopropanecarboxamide hydrochloride:
[0193]

Step 1: tert-Butyl((1R,2R)-1{[(cyclopropylsulfonyl)amino]carbonyl}-2-ethylcyclopropyl)carbamate:
[0194]

[0195] A hydrogenaton vessel was charged with a methanol (1000 mL) slurry of tert-butyl
((1R,2S)-1-{[(cyclopropylsulfonyl)amino]carbonyl}-2-vinylcyclopropyl)carbamate (164
g, 0.50 mol) (
Wang et al, US 6,995,174) and 5% Ru/C (dry, 7.5 wt%, 12.4 g) and set stirring. The vessel was placed under
nitrogen (20 psig) and vented to atmospheric pressure three times to remove residual
oxygen. The vessel was then placed under hydrogen (50 psig). After 20 hours, the vessel
was vented to atmospheric pressure. The reaction slurry was then transferred out of
the reaction and filtered through solka flok (34 grams, wetted w/100 mL methanol)
to yield a clear, light brown solution. The solka flok was rinsed with methanol (200
mL x 2). The combined methanol solutions were concentrated under reduced pressure
to yield crude product as a white solid (153 g). The crude product was slurried in
ethyl acetate (800 mL), warmed to 40 °C and aged 30 minutes. The solution was then
seeded, aged 30 minutes, and heptane (500 mL) was added via addition funnel over 30
minutes. The partially crystallized solid was cooled to room temperature and aged
overnight after which additional heptane (500 mL) was added. After one hour, additional
heptane (250 mL) was added via addition funnel, and the white slurry aged for one
hour. The solution was filtered and the solid was rinsed with heptane/EtOAc (500 mL,
4:1) and dried under reduced pressure to give tert-butyl ((1R,2R)-1-{[(cyclopropylsulfonyl)amino]carbonyl}-2-ethylcyclopropyl)carbamate
(125.9 g).
Step 2: (1R,2R)-1-amino-N-(cyclopropylsulfonyl)-2-ethylcyclopropanecarboxamide hydrochloride:
[0196] A solution of the product from Step 1 above (92 g, 0.28 mol) in DCM (1200 mL) was
cooled to 0°C and HCl bubbled through the solution for 10 min, the cooling bath removed
and the recatio mixture stirred for 2 h. Nitrogen was bubbled through the reaction
mixture for 5 min and the volatiles evaporated. The residue was azeotroped with DCM
(x3) to give an off white powder (75 g). LRMS (M+H)
+ Calcd. = 233; found 233.
Preparation of (2S)-cyclohexyl({[2,2-dimethylhex-5-en-1-yl)oxy]carbonyl}amino)acetic acid:
[0197]

[0198] (2
S)-Cyclohexyl({[(2,2-dimethylhex-5-en-1-yl)oxy]carbonyl}amino)acetic acid was prepared
according to the procedure for 3-methyl-
N-[(pent-4-enyloxy)carbonyl]-L-valine using (2
S)-amino(cyclohexyl)acetic acid and 2,2-dimethylhex-5-en-1-ol. LRMS (ESI)
mlz 312.3 [(M+H)
+; calcd for C
17H
30NO
4: 312.2].
EXAMPLE 22
HCV NS3 protease time-resolved fluorescence (TRF) assay
[0199] The NS3 protease TRF assay was performed in a final volume of 100µl in assay buffer
containing 50 mM HEPES, pH 7.5, 150 mM NaCl, 15 % glycerol, 0.15 % Triton X-100, 10
mM DTT, and 0.1 % PEG 8000. The NS3 protease was pre-incubated with various concentrations
of inhibitors for 10-30 minutes. The peptide substrate for the assay is Ac-C(Eu)-DDMEE-Abu-[COO]-XSAK(QSY7)-NH2,
where Eu is an europium-labeled group, Abu is 1-aminobutanoic acid which connects
an ester linkage with 2-hydroxy propanoic acid (X). Hydrolysis of the peptide by NS3
protease activity causes in separation of the fluorophore from the quencher, resulting
in an increase in fluorescence. Activity of the protease was initiated by adding the
TRF peptide substrate (final concentration 50-100 nM). The reaction was quenched after
1 hour at room temperature with 100 µl of 500 mM MES, pH 5.5. Product fluorescence
was detected using either a Victor V2 or Fusion fluorimeter (Perkin Elmer Life and
Analytical Sciiences) with excitation at 340 nm and emission at 615 nm with 50-400
µs delay. Testing concentrations of different enzyme forms was selected with a signal
to background ratio of 10-30. The inhibition constants were derived using a four-parameter
fit.
[0200] Compounds in Examples 1-21 were tested to have a Ki value of less than 100 nM (e.g.,
less than 1 nM) in the NS3 protease TRF assay as described above.