U.S. patent application number 10/445430 was filed with the patent office on 2003-11-27 for alkenyldiarylmethane non-nucleoside hiv-1 reverse transcriptase inhibitors.
Invention is credited to Casimiro-Garcia, Agustin, Cushman, Mark S., Rice, William G..
Application Number | 20030220315 10/445430 |
Document ID | / |
Family ID | 22103005 |
Filed Date | 2003-11-27 |
United States Patent
Application |
20030220315 |
Kind Code |
A1 |
Cushman, Mark S. ; et
al. |
November 27, 2003 |
Alkenyldiarylmethane non-nucleoside HIV-1 reverse transcriptase
inhibitors
Abstract
Alkenyldiarylmethane (ADAM) compounds have been found effective
as anti-HIV agents. Novel ADAM compounds, their pharmaceutical
formulations and a method of using same to treat viral infections
are described.
Inventors: |
Cushman, Mark S.; (West
Lafayette, IN) ; Casimiro-Garcia, Agustin; (West
Lafayette, IN) ; Rice, William G.; (Frederick,
MD) |
Correspondence
Address: |
BARNES & THORNBURG
11 SOUTH MERIDIAN
INDIANAPOLIS
IN
46204
|
Family ID: |
22103005 |
Appl. No.: |
10/445430 |
Filed: |
May 27, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10445430 |
May 27, 2003 |
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09581927 |
Jun 19, 2000 |
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6569897 |
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09581927 |
Jun 19, 2000 |
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PCT/US99/00916 |
Jan 15, 1999 |
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60071700 |
Jan 16, 1998 |
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Current U.S.
Class: |
514/183 ;
514/227.8; 514/254.03; 514/254.07; 514/362; 514/363; 514/364;
514/374; 514/381; 514/570; 544/1; 544/137; 544/138; 544/367;
544/60; 548/129; 548/131; 548/134; 548/221; 548/240; 548/257;
562/466; 562/899 |
Current CPC
Class: |
C07C 69/94 20130101;
C07C 323/62 20130101; C07D 263/32 20130101; A61P 31/12 20180101;
A61P 31/18 20180101; C07C 323/16 20130101; C07C 317/46 20130101;
A61P 43/00 20180101; C07C 65/28 20130101 |
Class at
Publication: |
514/183 ;
514/227.8; 514/254.03; 514/254.07; 514/362; 514/363; 514/364;
514/374; 514/570; 544/1; 544/60; 544/137; 544/138; 544/367;
548/131; 548/134; 548/129; 548/257; 548/221; 548/240; 562/466;
514/381; 562/899 |
International
Class: |
A61K 031/542; A61K
031/5377; A61K 031/496; A61K 031/433; A61K 031/4245; A61K 031/422;
A61K 031/421; C07D 419/02; C07D 417/02; C07D 43/02; C07D
413/02 |
Claims
1. A compound of the formula: 56wherein X is selected from the
group consisting of 57wherein R.sub.1 and R.sub.6 are H or halo;
R.sub.2 and R.sub.5 are independently OR.sub.11; R.sub.3 and
R.sub.4 are CO.sub.2R.sub.12 or Z; or R.sub.2 and R.sub.3 taken
together with the carbon atoms (C.sub.2, C.sub.3) to which they are
attached and R.sub.4 and R.sub.5 taken together with the carbon
atoms (C.sub.4, C.sub.5) to Which they are attached form a 5- or
6-membered ring of the formula 58wherein Q is O, S, or Se; R.sup.1
is C.sub.1-C.sub.4 alkyl, B is --OR.sup.1 or .dbd.O, and r is 1 or
0; provided that when B is .dbd.O, r is 1, and bond a is a single
bond, and when B is --OR.sup.1, r is 0 and bond a is a double bond;
R.sub.8 is selected from the group consisting of
(CH.sub.2).sub.mOR.sub.13, (CH.sub.2).sub.mN.sub.3,
(CH.sub.2).sub.mCOOR.sub.14, (CH.sub.2).sub.mZ, and
(CH.sub.2).sub.mNH.sub.2; R.sub.9 and R.sub.10 are independently
selected from the group consisting of H (C.sub.1-C.sub.5) alkyl,
(CH.sub.2).sub.nOR.sub.13, (CH.sub.2).sub.nN.sub.3,
(CH.sub.2).sub.nCOOR.sub.14, (CH.sub.2).sub.mZ and
(CH.sub.2).sub.nNH.sub.2; R.sub.11, R.sub.12, R.sub.13 and R.sub.14
are independently selected from the group consisting of H and
(C.sub.1-C.sub.5) alkyl; m is 1-4; n is 0-4; and Z is selected from
the following substituent groups: 596061with the proviso that when
X is 62R.sub.8 is not (CH.sub.2).sub.2OH.
2. The compound of claim 1 wherein X is 63R.sub.1 and R.sub.6 are
independently Br or Cl; R.sub.2 and R.sub.5 are each OCH.sub.3; and
R.sub.3 and R.sub.4 are each CO.sub.2CH.sub.3.
3. The compound of claim 2 wherein R.sub.8 is
(CH.sub.2).sub.mN.sub.3 or (CH.sub.2).sub.mCOOR.sub.14.
4. The compound of claim wherein R.sub.8 is (CH.sub.2).sub.mZ.
5. The compound of claim 1 wherein R.sub.2 and R.sub.3 taken
together with the carbon atoms to which they are attached, and
R.sub.4 and R.sub.5 taken together with the carbon atoms to which
they are attached each form a 5- or 6-membered ring.
6. A compound of the formula: 64wherein X is selected from the
group consisting of 65wherein R.sub.2 and R.sub.5 are independently
OR.sub.11; R.sub.3 and R.sub.4 are independently CO.sub.2R.sub.12
or Z; or R.sub.2 and R.sub.3 taken together with the carbon atoms
(C.sub.2, C.sub.3) to which they are attached and R.sub.4 and
R.sub.5 taken together with the carbon atoms (C.sub.4, C.sub.5) to
which they are attached form a 5- or 6-membered ring of the formula
66wherein Q is O, S, or Se; R.sup.1 is C.sub.1-C.sub.4 alkyl, B is
--OR.sup.1 or .dbd.O, and r is 1 or 0; provided that when B is
.dbd.O, r is 1, and bond a is a single bond, and when B is
--OR.sup.1, r is 0 and bond a is a double bond; R.sub.7 and R.sub.8
are independently selected from the group consisting of H,
(C.sub.1-C.sub.4) alky, (CH.sub.2).sub.mOR.sub.13,
(CH.sub.2).sub.mN.sub.3, (CH.sub.2).sub.mCOOR.sub.14,
(CH.sub.2).sub.mZ, and (CH.sub.2).sub.mNH.sub.2; R.sub.9 and
R.sub.10 are independently selected from the group consisting of H,
(C.sub.1-C.sub.5) alkyl, (CH.sub.2).sub.nOR.sub.13,
(CH.sub.2).sub.nN.sub.3, (CH.sub.2).sub.nCOOR.sub.14,
(CH.sub.2).sub.mZ and (CH.sub.2).sub.nNH.sub.2; R.sub.11, R.sub.12,
R.sub.13 and R.sub.14 are independently selected from the group
consisting of H and (C.sub.1-C.sub.5) alkyl; m is 1-3; n is 0-4;
and Z is selected from the following substituent groups: 676869
7. The compound of claim 6 wherein X is 70R.sub.2 and R.sub.5 are
each OCH.sub.3; and R.sub.3 and R.sub.4 are each
CO.sub.2CH.sub.3.
8. The compound of claim 7 wherein R.sub.8 is
(CH.sub.2).sub.mOR.sub.13, (CH.sub.2).sub.mN.sub.3,
(CH.sub.2).sub.mZ, or (CH.sub.2).sub.mCOOR.sub.1- 4.
9. The compound of claim 6 wherein R.sub.2 and R.sub.3 taken
together with the carbon atoms to which they are attached, and
R.sub.4 and R.sub.5 taken together with the carbon atoms to which
they are attached each form a 5- or 6-membered ring.
10. A composition comprising a reverse transcriptase inhibitory
effective amount of a compound of the formula: 71wherein X is
selected from the group consisting of 72wherein R.sub.1 and R.sub.6
are H or halo; R.sub.2 and R.sub.5 are independently OR.sub.11;
R.sub.3 and R.sub.4 are CO.sub.2R.sub.12 or Z; or R.sub.2 and
R.sub.3 taken together with the carbon atoms (C.sub.2, C.sub.3) to
which they are attached and R.sub.4 and R.sub.5 taken together with
the carbon atoms (C.sub.4, C.sub.5) to which they are attached form
a 5- or 6-membered ring of the formula 73wherein Q is O, S, or Se;
R.sup.1 is C.sub.1-C.sub.4 alkyl, B is --OR.sup.1 or .dbd.O, and r
is 1 or 0; provided that when B is .dbd.O, r is 1, and bond a is a
single bond, and when B is --OR.sup.1, r is 0 and bond a is a
double bond; R.sub.8 is selected from the group consisting of
(CH.sub.2).sub.mOR.sub.13, (CH.sub.2).sub.mN.sub.3,
(CH.sub.2).sub.mCOOR.sub.14, (CH.sub.2).sub.mZ, and
(CH.sub.2).sub.mNH.sub.2; R.sub.9 and R.sub.10 are independently
selected from the group consisting of H, (C.sub.1-C.sub.5) alkyl,
(CH.sub.2).sub.nOR.sub.13, (CH.sub.2).sub.nN.sub.3,
(CH.sub.2).sub.nCOOR.sub.14, (CH.sub.2).sub.mZ and
(CH.sub.2).sub.nNH.sub.2; R.sub.11, R.sub.12, R.sub.13 and R.sub.14
are independently selected from the group consisting of H and
(C.sub.1-C.sub.5) alkyl; m is 1-4; n is 0-4; and Z is selected from
the following substituent groups: 747576with the proviso that when
X is 77R.sub.8 is not (CH.sub.2).sub.2OH, and a pharmaceutically
acceptable carrier.
11. The composition of claim 10 wherein X is 78R.sub.1 and R.sub.6
are Br or Cl, R.sub.2 and R.sub.5 are each OCH.sub.3, R.sub.3 and
R.sub.4 are each CO.sub.2CH.sub.3 and R.sub.8 is
(CH.sub.2).sub.nOH, (CH.sub.2).sub.nCOOCH.sub.3, (CH.sub.2).sub.mZ
or (CH.sub.2).sub.nN.sub.3- .
12. A method of treating a warm-blooded vertebrate suffering from a
disease of viral origin, said method comprising the step-of
administering to said vertebrate a therapeutically effective amount
of an antiviral composition comprising a compound of the formula:
79wherein X is selected from the group consisting of 80wherein
R.sub.1 and R.sub.6 are H or halo; R.sub.2 and R.sub.5 are
independently OR.sub.11; R.sub.3 and R.sub.4 are CO.sub.2R.sub.12
or Z; or R.sub.2 and R.sub.3 taken together with the carbon atoms
(C.sub.2, C.sub.3) to which they are attached and R.sub.4 and
R.sub.5 taken together with the carbon atoms (C.sub.4, C.sub.5) to
which they are attached form a 5- or 6-membered ring of the formula
81wherein Q is O, S, or Se; R.sup.1 is C.sub.1-C.sub.4 alkyl, B is
--OR.sup.1 or .dbd.O, and r is 1 or 0; provided that when B is
.dbd.O, r is 1, and bond a is a single bond, and when B is
--OR.sup.1, r is 0 and bond a is a double bond; R.sub.8 is selected
from the group consisting of (CH.sub.2).sub.mOR.sub.13,
(CH.sub.2).sub.mN.sub.3, (CH.sub.2).sub.mCOOR.sub.14,
(CH.sub.2).sub.mZ, and (CH.sub.2).sub.mNH.sub.2; R.sub.9 and
R.sub.10 are independently selected from the group consisting of H,
(C.sub.1-C.sub.5) alkyl, (CH.sub.2).sub.nOR.sub.13,
(CH.sub.2).sub.nN.sub.3, (CH.sub.2).sub.nCOOR.sub.14,
(CH.sub.2).sub.mZ and (CH.sub.2).sub.nNH.sub.2; R.sub.11, R.sub.12,
R.sub.13 and R.sub.14 are independently selected from the group
consisting of H and (C.sub.1-C.sub.5) alkyl m is 1-4; n is 0-4; and
Z is selected from the following substituent groups: 828384and a
pharmaceutically acceptable carrier.
13. The method of claim 12 wherein X is 85R.sub.1 and R.sub.2 are
Br and Cl; R.sub.2 and R.sub.5 are each OCH.sub.3; and R.sub.3 and
R.sub.4 are each CO.sub.2CH.sub.3.
14. The method of claim 12 wherein R.sub.8 is
(CH.sub.2).sub.mOR.sub.13, (CH.sub.2).sub.mN.sub.3,
(CH.sub.2).sub.mZ, or (CH.sub.2).sub.mCOOR.sub.1- 4.
15. A compound of the formula 86wherein X is Cl or Br; and R.sub.3
and R.sub.4 are COOR.sup.1 or Z.
Description
FIELD OF THE INVENTION
[0001] This invention relates to compounds useful for antiviral
applications. More particularly, this invention relates to
non-nucleoside HIV-1 reverse transcriptase inhibitors having a
common alkenyldiarylmethane structure.
BACKGROUND AND SUMMARY OF THE INVENTION
[0002] The non-nucleoside HIV-1 reverse transcriptase inhibitors
(NNRTIs) are a structurally diverse set of compounds that inhibit
reverse transcriptase by an allosteric mechanism involving binding
to a site adjacent to the deoxyribonucleoside triphosphate binding
site of the enzyme. Familiar examples of NNRTIs include
hydroxyethoxymethylphenylthio- thymine (HEPT),
tetrahydroimidazobenzodiazepinone (TIBO), dipyridodiazepinone
(nevirapine), pyridinone, bis(heteroaryl)piperazine (BHAP),
tertbutyldimethylsilylspiroaminooxathiole dioxide (TSAO), and
.alpha.-anilinophenylacetamide (.alpha.-APA) derivatives.
Nevirapine has recently been approved for clinical use as an
anti-AIDS agent.
[0003] The use of NNRTIs as anti-AIDs agents has been limited by
the development of viral resistance to the NNRTIs. Although the
rapid emergence of resistant viral strains has hampered the
clinical development of the NNRTIs for the treatment of AIDS,
several strategies have emerged for overcoming resistance,
including switching to another NNRTI to which the virus has
remained sensitive, using higher doses of the NNRTI against the
resistant strain, employing of combinations of agents which elicit
mutations that counteract one another, and combining NNRTIs with
nucleoside reverse transcriptase inhibitors (RTIs). Accordingly, a
need remains for additional NNRTIs having unique patterns of
resistance mutations in order to facilitate the application of
these strategies.
[0004] The synthesis and biological evaluation of NNRTIs in the
alkenyldiarylmethane (ADAM) series has recently been reported.
Several of the alkenyldiarylmethane compounds were disclosed as
inhibiting the cytopathic effect of a wide variety of HIV-1 strains
in CEM, MT-4, and monocyte-macrophage cultures. (Cushman et al. J.
Med. Chem 1996, 39, 3217-3227) The most potent of these disclosed
compounds was ADAM I (1), which displayed anti-HIV activity vs. a
wide range of HIV-1 isolates and was synergistic with AZT. However,
the potency of ADAM I (1) against a variety of non-resistant HIV-1
strains was lower than that generally observed with many of the
known NNRTIs, ranging from 0.56 .mu.M vs. HIV-1.sub.6S in MT-4
cells to 151 .mu.M vs. HIV-1.sub.N119 in MT-4 cells.
[0005] The present invention is directed to a series of ADAM I
related compounds that are inhibitors of reverse transcriptase
activity and inhibit the cytopathic activity of HIV strains.
DETAILED DESCRIPTION OF THE INVENTION
[0006] The design of additional alkenyldiarylmethanes has been
aided by the availability of X-ray structures of HIV-1 reverse
transcriptase complexed with nevirapine, .alpha.-APA, and TIBO.
These structures reveal that nevirapine, .alpha.-APA, and TIBO
assume a similar butterfly shape and bind to the enzyme in a
similar manner with considerable overlap. Analysis of the X-ray
crystallography structures allows the construction of a
hypothetical model of the binding of ADAM I (1) to HIV-1 RT. The
model was constructed by overlapping the structure of ADAM I (1)
with that of nevirapine (2) in the binding pocket of HIV-1 RT
(Sculpt.TM. 2.0, Interactive Simulations, San Diego, Calif.).
During this process, it was assumed that the hexenyl side chain of
ADAM I (1) would point in the same direction as the cyclopropyl
substituent of nevirapine. The nevirapine structure was then
removed, the structure of the protein "frozen", and the energy of
the complex minimized while allowing the ligand to move. The
resulting hypothetical structure was consistent with the reported
structures of NNRTI enzyme complexes, and was also supported by
prior mutagenesis studies of the alkenyldiarylmethane binding site
of HIV-1 reverse transcriptase, in which it was determined that the
resistance mutations to ADAM 1 circumscribe a well-defined binding
pocket.
[0007] According to the model generated by this analysis, the end
of the ADAM I (1) side chain occupies a cavity formed by Glu 138,
Lys 103, Tyr 181, and Val 179 of the HIV-1 RT. Several functional
groups are present that would be capable of hydrogen bonding,
including the phenolic hydroxyl group of Tyr 181, the backbone
amide and side chain carboxylate of Glu 138, and the terminal amino
group of Lys 103. It was anticipated that the incorporation of
functional groups at the end of the alkenyl chain of the ligand
which are capable of hydrogen bonding might allow favorable
interactions with the adjacent residues of the RT. Therefore,
alkenyldiarylmethane related compounds were synthesized to
incorporate functionalities at the end of the alkenyl side chain
that would be capable of hydrogen bonding.
[0008] The present invention is directed to non-nucleoside
compounds that inhibit reverse transcriptase activity, their
pharmaceutical compositions and methods utilizing such
compounds/compositions for treating patients suffering from a viral
infection. More particularly the compounds of the present invention
are useful for treating patients suffering from a disease of
retroviral origin, such as AIDs.
[0009] The compounds of the present invention are
alkenyldiarylmethane compounds of formula I: 1
[0010] wherein X is selected from the group consisting of 2
[0011] wherein R.sub.1 and R.sub.6 are H or halo;
[0012] R.sub.2 and R.sub.5 are independently OR.sub.11;
[0013] R.sub.3 and R.sub.4 are CO.sub.2R.sub.12 or Z; or
[0014] R.sub.2 and R.sub.3 taken together with the carbon atoms
(C.sub.2, C.sub.3) to which they are attached and R.sub.4 and
R.sub.5 taken together with the carbon atoms (C.sub.4, C.sub.5) to
which they are attached form a 5- or 6-membered ring of the formula
3
[0015] wherein Q is O, S, or Se;
[0016] R.sup.1 is C.sub.1-C.sub.4 alkyl,
[0017] B is --OR.sup.1 or .dbd.O, and
[0018] r is 1 or 0;
[0019] provided that when B is .dbd.O, r is 1 and bond a is a
single bond and when B is --OR.sup.1, r is 0 and bond a is a double
bond;
[0020] R.sub.7 is hydrogen;
[0021] R.sub.8 is selected from the group consisting of
(CH.sub.2).sub.mOR.sub.13, (CH.sub.2).sub.mN.sub.3,
(CH.sub.2).sub.mCOOR.sub.14, (CH.sub.2).sub.mZ, and
(CH.sub.2).sub.mNH.sub.2;
[0022] R.sub.9 and R.sub.10 are independently selected from the
group consisting of H, (C.sub.1-C.sub.5) alkyl,
(CH.sub.2).sub.nOR.sub.13, (CH.sub.2).sub.nN.sub.3,
(CH.sub.2).sub.nCOOR.sub.14, (CH.sub.2).sub.mZ and
(CH.sub.2).sub.nNH.sub.2;
[0023] R.sub.11, R.sub.12, R.sub.13 and R.sub.14 are independently
selected from the group consisting of H and (C.sub.1-C.sub.5)
alkyl; m is 1-4; n is 0-4: and Z is selected from the following
subtituent groups: 456
[0024] with the proviso that when X is 7
[0025] R.sub.8 is not (CH.sub.2).sub.2OH.
[0026] In one embodiment of this invention there is provided a
compound of the above formula I, wherein X is 8
[0027] R.sub.1 and R.sub.6 are independently Br or Cl, R.sub.2 and
R.sub.5 are each OCH.sub.3, R.sub.3 and R.sub.4 are each
CO.sub.2CH.sub.3 or Z, R.sub.7 is H and R.sub.8 is
(CH.sub.2).sub.mOH, (CH.sub.2).sub.mCOOCH.sub- .3,
(CH.sub.2).sub.mZ, or (CH.sub.2).sub.mN.sub.3, wherein m is 2 or 3.
These compounds inhibit the cytopathic effect of HIV-1.sub.RF in
CEM-SS cells. (See Table 1 below).
[0028] In another embodiment of this invention there is provided a
reverse transcriptase inhibiting compound of the above formula I
wherein X is 9
[0029] R.sub.1 and R.sub.6 are halo;
[0030] R.sub.2 and R.sub.5 are OCH.sub.3;
[0031] R.sub.3 and R.sub.4 are CO.sub.2CH.sub.3;
[0032] R.sub.7 is H;
[0033] R.sub.8 is (C.sub.2-C.sub.5) alkyl,
(CH.sub.2).sub.mOR.sub.13, (CH.sub.2).sub.mN.sub.3,
(CH.sub.2).sub.mCOOR.sub.14, (CH.sub.2).sub.mZ, and
(CH.sub.2).sub.mNH.sub.3;
[0034] R.sub.13 and R.sub.14 are independently selected from the
group consisting of H and (C.sub.1-C.sub.5) alkyl; and m is
2-4.
[0035] In still another embodiment of the invention R.sub.1 and
R.sub.6 are both chloro or bromo.
[0036] The compounds of this invention are readily formulated into
pharmaceutical compositions, also within the scope of this
invention, for use in the presently described method for treatment
of patients suffering from a disease of retroviral origin. In one
embodiment of this invention, the pharmaceutical composition
comprises a reverse transcriptase inhibitory effective amount of a
compound of formula I: 10
[0037] wherein X is selected from the group consisting of 11
[0038] wherein R.sub.1 and R.sub.6 are H or halo;
[0039] R.sub.2 and R.sub.5 are independently OR.sub.11;
[0040] R.sub.3 and R.sub.4 are CO.sub.2R.sub.12 or Z; or
[0041] R.sub.2 and R.sub.3 taken together with the carbon atoms
(C.sub.2, C.sub.3) to which they are attached and R.sub.4 and
R.sub.5 taken together with the carbon atoms (C.sub.4, C.sub.5) to
which they are attached form a 5- or 6-membered ring of the formula
12
[0042] wherein Q is O, S, or Se;
[0043] R.sup.1 is C.sub.1-C.sub.4 alkyl,
[0044] B is --OR.sup.1 or .dbd.O, and
[0045] r is 1 or 0;
[0046] provided that when B is .dbd.O, r is 1, and bond a is a
single bond, and when B is --OR.sup.1, r is 0 and bond a is a
double bond;
[0047] R.sub.7 is hydrogen;
[0048] R.sub.8 is selected from the group consisting of
(CH.sub.2).sub.mOR.sub.13, (CH.sub.2).sub.mN.sub.3,
(CH.sub.2).sub.mCOOR.sub.14, (CH.sub.2).sub.mZ, and
(CH.sub.2).sub.mNH.sub.2;
[0049] R.sub.9 and R.sub.10 are independently selected from the
group consisting of H, (C.sub.1-C.sub.5) alkyl,
(CH.sub.2).sub.nOR.sub.13, (CH.sub.2).sub.nN.sub.3,
(CH.sub.2).sub.nCOOR.sub.14, (CH.sub.2).sub.mZ and
(CH.sub.2).sub.nNH.sub.2;
[0050] R.sub.11, R.sub.12, R.sub.13 and R.sub.14 are independently
selected from the group consisting of H and (C.sub.1-C.sub.5)
alkyl; m is 1-4; n is 0-4 and Z is as defined above; and
[0051] a pharmaceutically acceptable carrier.
[0052] Another pharmaceutical composition within the scope of this
invention comprises a reverse transcriptase inhibiting compound of
the above formula I wherein
[0053] X is 13
[0054] R.sub.1 and R.sub.6 are halo;
[0055] R.sub.2 and R.sub.5 are OCH.sub.3;
[0056] R.sub.3 and R.sub.4 are CO.sub.2CH.sub.3 or Z;
[0057] R.sub.8 is (C.sub.2-C.sub.4) alkyl,
(CH.sub.2).sub.mOR.sub.13, (CH.sub.2).sub.mN.sub.3,
(CH.sub.2).sub.mCOOR.sub.14, (CH.sub.2).sub.mZ and
(CH.sub.2).sub.mNH.sub.3;
[0058] R.sub.13 and R.sub.14 are independently selected from the
group consisting of H and (C.sub.1-C.sub.5) alkyl; and m is 2 or 3,
and a pharmaceutically acceptable carrier.
[0059] The present invention further provides pharmaceutical
formulations comprising an effective amount of an
alkenyldiarylmethane compound for use in the present method for
treating a patient suffering from a disease of retroviral origin.
As used herein, an effective amount of the alkenyldiarylmethane
compound is defined as the amount of the compound which, upon
administration to a patient, alleviates or eliminates symptoms of
the disease, or reduces or eliminates detectable levels of the
virus in the treated patient.
[0060] The effective amount to be administered to a patient is
typically based on body surface area, patient weight, patient
condition, and the potency, efficacy and therapeutic index of the
compound being administered. Body surface area may be approximately
determined from patient height and weight (see e.g., Scientific
Tables, Geigy Pharmaceuticals, Ardley, N.Y., pages 537-538 (1970)).
Effective doses will vary, as recognized by those skilled in the
art, dependent on route of administration, excipient usage and the
possibility of co-usage with other therapeutic treatments including
other anti-viral agents.
[0061] The pharmaceutical formulation may be administered via the
parenteral route, including subcutaneously, intraperitoneally,
intramuscularly and intravenously. Examples of parenteral dosage
forms include aqueous solutions of the active agent, in a isotonic
saline, 5% glucose or other well-known pharmaceutically acceptable
liquid carrier. In one aspect of the present embodiment, the
alkenyldiarylmethane compound is dissolved in a saline solution
containing 5% of dimethyl sulfoxide and 10% Cremphor EL (Sigma
Chemical Company). Additional solubilizing agents well-known to
those familiar with the art can be utilized as pharmaceutical
excipients for delivery of the present compounds.
[0062] The present compounds can also be formulated into dosage
forms for other routes of administration utilizing well-known
methods. The pharmaceutical compositions can be formulated, for
example, in dosage forms for oral administration in a capsule, a
gel seal or a tablet. Capsules may comprise any well-known
pharmaceutically acceptable material such as gelatin or cellulose
derivatives. Tablets may be formulated in accordance with
conventional procedure by compressing mixtures of the active
compound and solid carriers, and lubricants well-known to those
familiar with the art. Examples of solid carriers include starch,
sugar, bentonite. The compounds of the present invention can also
be administered in a form of a hard shell tablet or capsule
containing, for example, lactose or mannitol as a binder and
conventional fillers and tableting agents.
[0063] Typically, oral dosage levels range from 50-500 mg per dose,
administered from 1 to 4 times per day. More typically, oral dosage
levels ranging from 100 to 250 mg/dose are administered 1 to 4
times per day. With respect to parenteral dosing, levels between 25
and 250 mg are typically administered 1 to 4 times per day. Dosing
regimens with lower or higher amounts of drug may be indicated
depending upon the patients clinical state and the potency of the
compound being administered.
[0064] The anti-viral activity of the described compounds was
measured utilizing two different assays. The first assay measures
the effectiveness of the alkenyldiarylmethane compound to inhibit
reverse transcriptase activity, and the second assay measured the
compound's ability to inhibit the cytopathic activity of HIV-1 to
cells cultured in vitro. The mechanism of action for the disclosed
compounds' antiviral activities is believed to be due at least in
part to the compounds' ability to inhibit reverse transcriptase
(RT) activity. Several of the disclosed ADAM compounds have an
inhibitory effect on RT and yet do not exhibit a detectable
inhibitory effect on viral cytopathicity in the HIV cell assay.
This may be the result of the failure of the compound to penetrate
the cell membrane. For example, compound 21 has activity as an RT
inhibitor and yet no detectable inhibitory cytopathic effect was
detected. The inactivity of 21 might possibly be due to the fact
that the amino group is protonated at the pH of the assay medium
and therefore should be less able to penetrate cellular membranes.
Such compounds may have utility as antiviral agents, if they can be
formulated as prodrugs, using techniques known to those skilled in
the art, or through the use of other techniques that are known to
increase cellular uptake of compounds.
[0065] The following examples are provided to illustrate various
embodiments of the invention and are not intended to in any way
limit the scope of the invention as set forth in this specification
and appended claims.
EXAMPLE 1
[0066] Preparation of Congeners of Compound 1
[0067] Congeners of 1 were prepared in which the effect of alkenyl
chain length on activity could be investigated. Various analogs
were also prepared in which the bromines present in 1 were replaced
by chlorines, iodines, and hydrogens.
[0068] Considering first the compounds having the same substitution
in the aromatic rings as in 1, compounds 3-7 were prepared from the
inter-mediate benzophenone 10. The methylene and ethylene compounds
3 and 4 were prepared by the Wittig reactions of 10 starting from
methyltriphenylphosphonium bromide and ethyltriphenylphosphonium
bromide, respectively, using sodium bis(trimethylsilyl)amide as the
base. The methoxyethylene congener 5 was prepared in a similar
reaction employing methoxymethyl(triphenyl)phosphonium bromide.
Similarly, reaction of ketone 10 with the Wittig reagent derived
from 3-[tert-(butyldiphenylsily- loxy)propyl]triphenylphosphonium
bromide afforded intermediate 6. Removal of the protecting group
from 6 was accomplished with tetra-n-butylammonium fluoride in THF,
yielding the alcohol 7. Reaction of the alcohol 7 with mesyl
chloride in the presence of triethylamine gave the mesylate 8,
which was converted to the azide 9 on treatment with sodium azide
in DMF. 14
[0069] In order to convert 1 into a closely related compound
bearing a reactive functionality which might serve to alkylate the
enzyme, the conversion of the alkene moiety to an epoxide was
considered. Reaction of 1 with m-chloroperoxybenzoic acid in
methylene chloride afforded the desired epoxide 12.
[0070] A series of dichloro alkenyldiarylmethanes 13-23 bearing
alkenyl substituents capable of hydrogen bonding was prepared using
the substituted dichlorobenzophenone 11 as the starting material.
Using the Horner-Emmons reaction, compound 24 was reacted with NaH
at low temperature for 1 h to produce the corresponding anion,
which was then reacted with ketone 11 to afford, after
purification, ester 13 in good yield. The required reagent 24 was
prepared from diethylphosphonoacetic acid (25) and
2-(trimethylsilyl)ethanol (26). Deprotection of 13 was carried out
with tetra-n-butylammonium fluoride in THF, affording the
carboxylic acid 14 as a colorless crystalline solid. The methyl
ester 15 and tert-butyl ester 16 were also prepared by the
Horner-Emmons reaction starting from methyl diethylphosphonoacetate
(27) and tert-butyl diethylphosphonoacetate (28), respectively.
[0071] The Wittig reaction of the ketone 11 with the ylide derived
from 3-[tert(butyldiphenylsilyloxy)propyl]triphenylphosphonium
bromide afforded the alkene 17. Removal of the
tert-butyldiphenylsilyl protecting group was accomplished with
tetra-n-butylammonium fluoride in THF to provide the alcohol 18.
Oxidation of 18 under Jones conditions (Cro.sub.3, sulfuric acid,
acetone), followed by extraction with 3 M aqueous sodium hydroxide,
yielded the tricarboxylic acid 23, resulting from oxidation of the
primary alcohol in 18 and hydrolysis of the two ester groups during
extraction. On the other hand, reaction of the alcohol 18 with
methanesulfonyl chloride in dichloromethane, using triethylamine as
the base, afforded the mesylate 19. Reaction of the mesylate 19
with sodium azide in DMF gave the azide 20.
[0072] Various methods were considered for the reduction of the
azide 20 to the amine 21. The usual methods for conversion of
azides to amines involving lithium aluminum hydride or catalytic
hydrogenations are obviously not suitable for accomplishing the
conversion of 20 to 21 because of the other functionality in
addition to the azide present in 20. Following a protocol described
by Brown and Salunkhe, 1516
[0073] (Tetrahedron Lett. 1995, 36, 7987-7990) compound 20 was
reacted with dichloroborane dimethyl sulfide complex to produce,
after appropriate work up, amine 21 in low yield (15%).
Dichloroborane dimethyl sulfide complex has been reported to react
with olefins, but this process has been described to be slow and
incomplete in the absence of trichloroborane. However, it is likely
that the reactivity of the borane complex with olefins is at least
in part responsible for the poor yield observed in the conversion
of 20 to amine 21. Alternative methods were then considered in
order to reduce 20 to 21 in acceptable yield. Zwierzak et al.
reported the preparation of amines from azides using a variant of
the Staudinger reaction. (Synthesis, 1985, 202-204) They reacted
organic azides with trialkyl phosphates, instead of the commonly
used triphenylphosphine, to produce iminophosphorane intermediates,
which after treatment with hydrogen chloride afforded the
corresponding amine hydrochlorides. The higher reactivity of
trialkyl phosphates towards azides was the main assumption for the
modification. This methodology was considered to be the most
adequate for the conversion of azide 20 to amine 21, since the
other reactive functionality present in 21 would not be affected by
this method. In fact, the reaction of azide 20 with
triethylphosphite for 24 h, followed by hydrolysis of the
intermediate iminophosphorane with dry hydrogen chloride for 48 h,
afforded the amine 21 as its hydrochloride salt in 86% yield.
[0074] In order to synthesize an alkenyldiarylmethane having a
terminal methoxycarbonyl group at the end of an extended alkenyl
side chain, the benzophenone 11 was subjected to a Wittig reaction
with the ylide derived from the reaction of the phosphonium bromide
29 with sodium bis(nimethylsilyl)amide in THF. This resulted in the
formation of the desired compound 22, referred to herein as ADAM
II.
[0075] The effect of replacement of the methoxycarbonyl and methyl
ether groups by cyclic acetonides on biological activity could be
readily investigated by evaluating the known alcohol 30, prepared
previously from the ketone 32. The primary alcohol was converted to
the corresponding bromide 31 with carbon tetrabromide and
triphenylphosphine in acetonitrile.
[0076] In order to determine the importance of the two halogen
atoms for biological activity, congeners of ADAM I (1) were
prepared in which the halogens were replaced by iodines and by
hydrogens. The syntheses of these compounds is outlined in Scheme
1. Treatment of the known diphenylmethane 33 with dimethylsulfate
in refluxing acetone with potassium carbonate as the base resulted
in methylation of both carboxylic acids and both phenols to afford
compound 34, which was oxidized to the benzophenone 35 with
chromium trioxide in acetic anhydride. Reaction of the ketone
present in 35 with the Wittig reagent derived from
n-hexyltriphenylphosphonium bromide gave the desired analog 36, in
which the two chlorines present in 1 have been replaced by
hydrogens. 17
[0077] Nitration of 35 with nitric acid in acetic anhydride
afforded the dinitro intermediate 37. The two nitro groups present
in 37 were reduced to amines with Adam's catalyst in ethyl acetate
to provide the diamino compound 38. Treatment of the diamine 38
with nitrous acid resulted in the conversion of both amines to
diazonium groups, which were displaced by iodide in the presence of
potassium iodide and iodine under aqueous conditions to provide
intermediate 39. The reaction of 39 with the ylide derived from
n-hexyltriphenylphosphonium bromide afforded the desired analog
40.
EXAMPLE 2
[0078] Biological Results.
[0079] Nineteen new alkenyldiarylmethanes were tested for
prevention of the cytopathic effect of HIV-1.sub.RF in CEM-SS cells
and for cytotoxicity in uninfected CEM-SS, and the results are
listed in Table 1. In addition, they were tested as inhibitors of
HIV-1 reverse transcriptase, and the resulting IC.sub.50 values are
also listed in Table 1. The most potent of the new
alkenyldiarylmethanes, as well as the one with the highest
therapeutic index, proved to be ADAM II (22), which displayed an
EC.sub.50 for prevention of the cytopathic effect of HIV-1.sub.RF
of 0.013 .mu.M. This represents an approximately 700-fold increase
in potency over the lead compound 1, which was the most potent
alkenyldiarylmethane of the previously disclosed ADAM series.
Perhaps more importantly, the therapeutic index (the ratio of the
CC.sub.50 value to the EC.sub.50 value) increased by a factor of
162. The other new compounds which were equipotent or more potent
than 1 for the prevention of HIV-1 cytopathicity included the
primary alcohol 7 (EC.sub.50=6.0 .mu.M), the azide 9 (EC.sub.50=1.1
.mu.M), the primary alcohol 18 (EC.sub.50=8.6 .mu.M), and the azide
20 (EC.sub.50=0.27 .mu.M). No inhibitory effect was detected for
the remaining novel alkenyldiarylmethanes tested for inhibition of
HIV-1 cytopathicity at concentrations up to those resulting in
cytotoxicity in uninfected cells (see Table 1).
1TABLE 1 Anti-HIV-1 Activities of ADAMs. XTT Assay compd RT
(EC.sub.50 .mu.M).sup.a EC.sub.50 (.mu.M).sup.b CC.sub.50
(.mu.M).sup.e TI.sup.d 1 0.38 9.2 138 15 3 >100 .sup. NA.sup.e
316 -- 4 26 NA 10 -- 5 >100 NA 22 -- 7 31.6 6.0 >316 >52 9
94 1.1 >316 >278 12 5.6 NA >100 -- 14 >100 NA 48 -- 15
16 NA 25 -- 16 >100 NA >100 -- 18 >100 8.6 16.8 2 19 96 NA
6.9 -- 20 2.0 0.27 41.8 155 21 63 NA 15 -- 22 0.3 0.013 31.6 2430
23 >100 NA >316 -- 30 >100 NA 18.8 -- 31 >100 NA 176 --
36 3.2 NA 14 -- 40 11 NA >316 -- .sup.aInhibitory activity vs.
HIV-1 reverse transcriptase with rCdG as the template-primer.
.sup.bThe EC.sub.50 is the 50% inhibitory concentration for
cytopathicity of HIV-1.sub.RF in CEM-SS cells. .sup.cThe CC.sub.50
is the 50% cytotoxic concentration for mock-infected CEM cells.
.sup.dThe TI is the therapeutic index, which is the CC.sub.50
divided by the EC.sub.50. .sup.eNA means there was no observed
inhibition of HIV-1 cytopathicity up to the cytotoxic concentration
in uninfected cells.
[0080] The observed increase in antiviral potency of 22 relative to
1 did not correlate with inhibition of HIV-1 RT with
poly(rC).oligo(dG) as the template primer, since 1 (IC.sub.50 vs.
RT=0.38 .mu.M) was essentially equipotent as an enzyme inhibitor
than ADAM II (22) (IC.sub.50 vs. RT=0.30 .mu.M). All of the new
compounds displaying anti-HIV activity were also less potent than 1
as inhibitors of HIV-1 RT with poly(rC).oligo(dG) as the template
primer, and some of them were significantly less active. Examples
include compounds 7 (IC.sub.50 vs. RT=31.6 .mu.M), 9 (IC.sub.50 vs.
RT=94 .mu.M), and 18 (IC.sub.50 vs. RT>100 .mu.M). Conversely,
analogs 12 and 36 inhibit HIV-1 RT with IC.sub.50 values of 5.6
.mu.M and 3.2 .mu.M, respectively, and are inactive as inhibitors
of HIV-1mediated cytopathogenic effect.
[0081] Some of the closely related structural analogs of 1 are
revealing in their inactivity as inhibitors of the cytopathic
effect of HIV-1. Examples include the epoxide 12, the dechlorinated
analog 36, and the diiodo congener 40. These inactive compounds
emphasize the fact that there is a relatively high degree of
structural specificity associated with the antiviral activity of
the compounds in the series, so that even small changes in
structure can result in complete loss of activity. The inactivity
of 36 and 40 emphasize a significant role played by the two bromine
atoms present in 1. In this regard, the dichloro analog 41 was
reported previously to retain activity, although it was
approximately half as potent as 1. However, the effect of bromine
vs. chlorine substitution is not always consistent. For example, if
one considers the bromo alcohol 7 (EC.sub.50 6.0 .mu.M) vs. the
chloro alcohol 18 (EC.sub.50 of 8.6 .mu.M), they are approximately
equipotent as inhibitors of viral cytopathicity. On the other hand,
the chloro azide 20 (EC.sub.50 0.27 .mu.M) is four times more
potent than the bromo azide 9 (EC.sub.50 1.1 .mu.M).
[0082] The effect of replacement of the methoxycarbonyl and methyl
ether groups by cyclic acetonides can be seen by comparison of the
activities of the primary alcohols 18 and 30. While 18 inhibited
the cytopathic effect of HIV-1.sub.RF with an EC.sub.50 of 8.6, the
cyclic acetonide 30 was inactive. The corresponding primary bromide
31 was also inactive.
[0083] There appears to be an important effect of chain length of
the alkenyl appendage. Both of the active azides 9 and 20 have a
chain length which is identical with the alkenyl chain present in
ADAM 1, and in the most active compound, ADAM II (22), the chain
length is only slightly longer. 18
[0084] The effect of incorporation of hydrogen bonding groups at
the end of the alkenyl chain seems to be variable, depending on the
specific group incorporated and the length of the chain. For
example, the primary alcohol 18 was effective in preventing the
cytopathic effect of HIV-1.sub.RF with an EC.sub.50 of 8.6 .mu.M,
but the corresponding amine 21 was inactive at 100 .mu.M. The most
effective compounds for inhibition of viral cytopathicity were ADAM
II (22), having a methyl ester at the end of the chain, and the two
azides 9 and 20. Although these compounds do have hydrogen bond
accepting groups at the end of the alkenyl chain, it is difficult
to attribute their greater antiviral activity to a greater affinity
for the enzyme, because they are not more potent than 1 as enzyme
inhibitors, at least with poly(rC.oligo(dG) as the
template-primer.
[0085] In order to determine whether or not ADAM II was indeed
acting as an NNRTI compound 22 was tested in a number of assays
representative of important events in the replication cycle of
HIV-1. These included (in addition to RT inhibition with
poly(rA).oligo(dT) and poly(rC).oligo(dG) as template primers),
assays for inhibition of HIV-1 attachment/fusion to target cells
anid the activities of HIV-1 integrase and protease enzymes. The
effect of the compound on nucleocapsid protein was also
investigated. Compound 22 did not have any significant effect on
integrase, protease, or nucleocapsid protein. (See Table 2).
However, it inhibited RT with either poly(rA).oligo(dT) or
poly(rC).oligo(dG) as template primers. The greater sensitivity to
inhibition with poly(rC).oligo(dG) as the template/primer is
characteristic of the HIV-1-specific non-nucleoside reverse
transcriptase inhibitors. Interestingly, ADAM I 1, which had an
IC.sub.50 of 0.38 .mu.M with poly(rC).oligo(dG) as the template
primer did not inhibit the enzyme with poly(rA).oligo(dT) as the
template-primer. Thus there exists a clear distinction between the
abilities of ADAM I and ADAM II to inhibit RT activity with the
poly(rA).oligo(dT) template primer system.
[0086] In addition to the molecular target-based mechanistic
assays, ADAM II (22) was also evaluated in a time course assay to
determine the site of action of the compound during the early phase
of HIV-1 replication, as well as with latently HIV-1 infected U1
cells to probe for any antiviral actions during the post-infective
that phase of viral replication. The profile of inhibition of ADAM
II corresponded to that of an NNRTI, in which antiviral activity
was diminished when the compound was added to cultures four hours
after initiation of infection. This is analogous to the effects of
nevirapine, but unlike the action of dextran sulfate, which blocks
virus attachment at the cell surface and loses its effectiveness
within the first half hour after initiation of infection. PCR-based
analysis confirmed that ADAM II prevented formation of proviral DNA
during the early phase of infection. ADAM II had no effect on virus
production from U1 cells induced with TNF-.alpha. to produce virus
from integrated proviral DNA. Thus, ADAM II acted biologically to
inhibit replication by preventing reverse transcription.
[0087] The scope of the anti-HIV activity of ADAM II was
investigated in a number of laboratory-adapted strains,
lymphocyte-tropic clinical isolates, clade representatives, and
monocyte-tropic strains of HIV-1. Likewise, ADAM II was tested for
inhibitory activity against an array of HIV-1 strains containing
mutations in the reverse transcriptase enzyme. (See Tables 3, 4 and
5). The mutant viruses were obtained either by in vitro biological
selection in the presence of various NNRTIs, or by site directed
mutagenesis. In order to preserve continuity of data across the
surfeit of virus strains, a greater amount of virus (higher MOI)
was used in these competitive studies than was used in the original
screening assays. For this reason, the EC.sub.50 was generally
about 20-fold greater in these studies than in the original screen.
Nevertheless, ADAM II retained potent anti-HIV-1 activity against a
wide variety of laboratory and clinical HIV-1 strains in CEM-SS,
PBMC, and Mono/Mac cells (Table 3). Results from studies with the
panels of viruses having defined mutations in reverse transcriptase
are listed in Tables 4 and 5. Mutations conferring resistance to
ADAM II are clustered within the amino acid residues comprising the
NNRTI binding pocket. Resistance mutations to the NNRTI .alpha.-APA
are located close to the bound inhibitor, and it therefore seems
likely that the mutations conferring resistance to ADAM II may also
be located close to the bound ligand. The mutations conferring
greater than ten-fold resistance to ADAM II were located at
positions 103, 108, 110, 139, 181, and 188. In contrast, the A98G
and L1001 mutants were more sensitive to ADAM II than the
corresponding wild type virus (Table 5). The virus containing the
L100I mutation and the multiple mutations that confer resistance to
AZT also displayed increased sensitivity to ADAM II. This suggests
the possible clinical utility of ADAM II against AZT resistant
strains of HIV-1 and against strains of HIV-1 that express
L100I-based resistance to other NNRTIs.
2TABLE 2 Mechanistic Evaluations of ADAM II (22) Parameter
IC.sub.50 (.mu.M) Attachment/Fusion NI.sub.100.sup.a RT rAdT 1.9
rCdG 0.3 Integase NI.sub.100.sup.b Protease >100.sup.c
Nucleocapsin p7 Protein NI.sub.25 .sup.aA 20% inhibition was
observed at 100 .mu.M. .sup.bNI indicates that no inhibition of
activity was observed at the indicated high test concentration.
.sup.cA 30% inhibition of HIV-1 protease was observed at a
concentration of 100 .mu.M.
[0088]
3TABLE 3 ADAM II Activity Against HIV-1 Laboratory-Adapted Strains,
Lymphocyte-Tropic Clinical Isolates, Clades and Monocyte- tropic
Stains of HIV-1 Virus Isolate Virus Type Cell Type EC.sub.50
(.mu.M) HIV-1 RB WT-Laboratory.sup.a CEM-SS 0.91 HIV-1 IIIB
WT-Laboratory.sup.a CEM-SS 0.16 HIV-1 TEKI WT-Clinical.sup.b PBMC
2.20 HIV-1 ROJO WT-Clinical.sup.b PBMC 2.28 HIV-1 Clade A PBMC 1.09
HIV-1 Clade B PBMC 0.43 HIV-1 Clade C PBMC 1.04 HIV-1 Clade D PBMC
1.02 HIV-1 Clade E PBMC 0.40 HIV-1 Clade F PBMC 1.70 HIV-1 Ba-L
Monocyte-topic Mono/Mac 0.36 HIV-1 ADA Monocyte-topic Mono/Mac 0.28
.sup.aWild type laboratory-adapted strain. .sup.bWild type clinical
isolate.
[0089]
4TABLE 4 ADAM II Activity Against a Biological Panel of Resistant
Isolates in CEM-CC Cells Virus Isolate Mutation in RT EC.sub.50
(.mu.M) Fold Resistance HIV-1 IIIB Wild Type 0.39 OC.sup.a L100I
0.94 2.4 TSAO/Cost K110E 11.9 30 129/Cost K103N 10.8 28
Thiazol.sup.b V108I >50 >128 Calo.sup.c T139I 15 38 DPS.sup.d
Y181C >50 >128 3TC M184I 1.08 2.8 Cost Y188H >50 >128
HEPT.sup.e P236L 8.08 21 .sup.aOxathiin carboxanilide resistant.
.sup.bThiazolobenzimidazole resistant. .sup.cCananolide resistant.
.sup.dDiphenyl sulfonate resistant.
.sup.e(Hydroxyethoxy)methyl(phenylthio)thymine resistant.
[0090]
5TABLE 5 ADAM II Activity Against a Site Directed Panel of
Resistant Isolates in CEM-SS Cells Virus Isolate Mutation in RT
EC.sub.50 (.mu.M) Fold Resistance HIV-1 NL4-3 Wild type 1.78 Y188C
10.80 6.07 K103N >50 >28 K101E 3.05 1.71 L100I <0.16
S.sup.a 4XAZT/L100I.sup.b 0.27 S.sup.a A98G 0.84 S.sup.a V179D 50
28 Y181C >50 >28 L74V 2.23 1.25 4XAZT.sup.b 0.90 S.sup.a
V108I 12.0 6.74 V106A 12.7 7.13 4XAZT/Y181C.sup.b >50 >28
.sup.aEnhanced Sensitivity. .sup.bAZT resistant.
EXAMPLE 3
Experimental Section
[0091] General. Melting points were determined in capillary tubes
on a Mel-Temp apparatus and are uncorrected. Spectra were obtained
as follows: CI mass spectra on a Finnegan 4000 spectrometer, FAB
mass spectra and EI mass spectra on a Kratos MS50 spectrometer,
.sup.1H NMR spectra on Varian VXR-500S and Bruker ARX-300
spectrometers; IR spectra on a Beckman IR-33 spectrometer or on a
Perkin Elmer 1600 series FTIR. Microanalyses were performed at the
Purdue Microanalysis Laboratory, and all values were within
.+-.0.4% of the calculated compositions.
[0092]
3',3"-Dibromo-4',4"-dimethoxy-5',5"-bis(methoxycarbonyl)-1,1-diphen-
ylethene (3). Methyl(triphenyl)phosphonium bromide (132 mg, 0.36
mmol) was suspended in anhydrous THF (2 mL), the mixture was
stirred under Argon in an ice-bath, and sodium
bis(trimethylsilyl)amide (1.0 M in THF, 0.4 mL, 0.4 mmol) was added
by syringe. The mixture was stirred for 20 min, and a solution of
3,3'-dibromo-4,4'-dimethoxy-5,5'bis(methoxycarbonyl)diphenyl ketone
(10) (155 mg, 0.3 mmol) in anhydrous THF (3 mL) was slowly
injected. The ice bath was removed, and the reaction mixture was
stirred for 1 h at ambient temperature and 1 h at 60.degree. C.
overnight at ambient temperature. The reaction was quenched with
saturated ammonium chloride solution (3 mL). The yellow organic
layer was separated and the aqueous layer was extracted with
benzene (2.times.5 mL). The combined organic extracts were washed
with brine (2 mL) and dried (Na.sub.2SO.sub.4). The solvent was
evaporated and the residue was purified by flash chromatography on
silica gel (6 g), eluting with hexane-ethyl acetate (6:1) to give 3
(62 mg 42%) as a colorless solid: mp 144-145.degree. C.; .sup.1H
NMR (CDCl.sub.3, 300 MHz) .delta.7.70 (m, 2 H), 7.68 (m, 2 H), 5.53
(s, 2 H), 4.01 (s, 3 H), 4.00 (s, 3 H), 3.97 (s, 3 H), 3.96 (s, 3
H); IR (KBr) 2943, 1730, 1472, 1434, 1246, 1208, 1086, 992, 797,
726 cm.sup.-; CIMS m/z (rel intensity) 516 (27), 515 (96), 514
(21), 512 (M.sup.+, 5), 486 (10), 485 (45), 484 (21), 483 (100).
Anal. (C.sub.20H.sub.18Br.sub.2O.sub.6.1H.sub.2O) C, H.
[0093]
3',3"-Dibromo-4',4"-dimethoxy-5',5"-bis(methoxycarbonyl)-1,1-diphen-
ylpropene (4). Ethyl(triphenyl)phosphonium bromide (134 mg, 0.36
mmol) was suspended in anhydrous THF 2 mL, and the mixture was
stirred under Ar in an ice-bath. Sodium bis(trimethylsilyl)amide
(1.0 M in THF, 0.5 mL) was added by syringe. The mixture was
stirred for 20 min, and a solution of
3,3'-dibromo-4,4'-dimethoxy-5,5'bis(methoxycarbonyl)diphenyl ketone
(10) (155 mg, 0.3 mmol) in anhydrous THF (3 mL) was slowly
injected. The ice-bath was removed, and the reaction mixture was
stirred for 24 h at ambient temperature. The reaction was quenched
with saturated ammonium chloride solution (3 mL). The yellow
organic layer was separated and the aqueous layer was extracted
with benzene (2.times.5 mL). The combined organic extracts were
washed with brine (2 mL) and dried (Na.sub.2SO.sub.4). The solvent
was evaporated and the residue purified by flash chromatography on
silica gel (6 g), eluting with hexane-ethyl acetate (6:1), to
afford 4 (55 mg, 35%) as a colorless solid: MP 76-77.degree. C.;
.sup.1HNMR (CDCl.sub.3, 300 MHz) .delta.7.54 (m, 2 H), 7.51 (m, 1
H), 7.49 (m, 1 H), 6.18 (q, J=7.2 Hz, 1 H), 3.99 (s, 3 H), 3.93 (s,
6 H), 3.92 (s, 3 H), 1.77 (d, J=7.2 Hz, 3 H); IR (KBr) 2950, 1732,
1475, 1286, 1263, 1207, 997, 725 cm.sup.-1; CIMS m/z (rel
intensity) 531 (62), 530 (29), 529 (94), 528 (23), 527 (M+1, 57),
500 (12), 499 (53), 498 (24), 497 (100), 495 (53). Anal.
(C.sub.21H.sub.20OBr.sub.2O.sub.6) C, H.
[0094]
3',3"-Dibromo-4',4"-dimethoxy-5',5"-bis(methoxycarbonyl)-1-methoxy--
2,2-diphenylethene (5). Methoxymethyl(triphenyl)phosphonium bromide
(257 mg, 0.75 mmol) was suspended in anhydrous THF (4 mL), the
mixture was stirred under Ar in an ice bath, and sodium
bis(trimethylsilyl)amide (1.0 M in THF, 0.75 mL, 0.75 mmol) was
added by syringe. The mixture was stirred for 20 min, and a
solution of 3,3,'-dibromo-4,4'-dimethoxy-5,5'bi-
s(methoxycarbonyl)diphenyl ketone (10) (258 mg, 0.5 mmol) in
anhydrous THF (4 mL) was slowly injected. The ice bath was removed,
and the reaction mixture was stirred for 24 h at ambient
temperature, 3 h at 60.degree. C., and overnight at ambient
temperature. The reaction was quenched with saturated ammonium
chloride solution (5 mL). The yellow organic layer was separated
and the aqueous layer was extracted with benzene (2.times.10 mL).
The combined organic extracts were washed with brine (4 mL) and
dried (Na.sub.2SO.sub.4). The solvent was evaporated and the
residue purified by flash chromatography on silica gel (15 g),
eluting with hexane-ethyl acetate 6:1, to give compound 5 as a
colorless oil (76 mg, 28%) having an R.sub.f of 0.15 on silica gel
when hexane-ethyl acetate (6:1) was used as the sovent system:
.sup.1H NMR (CDCl.sub.3, 300 MHz) .delta.7.78 (d, J=2.1 Hz, 1 H),
7.74 (d, J=2.1 Hz, 1 H), 7.61(d, J=2.2 Hz, 1 H), 7.58 (d, J=2.2 Hz,
1 H), 6.51(s, 1 H), 4.01 (s, 6 H), 3.99 (s, 3 H), 3.97 (s, 3 H),
3.89 (s, 3 H); IR (neat) 2949, 1733,1636, 1475, 1436, 1289, 1255,
1208, 1124, 1081, 1049, 998 cm.sup.-1. HRFABMS calcd for
C.sub.21H.sub.21Br.sub.2O.sub.7 m/z 542.9654. Found m/z
542.9661.
[0095]
3'3"-Dibromo-4',4"-dimethoxy-5',5'-bis(methoxycarbony)-1,1-diphenyl-
-4-[(tert-butyldiphenylsilyl)oxy]-1-butene (6).
3-[tert-(Butyldiphenylsily- loxy)propyl]triphenylphosphonium
bromide (595 mg, 0.93 mmol) was suspended in anhydrous THF (6 mL)
and the mixture stirred under Ar on an ice bath. Sodium
bis(trimethylsilyl)amide (1.0 M in THF, 1 mL) was added by syringe.
The reaction mixture turned into a bright orange solution and was
stirred in an ice bath for 30 min. A solution of the ketone 10 (320
mg, 0.62 mmol) dissolved in anhydrous THF (5 mL) was added and the
reaction mixture was stirred at ambient temperature for 24 h. The
mixture was quenched with saturated ammonium chloride solution (10
mL), followed by ethyl acetate (10 mL). The organic layer was
separated and the aqueous layer was extracted with ethyl acetate
(3.times.20 mL). The combined organic extracts were washed with
brine (15 mL) and dried (Na.sub.2SO.sub.4). The solvent was
evaporated and the yellow oily residue purified by flash
chromatography on silica gel (20 g), eluting with hexane-ethyl
acetate 3:1, to give 6 (284 mg, 58%) as a yellow oil: .sup.1H NMR
(CDCI.sub.3, 300 MHz) .delta.7.67 (d, J=7.7 Hz, 4 H), 7.58 (m, 3
H), 7.51 (d, J=2.1 Hz, 1 H), 7.40 (m, 6 H), 6.10 (t, J=7.4 Hz, 1H),
4.03 (s, 3 H), 3.98 (s, 3 H), 3.95 (s, 3 H), 3.93 (s, 3 H) 3.79 (t,
J=6.1 Hz, 2 H), 2.39 (m, 2 H), 1.09 (s, 9 H); IR (neat) 2951, 2860,
1734, 1473, 1265, 1205, 998, 704 cm.sup.-1. Anal.
(C.sub.38H.sub.40Br.sub.2O.sub.7Si) C, H.
[0096]
3',3"-Dibromo-4-hydroxy-4',4"dimethoxy-5',5'-bis(methoxycarbony)-1,-
1-diphenyl-1-butene (7). Compound 6 (266 mg, 0.33 mmol) was
dissolved in dry THF (7 mL) and the solution stirred at 0.degree.
C. under Ar. A 1.0 M solution of tetrabutylammonium fluoride in THF
(0.7 mL, 0.7 mmol) was added. The solution turned yellow and was
stirred at 0.degree. C. for 5.5 h. Brine (10 mL) was added and the
phases were separated. The aqueous phase was extracted with ethyl
acetate (3.times.20 mL). The extract was dried (MgSO.sub.4) and the
solvent was evaporated. The residue was purified by flash column
chromatography on silica gel, eluting with hexane-ethyl acetate
3:1, 1:1 to give 7 (129 mg, 70%) as a yellowish oil: .sup.1H NMR
(CDCl.sub.3,300 MHz) .delta.7.58 (m, 3 H), 7.52 (m, 1 H), 6.14 (t,
J=7.1 Hz, 1 H), 4.00 (s, 3 H), 3.96 (s, 3 H), 3.94 (s, 3 H), 3.93
(s, 3 H), 3.77 (t, J=6.1 Hz, 2 H), 2.41 (q, J=6.3 Hz, 2 H), 1.59
(brs, 1 H); IR (neat) 3430 (broad band), 2951, 1732, 1474, 1265,
1208, 998 cm.sup.-1. Anal. (C.sub.22H.sub.22Br.sub.2O.sub.7) C,
H.
[0097]
3',3"-Dibromo-4-methanesulfonyloxy-4',4"dimethoxy-5',5'-bis(methoxy-
carbony)-1,1-diphenyl-1-butene (8). Compound (7) (310 mg, 0.55
mmol) and anhydrous triethylamine (0.23 mL, 1.7 mmol) were
dissolved in dry dichloromethane (7 mL) and the mixture was stirred
under Ar at 0.degree. C. Mesyl chloride (0.2 mL, 2.56 mmol) was
added and the mixture was stirred at 0.degree. C. for 3 h. The
reaction mixture was diluted with dichloromethane (10 mL) and
washed with 0.5 N HCI (2.times.20 mL, followed by sat NaHC0.sub.3
(1.times.20 mL) and brine (1.times.20 mL). The organic extract was
dried (MgSO.sub.4). The solvent was evaporated and the residue
purified on silica gel (16 g), eluting with hexane-ethyl acetate
3:1, to afford the mesylate 8 (270 mg, 77%) as a colorless oil:
.sup.1H NMR (CDCl.sub.3, 300 MHz) .delta.7.55 (m, 2 H),7.51 (m, 2
H), 6.14 (t, J=7.4 Hz, 1 H), 4.30 (t, J=6.2 Hz, 2 H), 3.99 (s, 3
H), 3.931 (s, 3 H), 3.927 (s, 3 H), 3.92 (s, 3 H), 3.04 (s, 3 H),
2.56 (q, J=7.0 Hz, 2 H); IR (neat) 2952, 1732, 1474, 1358, 1265,
1175, 1089, 995 cm.sup.-1. Anal. (C.sub.23H.sub.24Br.sub.2SO.sub.9)
C, H.
[0098]
4-Azido-3',3"-Dibromo-4',4"dimethoxy-5',5'-bis(methoxycarbony)-1,1--
diphenyl-1-butene (9). Compound 8 (231 mg, 0.363 mmol) was
dissolved in dry -N,N-dimethylformamide (5 mL). Sodium azide (120
mg, 1.82 mmol) was added and the mixture was stirred at
35-50.degree. C. for 3 h. The reaction mixture was allowed to reach
ambient temperature and then it was diluted with ethyl ether (42
mL). The ethereal solution was washed with water (2.times.35 mL),
brine (1.times.35 mL), and dried (MgSO.sub.4). The solvent was
evaporated and the residue was purified on silica gel (16 g),
eluting with hexane-ethyl acetate 3:1, to give 9 (132 mg, 62%) as a
colorless solid: mp 69-70.degree. C.; .sup.1H NMR (CDCl.sub.3, 300
MHz) .delta.7.56 (m, 3 H), 7.50 (m, 1 H), 6.05 (m, 1 H), 3.99 (m, 1
H), 3.93 (m, 3 H), 3.40 (m, 2 H), 2.41 (m, 2 H); IR (KBr) 2944,
2105, 1731, 1473, 1436, 1246, 1207, 1085, 999, 806, 726 cm.sup.1.
Anal (C.sub.22H.sub.21Br.sub.2N.sub.3O.sub.6) C, H.
[0099]
3',3"-Dibromo-4',4"-dimethoxy-5',5"-bis(methoxycarbonyl)-1,1,-diphe-
nyl-1-heptene Epoxide (12). Compound 1 (67 mg, 0.116 mmol) was
dissolved in dry methylene chloride (1 mL) and the solution cooled
in a freezer for 5 min. 3-Chloroperoxybenzoic acid [minimum 57% (70
mg)] was dissolved in CH.sub.2Cl.sub.2 (0.5 mL and injected into
the solution. The mixture was allowed to reach ambient temperature
and was stirred for 21 h. Solvent was evaporated and the residue
was purified by flash column chromatography on silica gel (7.5 g),
eluting with hexane-ethyl acetate 20:1 followed by 10:1, to give
the epoxide 12 (43 mg, 62%) as an oil: .sup.1H NMR (CDCI.sub.3, 300
MHz) .delta.7.74 (m, 2 H), 7.67 (m, 1 H), 7.65 (m, 1H), 3.97 (s, 3
H), 3.94 (s, 3 H), 3.93 (s, 3 H), 3.92 (s, 3 H), 3.35 (m, 1 H),
1.45 (m, 4 H) 1.27 (m, 5 H), 0.88 (m, 3 H). IR (neat) 2953, 1732,
1472, 1435, 1260, 1207, 1087, 999, 720 cm.sup.-1. Anal.
(C.sub.25H.sub.28Br.sub.2O.sub.7) C, H.
[0100] 2-(Trimethylsilyl)ethyl
3',3"-Dichloro-4',4"-dimethoxy-5,5"-bis(met-
hoxycarbonyl)-3,3-diphenylpropenoate (13). A suspension of sodium
hydride (0.018 g, 0.750 mmol) in dry THF (5 mL) was stirred in an
ice bath under an argon atmosphere. Phosphonoacetate 24 (0.21 mL,
0.70 mmol) was added and the mixture was stirred at 0.degree. C.
for 1 h. The initial suspension turned into a clear solution within
minutes. A solution of ketone 11 (0.200 g, 0.469 mmol) in dry THF
(4 mL) was then added dropwise. The ice bath was removed and the
reaction mixture was stirred at rt for 30 h. The solvent was
removed and the residue was taken up in cold water (30 mL) and
ethyl ether (30 mL). The layers were separated and the aqueous one
was extracted with ethyl ether (2.times.30 mL). The combined
organic fractions were washed with brine (2.times.30 mL), dried
over magnesium sulfate and filtered. The solvent was removed in
vacuo to give a residue. After flash chromatography (SiO.sub.2, 35
g; column dimensions: 3 cm.times.6.5 inch), eluting with
hexanes:ethyl acetate 5:1, compound 13 (0.216 g, 81.2%) was
obtained as a thick oil: .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta.7.60 (d, J=2.4 Hz, 1H), 7.53 (d, J=2.2 Hz, 1 H), 7.39 (d,
J=3.1 Hz, I H), 7.38 (d, J=2.5 Hz, I H), 6.31 (s, 1 H), 4.11 (m,
J=8.7 Hz, 2 H), 3.99 (s, 3 H), 3.95 (s, 3 H), 3.91 (s, 3 H), 3.90
(s, 3 H), 0.89 (m, J=8.7 Hz, 2 H), 0.01 (s, 9 H); IR (film) 2952,
1737, 1477, 1251, 1161, 997, 859, 744 cm.sup.-1; FABMS (m/z):
568.8[MH].sup.+. Anal. (C.sub.26H.sub.30Cl.sub.2O.sub.8Si) C,
H.
[0101]
3',3"-Dichloro-4',4"-dimethoxy-5',5"-bis(methoxycarbonyl)-3,3-diphe-
nylpropenoic Acid (14). A solution of ester 13 (0.198 g, 0.348
mmol) in dry THF (10 mL) was stirred under argon in an ice bath. A
1.0 M solution of TBAF (0.7 mL, 0.697 mmol) was added dropwise and
the reaction mixture stirred at 0.degree. C. for 1.5 h. The mixture
turned into a light yellowish solution. Brine (30 mL) was added and
the mixture stirred for 10 min. A 1.0 N solution of HCI (10 mL) was
then added and the product was extracted with ethyl ether
(3.times.30 mL). The combined organic extracts were washed with
brine (1.times.40 mL), dried over magnesium sulfate, filtered, and
the solvent removed. Pure 14 (0.135 g, 83%) was obtained as a
colorless crystalline solid after flash chromatography (SiO.sub.2,
20 g; column dimensions: 2.2 cm.times.6.5 inch), using
chloroform:methanol:formic acid (200:10:0.1 mL) as eluant: mp.
60-62.degree. C.; IR (film) 3400-2900, 2955, 1731, 1477, 1262,
1210, 1164, 994, 743 cm.sup.-1; .sup.1H NMR (300 NIHz, CDCl.sub.3)
.delta.7.59 (d, J=2.4 Hz, I H), 7.50 (d, J=2.2 Hz, 1 H), 7.37 (d,
J=2.2 Hz, 1 H), 7.35 (d, J=2.4 Hz, I H), 6.29 (s, 1 H), 3.97 (s, 3
H), 3.93 (s, 3 H), 3.89 (s, 3 H), 3.88 (s, 3 H); CIMS (m/z): 469
[MH, 25].sup.+, 451 [MH-18, 100].sup.+, 437 [MH-32, 23].sup.+.
Anal. (C.sub.21H.sub.18Cl.sub.2O.sub.8- 0.4H.sub.2O) C, H.
[0102] Methyl
3',3"-Dichloro-4',4"-dimethoxy-5,5"-bis(methoxycarbonyl)-3,3-
-diphenylpropenoate (15). A suspension of sodium hydride (0.012 g,
0.493 mmol) in dry THF (5 mL) was stirred in an ice bath under an
argon atmosphere. Methyl diethylphosphonoacetate (27) (0.09 m.L,
0.469 mmol) was added and the mixture was stirred at 0.degree. C.
for 1 h. The initial suspension turned into a clear solution within
minutes. A solution of ketone 11 (0.100 g, 0.235 mmol) in dry THF
(5 mL) was added dropwise. The ice bath was removed and the
reaction mixture was stirred at rt for 30 h. Cold water (15 mL) was
added and the mixture was stirred for 10 min. The product was
extracted with ethyl ether (3.times.25 mL). The combined organic
fractions were washed with brine (1.times.30 mL), dried over
magnesium sulfate, and filtered. The solvent was removed in vacuo
to give a residue. After purification by flash chromatography on
silica gel (35 g; column dimensions: 2 cm.times.6 inch), eluting
with hexanes:ethyl acetate 2:1, compound 15 (0.0948 g, 83.8%) was
obtained as a thick colorless oil: .sup.1H NMR (300 MHz,
CDCl.sub.3) .delta.7.59 (d, J=2.4 Hz, 1 H), 7.53 (d, J=2.2 Hz, 1
H), 7.37 (d, J=2.2 Hz, I H), 7.36 (d, J=2.4 Hz, 1 H), 6.32 (s, 1
H), 3.99 (s, 3 H), 3.94 (s, 3 H), 3.89 (s, 3 H), 3.88 (s, 3 H),
3.63 (s, 3 H); IR (film) 2951, 1731, 1477, 1435, 1264, 1164, 996
cm.sup.-1; CIMS (m/z): 483 [MH, 30].sup.+ and 451
[(MH).sup.+-CH.sub.3OH, 100]. Anal.
(C.sub.22H.sub.20Cl.sub.2O.sub.8) C, H.
[0103] tert-Butyl
3',3"-Dichloro-4',4"-dimethoxy-5,5"-bis(methoxycarbonyl)-
3,3-diphenylpropenoate (16). tert-Butyl diethylphosphonoacetate
(28) (0.44 mL, 1.872 mmol) was dissolved in dry THF (15 mL) and the
mixture was stirred under argon in an ice bath. A 1.0 M solution of
sodium bis(trimethylsilyl)amide (2.0 mL, 2.01 mmol) was added and
the solution was stirred at 0.degree. C. for 1 h. At this time, a
solution of the ketone 11.sup.34 (0.400 g, 0.936 mmol) in dry THF
(12 mL) was added dropwise and the resulting mixture was stirred at
rt for 24 h. The solvent was evaporated and the residue partitioned
between ethyl ether (40 mL and water (60 mL). The layers were
separated and the aqueous one was extracted with ethyl ether
(2.times.40 mL). The combined organic extracts were dried over
magnesium sulfate, filtered, and the solvent removed in vacuo to
give a yellowish oil. Purification by flash chromatography
(SiO.sub.2, 30 g), eluting with hexanes-ethyl acetate (3:1),
afforded pure 10 (0.229 g, 46.6%) as a white solid: mp
114-115.5.degree. C.; .sup.1H-NMR (300 MHz, CDCl.sub.3) .delta.7.57
(d, J=2.4 Hz, 1 H), 7.51 (d, J=2.3 Hz, 1 H), 7.37 (d, J=2.2 Hz, 1
H), 7.35 (d, J=2.4 Hz, 1H), 6.22 (s, 3 H), 3.98 (s, 3 H), 3.94 (s,
3 H), 3.90 (s, 3 H), 3.88 (s, 3 H), 1.29 (s, 3 H); IR (film) 2952,
1731, 1479, 1368, 1258, 1095, 998, 848, 744 cm.sup.-1; CIMS m/z
(relative intensity) 451 (MH.sup.+-C.sub.4H.sub.8, 100), 469
(MH.sup.+-C.sub.4H.sub.9OH, 92), 470 (MH.sup.+-C.sub.4H.sub.9O,
64). Anal. (C.sub.25H.sub.26Cl.sub.2O.sub.8,) C, H.
[0104]
3'3"-Dichloro-4',4"-dimethoxy-5',5'-bis(methoxycarbony)-1,1-dipheny-
l-4-[(tert-butyldiphenylsilyl)oxy]-1-butene (17).
3-[tert-(Butyldiphenylsi- lyloxy)propyl]triphenylphosphonium
bromide (0.45 g, 0.702 mmol) was suspended in dry THF (5 mL) and
stirred under argon. The suspension was cooled in an ice bath. A
1.0 M solution of NaHMDS (0.75 mL, 0.75 mmol) in THF was added
dropwise. The reaction mixture turned into a bright orange solution
and was stirreed in the ice bath for 30 min. A solution of the
ketone 11 (0.200 g, 0.468 mmol) in dry THF (2 mL) was added. The
solution was stirred at room temperature for 24 h. A saturated
solution of ammonium chloride (10 mL was then added, followed by
ethyl acetate (10 mL). The phases were separated and the aqueous
one was extracted with ethyl acetate (3.times.20 mL). The combined
organic extracts were washed with brine (1.times.40 mL), dried over
magnesium sulfate, filtered, and the solvent evaporated to give a
dark brown oil. Purification by flash chromatography (SiO.sub.2),
eluting with hexane/ethyl acetate (3:1), provided 17 (0.26 g, 76%)
as a yellowish oil: .sup.1H-NMR (300 Mz, CDCl.sub.3) .delta.7.63
(dd, J=1.4 Hz, 4 H), 7.49 (d, J=2.3 Hz, 1H), 7.46 (d, J=2.1 Hz,
1H), 7.31 (d, J=2.0 Hz, 1 H), 7.28 (d, J=2.3 Hz, 1H), 7.34 (m, 6
H), 6. 1 0 (t, J=7.4 Hz, 1H), 3.99 (s, 3 H), 3.94 (s, 3 H), 3.90
(s, 3 H), 3.88 (s, 3 H), 3.75 (t, J=6.1 Hz, 2 H), 2.36 (t, J=6.3
Hz, 1H), 2.34 (t, J=6.4 Hz, 1 H), 1.05 (s, 9 H); IR (film) 3050,
2951, 1737, 1475, 1428, 1270, 1202, 1093, 999, 910, 702 cm.sup.-1.
Anal. (C.sub.38H.sub.40Cl.sub.2O.sub.7Si) C, H.
[0105]
3',3"-Dichloro-4-hydroxy-4',4"dimethoxy-5',5'-bis(methoxycarbony)-1-
,1-diphenyl-1-butene (18). Silyl ether 17 (0.2 g, 0.282 mmol) was
dissolved in dry THF (6 mL) and stirred at 0.degree. C. under
argon. A 1.0 M solution of tetrabutylammonium fluoride in THF (0.6
mL, 0.6 mmol) was added and the initially yellow solution turned
into an orange solution which was stirred at 0.degree. C. for 5.5
h. Brine (10 mL) was added and the phases were separated. The
aqueous one was extracted with ethyl acetate (3.times.20 mL). The
combined organic extracts were washed with brine (1.times.40 mL),
dried over magnesium sulfate, filtered and the solvent evaporated
to give an orange oil. Purification by flash chromatography (silica
gel) using hexane/ethyl acetate 1:1 as the eluant afforded 18 (94.5
mg, 71.4%) as a colorless oil: .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta.7.52 (d, J=2.4 Hz, 1 H), 7.50 (d, J=2.1 Hz, 1H), 7.37 (d,
J=2.1 Hz, 1H), 7.31 (d, J=1.8 Hz, 1H), 6.13 (t, J=7.4 Hz, 1 H),
3.99 (s, 3 H), 3.93 (s, 3 H), 3.92 (s, 3 H), 3.91 (s, 3 H), 3.75
(t, J=6.3 Hz, 2 H), 2.38 (q, J=13.4 and J=6.7 Hz, I H), 1.66 (bs, 1
H); IR(film) 3542, 2951, 2874, 1732, 1477, 1428, 1268, 1210, 1093,
998, 866, 743 cm.sup.-1.
[0106]
3',3"-Dichloro-4',4"-dimethoxy-5',5"-bis(carboxy)-4,4-diphenyl-3-bu-
tenoic Acid (23). Chromium trioxide (0.64 g, 6.39 mmol) was
dissolved in 1.5 M sulfuric acid (9.3 mL, 13.9 mmol), and the
solution was stirred in an ice bath. A solution of alcohol 18
(0.50, 1.065 mmol) in acetone (15 mL) was then added and the ice
bath removed. The reaction mixture was stirred at room temperature
for 7 h. Ethyl ether (50 mL) was added and the phases separated.
The organic phase was washed with water (3.times.40 mL) and then
extracted with 3 M sodium hydroxide (3.times.20 mL). The combined
aqueous extracts were acidified with conc hydrochloric acid and the
cloudy solution was kept in the refrigerator overnight. Separation
of the precipitated solid by filtration, followed by washing with
water, afforded a yellowish solid (0.131 g), which was purified by
recrystallization from ethyl ether/dichloromethane to yield 19 (63
mg, 12.2%) as a pale yellow solid: mp 206-207.degree. C.; .sup.1H
NMR (300 MHz, acetone-d.sub.6) .delta.7.65 (d, J=2.3 Hz, 1 H), 7.62
(d, J=2.1 Hz, 1 H), 7.57 (d, J=2.1 Hz, 1 H), 7.52 (d, J=2.3 Hz, 1
H),6.42(t, J=7.4 Hz, 1 H), 3.97 (s, 6 H), 3.91 (s, 6 H), 3.20 (d,
J=7.4 Hz, 2 H); IR (film) 3400-2800, 2938, 1702, 1478, 1259, 996,
708 cn.sup.-1; FABMS m/z (rel intesity): 454 (M.sup.+, 45), 437
(M.sup.+-17, 100). Anal. (C.sub.20H.sub.16Cl.sub.2O.sub.80.4
H.sub.2O) C, H.
[0107]
3',3"-Dichloro-4-methanesulfonyloxy-4',4"dimethoxy-5',5'-bis(methox-
ycarbony)-1,1-diphenyl-1-butene (19). A solution of alcohol 18
(0.700 g, 1.491 mmol) and triethylamine (0.62 mL, 4.473 mmol) in
dry dichloromethane (20 mL was stirred under argon at 0.degree. C.
Mesyl chloride (0.35 mL, 4.473 mmol) was added and the mixture was
stirred at 0.degree. C. for 3 h. The reaction mixture was then
diluted with dichloromethane (30 mL) and washed with 0.5 N HCl
(2.times.40 mL), followed by sat NaHCO.sub.3 (40 mL) and brine (40
mL). The organic extract was dried over magnesium sulfate,
filtered, and the solvent removed in vacuo to produce a thick
yellow oil (0.77 g). Purification of a fraction of this oil (0.557
g) by flash chromatography (25 g SiO.sub.2) afforded pure 19 (0.521
g, 88.5%) as a thick oil, which slowly crystallized. The analytical
sample was obtained by recrystalization from chloroform-methanol:
mp. 83-85.degree. C.; IR (film) 2952, 1731, 1479, 1359, 1269, 1174,
995 cm.sup.-1; .sup.1H-NMR (300 MHz, CDCl.sub.3) .delta.7.49 (d,
J=2.4 Hz, 1 H), 7.47 (d, J=2.2 Hz, 1 H), 7.31 (d, J=2.1 Hz, 1 H),
7.29 (d, J=2.3 Hz, 1 H), 6.04 (t, J=7.4 Hz, 1 H), 4.27 (t, J=6.3
Hz, 2 H), 3.97 (s, 3 H), 3.91 (s, 3 H), 3.89 (s, 3 H), 3.88 (s, 3
H), 3.00 (s, 3 H), 2.53 (q, J=6.4 Hz and J=7.2 Hz, 2 H); CIMS m/z
(relative intensity) 547 (MH.sup.+, 46), 515 (100). Anal.
(C.sub.23H.sub.24Cl.sub.2O.sub.9S) C, H.
[0108]
4-Azido-3',3"-Dichloro-4',4"dimethoxy-5',5'-bis(methoxycarbony)1,1--
diphenyl-1-butene (20). Mesylate 19 (0.215 g, 0.393 mmol) was
dissolved in dry DMF (5 mL). Sodium azide (0.13 g, 1.965 mmol) was
added and the mixture was stirred at 3 5 to 50.degree. C. for 3 h.
The mixture was allowed to cool at room temperature, and then it
was diluted with ether (45 mL). The ethereal solution was washed
with water (2.times.40 mL), brine (1.times.40 mL), and dried over
magnesium sulfate. After filtration and evaporation of the solvent
in vacuo, a yellowish oil was obtained. Purification by flash
chromatography (SiO.sub.2, 16 g), eluting with hexane:ethyl acetate
3:1, afforded pure 20 (0.148 g, 76.1%) as an oil which slowly
crystallized. The analytical sample was obtained by
recrystallization from chloroform-pentane: mp 70-72.degree. C.;
.sup.1H NMR (300 MHz, CDCl.sub.3) .delta.7.50 (t, J=2.1 Hz, 2 H),
7.34 (d, J=2.2 Hz, 1 H), 7.30 (d, J=2.4 Hz, 1 H), 6.05 (t, J=7.4
Hz, 1 H), 3.99 (s, 3 H), 3.92 (s, 3 H), 3.91 (s, 3 H), 3.90 (s, 3
H), 3.39 (t, J=6.6 Hz, 2 H), 2.39 (q, J=6.7 Hz and J=7.2 Hz, 2 H);
IR (film) 2952, 2098, 1734, 1477, 1267, 997, 742 cm.sup.-1; CIMS
m/z (relative intensity) 494 (MH.sup.+, 100), 496 (70). Anal.
(C.sub.22H.sub.21Cl.sub.2N.sub.30.sub.6-0.5 H.sub.2O) C, H.
[0109]
4-Amino-3',3"-Dichloro-4',4"dimethoxy-5',5'-bis(methoxycarbony)-1,1-
-diphenyl-1-butene (21). A solution of azide 20 (0.228 g, 0.462
mmol) and EtO).sub.3P (0.24 mL, 1.387 mmol) in benzene (6 mL) was
stirred under argon at rt for 24 h. The reaction mixture was then
saturated with dry HCl for 10 min and stirred for 48 h at rt. The
solvent was removed and dry ethyl ether (15 mL) was added. The
solution was placed in the freezer for 48 h. The precipitated amine
hydrochloride was separated by filtration, washed with cold ethyl
ether, and dried under high vacuum overnight to provide 21 (0.200
g, 86%) as a white solid. The analytical sample was recrystallized
from ethanol-ethyl ether-pentane: mp 156-158.degree. C.; .sup.1H
NMR (300 MHz, CDCl.sub.3) .delta.7.49 (t, J=2.3 Hz, 2 H), 7.47 (d,
J=2.2 Hz, 1 H), 7.32 (d, J=2.2 Hz, 1 H), 7.29 (d, J=2.4 Hz, 1 H),
6.06 (t, J=7.4 Hz, 1 H), 3.96 (s, 3 H), 3.90 (s, 3 H), 3.89 (s, 3
H), 3.88 (s, 3H), 2.81(t, J=6.6 H.sub.2, 2H), 2.25 (q, J=7.1 Hz,
2H), 1.90 (bs, 2H, exchangeable with D.sub.2O; IR(film) 2951, 1731,
1476, 1435, 1261, 1208, 997, 742 cm.sup.-1; CIMS m/z (rel intesity)
467 (M.sup.+, 90), 466 (M.sup.+-1, 100). Anal.
(C.sub.22H.sub.23Cl.sub.2N- O.sub.6HCl) C, H, N.
[0110] Methyl
3',3"-Dichloro-4',4"-dimethoxy-5,5"-bis(methoxycarbonyl)-6,6-
-diphenylhexenoate (22).
(4-Methoxycarbonylbutyl)triphenylphosphonium bromide (29) (0.321 g,
0.704 mmol) was stirred in dry THF (15 mL) under argon at
-78.degree. C. A 1.0 M solution of NaN(SiMe.sub.3).sub.2 in THF
(0.78 mL, 0.78 mmol) was then added and the yellow solution was
stirred in a dry ice-acetone bath for 1 h. A -78.degree. C.
solution of ketone 11 (0.200 g, 0.469 mmol) in dry THF (5 mL) was
added and the reaction mixture was stirred at -78.degree. C. for 12
h, and then at rt for 12 h. Saturated NH.sub.4Cl (25 mL) was added
and the mixture stirred for 15 min. The layers were separated and
the aqueous phase was extracted with ethyl acetate (3.times.30 mL).
The combined organic extracts were washed with brine (1.times.40
mL), dried over magnesium sulfate, filtered, and the solvent
removed in vacuo to give a thick orange residue. Purification was
achieved by silica gel flash chromatography, eluting with
hexane:ethyl acetate 3:1, to provide 22 (0.110 g, 44.6%) as a pale
yellow oil: .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.7.51 (d, J=2.3
Hz, 1 H), 7.48 (d, J=2.2 Hz, 1 H), 7.33 (d, J=2.2 Hz, 1 H), 7.30
(d, J=2.3 Hz, 1 H), 6.05 (t, J=7.5 Hz, 1 H), 4.01 (s, 3 H), 3.95
(s, 3 H), 3.94 (s, 3 H), 3.93 (s, 3 H), 3.65 (s, 3H), 2.32 (t,
J=7.4 Hz, 2 H), 2.15 (q, J=7.4 Hz, 2 H), 1.80 (m, J=7.3 Hz, 2 H);
IR (film) 2951, 1736, 1477, 1261, 1208, 1093, 999, 743 cm.sup.-1;
FABMS m/z: (rel intensity) 525 (MH.sup.+, 30), 509
(M.sup.+-CH.sub.3, 28), 493 (M.sup.+-OCH.sub.3, 100). Anal.
(C.sub.25H.sub.26Cl.sub.20.sub.8) C, H.
[0111] Following a similar procedure using 4-carboxybutyltriphenyl
phosphonium bromide instead of 29 above, provided the corresponding
6,6-diphenyl hexanoic acid.
[0112] (2-Trimethylsilyl)ethyl Diethylphosphonoacetate (24). BOP-Cl
(2.53 g, 9.952 mmol) was added to a solution of
diethylphosphonoacetic acid (25) (1.6 mL, 9.952 mmol),
(2-trimethylsilyl)ethanol (26) (1.57 mL, 10.94 mmol), and
triethylamine (2.77 mL, 19.904 mmol) in dry dichloromethane (25
mL). The initial suspension turned into a clear solution within
minutes, which was stirred at rt under Ar for 1.2 h. Water (60 mL,
basified with sodium bicarbonate) was added and the phases were
separated. The organic phase was diluted with dichloromethane
(2.times.30 mL) and washed with water (1.times.50 mL and brine
(1.times.50 mL). The organic phase was dried over magnesium
sulfate, filtered and the solvent removed in vacuo to give a liquid
residue (6.89 g). Purification by flash chromatography (SiO.sub.2,
135 g) afforded 24 as a colorless liquid (2.47 g, 84%): bp
120-122.degree. C./0.05 mm Hg (Lit..sup.3 140-145.degree. C./0.1 mm
Hg); IR (film) 2981, 2954, 2904, 1737, 1268, 1027, 969, 838
cm.sup.-1; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.4.18 (m, J=8.6
Hz, 2 H), 4.14 (m, J=7.2 Hz, 4 H), 2.94 (s, 1 H), 2.87 (s, 1 H),
1.31 (t, J=7.1 Hz, 6 H), 0.98 (m, J=8.7 Hz, 2 H), 0.01 (s, 9 H).
Anal. (C.sub.11H.sub.25O.sub.5PSi) C, H.
[0113]
1-Bromo-4,4-bis(8',8"-dichloro-2',2',2",2"-tetramethyl-4',4"-dioxo--
6',6"-0(1,3-benzodioxyl)]-3-butene (31). A solution of the alcohol
31 (0.106 g, 0.215 mmol) and carbon tetrabromide (0.09. g, 0.271
mmol) in dry acetonitrile (8.5 mL) was stirred under argon. A
solution of triphenylphosphine (0.08 g, 0.305 mmol) in dry
acetonitrile (1.5 mL) was added dropwise. The reaction mixture was
stirred at reflux for 20 h. The solvent was removed in vacuo and
the resulting oil was extracted with ethyl ether (5.times.5 mL).
The solvent was evaporated from the combined organic extracts to
give a thick solid. Purification by flash chromatography
(SiO.sub.2, 25 g), eluting with hexane-ethyl acetate 5:1, afforded
the product 31 (66 mg, 56%) as a yellowish solid: mp
175-178.degree. C.; .sup.1H NMR (CDCl.sub.3, 300 MHz) .delta.7.70
(d, J=2.0 Hz, 1 H), 7.69 (d, J=1.8 Hz, 1 H), 7.45 (d, J=2.2 Hz, 1
H), 7.44 (d, J=2.1 Hz, I H), 6.09 (t, J=7.2 Hz, 1 H), 3.45 (t,
J=6.5 Hz, 2 H), 2.70 (q, J=6.7 Hz, 2 H), 1.84 (s, 6 H), 1.80 (s, 6
H); IR (film) 2999, 1745, 1607, 1483, 1283, 1199, 1063, 874, 756
cm.sup.-1; CIMS m/z (rel intensity) 555 (MH.sup.+, 55), 557
(MH.sup.++2, 100), 559 (MH.sup.++4, 40). Anal.
(C.sub.24H.sub.21BrCl.sub.2O.sub.6) C, H.
[0114] 4,4'-Dimethoxy-3,3'-bis(methoxycarbonyl)diphenylmethane
(34). A suspension of 3,3'-dicarboxy-4,4'-dihydroxydiphenylmethane
(33) (0.576 g, 2 mmol), dimethylsulfate (2 mL, 12 mmol), and
potassium carbonate (2.0 g) in acetone (20 mL) was heated at reflux
for 6 h. The solid mass was separated by filtration and washed with
acetone. Evaporation of the acetone gave the product 34 (0.51 g,
80%) as a brown oil: .sup.1H NMR (CDCl.sub.3,300 MHz) .delta.7.61
(d,J=1.8 Hz, 2 H), 7.25 (dd, J=1.8, 8.6 Hz, 2 H), 6.90 (d, J=8.5
Hz, 2H), 3.90 (s, 2 H), 3.87 (s, 6 H). Anal.
(C.sub.19H.sub.20O.sub.6) C, H.
[0115] 4,4'-Dimethoxy-3,3'-bis(methoxycarbonyl)benzophenone (35). A
solution of intermediate 34 (0.5 g, 1.8 mmol) in acetic anhydride
(25 mL) was cooled to 0.degree. C. in an ice bath containing NaCl.
Solid chromium (VI) oxide (3 g, 30 mmol) was added slowly to the
solution at 0.degree. C. After complete addition, the mixture was
stirred at 0.degree. C. for 1 h, and then room temperature for 12
h. The resulting viscous paste was broken up with ethyl acetate and
partitioned between 1 N HCl (200 mL) and ethyl acetate (200 mL).
The organic layer was washed with 1 N HCl (100 mL) and brine (100
mL), dried over Na2SO.sub.4, and the solvent removed under reduced
pressure. Trituration of the solid in 1 N HCl and filtering gave
the product 35 (0.12 g, 22%) as an off white solid: mp
142-144.degree. C.; .sup.1HNMR (acetone-d.sub.6) 8.14 (d, J=2.2 Hz,
2 H), 7.96 (dd, J=2.2, 8.7 Hz, 1 H), 7.30 (d, J=8.7 Hz, 1 H), 3.99
(s, 3 H), 3.84 (s, 3 H). FTIR (KBr) 2953, 2849, 1735, 1710, 1650,
1603, 1502, 1438, 1406, 1274, 1152, 1081, 1010, 950 cm.sup.-1.
Anal. (C.sub.19H.sub.18O.sub.7) C, H.
[0116]
4',4"-Dimethoxy-3',3"-bis(methoxycarbonyl)-1,1-diphenyl-1-heptene
(36). n-Hexyltriphenylphosphonium bromide (0.427 g, 1 mmol) was
dried by azeotropic distillation from a benzene solution and then
stirred in dry THF (10 mL) under nitrogen atmosphere. Sodium
bis(trimethylsilyl)amide (1 M in THF, 1 mL, 1 mmol) was added and
the ylide produced was stirred under nitrogen at 0.degree. C. for
30 min. Intermediate 35 (0.276 g, 0.77 mmol) was added as a
solution in THF (10 mL) under nitrogen. The mixture was stirred at
rt overnight and partitioned between 1 N HCl (100 mL) and ethyl
acetate (100 mL). The organic layer was dried over sodium sulfate,
and evaporated to afford an oil which was chromatographed on
SiO.sub.2 (230-400 mesh, 50 g), eluting with hexanes/ethyl acetate
(4:1) to give the product (36) (0.15 g, 46%) as an oil: .sup.1H NMR
(CDCl.sub.3, 300 MHz) .delta.7.63 (d, J=2.3 Hz, 1 H), 7.56 (d,
J=2.2 Hz, 1 H), 7.20 (dt, J=2.3 Hz, 1 H), 6.94 (d, J=8.6 Hz, 1 H),
6.82 (d, J=8.7 H, 1 H), 5.96(t, J=7.5 Hz, I H), 3.90 (s, 3 H), 3.84
(s, 3 H), 3.83 (s, 6 H), 2.05 (q, J=7.3 Hz, 2 H), 1.39(t, J=7.2 Hz,
2 H), 1.22 (m, J=1.9 Hz, 2 H), 0.83 (t, J=6.7 Hz, 3 H); IR (neat)
2927, 2853, 1732, 1606, 1500, 1435, 1263 cm.sup.-1. Anal.
(C.sub.25H.sub.300.sub.6) C, H.
[0117]
4,4'-Dimethoxy-3,3'-bis(methoxycarbonyl)-5,5'-dinitrobenzophenone
(37). A solution of compound 36 (1.07 g, 3 mmol) in acetic
anhydride (30 mL) was cooled to 0.degree. C. Nitric acid 90%, 20 mL
was added dropwise and the solution was stirred overnight while
warming to room temperature. The orange solution was poured onto
ice and water and extracted with ethyl acetate (3.times.100 mL).
The organic layer was washed with 5% KOH solution (3.times.50 mL),
dried over MgSO.sub.4, and evaporated. The oil (1.1 g) was flash
chromatographed on silica gel (250 g, 230-400 mesh), eluting with
hexanes-ethyl acetate (10:2), to afford 37 (0.41 g, 32%) as a
solid: mp 96-98.degree. C.; .sup.1H N MR(CDCl.sub.3, 300 MHz) 5
8.55 (s, 2 H), 8.41 (s, 2 H), 4.1 (s, 6H), 3.99 (s, 6 H). Anal.
(C.sub.19H.sub.16H.sub.2O.sub.7) C, H, N.
[0118] 3,3'-Diamino-5,5'-Bis(methoxycarbonyl)-4
4'-dinitroxybenzophenone (38). The dinitro compound 37 (0.33 g, 0.9
mmol) was hydrogenated at atmospheric pressure over platinum oxide
(0.2 g, 0.08 mmol) in ethyl acetate (50 mL). After tlc (SiO.sub.2,
hexanes-acetone, 10:2) had shown that all the starting material was
consumed, the catalyst was removed by filtration and the solvent
was removed at reduced pressure to give an oil. This oil was flash
chromatographed on SiO.sub.2 (30 g, 230-400 mesh) using ethyl
acetate. Evaporation of the solvent gave the product 38 (0.24 g,
80%) as a glassy solid; .sup.1H NMR (CDCl.sub.3, 300 MHz)
.delta.7.55 (d, J=2.1 Hz, 2 H), 7.31 (d, J=2.1 Hz, 2 H), 3.91 (s, 6
H), 3.89 (s, 6 H); IR (KBr) 3369, 2945,2837,1717, 1616 cm.sup.-1.
Anal. (C.sub.19H.sub.20N.sub.20.sub.7)
[0119]
3,3'-Diiodo-4,4',-dimethoxy-5,5'-bis(methoxycarbonyl)benzophenone
(39). A suspension of compound 38 (0.57 g, 1.4 mmol) in water (10
rnL) was cooled to 0.degree. C. Concentrated HCl (0.6 mL) was added
dropwise to give a yellow solution. After 10 min, sodium nitrite
(0.22 g, 3.2 mmol) dissolved in water (2 mL) was added and the
solution was stirred for 30 min at 0.degree.. The solution was then
poured into a solution of iodine (1 g, 3.9 mmol) and potassium
iodide (1.0 g, 5.9 mmol) in water (100 mL). The solution was
stirred at room temperature for 30 min. Extraction with ethyl
acetate (200 mL), washing with 10% sodium hydrosulfate solution
(100 mL), drying over MgSO.sub.4, and evaporation of the solvent
gave the crude dihodide (0.6 g). Recrystalization from
hexanes/methylene chloride gave the pure product 39 (0.54 g, 63%)
as a solid: mp 160-161.degree. C. .sup.1H NMR (300 MHz, CDCl.sub.3)
5 8.37 (d, J=2.5 Hz, 2 H), 8.27 (d, J=1.86 Hz, 2 H), 4.07 (s, 6 H),
3.95 (s, 6 H); IR (neat) 3078, 2954, 1734, 1670, 1608, 1540
cm.sup.-1. Anal. (C.sub.19H.sub.160.sub.7I.sub.2) C, H.
[0120]
3',3"-Diiodo-4',4"-Dimethoxy-5',5"-bis(methoxycarbonyl)-1,1-dipheny-
l-1-heptene (40). NaN(TMS).sub.2(1 mL, 1 mmol) was added to an ice
cold suspension of hexyltriphenylphosphonium bromide (0.427 g, 1
mmol) in THF (20 mL) and the solution was stirred for 30 min at
0.degree. C. A solution of the benzophenone 39 (0.2 g, 0.33 mmol)
in THF (5 mL) was added to the preformed ylide and the solution was
stirred at rt overnight. The mixture was partitioned between 1 N
HCl (100 mL) and ethyl acetate (100 mL). The organic layer was
evaporated and flash chromatographed on SiO.sub.2 (230-400 mesh,
50.0 g), eluting with hexanes-ethyl acetate (5:1). The fractions
which contained the product were pooled, evaporated, and
rechromatographed on silica gel (230-400 mesh, 20 g), eluting with
hexanes-ethyl acetate (5:1), to give the product 40 (0.022 g, 9%)
as an oil: .sup.1H NMR (CDCl.sub.3, 300 MHz) .delta.7.69 (s, 2 H),
7.54 (d, J=2.0 Hz, 1 H), 7.52 (d, J=2 Hz, 1 H), 6.01 (t, J=7.6 Hz,
1 H), 3.93 (s, 3 H), 3.89 (s, 3 H), 3.88 (s, 3 H), 3.85 (s, 3 H),
2.05 (q, J=7.3 Hz, 2 H), 1.42 (m, 2 H), 1.25 (m, 2 H), 0.85 (t,
J=6.7 Hz, 3 H); IR (neat) 2953, 2929, 1742, 1738, 1731, 1713
cm.sup.-1; HRFABMS calcd for C.sub.25H.sub.28I.sub.20.sub.6 m/z
677.9975 (M.sup.+). Found: m/z 677.9954. Anal.
(C.sub.25H.sub.28I.sub.2O.sub.60.5E- tOAc), C, H.
[0121] In Vitro Anti-HIV Assay. Anti-HIV screening of test
compounds against various viral isolates and cell lines was
performed as previously described. (Buckheit et al, Antiviral Res.
1995, 26, 117-132) This cell-based microliter assay quantitates the
drug-induced protection from the cytopathic effect of HIV. Data are
presented as the percent control of XTT values for the uninfected,
drug-free control. EC.sub.50 values reflect the drug concentration
that provides 50% protection from the cytopathic effect of HIV-1 in
infected cultures, while the IC.sub.50 reflects the concentration
of drug that causes 50% cell death in the uninfected cultures.
XTT-based results were confirmed by measurement of cell-free
supernatant reverse transcriptase and p24 levels.
[0122] Mechanistic Assays. The effects of inhibitors on the in
vitro activity of purified RT (kind gift of Steve Hughes,
NCI-FCRDC, Frederick, Md.) were determined by measurement of
incorporation of [.sup.32P]TTP into the poly(rA):oligo(dT) (rAdT)
homopolymer or [.sup.32P]GTP into the poly(rC):oligo(dG) (rCdG)
template/primer systems. Samples (5 .mu.L) were blotted onto DE81
paper, washed with 5% diabasic sodium phosphate, and then
quantitated on a Packard Matrix 9600 direct beta counter.
3'-Azido-2',3'-dideoxythymidine-5'-triphosphate (AZTTP) and NSC
629243 (UC38) served as a positive control for inhibition of
RT.
[0123] To determine if compounds affected the HIV-1 nucleocapsid
protein zinc fingers, fluorescence measurements of the Trp.sup.37
residue in the C-terminal zinc finger of the HIV-1 nucleocapsid
protein were performed as previously described. (Rice et al.
Science, 1995, 270, 1194-1197). The recombinant nucleocapsid
protein was prepared at 20 .mu.g/mL in 10 mM sodium phosphate
buffer (pH 7.0), treated with 25 .mu.M of test compound, then after
indicated time intervals the samples were diluted 1/10 in 10 mM
sodium phosphate buffer (pH 7.0) and the fluorescence intensity
measured. The excitation and emission wavelengths utilized with the
Shimadzu RF5000 spectrofluorimeter were 280 and 351 mm,
respectively. The analytical procedure employed to determine the
reagent-induced inhibition of HIV-1 protease activity has been
previously described. Recombinant HIV-1 protease (Bachem BioScience
Inc., King of Prussia, Pa.) and the substrate
(Val-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-NH.sub.2, Multiple Peptide
Systems, San Diego, Calif.) were utilized to determine the
concentration of test compound required to inhibit protease
activity by 50% (IC.sub.50). Briefly, HIV-1 protease (14.2 nM
final) was mixed with various concentrations of test compounds in
250 mM potassium phosphate buffer, pH 6.5, 2.5% (v/v) glycerol, 0.5
mM dithiothreitol, 0.5 mM EDTA and 375 mM ammonium sulfate, after
which the substrate was added (30 nmol) and the reaction incubated
at 37.degree. C. for 30 min. Reactions were terminated by the
addition of 20 pL of a mixture of 8 M guanidine-HCl to 10%
trifluoroacetic acid (8:1), and the reaction products were
separated by reverse-phase HPLC on a Nova-Pak C-18 column.
Absorbance was measured at 206 nm, peak areas quantitated, and the
percentage conversion to product used to calculate the percentage
of control cleavage in the presence of inhibitors. The 3'-cleavage
and integration activities of purified HIV-1 integrase were
quantitated as previously described. (Rice PNAS 1993, 90,
9721-9724).
[0124] The attachment/fusion assay was performed as described by
Ciminale (AIDS Res. Hum. Retrovir. 1990, 6, 1281-1287) with
modification. Briefly, HIV-1 envelope-expressing, Tat-producing
HL2/3 cells and CXCR-4 expressing, LTR-.beta.Gal-containing MAGI
cells (obtained from the AIDS Research and Reference Program,
National Institute of Allergy and Infectious Disease, NIH,
Bethesda; Md., USA) were preincubated separately with test compound
for 1 h at 37.degree. C., followed by admixture of the two cell
lines at a cell ratio of one to one. Incubations were then
continued for 16 h. The cells were then fixed and stained for the
expression of .beta.-gal with indolyl-.beta.-D-galactopyranoside
(X-Gal). The number of blue cells (indicating completion of
attachment and fusion of membranes) were counted by light
microscopy.
[0125] Other examples of the preparation of compounds in accordance
with this invention are illustrated in the following reaction
schemes: 19 20 21 22 23 24 25 26 27 2829 30 31 32 33 34 3536 37 38
39 40 41 42 43 4445 46 47 48
[0126] Using the foregoing reaction schemes and other
art-recognized synthetic procedures, one can prepare compounds of
the invention of the formula: 49
[0127] wherein X is Cl or Br and R.sub.3, R.sub.4, and R.sub.3' are
the same and are selected from the following substituents:
505152
[0128] In another embodiment, there are provided fused ring analogs
of the present alkenyldiarylmethane compounds, examples of which
are as follows: Fused Ring Analogs (R.sup.1=C.sub.1-C.sub.4 alkyl;
R=R.sub.3' above) 535455
[0129] The compounds of this embodiment can be synthesized using
the foregoing reaction schemes and other art-recognized synthetic
procedures.
* * * * *