U.S. patent application number 09/814074 was filed with the patent office on 2002-01-31 for calanolide analogues and methods of their use.
This patent application is currently assigned to Sarawak Medichem Pharmaceuticals,Inc.. Invention is credited to Flavin, Michael T., Xu, Ze-Qi, Zembower, David.
Application Number | 20020013478 09/814074 |
Document ID | / |
Family ID | 27497042 |
Filed Date | 2002-01-31 |
United States Patent
Application |
20020013478 |
Kind Code |
A1 |
Xu, Ze-Qi ; et al. |
January 31, 2002 |
Calanolide analogues and methods of their use
Abstract
Calanolide analogues that demonstrate potent antiviral activity
against many viruses are provided. Also provided is a method of
using calanolide analogues for treating or preventing viral
infections. The calanolide analogues provided are obtained via
syntheses employing chromene 4 and chromanone 7 as key
intermediates.
Inventors: |
Xu, Ze-Qi; (Naperville,
IL) ; Flavin, Michael T.; (Darien, IL) ;
Zembower, David; (Oak Park, IL) |
Correspondence
Address: |
MCDONNELL BOEHNEN HULBERT & BERGHOFF
300 SOUTH WACKER DRIVE
SUITE 3200
CHICAGO
IL
60606
US
|
Assignee: |
Sarawak Medichem
Pharmaceuticals,Inc.
|
Family ID: |
27497042 |
Appl. No.: |
09/814074 |
Filed: |
March 21, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09814074 |
Mar 21, 2001 |
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09173143 |
Oct 15, 1998 |
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6277879 |
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09173143 |
Oct 15, 1998 |
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08609537 |
Mar 1, 1996 |
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5892060 |
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08609537 |
Mar 1, 1996 |
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08510213 |
Aug 2, 1995 |
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6043271 |
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08510213 |
Aug 2, 1995 |
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08285655 |
Aug 3, 1994 |
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5489697 |
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Current U.S.
Class: |
549/277 |
Current CPC
Class: |
C07D 493/14 20130101;
C07D 311/16 20130101; C07D 493/04 20130101; A61K 45/06
20130101 |
Class at
Publication: |
549/277 |
International
Class: |
C07D 311/78 |
Claims
What is claimed is:
1. A compound of formula i: 16wherein R.sub.1 and R.sub.2 are
independently or .
2. The compound of claim 1, wherein R.sub.1=and R.sub.2=.
3. The compound of claim 1, wherein R.sub.1=and R.sub.2=.
4. The compound of claim 1, wherein R.sub.1=and R.sub.2=.
5. The compound of claim 1, wherein R.sub.1=and R.sub.2=.
6. 6,6,10,11-tetramethyl-4-propyl-2H,6H,12H-benzo[1,2-b:3,4-b':5,6-
b"]tripyran-2,12-dione.
7. A compound of formula ii: 17wherein R.sub.1, R.sub.2, and
R.sub.3 are independently H or CH.sub.3.
8. The compound of claim 7, wherein R.sub.1=H and
R.sub.2=R.sub.3=CH.sub.3- .
9. The compound of claim 7, wherein R.sub.1=R.sub.2=R.sub.3=H.
10. The compound of claim 7, wherein R.sub.1=R.sub.2CH.sub.3 and
R.sub.3=H.
11. A compound of formula iii: 18wherein R.sub.1 is C.sub.1-C.sub.6
linear or branched alkyl.
12. The compound of claim 11, wherein R.sub.1=ethyl.
13. The compound of claim 11, wherein R.sub.1=isopropyl.
14. The compound of claim 11, having formula vi: 19wherein R1 is
C.sub.1-C.sub.6 linear or branched alkyl.
15. The compound of claim 14, wherein R.sub.1=ethyl.
16. The compound of claim 14, wherein R.sub.1=isopropyl.
17. A compound of formula iv: 20wherein R.sub.1 is propyl or phenyl
and R.sub.2 isOH or OH; with the proviso that the compound of
formula iv is not (+)-costatolide, (+)-dihydrocostatolide,
(+)-soulattrolide, or (+)-calanolide A.
18. The compound of claim 17, wherein R.sub.1=propyl and
R.sub.2=OH.
19. The compound of claim 17, wherein R.sub.1=phenyl and
R.sub.2=OH.
20. The compound of claim 17, wherein R.sub.1=propyl and
R.sub.2=OH.
21. The compound of claim 17, having formula vii: 21wherein R.sub.1
is propyl or phenyl and R.sub.2 isOH orOH; with the proviso that
the compound of formula vii is not (+)-costatolide,
(+)-dihydrocostatolide, (+)-soulattrolide, or (+)-calanolide A.
22. The compound of claim 21, wherein R.sub.1=propyl and
R.sub.2=OH.
23. The compound of claim 21, wherein R.sub.1=phenyl and
R.sub.2=OH.
24. The compound of claim 21, wherein R.sub.1=propyl and
R.sub.2=OH.
25. A method of preventing or treating a viral infection comprising
administering to a mammal an effective, non-toxic amount of at
least one antiviral compound of the formula I: 22wherein R.sub.1 is
H, halogen, hydroxyl, amino, C.sub.1-6 alkyl, aryl-C.sub.1-6 alkyl,
mono-or poly-fluorinated C.sub.1-6 alkyl, hydroxy-C.sub.1-6 alkyl,
C.sub.1-6 alkoxy, amino-C.sub.1-8 alkyl, C.sub.1-6 alkylamino,
di(C.sub.1-6 alkyl)amino, C.sub.1-8 alkylamino-C.sub.1-8 alkyl,
di(C.sub.1-6 alkyl) amino-C.sub.1-8 alkyl, cyclohexyl, aryl, or
heterocycle, wherein aryl or heterocycle may each be unsubstituted
or substituted with one or more of the following: C.sub.1-6 alkyl,
C.sub.1-6 alkoxy, hydroxy-C.sub.1-4 alkyl, hydroxyl, amino,
C.sub.1-6 alkylamino, di(C.sub.1-6 alkyl) amino, amino-C.sub.1-8
alkyl, C.sub.1-8 alkylamino-C.sub.1-8 alkyl, di(C.sub.1-6
alkyl)amino-C.sub.1-8 alkyl, nitro, azido or halogen; R.sub.2 is H,
halogen, hydroxyl, C.sub.1-6 alkyl, aryl-C.sub.1-6 alkyl, mono- or
poly-fluorinated C.sub.1-6 alkyl, aryl or heterocycle; R.sub.3 and
R.sub.4 are independently selected from the group consisting of H,
halogen, hydroxyl, amino, C.sub.1-6 alkyl, aryl-C.sub.1-6 alkyl,
mono- or poly- fluorinated C.sub.1-6 alkyl, hydroxy-C.sub.1-6
alkyl, amino-C.sub.1-8 alkyl, C.sub.1-8 alkylamino-C.sub.1-8 alkyl,
di(C.sub.1-6 alkyl)amino-C.sub.1-8 alkyl, cyclohexyl, aryl or
heterocycle; and R.sub.3 and R.sub.4 can be taken together to form
a 5-7 membered saturated cycle ring or heterocyclic ring; R.sub.5
and R.sub.6 are independently selected from the group consisting of
H, C.sub.1-6 alkyl, aryl-C.sub.1-6 alkyl, mono- or poly-fluorinated
C.sub.1-6 alkyl, aryl or heterocycle; and R.sub.5 and R.sub.6 can
be taken together to form a 5-7 membered saturated cycle ring or
heterocycle ring; R.sub.7 is H, halogen, methyl, or ethyl; R.sub.8
and R.sub.9 are independently selected from the group consisting of
H, halogen, C.sub.1-6 alkyl, aryl-C.sub.1-6 alkyl, mono- or poly-
fluorinated C.sub.1-6 alkyl, hydroxy-C.sub.1-6 alkyl,
amino-C.sub.1-8 alkyl, C.sub.1-8 alkylamino-C.sub.1-8 alkyl,
di(C.sub.1-6 alkyl)amino-C.sub.1-8 alkyl, cyclohexyl, aryl or
heterocycle; and R.sub.8 and R.sub.9 can be taken together to form
a 5-7 membered saturated cycle ring or heterocyclic ring; R.sub.10
is halogen, O, OR.sub.11, NOR.sub.11, NHOR.sub.11, NOR.sub.12,
NHOR.sub.12, NR.sub.11R.sub.12, NR.sub.12, or NR.sub.12R.sub.13;
wherein R.sub.11 is H, acyl, P(O)(OH).sub.2, S(O)(OH).sub.2,
CO(C.sub.1-10 alkyl)CO.sub.2H, (C.sub.1-8 alkyl)CO.sub.2H,
CO(C.sub.1-10 alkyl)NR.sub.12R.sub.13, (C.sub.1-8 alkyl)
NR.sub.12R.sub.13; R.sub.12 and R.sub.13 are independently selected
from the group consisting of H, C.sub.1-6 alkyl, aryl, and
aryl-C.sub.1-6 alkyl; and R.sub.12 and R.sub.13 can be taken
together to form a 5-7 membered saturated heterocyclic ring
containing said nitrogen; or a pharmaceutically acceptable salt
thereof.
26. The method of claim 25, wherein the viral infection is from a
virus selected from the group consisting of Hepatitis B, a herpes
virus, or a respiratory virus.
27. The method of claim 26, wherein the herpes virus is Herpes
Simplex Type 1, Herpes Simplex Type 2, Cytomegalovirus, Varicella
Zoster Virus, or Epstein Barr Virus.
28. The method of claim 26, wherein the respiratory virus is
Influenza A, Influenza B, Parainfluenza, Adenovirus, Measles, or
Respiratory Syncytial Virus.
29. The method of claim 25, wherein the antiviral compound
comprises formula i: 23wherein R.sub.1 and R.sub.2 are
independently or .
30. The method of claim 29, wherein R.sub.1=and R.sub.2=.
31. The method of claim 29, wherein R.sub.1=and R.sub.2=.
32. The method of claim 29, wherein R.sub.1=and R.sub.2=.
33. The method of claim 29, wherein R.sub.1=and R.sub.2=.
34. The method of claim 29, wherein the viral infection is from a
virus selected from the group consisting of Hepatitis B, a herpes
virus, or a respiratory virus.
35. The method of claim 34, wherein the herpes virus is Herpes
Simplex Type 1, Herpes Simplex Type 2, Cytomegalovirus, Varicella
Zoster Virus, or Epstein Barr Virus.
36. The method of claim 34, wherein the respiratory virus is
Influenza A, Influenza B, Parainfluenza, Adenovirus, Measles, or
Respiratory Syncytial Virus.
37. The method of claim 30, wherein the viral infection is from a
virus selected from the group consisting of Hepatitis B,
Cytomegalovirus, Epstein Barr Virus, or measles.
38. The method of claim 25, wherein the antiviral compound
comprises 6,6,10,11 -tetramethyl-4-propyl-2H,6H,
12H-benzo[1,2-b:3,4-b':5,6-b"]trip- yran-2,12-dione.
39. The method of claim 38, wherein the viral infection is from a
virus selected from the group consisting of Hepatitis B, a herpes
virus, or a respiratory virus.
40. The method of claim 39, wherein the herpes virus is Herpes
Simplex Type 1, Herpes Simplex Type 2, Cytomegalovirus, Varicella
Zoster Virus, or Epstein Barr Virus.
41. The method of claim 39, wherein the respiratory virus is
Influenza A, Influenza B, Parainfluenza, Adenovirus, Measles, or
Respiratory Syncytial Virus.
42. The method of claim 25, wherein the antiviral compound
comprises formula ii: 24wherein R.sub.1, R.sub.2, and R.sub.3 are
independently H or CH.sub.3.
43. The method of claim 42, wherein R.sub.1=H and
R.sub.2=R.sub.3=CH.sub.3- .
44. The method of claim 42, wherein R.sub.1=R.sub.2=R.sub.3=H.
45. The method of claim 42, wherein R.sub.1=R.sub.2=CH.sub.3 and
R.sub.3=H.
46. The method of claim 42, wherein the viral infection is from a
virus selected from the group consisting of Hepatitis B, a herpes
virus, or a respiratory virus.
47. The method of claim 46, wherein the herpes virus is Herpes
Simplex Type 1, Herpes Simplex Type 2, Cytomegalovirus, Varicella
Zoster Virus, or Epstein Barr Virus.
48. The method of claim 46, wherein the respiratory virus is
Influenza A, Influenza B, Parainfluenza, Adenovirus, Measles, or
Respiratory Syncytial Virus.
49. The method of claim 44, wherein the viral infection is from
Hepatitis B.
50. The method of claim 45, wherein the viral infection is from
Hepatitis B.
51. The method of claim 25, wherein the antiviral compound
comprises formula iii: 25wherein R.sub.1 is C.sub.1-C.sub.6 linear
or branched alkyl.
52. The method of claim 51, wherein R.sub.1=ethyl.
53. The method of claim 51, wherein R.sub.1=isopropyl.
54. The method of claim 51, wherein the viral infection is from a
virus selected from the group consisting of Hepatitis B, a herpes
virus, or a respiratory virus.
55. The method of claim 54, wherein the herpes virus is Herpes
Simplex Type l, Herpes Simplex Type 2, Cytomegalovirus, Varicella
Zoster Virus, or Epstein Barr Virus.
56. The method of claim 54, wherein the respiratory virus is
Influenza A, Influenza B, Parainfluenza, Adenovirus, Measles, and
Respiratory Syncytial Virus.
57. The method of claim 51, wherein the antiviral compound
comprises formula vi: 26wherein R.sub.1 is C.sub.1-C.sub.6 linear
or branched alkyl.
58. The method of claim 57, wherein R.sub.1=ethyl.
59. The method of claim 57, wherein R.sub.1=isopropyl.
60. The method of claim 57, wherein the viral infection is from a
virus selected from the group consisting of Hepatitis B, a herpes
virus, or a respiratory virus.
61. The method of claim 60, wherein the herpes virus is Herpes
Simplex Type 1, Herpes Simplex Type 2, Cytomegalovirus, Varicella
Zoster Virus, or Epstein Barr Virus.
62. The method of claim 60, wherein the respiratory virus is
Influenza A, Influenza B, Parainfluenza, Adenovirus, Measles, and
Respiratory Syncytial Virus.
63. The method of claim 25, wherein the antiviral compound
comprises formula iv: 27wherein R.sub.1 is propyl or phenyl and
R.sub.2 is OH or OH.
64. The method of claim 63, wherein R.sub.1=propyl and
R.sub.2=OH.
65. The method of claim 63, wherein R.sub.1=phenyl and
R.sub.2=OH.
66. The method of claim 63, wherein R.sub.1=propyl and
R.sub.2=OH.
67. The method of claim 63, wherein the viral infection is from a
virus selected from the group consisting of Hepatitis B, a herpes
virus, or a respiratory virus.
68. The method of claim 67, wherein the herpes virus is Herpes
Simplex 1 0 Type 1, Herpes Simplex Type 2, Cytomegalovirus,
Varicella Zoster Virus, or Epstein Barr Virus.
69. The method of claim 67, wherein the respiratory virus is
Influenza A, Influenza B, Parainfluenza, Adenovirus, Measles, and
Respiratory Syncytial Virus.
70. The method of claim 64, wherein the viral infection is from a
virus selected from the group consisting of Hepatitis B or
Cytomegalovirus.
71. The method of claim 63, wherein the antiviral compound
comprises formula vii: 28wherein R.sub.1 is propyl or phenyl and
R.sub.2 is OH or OH.
72. The method of claim 71, wherein R.sub.1=propyl and
R.sub.2=OH.
73. The method of claim 71, wherein R.sub.1=phenyl and
R.sub.2=OH.
74. The method of claim 71, wherein R.sub.1=propyl and
R.sub.2=OH.
75. The method of claim 71, wherein the viral infection is from a
virus selected from the group consisting of Hepatitis B, a herpes
virus, or a respiratory virus.
76. The method of claim 75, wherein the herpes virus is Herpes
Simplex Type 1, Herpes Simplex Type 2, Cytomegalovirus, Varicella
Zoster Virus, or Epstein Barr Virus.
77. The method of claim 75, wherein the respiratory virus is
Influenza A, Influenza B, Parainfluenza, Adenovirus, Measles, and
Respiratory Syncytial Virus.
78. The method of claim 72, wherein the viral infection is from a
virus selected from the group consisting of Hepatitis B or
Cytomegalovirus.
79. The method of claim 25, which further comprises
co-administering an effective therapeutic amount of at least one
compound selected from the group consisting of a second antiviral
compound, an immunostimulant, an immunomodulator, an antibiotic, or
a chemokine inhibitor.
80. The method of claim 79, wherein the second antiviral compound
is a protease inhibitor.
81. The method of claim 80, wherein the protease inhibitor is
selected from the group consisting of indinavir, saquinavir,
ritonavir, and nelfmavir.
82. The method of claim 79, wherein the second antiviral compound
is a biflavanoid.
83. The method of claim 82, wherein the biflavanoid is selected
from the group consisting of robustaflavone, amentoflavone, and a
derivate or salt thereof.
84. The method of claim 79, wherein the second antiviral compound
is selected from the group consisting of AZT, ddC, ddI, D4T, 3TC,
acyclovir, gancyclovir, fluorinated nucleosides and nonnucleoside
analog compounds such as TIBO derivatives and nevirapine,
.alpha.-interfon, recombinant CD4, amantadine, rimantadine,
ribavirin, and vidarabine.
85. The method of claim 79, wherein the immunostimulant is an
interleukin or cytokine.
86. The method of claim 79, wherein the antibiotic is an
antibacterial agent, antifungal agent, or anti-pneumocysitis
agent.
87. A compound of the formula I: 29wherein R.sub.1 is H, halogen,
hydroxyl, amino, C.sub.1-6 alkyl, aryl-C.sub.1-6 alkyl, mono-or
poly-fluorinated C.sub.1-6 alkyl, hydroxy-C.sub.1-6 alkyl,
C.sub.1-6 alkoxy, amino-C.sub.1-8 alkyl, C.sub.1-6 alkylamino,
di(C.sub.1-6 alkyl)amino, C.sub.1-8 alkylamino-C.sub.1-8 alkyl,
di(C.sub.1-6 alkyl) amino-C.sub.1-8 alkyl, cyclohexyl, aryl, or
heterocycle, wherein aryl or heterocycle may each be unsubstituted
or substituted with one or more of the following: C.sub.1-6 alkyl,
C.sub.1-6 alkoxy, hydroxy-C.sub.1-4 alkyl, hydroxyl, amino,
C.sub.1-6 alkylamino, di(C.sub.1-6 alkyl) amino, amino-C.sub.1-8
alkyl, C.sub.1-8 alkylamino-C.sub.1-8 alkyl, di(C.sub.1-6
alkyl)amino-C.sub.1-8 alkyl, nitro, azido or halogen; R.sub.2 is H,
halogen, hydroxyl, C.sub.1-6 alkyl, aryl-C.sub.1-6 alkyl, mono- or
poly-fluorinated C.sub.1-6 alkyl, aryl or heterocycle; R.sub.3 and
R.sub.4 are independently selected from the group consisting of H,
halogen, hydroxyl, amino, C.sub.1-6 alkyl, aryl-C.sub.1-6 alkyl,
mono- or poly- fluorinated C.sub.1-6 alkyl, hydroxy-C.sub.1-6
alkyl, amino-C.sub.1-8 alkyl, C.sub.1-8 alkylamino-C.sub.1-8 alkyl,
di(C.sub.1-6 alkyl)amino-C.sub.1-8 alkyl, cyclohexyl, aryl or
heterocycle; and R.sub.3 and R.sub.4 can be taken together to form
a 5-7 membered saturated cycle ring or heterocyclic ring; R.sub.5
and R.sub.6 are independently selected from the group consisting of
H, C.sub.1-6 alkyl, aryl-C.sub.1-6 alkyl, mono- or poly-fluorinated
C.sub.1-6 alkyl, aryl or heterocycle; and R.sub.5 and R.sub.6 can
be taken together to form a 5-7 membered saturated cycle ring or
heterocycle ring; R.sub.7 is H, halogen, methyl, or ethyl; R.sub.8
and R.sub.9 are independently selected from the group consisting of
H, halogen, C.sub.1-6 alkyl, aryl-C.sub.1-6 alkyl, mono- or poly-
fluorinated C.sub.1-6 alkyl, hydroxy-C.sub.1-6 alkyl,
amino-C.sub.1-8 alkyl, C.sub.1-8 alkylamino-C.sub.1-8 alkyl,
di(C.sub.1-6 alkyl)amino-C.sub.1-8 alkyl, cyclohexyl, aryl or
heterocycle; and R.sub.8 and R.sub.9 can be taken together to form
a 5-7 membered saturated cycle ring or heterocyclic ring; R.sub.10
is halogen, O, OR.sub.11, NOR.sub.11, NHOR.sub.11, NOR.sub.12,
NHOR.sub.12, NR.sub.11, R.sub.12, NR.sub.12, or NR.sub.12R.sub.13;
wherein R.sub.11 is H, acyl, P(O)(OH).sub.2, S(O)(OH).sub.2,
CO(C.sub.1-10 alkyl)CO.sub.2H, (C.sub.1-8 alkyl)CO.sub.2H,
CO(C.sub.1-10 alkyl)NR.sub.12R.sub.13, (C.sub.1-8 alkyl)
NR.sub.12R.sub.13; R.sub.12 and R.sub.13 are independently selected
from the group consisting of H, C.sub.1-6 alkyl, aryl, and
aryl-C.sub.1-6 alkyl; and R.sub.12 and R.sub.13 can be taken
together to form a 5-7 membered saturated heterocyclic ring
containing said nitrogen; or a pharmaceutically acceptable salt
thereof, alone or in combination with a carrier.
88. The compound of claim 87, having formula viii: 30wherein
R.sub.1 is H, halogen, hydroxyl, amino, C.sub.1-6 alkyl,
aryl-C.sub.1-6 alkyl, mono- or poly-fluorinated C.sub.1-6 alkyl,
hydroxy-C.sub.1-6 alkyl, C.sub.1-6 alkoxy, anino-C.sub.1-8 alkyl,
C.sub.1-6 alkylamino, di(C.sub.1-6 alkyl)amino, C.sub.1-8
alkylamino-C.sub.1-8 alkyl, di(C.sub.1-6 alkyl)amino-C.sub.1-8
alkyl, cyclohexyl, aryl or heterocycle, wherein aryl or heterocycle
may each be unsubstituted or substituted with one or more of the
following: C.sub.1-6 alkyl, C.sub.1-6 alkoxy, hydroxy-C.sub.1-4
alkyl, hydroxyl, amino, C.sub.1-6 alkylamino, di(C.sub.1-6
alkyl)amino, amino-C.sub.1-8 alkyl, C.sub.1-8 alkylamino-C.sub.1-8
alkyl, di(C.sub.1-6 alkyl)amnino-C.sub.1-8 alkyl, nitro, azido or
halogen; R.sub.2 is H, halogen, hydroxyl, C.sub.1-6 alkyl,
aryl-C.sub.1-8 alkyl, mono- or poly-fluorinated C.sub.1-6 alkyl,
aryl or heterocycle; R.sub.3 and R.sub.4 are independently selected
from the group consisting of H, halogen, hydroxyl, amino, C.sub.1-6
alkyl, aryl-C.sub.1-6 alkyl, mono- or poly- fluorinated C.sub.1-6
alkyl, hydroxy-C.sub.1-6 alkyl, amino-C.sub.1-8 alkyl, C.sub.1-8
alkylamino-C.sub.1-8 alkyl, di(C.sub.1-6 alkyl) amino-C.sub.1-8
alkyl, cyclohexyl, aryl or heterocycle; and R.sub.3 and R.sub.4 can
be taken together to form a 5-7 membered saturated cycle ring or
heterocyclic ring; R.sub.5 and R.sub.6 are independently selected
from the group consisting of H, C.sub.1-6 alkyl, aryl-C,6 alkyl,
mono- or poly- fluorinated C.sub.1-6 alkyl, aryl or heterocycle;
R.sub.7 is H, halogen, methyl, or ethyl; R.sub.8 and R.sub.9 are
independently selected from the group consisting of H, halogen,
C.sub.1-6 alkyl, aryl-C.sub.1-6 alkyl, mono- or poly- fluorinated
C.sub.1-6 alkyl, hydroxy-C.sub.1-6 alkyl, mono- or poly-fluorinated
C.sub.1-6 alkyl, hydroxy-C.sub.1-6 alkyl, amino-C.sub.1-8 alkyl,
C.sub.1-8 alkylamino-C.sub.1-8 alkyl, di(C.sub.1-6
alkyl)amino-C.sub.1-8 alkyl, cyclohexyl, aryl or heterocycle; and
R.sub.8 and Rg can be taken together to form a 5-7 membered
saturated cycle ring or heterocyclic ring; R.sub.10 is halogen,
OR.sub.11, NHOR.sub.11, NHOR.sub.12, NR.sub.11R.sub.12,
NR.sub.12R.sub.13; wherein R.sub.11 is H, acyl, P(O)(OH).sub.2,
S(O)(OH).sub.2, CO(C.sub.1-10 alkyl)CO.sub.2H, (C.sub.1-8
alkyl)CO.sub.2H, CO(C.sub.1-10 alkyl)NR.sub.12R.sub.13, (C.sub.1-8
alkyl) NR.sub.12R.sub.13; R.sub.12 and R.sub.13 are independently
selected from the group consisting of H and C.sub.1 alkyl; and
R.sub.12 and R.sub.13 can be taken together to form a 5-7 membered
saturated heterocyclic ring containing said nitrogen; or a
pharmaceutically acceptable salt thereof, alone or in combination
with a carrier; with the proviso that when R.sub.1 is n-propyl or
phenyl, R.sub.2, R.sub.7, and R.sub.8 are H, R.sub.5, R.sub.6, and
R.sub.9 are methyl, and R.sub.10 is OH, then R.sub.3 and R.sub.4
cannot both be H; when R.sub.1 is n-propyl or phenyl, R.sub.2,
R.sub.4, R.sub.7, and R.sub.8 are H, R.sub.5, R.sub.6, and R.sub.9
are methyl, and R.sub.10 is OH, then R.sub.3 cannot be methyl; when
R.sub.1 is n-propyl or phenyl, R.sub.2, R.sub.3, R.sub.7, and
R.sub.9 are H, R.sub.5, R.sub.6, and R.sub.8 are methyl, and
R.sub.10 is OH, then R.sub.4 cannot be methyl.
89. The compound of claim 88 wherein R.sub.3is methyl.
90. The compound of claim 89 wherein R.sub.4is methyl.
91. The compound of claim 90 wherein R.sub.8 is H.
92. The compound of claim 91 wherein R.sub.9 is methyl.
93. The compound of claim 92 wherein R.sub.1 is phenyl.
94. The compound of claim 92 wherein R.sub.1 is n-propyl.
95. The compound of claim 88, wherein R.sub.1=n-propyl;
R.sub.3=R.sub.4=R.sub.5=R.sub.6=R.sub.9=methly;
R.sub.2=R.sub.7=R.sub.8=H- ; and R.sub.10=OH.
96. The compound of claim 88, wherein R.sub.1=n-propyl;
R.sub.3=R.sub.4=R.sub.5=R.sub.6=R.sub.8=methyl;
R.sub.2=R.sub.7=R.sub.9=H- ; and R.sub.10=OH.
97. The compound of claim 89 wherein R.sub.4 is H.
98. The compound of claim 97 wherein R.sub.8 is H.
99. The compound of claim 98 wherein R.sub.9 is ethyl.
100. The compound of claim 99 wherein R.sub.1 is phenyl.
101. The compound of claim 99 wherein R.sub.1 is n-propyl.
102. The compound of claim 88, wherein R.sub.1=n-propyl;
R.sub.2=R.sub.4=R.sub.7 =R.sub.8=H; R.sub.3=R.sub.5=R.sub.6=methyl;
R.sub.10=ethyl; and R.sub.10=OH.
103. The compound of claim 88, wherein R.sub.1=n-propyl;
R.sub.2=R.sub.3=R.sub.7=R.sub.9=H; R.sub.4=R.sub.5=R.sub.6=methiyl;
R.sub.8=ethyl; and R.sub.10=OH.
104. The compound of claim 88, wherein R.sub.1=n-propyl,
R.sub.2=R.sub.3=R.sub.7=R.sub.8=H; R.sub.4=R.sub.5=R.sub.6=methyl;
R.sub.8=ethyl; and R.sub.10=OH.
105. The compound of claim 88, wherein R.sub.1=n-propyl,
R.sub.2=R.sub.4=R.sub.7=R.sub.9=H; R.sub.3=R.sub.5=R.sub.6=methyl;
R.sub.8=ethyl; and R.sub.10=OH.
106. The compound of claim 89 wherein R.sub.8 is methyl.
107. The compound of claim 106 wherein R.sub.9 is methyl.
108. The compound of claim 107 wherein R.sub.4 is H.
109. The compound of claim 108 wherein R.sub.1 is phenyl.
110. The compound of claim 108 wherein R.sub.1 is n-propyl.
111. The compound of claim 108, wherein R.sub.1=n-propyl;
R.sub.2=R.sub.4=R.sub.7=H;
R.sub.3=R.sub.5=R.sub.6=R.sub.8=R.sub.9=metiyl- ; and
R.sub.10=OH.
112. The compound of claim 88, wherein R.sub.1=n-propyl;
R.sub.2=R.sub.3=R.sub.7=H;
R.sub.4=R.sub.5=R.sub.6=R.sub.8=R.sub.9=methyl- ; and
R.sub.10=OH.
113. The compound of claim 87, having formula ix: 31wherein R.sub.1
is H, halogen, hydroxyl, amino, C.sub.1-6 alkyl, aryl-C.sub.1-6
alkyl, mono- or poly-fluorinated C.sub.1-6 alkyl, hydroxy-C.sub.1-6
alkyl, C.sub.1-6 alkoxy, amino-C.sub.1-8 alkyl, C.sub.1-6
alkylamino, di(C.sub.1-6 alkyl)amino, C.sub.1-8
alkylamino-C.sub.1-8 alkyl, di(C.sub.1-6 alkyl)amino-C.sub.1-8
alkyl, cyclohexyl, aryl or heterocycle, wherein aryl or heterocycle
may each be unsubstituted or substituted with one or more of the
following: C.sub.1-6 alkyl, C.sub.1-6 alkoxy, hydroxy-C.sub.1-4
alkyl, hydroxyl, amino, C.sub.1-6 alkylamino, di(C.sub.1-6
alkyl)amino, amino-C.sub.1-8 alkyl, C.sub.1-8 alkylamino-C.sub.1-8
alkyl, di(C, alkyl)amino-C.sub.1-8 alkyl, nitro, azido or halogen;
R.sub.2 is H, halogen, hydroxyl, C.sub.1-6 alkyl, aryl-C.sub.1-6
alkyl, mono- or poly-fluorinated C.sub.1-6alkyl, aryl or
heterocycle; R.sub.3 and R.sub.4 are independently selected from
the group consisting of H, halogen, hydroxyl, amino, C .sub.1-6
aLkyl, aryl-Cl alkyl, mono- or poly- fluorinated C.sub.1-6 alkyl,
hydroxy-C.sub.1-6 alkyl, amino-C.sub.1-8 alkyl, C.sub.1-8
alkylamino-C.sub.1-8 alkyl, di(C.sub.1-6 alkyl)amino-C.sub.1-8
alkyl, cyclohexyl, aryl or heterocycle; and R.sub.3 and R.sub.4 can
be taken together to form a 5-7 membered saturated cycle ring or
heterocyclic ring; R.sub.5 and R.sub.6 are independently selected
from the group consisting of H, C.sub.1-6 alkyl, aryl-C, alkyl,
mono- or poly- fluorinated C.sub.1-6 alkyl, aryl or heterocycle;
and R.sub.5 and R.sub.6 can be taken together to form a 5-7
membered saturated cycle ring or heterocyclic ring; R.sub.7 is H,
halogen, methyl, or ethyl; R.sub.8 and R.sub.9 are independently
selected from the group consisting of H, halogen, C.sub.1-6 alkyl,
aryl-C.sub.1-6 alkyl, mono- or poly-fluorinated C.sub.1-6 alkyl,
hydroxy-C.sub.1-6 alkyl, amino-C.sub.1-8 alkyl, C.sub.1-8
alkylamino-C.sub.1-8 alkyl, di(C.sub.1-6 alkyl)amino-C.sub.1-8
alkyl, cyclohexyl, aryl or heterocycle; and R.sub.8 and R.sub.9 can
be taken together to form a 5-7 membered saturated cycle ring or
heterocyclic ring; R.sub.10 is halogen, OR.sub.11, NHOR.sub.11,
NHOR.sub.12, NR.sub.11R.sub.12, NR12R.sub.13; wherein R.sub.11 is
H, acyl, P(O)(OH).sub.2, S(O)(OH).sub.2, CO(C.sub.1-10
alkyl)CO.sub.2H, (C.sub.1-8 alkyl)CO.sub.2H, CO(C.sub.1-10
alkyl)NR.sub.12R.sub.13, (C.sub.1-8 alkyl) NR.sub.12R.sub.13;
R.sub.12 and R.sub.13 are independently selected from the group
consisting of H and C.sub.1-6 alkyl; and R.sub.12 and R.sub.13 can
be taken together to form a 5-7 membered saturated heterocyclic
ring containing said nitrogen; or a pharmaceutically acceptable
salt thereof, alone or in combination with a carrier; with the
proviso that when RI is n-propyl, R.sub.2, R.sub.4, R.sub.7, and
R.sub.8 are H, R.sub.5, R.sub.6, and R.sub.9 are methyl, and
R.sub.10 is OH, then R.sub.3 cannot be methyl; when R.sub.1 is
n-propyl, R.sub.2, R.sub.3, R.sub.7, and R.sub.9 are H, R.sub.5,
R.sub.6, and R.sub.8 are methyl, and R.sub.10 is OH, then R.sub.4
cannot be methyl.
114. The compound of claim 87, having formula x: 32wherein
R.sub.1is H, halogen, hydroxyl, amino, C.sub.1-6 alkyl,
aryl-C.sub.1-6 alkyl, mono- or poly-fluorinated C.sub.1-6 alkyl,
hydroxy-C.sub.1-6 alkyl, C.sub.1-6 alkoxy, amino-C.sub.1-8 alkyl,
C.sub.1-6 alkylamino, di(C.sub.1-6 alkyl)amino, C.sub.1-8
alkylamino-C.sub.1-8 alkyl, di(C.sub.1-6 alkyl) amino-C.sub.1-8
alkyl, cyclohexyl, aryl, or heterocycle, wherein aryl or
heterocycle may each be unsubstituted or substituted with one or
more of the following: C.sub.1-6 alkyl, C.sub.1-6 alkoxy,
hydroxy-C.sub.1-4 alkyl, hydroxyl, amino, C.sub.1-6 alkylamino,
di(C.sub.1-6 alkyl)amino, arino-C.sub.1-8 alkyl, C.sub.1-8
alkylamono-C.sub.1-8 alkyl, di(C.sub.1-6 alkyl)amino-C.sub.1-8
alkyl, nitro, azido or halogen; R.sub.2 is H, halogen, hydroxyl,
C.sub.1-6 alkyl, aryl-C.sub.1-6 alkyl, mono- or poly-fluorinated
C.sub.1-6 alkyl, aryl or heterocycle; R.sub.3 and R.sub.4 are
independently selected from the group consisting of H, halogen,
hydroxyl, amino, C.sub.1-6 alkyl, aryl-C.sub.1-6 alkyl, mono- or
poly- fluorinated C.sub.1-6 alkyl, hydroxy-C.sub.1-6 alkyl
amno-C.sub.1-8 alkyl, C.sub.1-8 alkylamino-C.sub.1-8 alkyl,
di(C.sub.1-6 alkyl)amino-C.sub.1-8 alkyl, cyclohexyl, aryl or
heterocycle; and R.sub.3 and R.sub.4 can be taken together to form
a 5-7 membered saturated cycle ring or heterocyclic ring; R.sub.5
and R.sub.6 are independently selected from the group consisting of
H, C.sub.1-6 alkyl, aryl-C,.sub.6 alkyl, mono- or poly-fluorinated
C.sub.1-6 alkyl, aryl or heterocycle; and R.sub.5 and R.sub.6 can
be taken together to form a 5-7 membered saturated cycle ring or
heterocycle ring; R.sub.7 is H, halogen, methyl, or ethyl; R.sub.8
and R.sub.9 are independently selected from the group consisting of
H, halogen, C.sub.1-6 alkyl, aryl-C.sub.1-6 alkyl, mono- or poly-
fluorinated C.sub.1-6 alkyl, hydroxy-C.sub.1-6 alkyl,
amino-C.sub.1-8 alkyl, C.sub.1-8 alkylamlno-C.sub.1-8 alkyl,
di(C.sub.1-8 alkyl)amino-C.sub.1-8 alkyl, cyclohexyl, aryl or
heterocycle; and R.sub.8 and R.sub.9 can be taken together to form
a 5-7 membered saturated cycle ring or heterocyclic ring; R.sub.10
is H, C.sub.1-6 alkyl, aryl and aryl-C.sub.1-6 alkyl, acyl,
P(O)(OH).sub.2, S(O)(OH).sub.2, CO(C.sub.1-10 alkyl)CO.sub.2H,
(C.sub.1-8 alkyl)CO.sub.2H, CO(C.sub.1-10 alkyl)NR.sub.11R.sub.12,
(C.sub.1-8 alkyl) NR.sub.11R.sub.12; wherein R.sub.11 and R.sub.12
are independently selected from the group consisting of H,
C.sub.1-6 alkyl, aryl, and aryl-C.sub.1-6alkyl; and R.sub.11 and
R.sub.12 can be taken together to form a 5-7 membered saturated
heterocyclic ring containing said nitrogen; or a pharmaceutically
acceptable salt thereof, alone or in combination with a carrier.
Description
CROSS-REFERENCE
[0001] This is a continuation-in-part of co-pending U.S. patent
application Ser. No. 08/609,537, filed Mar. 1, 1996, which is a
continuation-in-part of co-pending U.S. patent application Ser. No.
08/510,213, filed Aug. 2, 1995, which in turn is a
continuation-in-part of U.S. patent application Ser. No.
08/285,655, filed Aug. 3, 1994, now U.S. Pat. No. 5,489,697 issued
Feb. 6, 1996.
FIELD OF THE INVENTION
[0002] This invention relates to calanolide analogues that
demonstrate potent antiviral activity against a spectrum of
viruses. This invention also relates to the use of calanolide
analogues for treating or preventing viral infections.
BACKGROUND OF THE INVENTION
[0003] Viruses, an important etiologic agent in infectious disease
in humans and other mammals, are a diverse group of infectious
agents that differ greatly in size, shape, chemical composition,
host range, and effects on hosts. After several decades of study,
only a limited number of antiviral agents are available for the
treatment and/or prevention of diseases caused by viruses such as
HIV, hepatitis B, herpes simplex type 1 and 2, cytomegalovirus,
varicella zoster virus, Epstein Barr virus, influenza A and B,
parainfluenza, adenovirus, measles, and respiratory syncytial
virus. Because of their toxic effects on a host, many antiviral
agents are limited to topical applications. Accordingly, there is a
need for safe and effective antiviral agents with a wide-spectrum
of anti-viral activity with reduced toxicity to the host.
[0004] A. Human Immunodeficiency Virus (HIV)
[0005] Human immunodeficiency virus (HIV), which was also called
human T-lymphotropic virus type III (HTLV-III),
lymphadenopathy-associated virus (LAV) or AIDS-associated
retrovirus (ARV), was first isolated in 1982 and has been
identified as the etiologic agent of the acquired immunodeficiency
syndrome (AIDS) and related diseases. Since then, chemotherapy of
AIDS has been one of the most challenging scientific endeavors. So
far, eleven drugs have been approved by FDA and are being
clinically used as drugs for the treatment of AIDS and AIDS-related
complex. Although these FDA-approved drugs can extend the life of
AIDS patients and improve their quality of life, none of these
drugs are capable of curing the disease. Bone-marrow toxicity and
other side effects as well as the emergence of drug-resistant viral
strains limit the long-term use of these agents..sup.1 On the other
hand, the number of AIDS patients worldwide has increased
dramatically within the past decade and estimates of the reported
cases in the very near future also continue to rise dramatically.
It is therefore apparent that there is a great need for other
promising drugs having improved selectivity and activity to combat
AIDS..sup.1 Several approaches including chemical synthesis,
natural products screening, and biotechnology have been utilized to
identify compounds targeting different stages of HIV replication
for therapeutic intervention..sup.2
[0006] Very recently, the screening program at the National Cancer
Institute has discovered a class of remarkably effective anti-HIV
natural products, named calanolides, from the rain forest tree
Calophyllum lanigerum, with calanolide A, 1, being the most potent
compound in the reported series..sup.3 For example, calanolide A
demonstrated 100% protection against the cytopathic effects of
HIV-1, one of two distinct types of HIV, down to a concentration of
0.1 .mu.M. This agent also halted HIV-1 replication in human
T-lymphoblastic cells (CEM-SS)(EC.sub.50=0.1 .mu.M/IC.sub.50=20
.mu.M)..sup.3 More interestingly and importantly, calanolide A was
found to be active against both the AZT-resistant G-9106 strain of
HIV as well as the pyridinone-resistant A17 virus..sup.3 1
[0007] Thus, the calanolides, known as HIV-1 specific reverse
transcriptase inhibitors, represent novel anti-HIV chemotherapeutic
agents for drug development.
[0008] B. Hepatitis B Virus (HBV)
[0009] The hepatitis B virus (HBV) infects people of all ages. It
is one of the fastest-spreading sexually trrnsmitted diseases, and
also can be transmitted by sharing needles or by behavior in which
a person's mucus membranes are exposed to an infected person's
blood, semen, vaginal secretions, or saliva. While the initial
sickness is rarely fatal, ten percent of the people who contract
hepatitis are infected for life and run a high risk of developing
serious, long-term liver diseases, such as cirrhosis of the liver
and liver cancer, which can cause serious complications or
death..sup.21 The World Health Organization lists HBV as the ninth
leading cause of death. It is estimated that about 300 million
persons are chronically infected with HBV worldwide, with over 1
million of those in the United States. The Center for Disease
Control and Prevention estimates that over 300,000 new cases of
acute HBV infection occurs in the United States each year,
resulting in 4,000 deaths due to cirrhosis and 1,000 due to
hepatocellular carcinoma..sup.22 The highest rates of HBV
infections occur in Southeast Asia, South Pacific Islands,
Sub-Saharan Africa, Alaska, Amazon, Bahai, Haiti, and the Dominican
Republic, where approximately 20% of the population is chronically
infected..sup.23
[0010] Hepatitis B virus (HBV) infection is currently the most
important chronic virus infection, but no safe and effective
therapy is available at present. The major therapeutic option for
carriers of HBV is alpha interferon, which can control active virus
replication. However, even in the most successful studies, the
response rate in carefully selected patient groups has rarely
exceeded 40%..sup.24, 25 One of the reasons cited for interferon
failure is the persistence of viral supercoiled DNA in the
liver..sup.26 Clinical exploration of many promising antiviral
agents such as nucleoside analogues is hampered because their
aspecific body distribution leads to significant toxic side
effects. Recently, however, a new nucleoside analogue,
2',3'-dideoxy-3'-thiacytidine (3TC), was discovered and found to be
extremely potent against HBV replication with only minimal side
effects. .sup.27-29
[0011] C. Influenza Virus
[0012] Influenza is a viral infection marked by fever, chills, and
a generalized feeling of weakness and pain in the muscle, together
with varying signs of soreness in the respiratory tract, head, and
abdomen. Influenza is caused by several types of myxoviruses,
categorized as groups A, B, and C.sub.4. These influenza viruses
generally lead to similar symptoms but are completely unrelated
antigenically, so that infection with one type confers no immunity
against the other. Influenza tends to occur in wavelike epidemics
throughout the world; influenza A tends to appear in cycles of two
to three years and influenza B in cycles of four to five years.
Influenza is one of the few common infectious diseases that are
poorly controlled by modern medicine. Its annual epidemics are
occasionally punctuated by devastating pandemics. For example, the
influenza pandemic of 1918, which killed over 20 million people and
affected perhaps 100 times that number, was the most lethal plague
ever recorded. Since that time, there have been two other pandemics
of lesser severity, the so-called Asian flu of 1957 and the Hong
Kong flu of 1968. All of these pandemics were characterized by the
appearance of a new strain of influenza virus to which the human
population had little resistance and against which previously
existing influenza virus vaccines were ineffective. Moreover,
between pandemics, influenza virus undergoes a gradual antigenic
variation that degrades the level of immunological resistance
against renewed infection..sup.30
[0013] Anti-influenza vaccines, containing killed strains of types
A and B virus currently in circulation, are available, but have
only a 60 to 70% success rate in preventing infection. The standard
influenza vaccine has to be redesigned each year to counter new
variants of the virus. In addition, any immunity provided is
short-lived. The only drugs currently effective in the prevention
and treatment of influenza are amantadine hydrochloride and
rimantadine hydrochloride..sup.31-33 While the clinical use of
amantadine has been limited by the excess rate of CNS side effects,
rimantadine is more active against influenza A both in animals and
human beings, with fewer side effects..sup.34, 35 It is the drug of
choice for the chemoprophylaxis of influenza A..sup.30, 36, 37
However, the clinical usefulness of both drugs is limited by their
effectiveness against only influenza A viruses, by the uncertain
therapeutic efficacy in severe influenza, and by the recent
findings of recovery of drug-resistant strains in some treated
patients..sup.38-42 Ribavirin has been reported to be
therapeutically active, but it remains in the investigational stage
of development..sup.43, 44
[0014] D. Cytomegalovirus (CMV)
[0015] Cytomegalovirus (CMV) is a member of the herpes virus
family, other well-known members of which include herpes simplex
virus, types I and II, Epstein Barr virus and Varicella Zoster
virus. Although these viruses are related taxonomically, all
comprising double-stranded DNA viruses, infections due to these
viruses manifest in clinically distinct ways. In the case of CMV,
medical conditions arising from congenital infection include
jaundice, respiratory distress and convulsive seizures that may
result in mental retardation, neurologic disability or death.
Infection in adults is frequently asymptomatic, but may manifest as
mononucleosis, hepatitis, pneumonitis or retinitis, particularly in
immunocompromised patients such as AIDS sufferers, chemotherapy
patients and organ transplant patients undergoing tissue rejection
therapy.
[0016] Up to 45% of all HIV-infected persons will develop
cytomegalovirus-induced disease before their lives end..sup.45
Although two antiviral agents-ganciclovir and foscavir--are
available to treat human cytomegalovirus (HCMV), they act as
virustatic agents to slow but not halt progression of disease;
hence, disease routinely progresses despite daily maintenance with
either agent. Moreover, therapy using either agent is problematic
because both agents are associated with serious
toxicities..sup.46
[0017] Classical drug therapies have generally focused upon
interactions with proteins in efforts to modulate their
disease-causing or disease-potentiating functions. Such therapeutic
approaches have failed for cytomegalovirus infections. Effective
therapy for CMV has not yet been developed despite studies on a
number of antiviral agents. Interferon, transfer factor, adenine
arabinoside (Ara-A), acycloguanosine (Acyclovir) and certain
combinations of these drugs have been ineffective in controlling
CMV infections. Based on preclinical and clinical data, foscarnet
and ganciclovir show limited potential as antiviral agents.
Foscarnet treatment has resulted in the resolution of CMV retinitis
in five AIDS patients to date. Ganciclovir studies have shown
efficacy against CMV retinitis and colitis. However, though
ganciclovir seems to be well tolerated by most treated individuals,
the appearance of a reversible neutropenia, the emergence of
resistant strains of CMV upon long-term administration, and the
lack of efficacy against CMV pneumonitis limit the long term
applications of this compound. The development of more effective
and less toxic therapeutic compounds and methods is needed for both
acute and chronic use.
[0018] Several HCMV vaccines have been developed or are in the
process of development. Vaccines based on live attenuated strains
of HCMV have been described. A proposed HCMV vaccine using a
recombinant vaccinia virus expressing HCMV glycoprotein B has also
been described. However, vaccinia models for vaccine delivery are
believed to cause local reactions. Additionally, vaccinia vaccines
are considered possible causes of encephalitis.
[0019] E. Other Herpes Viruses
[0020] Varicella zoster virus (VZV) is the etiologic agent that
produces both varicella (chickenpox) and zoster (shingles). As with
other herpes viruses, VZV causes both an acute illness and lifelong
latent infection. Acute primary infection (varicella) typically
occurs during childhood, where the resulting infection is
relatively mild. Conversely, primary infection in adults can be
more severe. Herpes zoster cutaneous eruptions are caused by
reactivation of VZV present in sensory ganglia..sup.47 Herpes
zoster occurs more frequently with elderly and immunosuppressed
individuals, and is eight times more likely to develop in
HIV-infected individuals than in other individuals in comparable
age groups..sup.48
[0021] Along with other immunosuppressed patients, HIV-infected
patients may develop severe and in certain cases life-threatening
illnesses following either primary or recurrent VZV infection.
Therapy for HlV-infected patients experiencing VZV infection
generally involves administering acyclovir or vidarabine (Ara-A),
with hospitalization required in many instances. To inhibit VZV
replication, serum levels of acyclovir are about ten times greater
than those needed to to inhibit Herpes Simplex Type 1 and 2.
[0022] Herpes simplex virus type I and type 2 (HSV-1 and HSV-2) can
establish latency following primary infection and can thus
subsequently reactivate to induce recurrent disease. Upon primary
infection, herpes simplex type 1 induces diseases including primary
gingivostomatitis, encephalitis, and kerato-conjunctivitis, while
herpes simplex type 2 induces primary genital herpes and neonatal
herpes. Upon recurrence, herpes simplex type 1 induces diseases
including recurrent oral herpes and recurrent
kerato-conjunctivitis, while herpes simplex type 2 induces
recurrent genital herpes..sup.49 HSV infection in HIV-infected
patients can produce widespread and occasionally life-threatening
lesions.
[0023] Acyclovir, delivered either intravenously, orally, or
topically, shortens clinical illness in both immunocompetent and
imrnunosuppressed patients. Vidarabine also has been used in
treating HSV. Some vaccine strategies have been investigated with a
view towards preventing initial primary infection. However,
protecting only against primary disease but not protecting against
latency and subsequent recurrence is inadequate for those persons
already initially infected. Moreover, acyclovir-resistant HSV
infections recently have been observed, in many cases occurring
among HIV-infected patients treated successfully with acyclovir in
the past. The existence of such acyclovir-resistant infections in
HIV-infected patients is troubling in view of the limited number of
alternative therapeutic options available.
[0024] Respiratory Syncytial Virus (RSV) is the prime etiologic
agent producing lower respiratory tract disease. RSV causes
extensive yearly epidemics during which there is a marked increase
in hospital admissions of patients, especially infants and young
children, experiencing severe lower respiratory tract disease.
Immunosuppressed patients infected with RSV are at high risk of
mortality. Ribavirin is the only currently approved drug for
treating RSV infections. However, this drug appears to have limited
efficacy. Additionally, development of effective vaccines has
proven difficult to date.
[0025] F. Opportunistic Infections
[0026] The viruses above can act as sole causes of infection or can
act to produce opportunistic infections in patients already
battling immunosuppressing infections such as HIV. Acting by
themselves, these viruses can present therapeutic challenges. But
when acting to produce opportunistic infections in HIV-infected or
otherwise immunosuppressed patients, these viruses dramatically
increase the difficulty and complexity of successful treatment.
[0027] In addition to the viruses discussed above, other viral,
bacterial, fungal, and protozoal pathogens can induce opportunistic
infections. Common opportunistic pathogens in addition to those
described above include Mycobacterium avium complex (MAC),
Pneumocystis carinii (PC), and M. tuberculosis.
[0028] Present therapies for HIV-infected patients also suffering
from opportunistic infection generally involve administering a
plurality of antiviral compounds. In such a treatment regimen,
termed combination therapy, each antiviral compound employed
demonstrates best antiviral activity against a distinct viral
infection. For example, a combination therapy of AZT and
ganciclovir can be used for an HIV-infected patient also
experiencing CMV retinitis, where AZT targets the HIV infection and
ganciclovir targets the CMV infection. Thus, combination therapies
can be powerfiil therapeutic tools. Even more powerful and
desirable, however, would be a single antiviral compound that
demonstrates antiviral activity against both HIV and other
viruses.
[0029] While some limited success has been realized in the search
for viable therapeutics for treatment of the viral infections
discussed above, therapeutic agents for many viruses remain
severely limited. Furthermore, there are no known safe and
therapeutic treatments for HBV, influenza and HIV. In HBV, with the
possible exception of the drug 3TC, the use of nucleoside-based
antiviral agents leads to toxicity, probably due to
cross-inhibition of cellular mitchondrial DNA. Clearly, there is a
need for a new class of antiviral agents which could minimize the
toxicity associated with cross-inhibition. In influenza, amantadine
and rimantadine have been shown to be moderately effective against
only influenza A viruses, with amantadine having excessive side
effects. Recently, strains of influenza A resistant to amantadine
and rimantadine have been isolated. Accordingly, there is a need
for new types of therapeutic antiviral agents particularly against
both influenza A and influenza B, as well as against HIV, HBV and
HIV and other viruses. Furthermore, due to the loss of CD4 T
lymphocytes in an HIV infected person, leading to immunodeficiency
and thus increasing susceptibility to a broad range of
opportunistic viral, bacterial, fungal, and protozoal pathogens,
identifying anti-HIV agents having a spectrum of antiviral and
antimicrobial activities is of particular interest. These agents
would be not only effective against HIV infection, but also
effective against or preventive of opportunistic infections in AIDS
patients.
[0030] A natural source of calanolide A is limited..sup.4 This
limited availability fueled the desire to develop practical
synthesis routes to enable further study and development to be
carried out on this active and promising series of compounds.
[0031] Herein, we describe synthetic calanolide analogues
demonstrating potent antiviral activity against many viruses. We
also describe methods of using such calanolide analogues for
treating or preventing viral infections.
SUMMARY OF THE INVENTION
[0032] The present invention relates to calanolide analogues and
methods of using such compounds for treating or preventing viral
infections, especially infections caused by Hepatitis B, herpes
viruses, or respiratory viruses. Herpes viruses against which
calanolide analogues of the invention act as antiviral agents
include Herpes Simplex Type 1, Herpes Simplex Type 2,
Cytomegalovirus, Varicella Zoster Virus, or Epstein Barr Virus.
Respiratory viruses against which calanolide analogues of the
invention act as antiviral agents include Influenza A, Influenza B,
Parainfluenza, Adenovirus, Measles, or Respiratory Syncytial
Virus.
[0033] Calanolide analogues of the invention can be used alone or
in combination with other therapeutic agents, including but not
limited to Intron A and 3TC for Hepatitis B; gancyclovir,
progancyclovir, famcyclovir, foscarnet, vidarabine, cidovir, and
acyclovir for herpes viruses; and ribavarin, amantidine, and
rimantidme for respiratory viruses.
[0034] Calanolide analogues of the invention also demonstrate
activity against multi-viral infections. For example, they are
effective against both HIV and other opportunistic infections to
which people with HIV are especially prone. Among all known HIV
antiviral agents, the calanolide analogues described herein are
unique in their ability to combat not only BIV but also other
opportunistic infections.
[0035] The present invention provides calanolide analogues obtained
via syntheses employing chromene 4 and chromanone 7 as key
intermediates, which is described in U.S. patent application Ser.
No. 08/510,213, filed Aug. 2, 1995, herein incorporated by
reference in its entirety. Chromene 4 is synthesized by the
sequence depicted in Scheme I. Thus, 5,7-dihydroxy4-propylcoumarin,
2,.sup.5 was prepared quantitatively from ethyl butyrylacetate and
phloroglucinol under Pechmann conditions..sup.6 Product yield and
purity were dependent on the amount of sulfuric acid used. The
8-position of 5,7-dihydroxy-4-propylcoumarin, 2, was then
selectively acylated at 8-10.degree. C. by propionyl chloride and
AlCl.sub.3 in a mixture of carbon disulfide and nitrobenzene to
afford 5,7-dihydroxy-8-propionyl4-propylcoumarin, 3.
[0036] In an alternative and preferred reaction, coumarin
intermediate 3 may be produced in large scale quantities and with
minimal formation of undesirable 6-position acylated product and
6,8-bis-acylated product by selective acylation of
5,7-dihydroxy4-propylcoumarin 2 with a mixture of propionic
anhydride and AlCl.sub.3 at about 70-75 .degree. C.
[0037] The chromene ring was introduced upon treatment of compound
3 with 4,4-dimethoxy-2-methylbutan-2-ol, providing 4 in 78% yield
(Scheme I). Chlorotitanium-mediated aldol reaction of chromene 4
with acetaldehyde led to formation of (.+-.)-8a and (.+-.)-8b in a
ratio of 95:5. The racemic syn aldol product [(.+-.)-8a] was
resolved by enzyme-catalyzed acylation. Thus, in the presence of
lipase and vinyl acetate, (-)-8a was selectively acylated and the
desired enantiomer (+)-8a was unreacted. The purified (+)-8a was
subjected to a Mitsunobu.sup.7a-c reaction, exclusively leading to
(+)-trans-chromanone [(+)-7].
[0038] Finally, Luche reduction.sup.8 on (+)-7 led to formation of
(+)-calanolide A [(+)-1] which contained 10% of (+)-calanolide B
(see Scheme III). (+)-Calanolide A [(+)-1] was further separated
from (+)-calanolide B by preparative normal phase HPLC and was
identical with an authentic sample.
[0039] If desired, the racemic anti aldol product [(.+-.)-8b] may
also be resolved by enzyme-catalyzed acylation into (+)-8b and the
ester 10 from (-)-8b (Scheme IV). Mitsunobu reaction on (+)-8b
would lead to formation of the cis-chromanone 7a which could then
be reduced to produce calanolide C.
[0040] The synthetic sequence for (+)-calanolide A was extended to
the synthesis of calanolide analogues. Thus, Pechmann reaction of
phloroglucinol with various .beta.-ketoesters yields substituted
5,7-dihydroxycoumarin 11 (Scheme V). Friedel-Crafts acylation of
substituted 5,7-hydroxycoumarin 11 leads to formation of 8-acylated
5,7-dihydroxycoumarin 12. Chromenylation of 12 can be achieved by
reacting with substituted .beta.-hydroxyaldehyde dimethylacetal,
affording chromenocoumarin 13. Aldol reaction of chromenocoumarin
13 with carbonyl compounds in the presence of LDA with or without
metal complexing agents forms the racemic aldol product (+)-14.
Cyclization of (.+-.)-14 under Mitsunobu conditions, by using
triphenylphosphine and diethyl azodicarboxylate (DEAD), leads to
formation of chromanone analogue (.+-.)-15. Reduction of (.+-.)-15
with sodium borohydride with or without cerium chloride yields the
12-hydroxy analogue (.+-.)-16 (Scheme V).
[0041] Catalytic hydrogenation of both (.+-.)-15 and (.+-.)-16
produces 7,8-dihydro derivatives (.+-.)-17 and (.+-.)-18 (Scheme
VI). Treatment of (.+-.)-15 with hydroxylamine or alkoxyamine
affords oxime derivatives (.+-.)-19 (Scheme VI). Reduction of
(.+-.)-19 under different conditions.sup.9 should selectively yield
hydroxylamino or amino compounds (20 and 21).
[0042] Optically active forms of 14-21 would be obtained by
employing enzymatic acylation, as described in Scheme III for
(+)-calanolide A [(+)-1]. Thus, enzyme-catalyzed acylation of the
racemic aldol product (.+-.)-14 would selectively acylate one
enantiomer [i.e. (-)-14] and leave the other enantiomer [i.e.
(+)-14] unreacted, which would be easily separated by conventional
methods such as silica gel column chromatography. The acylated
enantiomer [i.e. (-)-14] may be hydrolyzed to form the pure
enantiomer [i.e. (-)-14]. The optically pure enantiomers thus
obtained [(+)-14 and (-+14] will be cyclized to (+)-15 and (-)-15,
respectively, by Mitsunobu reaction. Reduction of (+)-15 and (-)-15
would lead to formation of (+)-16 and (-)-16. Hydrogenation of
optically active forms of 15 and 16 would provide pure enantiomers
of 17 and 18 [(+)-and (-)-17; (+)-and (-)-18]. Treatment of pure
enantiomers of 15 with hydroxylamine and alkoxylamine affords
enantiomerically pure oxime 19 [(+)-and (-)-19]. If desired, (+)-19
and (-)-19 may be reduced to produce enantiomerically pure 20 and
21 [(+)-and (-)-20; (+)-and (-)-21].
[0043] The 12-hydroxyl group in compound 1, 16, and 17 as well as
their optically active forms can be epimerized by a number of
methods including acidic conditions, neutral Mitsunobu
conditions.sup.7a-c, or with DAST..sup.7d An example showing
conversion of (-)calanolide A [(-)-1] into (-)-calanolide B is
depicted in Scheme VII.
[0044] The process used to produce compounds of the present
invention may be utilized to prepare a wide variety of calanolide
analogues such as Formulas i-v shown in Scheme VIII and Formulas
vi-vii shown in Scheme IX.
[0045] For Formula i, R.sub.1 and R.sub.2 are independently or
.
[0046] For Formula ii, R.sub.1, R.sub.2, and R.sub.3 are
independently H or CH3.
[0047] For Formula iii, R.sub.1 is C.sub.1 -C.sub.6 linear or
branched alkyl.
[0048] For Formula iv, R.sub.1 is propyl or phenyl and R.sub.2 is
OH or OH.
[0049] For Formula vi, R.sub.1 is C.sub.1-C.sub.6 linear or
branched alkyl.
[0050] For Formula vii, R.sub.1 is propyl or phenyl and R.sub.2 is
OH or OH.
[0051] Additional exemplary calanolide analogues include but are
not limited to Formulas 15 and 16 shown in Scheme V, and Formulas
17 and 18 shown in Scheme VI.
[0052] Methods for treating and/or preventing viral infections
using compounds of the invention are also described. Representative
viral infections include HIV, hepatitis B, herpes simplex type 1
and 2, cytomegalovirus, varicella zoster virus, Epstein Barr virus,
influenza A and B, parainfluenza, adenovirus, measles, and
respiratory syncytial virus.
[0053] Accordingly, it is an object of the invention to provide
calanolide analogues obtained via syntheses employing chromene 4
and chromanone 7 as key intermediates.
[0054] A further object of the invention is to provide a method for
treating or preventing viral infections using calanolide analogues
of the formula I: 2
[0055] whrein
[0056] R.sub.1 is H, halogen, hydroxyl, amino, C.sub.1-6 alkyl,
aryl-C.sub.1-6 alkyl, mono-or poly-fluorinated C.sub.1-6 alkyl,
hydroxy-C.sub.1-6 alkyl, C.sub.1-6 alkoxy, amino-C.sub.1-8 alkyl,
C.sub.1-6 alkyl amino, di(C.sub.1-6 alkyl)amino, C.sub.1-8
alkylamino-C.sub.1-8 alkyl, di(C.sub.1-6 alkyl) amino-C.sub.1-8
alkyl, cyclohexyl, aryl, or heterocycle, wherein aryl or
heterocycle may each be unsubstituted or substituted with one or
more of the following: C.sub.1-6 alkyl, C.sub.1-6 alkoxy,
hydroxy-C.sub.1-4 alkyl, hydroxyl, amino, C.sub.1-6 alkylamino,
di(C.sub.1-6 alkyl) amino, amino-C.sub.1-8 alkyl, C.sub.1-8
alkylamino-C.sub.1-8 alkyl, di(C.sub.1-6 alkyl)amino-C.sub.1-8
alkyl, nitro, azido or halogen;
[0057] R.sub.2 is H, halogen, hydroxyl, C.sub.1-6 alkyl,
aryl-C.sub.1-6 alkyl, mono- or poly-fluorinated C.sub.1-6 alkyl,
aryl or heterocycle;
[0058] R.sub.3 and R.sub.4 are independently selected from the
group consisting of H, halogen, hydroxyl, amino, C.sub.1-6 alkyl,
aryl-C.sub.1-6 alkyl, mono- or poly- fluorinated C.sub.1-6 alkyl,
hydroxy-C.sub.1-6 alkyl, amino-C.sub.1-8 alkyl, C.sub.1-8
alkylamino-C.sub.1-8 alkyl, di(C.sub.1-6 alkyl) amino-C.sub.1-8
alkyl, cyclohexyl, aryl or heterocycle; and R.sub.3 and R.sub.4 can
be taken together to form a 5-7 membered saturated cycle ring or
heterocyclic ring;
[0059] R.sub.5 and R.sub.6 are independently selected from the
group consisting of H, C.sub.1-6 alkyl, aryl-C.sub.1-6 alkyl, mono-
or poly-fluorinated C.sub.1-6 alkyl, aryl or heterocycle; and
R.sub.5 and R.sub.6 can be taken together to form a 5-7 membered
saturated cycle ring or heterocycle ring;
[0060] R.sub.7 is H, halogen, methyl, or ethyl;
[0061] R.sub.8 and R.sub.9 are independently selected from the
group consisting of H, halogen, C.sub.1-6 alkyl, aryl-C.sub.1-6
alkyl, mono- or poly- fluorinated C.sub.1-6 alkyl,
hydroxy-C.sub.1-6 alkyl, amino-C.sub.1-8 alkyl, C.sub.1-8
alkylamino-C.sub.1-8 alkyl, di(C.sub.1-6 alkyl)amino-C.sub.1-8
alkyl, cyclohexyl, aryl or heterocycle; and R.sub.8 and R.sub.9 can
be taken together to form a 5-7 membered saturated cycle ring or
heterocyclic ring;
[0062] R.sub.10 is halogen, O, OR.sub.11, NOR.sub.11, NHOR.sub.11,
NOR.sub.12, NHOR.sub.12, NR.sub.11,R.sub.12, NR.sub.12, or
NR.sub.12R.sub.13; wherein R.sub.11 is H, acyl, P(O)(OH).sub.2,
S(O)(OH).sub.2, CO(C.sub.1-10 alkyl)CO.sub.2H, (C.sub.1-8
alkyl)CO.sub.2H, CO(C.sub.1-10 alkyl)NR.sub.12R.sub.13, (C.sub.1-8
alkyl) NR.sub.12R.sub.13; R.sub.12 and R.sub.13 are independently
selected from the group consisting of H, C.sub.1-6 akyl, aryl, and
aryl-C.sub.1-6 alkyl; and R.sub.12 and R.sub.13 can be taken
together to form a 5-7 membered saturated heterocyclic ring
containing said nitrogen;
[0063] or a pharmaceutically acceptable salt thereof.
[0064] These and other objects of the invention will become
apparent in view of the detailed description below. 3 4 5 6 7 8 9
10 11
DESCRIPTION OF THE DRAWINGS
[0065] FIGS. 1(a) to 1(e) illustrate in vitro MTT assay results, as
described in Example 37, using G910-6 HIV viral strain which is
AZT-resistant.
[0066] FIGS. 2(a) to 2(e) illustrate in vitro MTT assay results, as
described in Example 37, using H112-2 HIV viral strain which was
not pre-treated with AZT.
[0067] FIGS. 3(a) to 3(e) illustrate in vitro MTT assay results, as
described in Example 37, using A-17 HIV viral stain which is
resistant to non-nucleoside inhibitors such as TIBO but is
sensitive to AZT.
[0068] FIGS. 4(a) to 4(d) illustrate in vitro MTT assay results, as
described in Example 37, using IIIB cultivated HIV viral
strain.
[0069] FIGS. 5(a) to 5(d) illustrate in vitro MTT assay results, as
described in Example 37, using RF cultivated HIV viral strain.
[0070] FIG. 6 is an HPLC chromatograrn of (a) (.+-.)-calanolide A
on normal phase column; (b) (.+-.)calanolide A on a chiral HPLC
column; (c) (+)-calanolide A on a chiral HPLC column and (d)
(-)-calanolide A on a chiral HPLC column. The HPLC conditions are
described in Example 13.
[0071] FIG. 7 illustrates representative examples of inventive
compounds that were evaluated in the in vitro MTT assay of Example
38.
[0072] FIG. 8 illustrates representative examples of inventive
compounds that were evaluated in the in vitro assays of Examples 39
- 41.
DETAILED DESCRIPTION OF THE INVENTION
[0073] All patents, patent applications, and literature references
cited herein are incorporated by reference in their entirety.
[0074] The present invention relates to calanolide analogues and
methods of using such compounds for treating or preventing viral
infections. In one embodiment, the invention provides calanolide
analogues obtained via syntheses employing chromene 4 and
chromanone 7 as key intermediates, as shown in Schemes I and III.
According to this synthetic scheme, chromene 4 may be prepared from
5,7-dihydroxy-4-propylcoumarin, 2, as shown in Scheme I. According
to this synthetic scheme, 5,7-dihydroxy-4-propylcouma- rin,
2,.sup.5 was prepared quantitatively from ethyl butyryacetate and
pbloroglucinol under Pechmann conditions..sup.6
[0075] In conducting ths reaction, a volume of a concentrated acid
is added in a dropwise manner to a stirring mixture of ethyl
butyrlacetate and phloroglucinol with a molar ratio ranging between
about 3:1 and about 1:3, with a preferable range being about
0.9:1.0. The dropwise addition of an acid was conducted at a rate
such that the temperature of the reaction mixture is maintained at
a temperature ranging between about 0.degree. C. and about
120.degree. C., preferably about 90.degree. C.
[0076] Suitable, but not limiting, examples of concentrated acid
include sulurc acid, trfluoroacetic acid, and methanesulfonic acid.
In making compounds of the invention, concentrated sulfuric acid is
particularly preferred. As the product yield and purity appear to
be dependent on the amount of concentrated sulfurfic acid used, it
is preferred that the amount of concentrated sulfuric acid ranges
between about 0.5 and 10 mole, most preferably ranging between
about 2 and about 3.5 mole, per mole of ethyl butyrylacetate.
[0077] The reaction mixture is then heated to a temperature ranging
between about 40.degree.C. and about 150.degree. C., preferably
about 90.degree. C., until the reaction reaches completion as
determined by TLC analysis. The reaction mixture is then poured
onto ice and the precipitated product is collected by filtration
and dissolved in an organic solvent. Suitable, but non-limiting,
examples of organic solvents include ethyl acetate, chloroform, and
tetraydrofua. A preferred solvent is ethyl acetate. The resulting
solution is then washed with brine and dried over a suitable drying
agent, e.g., sodium sulfate. The yields of this reaction are
generally quantitative.
[0078] Thereafter, 5,7diydroxy-8-propionyl-4-propylcoumarin, 3, was
prepared by selectively acylating the 8-position of
5,7diydroxyffpropylcoumarin, 2, with propionyl chloride in the
presence of a Lewis acid catalyst (Friedal-Crafts acylation). In
conducting thiis reaction, a solution of propionyl chloride in a
suitable solvent, e.g., carbon disulfide, was added in a dropwise
manner to a vigorously stirred solution of
5,7-dihydroxy4-propylcoumnarin, 2, a Lewis acid and an organic
solvent cooled in an ice bath. Dropwise addition of propionyl
chloride is conducted such that the temperature of the reaction
mixture is maintained at a temperature ranging between 0.degree. C.
and about 30 .degree. C., preferably between about 8.degree. C. and
10.degree. C.
[0079] In makting compounds of the invention, the amount of
propionyl chloride used generally ranges between about 0.5 and
about 6 moles, preferably ranging between about 1 and about 2
moles, per mole of 5,7-dihydroxy4propylcoumarin, 2.
[0080] Non-limting examples of Lewis acid catalysts useful in the
acylation reaction include AlCl.sub.3, BF.sub.3, SNCl.sub.4,
ZnCl.sub.2, POCl.sub.3, and TICl.sub.4. A preferred Lewis acid
catalyst is AlCl.sub.3. The amount of Lewis acid catalyst relative
to 5,7diydroxy-4-propylcoumarin, 2, ranges between about 0.5 and
about 12 moles, preferably ranging between about 2 and about 5
moles, per mole of 5,7-diydroxy-4-fiprpylcoumarin 2.
[0081] Non-limiting examples of organic solvent for use in
preparing the 5,7-dihydroxy- 4-propylcoumarin, 2, solution include
nitrobenzene, nitromethane, chlorobenzene, or toluene and mixtures
thereof. A preferred organic solvent for use in this invention is
nitrobenzene.
[0082] Upon completion of the addition of propionyl chloride, the
vigorously stirred reaction mixture is maintained at a temperature
ranging between about 0.degree. C. and about 120.degree. C.,
preferably ranging between about 25.degree. C. and 80.degree. C.,
until the reaction reaches completion as monitored by conventional
means such as TLC analysis. The reaction mixture is then poured
onto ice and extracted several times with a suitable solvent such
as ethyl acetate, chloroform, methylene chloride, tetrahydrofuran,
or a mixture of chloroform/methanol. A preferred solvent for this
extraction is ethyl acetate. The extracts are then dried over a
suitable drying agent, e.g., sodium sulfate, and the product may be
purified by conventional means such as silica gel column
chromatography.
[0083] On small scale (<1 gram), the yield of
5,7-dihydroxy-8-propionyl- -4-propylcoumarin 3, produced by the
above described reaction is generally quantitative. However, on
larger scale (>1 gram), thie reaction was very difficult to
control and did not exclusively afford the desired product as the
desired 8-position acylated product 3 was accompanied by the
formation of undesired 6-position acylated product and
6,8-bis-acylated product. Thus, an alternative and preferred route
for preparing 5,7-dihydroxy-8-propionyl4-propylcoumarin 3 in large
scale quantities was devised.
[0084] Preparation of 8-acylated coumarin 3 on a 5 gram scale as a
single product (45% yield) has been achieved by adding a rnixture
of propionic anhydride, a Lewis acid, e.g., AlCl.sub.3, and
suitable solvent, e.g., 1,2-dchloroedmae, into a vigorously
stirring pre-heated mixture of coumarin, a Lewis acid, e.g.,
AlCl.sub.3, and suitable solvent, e.g., 1,2-dichloroethane, at a
temperature ranging between about 40.degree. C. and about
160.degree. C., preferably ranging between about 70.degree. C. and
about 75.degree. C. Dropwise addition of the propionic anhydride
solution is conducted at a rate such that the temperature of the
reaction mixture is maintained within the desired temperature
range.
[0085] The amount of propionic anhydride used in the reaction
generally ranges between about 0.5 and about 10 moles, preferably
ranging between about 1 and about 2 moles, per mole of
5,7-dihydroxy~propylcoumarin 2.
[0086] Non-limiting examples of Lewis acid catalysts useful in the
acylation reaction include AlCl.sub.3, BF.sub.3, POCl.sub.3,
SnCl.sub.4, ZnCl.sub.2 and TICl.sub.4. A preferred Lewis acid
catalyst is AlCl.sub.3. The amount of Lewis acid catalyst relative
to 5,7-diydroxy-4-propylcoumar- n 2, ranges between about 0.5 and
about 12 moles, preferably ranging between about 2 and about 4
moles, per mole of 5,7-dihydroxy-4-propylcoum- arin, 2.
[0087] Suitable but nonlmting examples of solvents for use in
making compounds of the invention include diglyme, nitromethane,
1,1,2,2-tetrachloroethane, and 1,2-dichloroethane (preferred).
Uponl completion of the addition of propionyl anhydride, the
vigorously stirred reaction mixture is maintained at a temperature
ranging between about 40.degree. C. and about 160.degree. C.,
preferably ranging between about 70.degree. C. and 75.degree. C.,
until the reaction reaches completion as monitored by conventional
means such as TLC analysis. The workup procedure is the same as
described above.
[0088] The product was purified without the use of column
chromatography to afford the desired product 3. This procedure has
been scaled-up to 1.7 kg of coumarin (for details see experimental
section) and the yield for 8-acylated coumarin 3 was 29% after
recrystallization. The yield for 8-acylated coumarin 3 may be futer
improved by changing the purification processing. For example, the
crude product may be recrystallized from solvent(s) other than
dioxane, or a simple washing with an appropriate solvent may lead
to product pure enough for the next reaction step.
[0089] Thereafter, chromene 4 was prepared by introducing the
chromene ring into 5,7-dihydroxy-8-propionyl-4-propylcoumarin, 3,
using 4,4-dimethoxy-2-methylbutan-2-ol. A solution of
5,7-dihydroxy-8-propionyl- -4-propylcoumarin, 3, and
4,4-dimethoxy-2-methylbutan-2-ol in a suitable organic solvent in
the presence of a base was reacted at a temperature ranging between
about 40.degree. C. and about 180.degree. C., preferably ranging
between about 100.degree. C. and about 120.degree. C., until the
reaction reached completion as determined by conventional means
such as TLC analysis. Water and methanol formed during the reaction
were removed azeotropically via a Dean-Stark trap.
[0090] In making compounds of the invention, the amount of
4,4-dimethoxy-2-methylbutan-2-ol employed in the reaction generally
ranges between about 0.5 and about 8 moles, preferably ranging
between about 2 and about 4 moles, per mole of
5,.sup.7-dihydroxy-8-propionyl-4-p- ropylcoumarin 3.
[0091] Suitable, but not limiting examples of organic solvents
include pyridine, triethylamine, N,Ndimethylformamide (DMF),
toluene, tetrahydrofaran (THF) or 1,2-dichloroethane. Suitable, but
non-limiting examples of the bases include pyridine,
4-direthylaminopyridine, triethylamine, N,N-diethylaniline,
1,5-diazabicyclo-[4,3,0]-non-5-ene (DBN),
1,8-diazabicyclo-[5,4,0]undec-7-ene (DBU), sodium carbonate and
sodium bicarbonate. Pyridine was used as both base and solvent in
this invention on a small scale; for scale-up, however, pyridine
was used as a base and toluene was used as a solvent.
[0092] Upon completion of the reaction, the solvent is removed
under reduced pressure and the reaction product is dissolved in a
suitable solvent, e.g., ethyl acetate.
[0093] The solution is then washed sequentially with water and
brine and dried over a suitable drying agent, e.g., sodium sulfate.
Thereafter, the crude chromene 4 product can be purified by
conventional means such as silica gel column chromatography using
25% ethyl acetatelhexane as the elution solvent. The yields of
chromene 4 generally fall with the range of about 60% and about
85%, usually resulting in about 78% yield. Chromene 4 was then used
to prepare chromanone 7.
[0094] A number of alternative routes were devised for preparing
chromanone 7 from chromene 4 in large scale quantities. These
routes were described in U.S. patent application Ser. No.
08/510,213, filed Aug. 2, 1995, the disclosure which is
incorporated herein in its entirety. For instance, U.S. patent
application Ser. No. 08/510,213 describes a one-step reaction
process (paraldehyde one-step reaction), shown in Scheme II, and a
two-step reaction process (LDA/sulfuric acid process or
LDA/Mitsunobu process) for preparing chromanone 7 from chromene 4.
Examples of these reactions are provided in the Examples below. In
this invention, a new route for preparing chromanone 7 from
chromene 4 was devised, shown in Scheme III, which introduces a
chiral resolution step between the two step LDA/Mitsunobu process
described in the 08/510,213 application and illustrated below. One
of the benefits for including the enzyme acylation/resolution step
at this stage of the process is that it provides a more practical
and economical means for producing large scale amounts of
chromanone (+)-7, which would lead to formation of (+)-calanolide A
after reduction without the subsequent need for chiral HPLC
resolution of the racemic calanolide A.
[0095] According to Scheme HI, (+)-chromanone 7 was prepared by a
chlorotitanium-mediated aldol condensation reaction of chromene 4
with acetaldehyde which led to formation of aldol products
(.+-.)-8a and (.+-.)8b in a ratio of 95:5, respectively. In
conducting the aldol condensation reaction, a solution of LDA was
added dropwise to a solution of chromene 4 dissolved in a solvent
at a temperature ranging between about -78.degree. C. and about
0.degree. C., preferably about -30.degree. C. and about -78.degree.
C. Thereafter, a solution of titanium tetrachloride was added
dropwise to the stirring reaction mixture. The resulting solution
was then warmed to a temperature ranging between about -78.degree.
C. and about 40.degree. C., preferably about 40.degree. C., and
allowed to stir for about 45 minutes to allow for transmetallation.
Thereafter, the solution was recooled to -78.degree. C.
[0096] The amount of LDA added per mole of chromene 4 ranged
between about 1 and about 4 moles, preferably ranging between about
2 and about 3 per mole of chromene 4. Dropwise addition LDA is
conducted such that the reaction temperature is maintained within
the desired range.
[0097] The amount of titanium tetrachloride ranges between about
0.5 and about 10 moles, preferably ranging between about 2 and
about 4 moles per mole of chromene 4.
[0098] Suitable, but not limiting examples of solvent include
methylene chloride, TIF, diethyl ether, dioxane, etc.
[0099] Acetaldehyde was then added dropwise to the reaction mixture
in amounts ranging between about 1 and about 12 moles, preferably
ranging between about 4 and about 6 moles per mole of chromene 4.
Dropwise addition of acetaldehyde is conducted such that the
reaction temperature is maintained within the aforementioned range.
The reaction was monitored by conventional means, e.g., TLC
analysis, until it reached completion.
[0100] The aldol reaction of chromene 4 with acetaldehyde may be
carried out under conditions which employs bases other than LDA.
For example, metal hydroxides such as NaOH, KOH and Ca(OH).sub.2,
metal alkoxides such as MeONa, EtONa and t-BuOK, and amines such as
pyrrolidine, piperidine, diisopropylethylamine,
1,5-diazabicyclo[4,3,0]non-5-ene (DBN),
1,8-diazabicyclo[5,4,0]undec-7-ene (DBU), NaNH.sub.2 and LiHMDS as
well as hydrides such as NaH and KH can all be employed for the
aldol reactions..sup.10 Also, aldol reactions can be mediated by
metal complexes of Al, B, Mg, Sn, Zn, Zr and other Ti compounds
such as (i-PrO).sub.3TiCl, (i-PrO).sub.4Ti, PhBCl.sub.2,
(n-Bu).sub.2BCl, BF.sub.3, (n-Bu).sub.3SnCl, SnCl.sub.4,
ZnCl.sub.2, MgBr.sub.2, Et.sub.2AlCl with or without chiral
auxiliaries such as 1,1'-binaphthol, norephedrinesulfonate,
camphanediol, diacetone glucose and dialkyl tarrate..sup.11-13
[0101] Thereafter, the reaction mixture was quenched at -30.degree.
C. to -10.degree. C. with saturated aqueous ammonium chloride
solution and extracted with a suitable solvent, e.g., ethyl
acetate. The pooled extracts were washed with brine and dried over
a suitable drying agent, e.g., sodium sulfate. The yields of aldol
product generally range between about 40% and about 80%, usually
about 70%.
[0102] It should be noted that the aldol reaction of chromene 4
results in a product having two asyrnmetric centers which in turn
would result in a diastereomeric mixture of two sets of enantiomers
(four optically active forms). The mixture may be separated by
conventional means to produce racemic syn aldol product (.+-.)-8a
and racemic anti aldol product (.+-.)8b which may be resolved into
optically active forms. Conventional resolution methods may be used
such as chromatography or fractional crystallization of suitable
diastereoisomeric derivatives such as salts or esters with
optically active acids (e.g., camphor-10-sulfonic acid, camphoric
acid, methoxyacetic acid, or dibenzoyltartaric acid) or
enzymatically catalyzed acylation or hydrolysis of the racemic
esters. The resultant or synthetic enantiomer may then be
transformed to enantioselective synthesis of (+)-calanolide A and
its congeners.
[0103] In one method, the racemic aldol product may be resolved by
high performance liquid chromatography (HPLC) with organic solvent
system as a mobile phase. HPLC is performed on a column packed with
chiral packing material. Suitable, but not limiting, examples of
chiral packing material include amylose carbamate, D-phenylglycine,
L-phenylglycine, D-leucine, L-leucine, D-naphthylalanine,
L-naphthylalanine, or L-naphthylleucine. These materials may be
bounded, either ionically or covalently, to silica sphere which
particle sizes ranging between about 5 m and about 20 m. Suitable,
but non-limiting, mobile phase includes hexane, heptane,
cyclohexane, ethyl acetate, methanol, ethanol, or isopropanol and
mixtures thereof. The mobile phase may be employed in isocratic,
step gradient or continuous gradient systems at flow rates
generally ranging between about 0.5 mL/min. and about 50
mL/min.
[0104] In making compounds of the invention, the racemic product,
i.e., syn aldol product [(.+-.)8a], is resolved preferably by
enzyme-catalyzed acylation. Enzymatic resolution may employ enzymes
such as lipase CC (Candida cylindracea), lipase AK (Candida
cylindracea), lipase AY (Candida cylindracea), lipase PS
(Pseudomonas Species), lipase AP (Aspergillus niger), lipase N
(Rhizopus nieveuis), lipase FAP (Rhizopus nieveus), lipase PP
(Porcine Pancrease), pig (porcine) liver esterase (PLE), pig liver
acetone powder (PLAP), or subtilisin. Immobilized forms of the
enzyme on cellite, molecular sieves, or ion exchange resin are also
contemplated for use in this method. The amount of enzyme used in
the reaction depends on the rate of chemical conversion desired and
the activity of the enzyme. The preferred enzyme for use in the
enzyme-catalyzed acylation reaction is lipase.
[0105] The enzymatic acylation reaction is carried out in the
presence of an acylating agent. Suitable, but not limiting,
examples of acylating agents include vinyl acetate, vinyl
propionate, vinyl butyrate, vinyl stearate, acetic anhydride,
propionic anhydride, phthalic anhydride, acetic acid, propionic
acid, hexanoic acid or octanoic acid. The enzymatic reaction
employs at least one mole of acylating agent per mole of aldol
product. Acylating agent can be used as a solvent in the acylation
reaction or in solution with another solvent such as hexanes,
chloroform, benzene, tert-butylmethyl ether, and THF. The preferred
solvent and acylating agent for use in the enzyme-catalyzed
acylation are tert-butylmethyl ether and vinyl acetate,
respectively.
[0106] Suitable, but not limiting examples of solvents for use in
the enzymatic hydrolysis reaction include water, suitable aqueous
buffers such as sodium phosphate buffers, or alcohols such as
methanol or ethanol.
[0107] One skilled in the art will appreciate that racemic esters
of aldol products can be made by conventional esterification means
and selectively hydrolyzed by enzymes so as to produce, in high
enantiomeric excess, optically active aldol product, i.e., (+)-8,
in free or esterified form.
[0108] The purified (+)-8a was subjected to a neutral Mitsunobu
reaction, selectively leading to (+)-trans-chromanone [(+)-7]. In
performing this reaction, diethyl azodicarboxylate (DEAD) was added
dropwise to a solution containing (+)-8a and triphenylphosphine at
a temperature ranging between about -10.degree. C. and about
40.degree. C., preferably about ambient temperature. The amount of
DEAD used in the reaction generally ranges between about 1 mole and
about 10 moles preferably about 1 mole and about 4 moles, per mole
of aldol (+)8a. The amount of triphenylphosphine used in the
reaction generally ranged between about 1 mole and about 10 moles,
preferably ranging between about 1 mole and about 4 moles, per mole
of aldol (+)-8a.
[0109] Instead of DEAD, other suitable azo reagents reported in the
literature can be employed such as diisopropyl azodicarboxylate
(DIAD), dibutyl azodicarboxylate (DBAD),
dipiperidinoazodicarboxamide,
bis(N.sup.4-methylpiperazin-1-yl)azodicarboxamide,
dimorpholinoazodicarboxamide, N,N,N',N'-tetramethylazodicarboxamide
(TMAD).sup.14. Also, in addition to triphenylphosphine, other
phosphine derivatives such as tri-n-butylphosphine,.sup.14
triethylphosphine, trimethylphosphine and
tris(dimethylamino)phosphine may be used.
[0110] Thereafter, the reaction was quenched with saturated
ammonium chloride upon completion and extracted with a suitable
solvent, e.g., ethyl acetate. The pooled organic layers were washed
with brine, concentrated in vacuo and the crude chromanone (+)-7
was purified by conventional means as discussed above. The yields
of chromanone (+)-7 from the Mitsunobu reaction generally range
between about 60% and about 80%, usually about 70%.
[0111] Finally, mild borohydride reduction of chromanone (+)-7 in
the presence of CeCl.sub.3(H.sub.2O).sub.7 (Luche reduction)
produced (+)-calanolide A with the desired stereochemical
arrangement. In conducting the reduction reaction, a solution of
chromanone (+)-7 was added dropwise into a solution of reducing
agent, e.g., sodium borohydride and a metal additive, e.g.,
CeCl.sub.3(H.sub.2O).sub.7 in ethanol. The rate of addition is such
that the reaction mixture temperature is maintained within a range
of between about 40.degree..degree.C. and about 60.degree. C.,
preferably ranging between about -10.degree. C. and about
-30.degree. C. Thereafter, the reaction mixture was stirred at a
temperature ranging between about 40.degree. C. and about
60.degree. C.
[0112] In general, the amount of metal additive, e.g.,
CeCl.sub.3(H.sub.2O).sub.7 present in the reaction mixture ranged
between about 0.1 and about 2 moles, preferably ranging between
bout 0.5 and about 1 mole, per mole of sodium borohydride. In
addition, the amount of reducing agent, e.g., sodium borohydride
employed in the reaction generally ranged between about 0.1 and
about 12 moles, preferably ranging between about 2 and about 4
moles, per mole of chromanone (+)-7. Suitable, but non-limiting,
examples of reducing agents include NaBH.sub.4
LiAlH.sub.4,(i-Bu).sub.2AlH,(n-Bu).sub.3SnH,9-BBN, Zn(BH4).sub.2,
BH.sub.3, DIP-chloride, selectrides and enzymes such as baker
yeast. Suitable, but non-limiting, examples of metal additives
include CeCl.sub.3, ZnCl.sub.2, AlCl.sub.3, TiCl.sub.4, SnCl.sub.3,
and LnCl.sub.3 and their mixture with triphenylphosphline oxide. In
practicing this invention, sodium borohydride as reducing agent and
CeCl.sub.3(H.sub.2O).sub.7 as metal additive are preferred.
[0113] Thereafter, the reduction mixture was diluted with water and
extracted with a suitable solvent, e.g., ethyl acetate. The extract
was dried over a suitable drying agent, e.g., sodium sulfate, and
concentrated. The resulting residue was then purified by
conventional means such as silica gel chromatography, using ethyl
acetate/hexane solvent mixtures. Luche reduction on (+)-7 led to
formation of (+)-calanolide A [(+)-1] which contained 10% of
(+)-calanolide B. (+)-Calanolide A [(+)-1] was further separated
from (+)-calanolide B by preparative normal phase HPLC and was
identical with an authentic sample.
[0114] Thus, (+)-calanolide A, 1, was successfully prepared with
the desired stereochemical arrangement by treatment of the key
intermediate chromene 4 with chlorotitanium catalyzed aldol
reaction to produce (.+-.)8a, enzyme resolution of the racemate to
produce (+)8a, and neutral Mitsunobu reaction of (+)8a to produce
chromanone (+)-7, followed by Luche reduction via chromanone (+)7
(see Scheme III).
[0115] Enzyme resolution of transe8b racemate with vinyl acetate
and lipase allowed for the separation of (+)-8b, which, following
treatment under neutral Mitsunobu reaction with triphenylphosphine
and DEAD and subsequent Luche reduction, would result in calanolide
C (Scheme IV).
[0116] In another embodiment of the invention, analogues of
calanolide A are provided by extension of the aforementioned
synthetic sequence for (+)-calanolide A. Pechmann reaction of
phloroglucinol with substituted .beta.-ketoesters yields
substituted 5,7-dihydroxycoumarin 1 as shown in Scheme V. The
conditions and reagents used in the Pechmann reaction are described
above.
[0117] Suitable, but non-limiting, .beta.-ketoesters include those
of formula .alpha.: 12
[0118] wherein R.sub.1 is H, halogen, hydroxyl, amino, C.sub.1-6
alkyl, aryl-C.sub.1-6 alkyl, mono- or poly-fluorinated C.sub.1-6
alkyl, hydroxy-C.sub.1-6 alkyl, C.sub.1-6 alkoxy, amino-C.sub.1-8
alkyl, C.sub.1-6 alkylamino, di(C.sub.1-6 alkyl)amino, C.sub.1-8
alkylamino-C.sub.1-8 alkyl, di(C.sub.1-6 alkyl) amino-C.sub.1-8
alkyl, cyclohexyl, aryl, or heterocycle, wherein aryl or
heterocycle may each be unsubstituted or substituted with one or
more of the following: C.sub.1-6 alkyl, C.sub.1alkoxy,
hydroxy-C.sub.1-4 alkyl, hydroxyl, amino, C.sub.1-6 alkylamino,
di(C.sub.1-6 alkyl)amino, amino-C.sub.1-8 alkyl, C.sub.1-8
alkylamino-C.sub.1-8 alkyl, di(C.sub.1-6 alkyl-amino-C.sub.1-8
alkyl, nitro, azido or halogene; and R.sub.2 is H, halogen,
hydroxyl, C.sub.1-6 alkyl, aryl-C.sub.1-8 alkyl, mono- or
poly-fluorinated C.sub.1-6 alkyl, aryl or heterocycle.
[0119] Friedel-Crafts acylation of substituted
5,7-dihydroxycoumarin 11 leads to formation of 8-acylated
5,7-dihydroxycoumarin 12. The conditions and reagents used in the
Friedel-Crafts acylation reaction are described above.
[0120] Non-limiting examples of carboxylic acid anhydrides and
halides include formula b carboxylic acid anhydrides and halides:
13
[0121] wherein X is halogen (e.g. chloro) or OCOCHR.sub.3R.sub.4
wherein R.sub.3 and R.sub.4 are independently selected from the
group consisting of H, halogen, hydroxyl C.sub.1-6 alkyl,
aryl-C.sub.1-6 alkyl, mono- or poly-fluorinated C.sub.1-6 alkyl,
hydroxy-C.sub.1-6 alkyl, amino-C.sub.1-8 alkyl, C.sub.1-8
alkylamino-C.sub.1-8 alkyl, di(C.sub.1-6 alkyl)amio-C.sub.1-8
alkyl, cyclohexyl, aryl or heterocycle; and R.sub.3 and R.sub.4 can
be taken together to form a 5-7 membered saturated cycle ring or
heterocyclic ring.
[0122] Chromenylation of 12 can be achieved by reacting with
substituted .beta.-hydroxyaldehyde dimethylacetal, affording
chromenocoumarin 13. The conditions and amounts of reagents are
described above. Representative examples of substituted
.beta.-hydroxyaldehyde dimethylacetals of formula c comprise:
14
[0123] wherein R.sub.5 and R.sub.6 are independently selected from
the group consisting of H, C.sub.1-6 alkyl, aryl-C.sub.1-6 alkyl,
mono- or poly-fluorinated C.sub.1-6 alkyl, aryl or heterocycle;
R.sub.5 and R.sub.6 can be taken together to form a 5-7 membered
saturated cycle ring or heterocycle ring; and R.sub.7 is H,
halogen, methyl, ethyl.
[0124] Aldol condensation reaction of chromene 13 with carbonyl
compounds in the presence of LDA forms the racemic aldol product
(.+-.)-14. According to the present invention, a solution of LDA in
THF was added dropwise to a solution of chromene 13 in THF at a
temperature ranging between about -78.degree. C. and about
0.degree. C., preferably about -30.degree. C. and about -78.degree.
C. The amount of LDA added per mole of chromene 13 ranged between
about 1 and about 4 moles, preferably ranging between about 2 and
about 3 moles per mole of chromene 13. Dropwise addition of LDA is
conducted such that the reaction temperature is maintained within
the desired range.
[0125] A carbonyl compound of formula iv was then added dropwise to
the reaction mixture in amounts ranging between about 1 and about
12 moles, preferably ranging between about 4 and about 6 moles per
mole of chromene 13. Dropwise addition of carbonyl compound is
conducted such that the reaction temperature is maintained within
the aforementioned range. The reaction was monitored by
conventional means, e.g., TLC analysis, until it reached
completion.
[0126] Representative examples of formula d carbonyl compounds
comprise: 15
[0127] wherein R.sub.8 and R.sub.9 are independently selected from
the group consisting of H, halogen, C.sub.1-6 alkyl, aryl-C.sub.1-6
alkyl mono- or poly-fluorinated C.sub.1-6 alkyl, hydroxy-C.sub.1-6
alkyl amino-C.sub.1-8 alkyl C.sub.1-8 alkylamino-C.sub.1-8 alkyl,
di(C.sub.1-6 alkyl)amino-C.sub.1-8 alkyl, cyclohexyl, aryl or
heterocycle; and R.sub.8 and R.sub.9 can be taken together to form
a 5-7 membered saturated cycle ring or heterocyclic ring.
[0128] One skilled in the art will appreciate that the aldol
reaction of chromene 13 with carbonyl compounds of formula d to
form 14 can be carried out under conditions which employs bases
other than LDA. For example, metai hydroxides such as NaOH, KOH and
Ca(OH).sub.2, metal alkoxides such as MeONa, EtONa and t-BuOK, and
amines such as pyrrolidine, piperidine, diisopropylethylamine,
1,5-diazabicyclo[4,3,0]no- n-5-ene (DBN),
1,8-diazabicyclo[5,4,0]undec-7-ene (DBU), NaNH.sub.2 and LiHMDS as
well as hydrides such as NaH and KH can all be employed for the
aldol reactions..sup.10 Also, aldol reactions can be mediated by
metal complexes of Al, B, Mg, Sn, Ti, Zn and Zr compounds such as
TiCl.sub.4, (i-PrO).sub.3TiCl, (i-PrO).sub.4Ti, PhBCl.sub.2,
(n-Bu).sub.2BCl, BF.sub.3, (n-Bu).sub.3SnCl,SnCl.sub.4, ZnCl.sub.2,
MgBr.sub.2, Et.sub.2AlCl with or without chiral auxiliaries such as
1,1'-binaphthol, norephedrinesulfonate, camphanediol, diacetone
glucose and dialkyl tartrate..sup.11-13
[0129] Thereafter, the reaction mixture was quenched at -30.degree.
C. to -10.degree. C. with saturated aqueous ammonium chloride
solution and extracted with a suitable solvent, e.g., ethyl
acetate. The pooled extracts were washed with brine and dried over
a suitable drying agent, e.g., sodium sulfate. The yields of aldol
product (.+-.)-14 generally range between about 40% and about 80%,
usually about 70%.
[0130] Cyclization of (.+-.)-14 under neutral Mitsunobu conditions,
by using triphenylphosphine and diethyl azodicarboxylate (DEAD),
leads to formation of chromanone analogue (.+-.)-15. Reduction of
(.+-.)15 with sodium borohydride with or without metal additives
such as cerium chloride yields the 12-hydroxy analogue (.+-.)-16
(Scheme V). The conditions and amounts of reagents used in the
Mitsunobu and borohydride reduction reactions are described
above.
[0131] Catalytic hydrogenation of both (.+-.)-15 and (.+-.)-16
produces 7,8-dihydro derivatives (.+-.)-17 and (.+-.)18 (Scheme
VI). To a solution of U+-15 or (.+-.)-16 in ethanol or
ethanolimethylene chloride mixtures in a conventional Parr
apparatus under H.sub.2, hydrogenation catalyst was added at
ambient temperature. The mixture was shaken under hydrogen for a
time sufficient to complete the hydrogenation reaction. The
solution was then gravity filtered to remove catalyst and solvent
was evaporated.
[0132] Suitable, but non-limiting, hydrogenation catalysts for use
in the invention include Pd/C, PtO.sub.2 and Rh/C, Raney-Ni. In
making compounds of the invention, 10% palladium/carbon is
preferred. The amount of catalyst employed generally ranges between
about 0.01 and about 0.5 mole, preferably ranging between about
0.05 and about 0.1 mole per mole of (.+-.)-15 or (.+-.)16.
[0133] In yet another embodiment of the invention, intermediate
chromanones (.+-.)7, (+)-7, (+)-7a and (.+-.)-15 can be used to
prepare oxime, hydroxyamino, alkoxyamino or amino calanolide
derivatives. Treatment of the said chromanones with hydroxylamine
or alkoxyamine affords oxime derivatives (.+-.)-19 (Scheme VI).
[0134] Representative amines for preparing oxime derivatives
comprise NH.sub.2OR.sub.10 wherein R.sub.10 is H, C.sub.1-8 alkyl,
phenyl, benzyl, acyl P(O)(OH).sub.2, S(O)(OH).sub.2, CO(C.sub.1-10
alkyl)CO.sub.2H, (C.sub.1-8 alkyl)CO.sub.2H, CO(C.sub.1-10
alkyl)NR.sub.12 R.sub.13, (C.sub.1-8 alkyl) NR.sub.12R.sub.13;
wherein R.sub.12 and R.sub.13 are independently selected from the
group consisting of H, C.sub.1-6 alkyl; and R.sub.12 and R.sub.13
can be taken together to form a 5-7 membered saturated heterocyclic
ring containing said nitrogen. Examples of useful alkoxyamines
include methoxyamine and benzyloxyamine.
[0135] The oxime derivatives may be prepared by refluxing a
mehianolic solution of the chromanone with hydroxyl amine or
alkoxyamine in the presence of a metal carbonate such as potassium
carbonate or pyrdine until the reaction reaches completion. The
amount of amine generally ranges between about 1 and about 20
moles, preferably between about 3 and about 6 moles, per mole of
chromanone.
[0136] Upon completion of the reaction, filtration of the solution
to remove solids and removal of solvent resulted in an oil which
was purified via silica gel chromatography.
[0137] The yields of oximes generally range between about 30% and
about 80%, usually about 50%.
[0138] If desired, oxime derivatives (.+-.)-19 may be reduced under
different conditions.sup.9 to yield hydroxyamino or amino compounds
(20 and 21).
[0139] Thus, optically active forms of 14 -21 (Scheme V and VI)
would be obtained by employing enzymatic acylation, as described
above, in the procedure outlined in Scheme III for (+)-calanolide A
[(+)-l]. Enzyme-catalyzed acylation of the racemic aldol product
(.+-.)14 would selectively acylate one enantiomer [i.e. (-)-14] and
leave the other enantiomer [i.e. (+)-14] unreacted, which would be
easily separated by conventional methods such as silica gel column
chromatography. The acylated enantiomer [i.e. (-)-14] may be
hydrolyzed to form the pure enantiomer [(i.e. (-)-14]. The
optically pure enantiomers thus obtained [(+)-14 and (-)-14] will
be cyclized to (+)-15 and (-)-15, respectively, by the Mitsunobu
reaction as described above. Subsequent reduction of (+)-15 and
(-)-15 would lead to formation of (+)16 and (-)-16, respectively.
Hydrogenation of optically active forms of 15 and 16 would provide
pure enantiomers of 17 and 18 [(+)-and (-)-17; (+)-and (-)-18],
respectively. Treatment of pure enantiomers of 15 with
hydroxylamine or alkoxyamine, as described above, should afford
enantiomerically pure oxime 19 [(+)-and (-)-19]. Reduction of (+)19
and (-)-19 would lead to formation of enantiomerically pure 20 and
21 [(+)-and (-)-20; (+)-and (-)-21].
[0140] The 12-hydroxyl group in compound 1, 16, and 17 as well as
their optically active forms can be epimerized under a variety of
conditions including acidic conditions, neutral Mitsunobu
conditions.sup.7a-c, or with DAST..sup.7d An example showing
conversion of (-)calanolide A [(-)-1] into (-)calanolide B using
DAST.sup.7d is depicted in Scheme VII.
[0141] Thus, the process used to produce compounds of the present
invention may be utilized to prepare a wide variety of calanolide
analogues such as Formulas i-v shown in Scheme VIII and Formulas
vi-vii shown in Scheme IX.
[0142] For Formula i, R.sub.1 and R.sub.2 are independently or
.
[0143] For Formula ii, R.sub.1, R.sub.2, and R.sub.3 are
independently H or CH.sub.3.
[0144] For Formula iii, R.sub.1 is C.sub.1-C.sub.6 linear or
branched alkyl.
[0145] For Formula iv, R.sub.1 is propyl or phenyl and R.sub.2 is
OH or OH.
[0146] For Formula vi, R.sub.1 is C.sub.1-C.sub.6 linear or
branched alkyl.
[0147] For Formula vii, R.sub.1 is propyl or phenyl and R.sub.2 is
OH or OH.
[0148] Additional exemplary calanolide analogues include but are
not limited to Formulas 15 and 16 shown in Scheme V, and Formulas
17 and 18 shown in Scheme.
[0149] In another embodiment of the invention, (-)-calanolide B,
obtained via conversion of (-)calanolide A, is provided. It has
been discovered that (-)-calanolide A may be converted readily to
(-)-calanolide B using diethylamidosulfur trifluoride (DAST) or the
Mitsunobi reaction, e.g., diethyl azodicarboxylate and
triphenylphosphine, under the conditions and ranges described
above.
[0150] The amount of DAST employed in the inversion reaction
generally ranges between about 0.5 and about 5.0 moles, preferably
ranging between about 1 and about 2.0 moles, per mole of
(-)-calanolide A. Suitable, but non-limiting, reaction solvents for
use in the invention include methylene chloride, THF, diethyl
ether, or chloroform. In practicing the invention, the preferred
solvent is methylene chloride. The reaction may be conducted at a
temperature ranging between about -78.degree. C. and about
50.degree. C., preferably about -78.degree. C., until the reaction
is complete as determined by usual methods such as thin layer
chromatography.
[0151] In yet another embodiment of the invention, a method for
treating or preventing viral infections in mammals using calanolide
analogues of the invention is presented. Examples of mammals
include humans, primates, bovines, ovines, porcines, felines,
canines, etc. Examples of viruses may include, but not be limited
to, HIV-1, HV-2, herpes simplex virus (type 1 and 2) (HSV-1 and 2),
varicella zoster virus (VZV), cytomegalovirus (CMV), papilloma
virus, HTLV-1, HTLV-2, feline leukemia virus (FLV), Epstein Barr
virus, avian sarcoma viruses such as rous sarcoma virus (RSV),
hepatitis types A-E, equine infections, influenza A and B virus,
parainfluenza, adenovirus, arboviruses, respiratory syncytial
virus, measles, mumps and rubella viruses. More preferably the
compounds of the present invention will be used to treat a human
infected with HIV, Hepatitis B, cytomegalovirus, Epstein Barr
virus, or measles.
[0152] Hence the compounds of the present invention are
particularly useful in the prevention or treatment of infection by
the human immunodeficiency virus and also in the treatment of
consequent pathological conditions associated with AIDS. Treating
AIDS is defined as including, but not limited to, treating a wide
range of states of HIV infection: AIDS, ARC, both symptomatic and
asymptomatic, and actual or potential exposure to HIV. For example,
the compounds of this invention are useful in treating infection of
HIV after suspected exposure to HIIV by e.g., blood transfusion,
exposure to patient blood during surgery or an accidential needle
stick.
[0153] Antiviral calanolide analogues of the invention may be
formulated as a solution of lyophilized powders for parenteral
administration. Powders may be reconstituted by addition of a
suitable diluent or other pharmaceutically acceptable carrier prior
to use. The liquid formulation is generally a buffered, isotonic,
aqeuous solution. Examples of suitable diluents are normal isotonic
saline solution, standard 5% dextrose in water or in buffered
sodium or ammonium acetate solution. Such formulation is especially
suitable for parenteral administration, but may also be used for
oral administration. It may be desirable to add excipients such as
polyvinylpyrrolidone, gelatin, hydroxy cellulose, acacia,
polyethylene glycol, mannitol, sodium choride or sodium
citrate.
[0154] Alternatively, the compounds of the present invention may be
encapsulated, tableted or prepared in an emulsion (oil-in-water or
water-in-oil) syrup for oral administration. Pharmaceutically
acceptable solids or liquid carriers, which are generally known in
the pharmaceutical formulary arts, may be added to enhance or
stabilize the composition, or to facilitate preparation of the
composition. Solid carriers include starch (corn or potato),
lactose, calcium sulfate dihydrate, terra alba, croscarmellose
sodium, magnesium stearate or stearic acid, talc, pectin, acacia,
agar, gelatin, maltodextrins and microcrystalline cellulose, or
collodial silicon dioxide. Liquid carriers include syrup, peanut
oil, olive oil, corn oil, sesame oil, saline and water. The carrier
may also include a sustained release material such as glyceryl
monostearate or glyceryl distearate, alone or with a wax. The
amount of solid carrier varies but, preferably, will be between
about 10 mg to about 1 g per dosage unit.
[0155] The dosage ranges for administration of antiviral calanolide
analogues of the invention are those to produce the desired affect
whereby symptoms of infection are ameliorated. For example, as used
herein, a pharmaceutically effective amount for an HIV or other
viaal infection refers to the amount administered so as to maintain
an amount which suppresses or inhibits secondary infection by
syncytia formation or by circulating virus throughout the period
during which the HIV or other viral infection is evidenced, such as
by presence of antiviral antibodies, presence of culturable virus
and presence of antigen in patient sera. For example, the presence
of anti-HIV antibodies can be determined through use of standard
ELISA or Western blot assays, e.g., anti-gp 120, anti-gp41,
anti-tat, anti-p55, anti-p17, antibodies, etc. The dosage will
generally vary with age, extent of the infection, the body weight
and counterindications, if any, for example, immune tolerance. The
dosage will also be determined by the existence of any adverse side
effects that may accompany the compounds. It is always desirable,
whenever possible, to keep adverse side effects to a minimum.
[0156] One skilled in the art can easily determine the appropriate
dosage, schedule, and method of administration for the exact
formulation of the composition being used in order to achieve the
desired effective concentration in the individual patient. However,
the dosage can vary from between about 0.001 mg/kg/day to about 50
mg/kg/day, but preferably between about 0.01 to about 1.0
mg/kg/day.
[0157] For viral infections other than HIV, antiviral activity can
be shown via other standard assays. For example, calanolide
analogue antiviral efficacy against HSV, CMV, and VZV can be
determined by cytopathic effect (CPE) inhibition assay. Similarly,
calanolide analogue efficacy against HSV-1, HSV-2, VZV, CMV can be
determined by plaque reduction assay. In this method, the reduction
of plaque on a treated agar plate is compared to an untreated
control. Calanolide analogue efficacy against EBV can be
determrined by immunofluoresence assay, where monoclonal antibodies
and fluorescin conjugated anti-mouse antibody are sequentially
added to incubated cell cultures infected with EBV, with the number
of fluoresence positive cells in smears ultimately counted.
[0158] The pharmaceutical composition may contain other
pharmaceuticals in conjunction with the antiviral calanolide
analogues of the invention. For example, other pharmaceuticals may
include, but are not limited to, other antiviral compounds (e.g.,
AZT, ddC, ddI, D4T, 3TC, acyclovir, gancyclovir, fluorinated
nucleosides and nonnucleoside analog compounds such as TIBO
derivatives and nevirapine, .alpha.-interfon and recombinant CD4),
protease inhibitors (e.g., indinavir, saquinavir, ritonavir, and
nelfinavir), immunostimulants (e.g., various interleukins and
cytokines), immunomodulators, antibiotics (e.g., antibacterial,
antifungal, anti-pneumocysitis agents), and chemokine inhibitors.
Administration of the inhibitory compounds with other
anti-retroviral agents that act against other HIV proteins such as
protease, intergrase and TAT will generally inhibit most or all
replicative stages of the viral life cycle.
[0159] The calanolide analogues described herein can be used either
alone or in conjunction with other pharmaceutical compounds to
effectively combat a single infection. For example, calanolide
analogues of the invention can be used either alone or combined
with acyclovir in a combination therapy to treat HSV-1. Calanolide
analogues can also be used either alone or in conjunction with
other pharmaceutical compounds to combat multiple infections. For
example, calanolide analogues can be used in combination with
Intron A and/or a biflavanoid for treating Hepatitis B; with
gancyclovir, progancyclovir, famcyclovir, foscarnet, vidarabine,
cidovir, and/or acyclovir for treating herpes viruses; and with
ribavarin, amantidine, and/or rimantidine for treating respiratory
viruses.
[0160] In addition, the compounds of the present invention are
useful as tools and/or reagents to study inhibition of retroviral
reverse transcriptases. For example, the instant compounds
selectively inhibit HIV reverse transcriptase. Hence, the instant
compounds are useful as a structure/activity relationship (SAR)
tool to study, select and/or design other molecules to inhibit
HIV.
[0161] The following examples are illustrative and do not serve to
limit the scope of the invention as claimed. The inhibitory
activities against HIV and other viruses including hepatitis B,
herpes viruses (HSV-1, HSV-2, HCMV, VZV, and Epstein Barr virus),
and respiratory viruses (influenza A, influenza B, parainfluenza,
adenovirus, measles, and respiratory syncytial virus) were
investigated.
EXPERIMENTAL
[0162] All chemical reagents and solvents referred to herein are
readily available from a number of commercial sources including
Aldrich Chemical Co. or Fischer Scientific. NMR spectra were run on
a Hitachi 60 MHz R-1200 NMR spectrometer or a Varian VX-300 NMR
spectrometer. IR spectra were obtained using a Midac M series FT-IR
instrument. Mass spectral data were obtained using a Finnegan MAT
90 mass spectrometer. All melting points are corrected.
EXAMPLE 1
[0163] 5,7-Dihydroxy4-propylcoumarin.sup.5 (2)
[0164] Concentrated sulfuric acid (200 mL) was added into a mixture
of phloroglucinol dihydrate (150 g, 0.926 mol) and ethyl
butyrylacetate (161 g, 1.02 mol). The resulting mixture was stirred
at 90.degree. C. for two hours whereupon it was poured onto ice.
The solid product was collected by filtration, and then dissolved
in ethyl acetate. The solution was washed with brine and dried over
Na.sub.2SO.sub.4. After removal of the solvent in vacuo, the
residue was triturated with hexane to provide essentially pure
compound 2 (203 g) in quantitative yield, mp 233-235.degree. C.
(Lit..sup.5 236-238.degree. C). .sup.1H-NMR.sup.5 (DMSO-d.sub.6)
.delta.0.95 (3H, t, J=6.9 Hz, CH.sub.3); 1.63 (2H, apparent sextet,
J=7.0 Hz, CH.sub.2); 2.89 (2H, t, J=7.5Hz,CH.sub.2); 5.85 (1H, s,
H.sub.3); 6.22 (1H, d, J=2.0 Hz, H.sub.6); 6.31 (1H, d, J=2.0 Hz,
H.sub.8); 10.27 (1H, s, OH); 10.58 (1H, s, OH); MS EI); 220(100,
M.sup.+); 205 (37.9, M-CH.sub.3); 192 (65.8, M-C.sub.2H.sub.4); 177
(24.8, M-C.sub.3H.sub.7); 164 (60.9, M-CHCO.sub.2+1); 163 (59.6
M-CHCO.sub.2); IR (KBr): 3210 (vs and broad, OH); 1649 (vs, sh);
1617 (vs, sh); 1554 (s) cm.sup.-1; Anal. calcd. for
C.sub.12H.sub.24O.sub.4: C, 65.45; H, 5.49; Found: C, 65.61; H,
5.44.
EXAMPLE 2
[0165] 5,7-Dihydroxy-8-propionyl-4-propylcoumarin (3)
[0166] A three-neck flask (500 mL) equipped with an efficient
methanical stirrer, thermometer and addition funnel was charged
with 5,7-dihydroxy4propylcoumarin, 2, (25.0 g, 0.113 mol), aluminum
chloride (62.1 g; 0.466 mol), and nitrobenzene (150 mL) and the
mixture was stirred until a solution was obtained, which was cooled
to 0.degree. C. in an ice bath. A solution of propionyl chloride
(15.2 g; 0.165 mol) in carbon disulfide (50 mL) was added dropwise
at such a rate that the reaction temperature was maintained at
8-10.degree. C. Addition was completed over a period of 1 hour with
vigorous stirring. The reaction was monitored by TLC using a mobile
phase of 50% ethyl acetate/hexane. After three hours, an additional
portion of propionyl chloride (2.10 g; 0.0227 mol) in carbon
disulfide (10 mL) was added. Immediately after the TLC analysis
indicated the total consumption of starting material, the reaction
mixture was poured onto ice, and allowed to stand overnight. The
nitrobenzene was removed by steam distillation, and the remaining
solution was extracted several times with ethyl acetate. The
extracts were combined and dried over Na.sub.2SO.sub.4. The crude
product obtained by evaporation in vacuo was purified by
chromatography on a silica gel column eluting with 50% ether/hexane
to provide the desired propionylated coumarin 3, mp 244-246.degree.
C. .sup.1H-NMR (DMSO.sub.6) .delta.0.96 (3H, t, J=7.3 Hz,
CH.sub.3); 1.10 (3H, t, J=7.2 Hz, CH.sub.3); 1.60 (2H, m,
CH.sub.2); 2.88 (2H, t, J=7.7 Hz, CH.sub.2); 3.04 (2H, q, J=7.2 Hz,
CH.sub.2); 5.95 (1H, s, H.sub.3); 6.31 (1H, S, H.sub.6); 11.07 (1H,
s, OH); 11.50 (1H, s, OH); MS (EI): 277 (6.6, M+1); 276 (9.0,
M.sup.+) ; 247 (100, M-C.sub.2H.sub.5); IR (KBr): 3239 (s and
broad, OH); 1693 (s, C.dbd.O), 1625 and 1593 (s) cm.sup.-1; Anal.
calcd. for C.sub.15H.sub.16O.sub.5: C, 65.21; H, 5.84; Found: C,
64.92; H, 5.83. The isomer assignment was made by analogy to
precedent..sup.15
EXAMPLE 3
[0167]
2,2-Dimethyl-5-hydroxy-6proplonyl-10-propyl-2H,8H-benzot[1,2-b:3,4--
b']dipyran-8-one (4)
[0168] A mixture of 3 (2.60 g, 9.42 mmol) and
4,4-dimethoxy-2-methylbutan-- 2-ol (5.54 g, 37.7 mmol) were
dissolved in anhydrous pyridine (6.5 mL). The mixture was refluxed
under nitrogen for three days. After removal of the solvent in
vacuo, the residue was dissolved in ethyl acetate. The ethyl
acetate was washed several times with 1 N HCl and brine. It was
then dried over Na.sub.2SO.sub.4. The crude product obtained by
evaporation in vacuo was purified by silica gel column
chromatography, eluting with 25% ethyl acetate/hexane to afford
2.55 g of 4 in 78.6% yield, mp 96-98.degree. C. .sup.1H-NMR
(CDCl.sub.3) .delta.1.05 (3H, t, J=7.3 HZ, CH.sub.3); 1.22 (3H,
J=7.5 Hz, CH.sub.3); 1.53 (6H, s, 2 CH.sub.3); 1.75 (2H, m,
CH.sub.2); 2.92 (2H, t, J=7.1 Hz, CH.sub.2); 3.35 (2H, q, J=7.1 Hz,
CH.sub.2); 5.56 (1H, d, J=10.0 Hz, H.sub.3); 5.98 (1H, s, H.sub.9);
6.72 (1H, d, J=10.0 Hz, H,); MS (EI): 343 (5.7, M+1); 342 (22.5,
M.sup.+); 327 (100, M-CH.sub.3); IR (KBr): 1728 (vs, C.dbd.O)
cm.sup.-1; Anal. calcd. for C.sub.20H.sub.22O.sub.5; C, 70.16; H,
6.48; Found: C, 70.45; H, 6.92.
EXAMPLE 4
[0169] 10,11-Didehydro-12-oxocalanolide A (5)
[0170] A mixture of 4 (1.76 g, 5.11 mmol) and sodium acetate (0.419
g, 5.11 mmol) in acetic anhydride (12 mL) were refluxed for 10
hours whereupon the solvent was removed in vacuo. The residue was
purified by silica gel column chromatography, eluting first with
25% ethyl acetate/hexane followed by 50% ethyl acetatelhexane to
provide 1.16 g (62% yield) of enone 5
(6,6,10,11-tetramethyl-4-propyl-2H,6H,12H-benzo[1,-
2-b:3,4-b':5,6-b"]tripyran-2,12-dione) as a white solid, mp
209-209.5 .degree. C. .sup.1H-NMR (CDCl.sub.3) .delta.1.05 (3H, t,
J=6.6 Hz, CH.sub.3); 1.56 (6H, s, 2 CH.sub.3); 1.73 (2H, m,
CH.sub.2); 1.98 (3H, s, CH.sub.3); 2.38 (3H, s, CH.sub.3); 2.91
(2H, t, J=7.5 Hz, CH.sub.2); 5.69 (1H, d, J=10.0 Hz, H.sub.7); 6.11
(1H, s, H.sub.3); 6.71 (1H, d, J=10 Hz, H.sub.8); MS (EI): 366
(29.6, M.sup.+); 351 (100, M-CH.sub.3); 323 (16.5,
M-C.sub.3H.sub.7); IR (KBr): 1734 (vs, C.dbd.O), 1657, 1640, 1610,
and 1562 cm.sup.-1; Anal. calcd. for C.sub.22H.sub.22, O.sub.5:
72.12; H, 6.05; Found: C, 72.14; H, 6.15.
EXAMPLE 5
[0171] 10,11-Didehydrocalanolide A (6)
[0172] A mixture of enone 5 (160 mg, 0.437 mmol) and tri-n-butyltin
hydride (0.318 g, 1.09 mmol) in dry dioxane (2.0 mL) was refluxed
under nitrogen for 12 hours. The solvent was then removed in vacuo
and the residue was purified by preparative TLC using 25% ethyl
acetate in hexane as the mobile phase. The product exhibited an
R.sub.f of about 0.4. Enol 6
(12-hydroxy-6,6,10,11-tetramethyl-4-propyl-2H,6H,12H-benzo[1,2-b:3,4-b'-
:5,6-b"]tripyran-2-one) (13.3 mg, 8%) was isolated as an oil from
the plate by ethyl acetate elution. This elution may have been
inefficient, and the actual yield higher, as indicated by
analytical TLC of the crude product. .sup.1H-NMR (CDCl.sub.3)
.delta.0.92 (3H, t, J=6.0 Hz, CH.sub.3); 1.26 (3H, s, CH.sub.3);
1.39 (3H, s, CH.sub.3); 1.63 (2H, m, CH.sub.2); 1.96 (3H, s,
CH.sub.3); 2.36 (3H, s, CH.sub.3); 2.45 (2H, t, J=6.0 Hz,
CH.sub.2); 3.65 (1H, s, H.sub.12); 5.51 (1H, d, J=10.0 Hz,
H.sub.7); 6.06 (1H, S, H.sub.3); 6.67 (1H, d, J =10.0 Hz, H.sub.8);
13.25 (1H, br s, OH); MS (EI): 369 (3.8, M+1), 368 (4.4, M.sup.+),
367 (8.3, M-1) 366 (28.4, M-2), 351 (100, M-OH); IR(KBr): 1651 (s),
1589 (m)cm.sup.-1.
EXAMPLE 6
[0173] 12-Oxocalanolide A [(.+-.)-(7)
[0174] A solution containing chromene 4 (344 mg, 1.0 mmol),
acetaldehyde diethylacetal (473 mg, 4.0 mmol), trifluoroacetic acid
(1.5 mL, 19.4 mmol) and anhydrous pryidine (0.7 mL) was heated at
140.degree. C. under N.sub.2. The reaction was monitored by TLC
analysis. After 4 hours, the reaction mixture was cooled to room
temperature, diluted with ethyl acetate and washed several times
with 10% aqueous NaHCO.sub.3 and brine. The organic layer was
separated and dried over Na.sub.2SO.sub.4. The solvent was removed
in vacuo and the crude product was purified by silica gel column
chromatography eluting with ethyl acetate/hexane (2:3). Chromanone
(.+-.)-7 (10,11-trans-dihydro-4-propyl-6,6,10,11-tetramethyl-2-
H,6H,12H-benzo[1,2-b:3,4-b':5,6-b"]-tripyran-2,12-dione) (110 mg,
30% yield) was obtained m.p. 176-177.degree. C. (Lit..sup.5
130-132.degree. C.). .sup.1HNMR.sup.5 (CDCl.sub.3) .delta.1.02 (3H,
t, J=7.5 Hz, CH.sub.3); 1.21 (3H, d, J=6.8 Hz, CH.sub.3); 1.51 (3H,
d, J=7.0 Hz, CH.sub.3); 1.55 (6H, 2s, 2 CH.sub.3); 1.63 (2H,
sextet, J=7.0 Hz, CH.sub.2); 2.55 (1H, dq, J=6.9 Hz, J=11.0 Hz,
H.sub.11); 2.88 (2H, t, J=7.6 Hz, CH.sub.2); 4.28 (1H, dq, J=6.3
Hz, J=11.0 Hz, H.sub.10); 5.60 (1H, d, J=9.9 Hz, H.sub.7); 6.04
(1H, s, H.sub.3); 6.65 (1H, d, J=11.8 Hz, H.sub.8); MS (CI): 369
(100, M+1).
EXAMPLE 7
[0175] (.+-.)-Calanolide A (1):
[0176] To a solution of chromanone (.+-.)-7 (11 mg, 0.03 mmol) in
ethanol (0.4 nmL) was added sodium borohydride (2.26 g, 0.06 mmol)
and CeCl.sub.3(H.sub.2O).sub.7 (11.2 mg, 0.03 mmol) in ethanol (5
mL) at room temperature. After stirring for 45 minutes, the mixture
was diluted with H.sub.2O and extracted with ethyl acetate. The
organic layer was dried over Na.sub.2SO.sub.4 and concentrated. The
crude product was purified by preparative TLC eluting with ethyl
acetate/hexane (1:1) to afford (.+-.)-calanolide A (1) (10.5 mg,
94%). m.p. 52-54.degree. C., which increased to 102.degree. C.
after it was dried thoroughly (Lit.sup.5. 56-58.degree. C.).
.sup.1H NMR (CDCl.sub.3): .delta.1.03 (3H, t, J=7.3Hz, CH.sub.3),
1.15 (3H, d, J=6.8Hz, CH.sub.3), 1.46 (3H, d, J=6.8Hz, CH.sub.3),
1.47 (3H, s, CH.sub.3), 1.51 (3H, s, CH.sub.3), 1.66 (2H, m,
CH.sub.2), 1.93 (1H, m, H.sub.11), 2.89 (2H, m, CH.sub.2), 3.52
(1H, broad-s, OH), 3.93 (1H, m, H.sub.10), 4.72 (1H, d, J=7.8Hz,
H.sub.12), 5.54 (1H, d, J=10.0 Hz, H.sub.7), 5.94 (1H, s, H.sub.3),
6.62 (1H, d, J=9.9Hz, H.sub.8); MS (CI): 371 (75.4, M+1), 370
(16.1, M.sup.+), 353 (100, M-OH); Anal. calcd. for
C.sub.22H.sub.25O.sub.5: C, 71.33; H, 7.07; Found: C, 71.63; H,
7.21.
EXAMPLE 8
[0177] 5,7-Dihydroxy-4-propylcoumarin (2):
[0178] In this Example, kilogram scale preparation of intermediate
2 is described. Into a stirring suspension of phloroglucinol
(3574.8 g, 28.4 mol, pre-dried to constant weight) and ethyl
butyrylacetate (4600 mL, 28.4 mol) was added concentrated sulfric
acid dropwise at such a rate that the internal temperature did not
exceed 40.degree. C. After 100 mL of sulfuric acid was added, the
temperature rose to 70.degree. C. and the suspension turned into a
yellow solid. Analysis of TLC indicated that the reaction had
proceeded to completion. The reaction mixture was diluted with
water (10 L) and stirred at ambient temperature overnight. The
precipitated product was collected by filtration and then rinsed
with water until the filtrate was neutral. A quantity of 4820 g
(77% yield) of 5,7-dihydroxy-4-propylcoumarin 2 was obtained after
being dried, which was identical with an authentic sample by
comparsion of TLC, melting point and spectroscopic data.
EXAMPLE 9
[0179] 5,7-Dihydroxy-8-propionyl-4-propylcoumarin (3)
[0180] In this Example, kilogram quantities of intermediate 3 were
synthesized using propionic anhydride instead of propionyl
chloride. 5,7-dihydroxy-4-propylcoumarin 2 (1710 g, 7.77 mol) and
AlCl.sub.3 (1000 g, 7.77 mol) were mixed in 1,2-dichloroethane (9
L). The resulting orange suspension was stirred and heated to
70.degree. C. until a solution was obtained. Then, a mixture of
propionic anhydride (1010 g. 7.77 mol) and AlCl.sub.3 (2000 g,
15.54 mol) in 1,2-dichloroethane (3.4 L) was added dropwise over 3
h. The reaction was allowed to stir at 70.degree. C. for an
additonal hour. After being cooled down to room temperature, the
reaction mixture was poured into a rapidly stirring mixture of ice
water and 1N HCl. The precipitated product was taken into ethyl
acetate (30 L) and the aqueous solution was extracted with the same
solvent (10 L.times.2). The combined extracts were successively
washed with 1 N HCl (10 L), saturated aq. NaHCO.sub.3 (10 L), and
water (10 L). After being dried over MgSO.sub.4 and concentrated in
vacuo, a solid product (1765 g) was obtained which was washed with
ethyl acetate (15 L) and recrystallized from dioxane (9.5 L) to
provide 514 g of pure compound 3. From the ethyl acetate washings,
an additional 100 g of compound was obtained after
recrystallization from dioxane. Thus, the combined yield for
compound 3, which was identical with an authentic sample by
comparison of TLC, melting point and spectroscopic data, was
29%.
EXAMPLE 10
[0181] 2,2-Dimethyl-5-hydroxy-6propionyl-10-propyl-2H,
8H-benzo[1,2-b:3,4-b']dipyran-8-one (4):
[0182] In this Example, intermediate 4 was prepared in half
kilogram quantities from 3 via modification of the reaction
conditions described in Example 3. A mixture of compound 3 (510.6
g, 1.85 mol) and 4,4-dimethoxy-2-methylbutan-2-ol (305.6 g, 2.06
mol) were dissolved in a mixture of toluene (1.5 L) and dry
pyridine (51 mL). This mixture was stirred and refluxed; water and
methanol formed during the reaction were removed azeotropically via
a Dean-Stark trap. The reaction was monitored by TLC. After 6 days,
the reaction had proceeded to completion. The mixture was then
cooled to ambient temperature and diluted with ethyl acetate (2 L)
and 1 N HCl (1 L). The ethyl acetate solution was separated and
washed with 1N HCl (500 mL) and brine (1 L). After being dried over
Na.sub.2SO.sub.4 and evaporated in vacua, a quantity of 590 g (93%
yield) of compound 4 was obtained which was greater than 95% pure
without further purification and was compared with an authentic
sample by TLC and spectroscopic data.
EXAMPLE 11
[0183] 12-Oxocalanolide A ((.+-.)-7):
[0184] In this Example, chromanone (.+-.)-7 was prepared from two
alternative pathways involving either a one-step paraldehyde
reaction (procedure A) or a two-step reaction process (procedures B
and C).
[0185] Procedure A. Paraldehyde One-Step Reaction: To a stirring
solution of chromene 4 (350 mg, 1.0 mmol) and PPTS (250 mg, 1.0
mmol) in 1,2-dichloroethane (2 mL) at ambient temperature under
N.sub.2 was added 3 mL paraldehyde (22.5 mmol). The resulting
mixture was refluxed for 7 h. Then, CF.sub.3CO.sub.2H (1 mL), an
additional equivalent of PPTS and 1 mL of paraldehyde were added;
the mixture was refluxed overnight. The reaction mixture was
neutralized with saturated aqueous NaHCO.sub.3 and extracted with
ethyl acetate (50 mL.times.3). The crude product obtained by
evaporation under reduced pressure was washed with hexane. The
residue was purified by column chromatography eluting with ethyl
acetate/hexane (1:2) to afford 100 mg (27% yield) of chromanone
(.+-.)-7 and 30 mg (8% yield) of (.+-.)-7a. Chromanone (.+-.)-7
(10,11-trans-dihydro-4-propyl-6,-
6,10,11-tetramethyl-2H,6H,12H-benzo[1,2-b:3,4-b':5,6-b"]tripyran-2,12-dion-
e) obtained by this method was identical with an authentic sample
by comparison of TLC, HPLC and spectroscopic data.
[0186] Procedure B LDA/Sulfuric Acid Two-Step Reaction: To a
stirring solution of chromene 4 (5.0 g, 14.6 mmol) in THF (75 mL)
at -30.degree. C. under N.sub.2 was added 18.3 mL (36.5 mmol) of 2
M LDA in TBF. After 15 min at the same temperature, acetaldehyde
(5.0 mL, 89.5 mmol) was added via syringe. The reaction was
monitored by TLC analysis. After 1 h, the reaction mixture was
quenched at -10.degree. C. with saturated aqueous NH.sub.4Cl (75
mL) and extracted with ethyl acetate (125 mL.times.3). The combined
extracts were washed with brine (125 mL) and dried over
Na.sub.2SO.sub.4. Removal of solvents in vacuo afforded a reddish
oil of (.+-.) -8a and (.+-.)-8b (8.5 g).
[0187] The crude (.+-.)-8a and (.+-.)-8b was dissolved in acetic
acid (100 mL) and then 50% H.sub.2SO.sub.4 (100 mL) was added with
stirring. The resulting mixture was heated at 75.degree. C. for 2.5
h and then at 50.degree. C. for 4 h. TLC analysis indicated that
the starting material had been consumed. The reaction mixture was
determined to contain both chromanone (.+-.)-7 and
10,11-cis-dimethyl derivative (.+-.)-7a in a 1:1 ratio. After
cooling to ambient temperature, the reaction mixture was poured
into a mixture of ice water (500 mL) and ethyl acetate (500 mL).
The layers were separated and the aqueous layer was extracted with
ethyl acetate (200 mL.times.3). The ethyl acetate solutions were
combined and washed with saturated aqueous NaHCO.sub.3 and brine.
After being concentrated in vacuo, the product was purified by
chromatography on a silica gel column eluting with ethyl
acetate/hexane (2:3) to provide 850 mg (16% yield) of chromanone
(.+-.)-7, which was further purified by recrystallation from ethyl
acetate/hexane and was identical with an authentic sample by
comparison of TLC, HPLC and spectroscopic data.
[0188] Procedure C. LDA/Mitsunobu Two-Step Reaction: Into a
stirring solution of THF (10 mL) containing triphenylphosphine
(1.27 g, 4.80 mmol) and the crude mixture of (.+-.)-8a and
(.+-.)-8b, obtained from chromene 4 (1.0 g, 2.34 mmol), 2.5
equivalents of LDA and 6.0 equivalents of acetaldehyde by the
procedure described above, was added dropwise diethyl
azodicarboxylate (DEAD, 0.77 mL, 4,89 mmol). The resulting reddish
solution was stirred at ambient temperature under N.sub.2 for I h,
after which the reaction mixture was quenched with saturated
aqueous NH.sub.4Cl and extracted with ethyl acetate (50
mL.times.3). The extracts were washed with brine and dried over
Na.sub.2SO.sub.4. After removal of solvents, the crude product was
purified by column chromatography on silica gel eluting with ethyl
acetate/hexane (2:3) to provide 412 mg (48% yield, based on
chromene 4) of chromanone (.+-.)-7, the predominant product of the
reaction, which was identical with an authentic sample by
comparison of TLC, HPLC and spectroscopic data.
EXAMPLE 12
[0189] (.+-.)-Calanolide A (1):
[0190] In this Example, (.+-.)-calanolide A was prepared in
multi-gram scale using the procedure described in Example 7. To a
stirring solution of chromanone (.+-.)-7 (51.5 g, 0.14 mol) in
ethanol (1.5 L) was added CeCl.sub.3(H.sub.2O).sub.7 (102 g, 274
mmol). The mixture was stirred for 1.5 h at room temperature under
N.sub.2 and then cooled to -30.degree. C. with an ethylene
glycol/H.sub.2O (1:2 w/w) dry ice bath. After the temperature was
equilibrated to -30.degree. C., NaBH.sub.4 (21.3 g, 563 mmol) was
added and stirred at the same temperature for 8.5 h, at which time
the reaction was quenched with H.sub.2O (2 L) and extracted with
ethyl acetate (2 L.times.3). The extracts were combined, washed
with brine (2 L) and dried over Na.sub.2SO.sub.4. The crude product
obtained by removal of solvent under reduced pressure was passed
through a short silica gel column to provide 53 g of mixture which
contained 68% of (.+-.)-calanolide A, 14% of calanolide B and 13%
of chromanone (.+-.)-7 as shown by HPLC. This material was
subjected to further purification by preparative HPLC to afford
pure (.+-.)-calanolide A (1).
EXAMPLE 13
[0191] Chromatographic Resolution of Synthetic (.+-.)-Calanolide
A
[0192] The synthetic (.+-.)-1 was resolved into enantiomers,
(+)-calanolide A and (-)-calanolide A, by preparative HPLC.sup.16.
Thus, using a normal phase silica gel HPLC column (250 mm.times.4.6
mm I. D. Zorbasil, 5 .mu.m particle size, MAC-MOD Analytical, Inc.,
PA, USA), the synthetic (.+-.)-1 appeared as one peak with a
retention time of 10.15 minutes when hexane/ethyl acetate (70:30)
was used as the mobile phase at a flow rate of 1.5 mL/min and a
wavelength of 290 nm was used as the uv detector setting. However,
on a chiral HPLC column packed with amylose carbamate (250
mm.times.4.6 mm I. D. Chiralpak AD, 10 .mu.m particle size, Chiral
Technologies, Inc., PA, USA), two peaks with retention times of
6.39 and 7.15 minutes in a ratio of 1:1 were observed at a flow
rate of 1.5 mL/min. The mobile phase was hexane/ethanol (95:5) and
the uv detector was set at a wavelength of 254 nm. These two
components were separated using a semi-preparative chiral HPLC
column, providing the pure enantiomers of calanolide A. The
chemical structures of the separated enantiomers, which were
assigned based on their optical rotations and compared with the
reported natural product, were characterized by spectroscopic data.
HPLC chromatograms of (.+-.)-calanolide A and its optical forms are
shown in FIG. 6.
[0193] (+)-Calanolide A (1): mp 47-50.degree. C. (Lit..sup.17
45-48.degree. C.);[.alpha.].sup.25.sub.D=+68.8.degree. (CHCl.sub.3,
c 0.7) (Lit..sup.17 [.alpha.].sup.25.sub.D+66.60) (CHCl.sub.3; c
0.5); .sup.1H NMR (CDCl.sub.3) .delta.1.03 (3H, t, J=7.3 Hz,
CH.sub.3), 1.15 (3H, d, J=6.8 Hz, CH.sub.3), 1.46 (3H, d, J=6.4 Hz,
CH.sub.3), 1.47 (3H, s, CH.sub.3), 1.51 (3H, s, CH.sub.3), 1.66
(2H, m, CH.sub.2), 1.93 (1H, m, H.sub.11), 2.89 (2H, m, CH.sub.2),
3.52 (1H, d, J=2.9 Hz, OH), 3.93 (1H, m, H.sub.10), 4.72 (1H, dd,
J=7.8 Hz, J=2.7 Hz, H.sub.12), 5.54 (1H, d, J=9.9 Hz, H.sub.7),
5.94 (1H, s, H.sub.3), 6.62 (1H, d, J=9.9 Hz, H.sub.8); .sup.13C
NMR (CDCl.sub.3) 13.99 (CH.sub.3), 15.10 (CH.sub.3), 18.93
(CH.sub.3), 23.26 (CH.sub.2), 27.38 (CH.sub.3), 28.02 (CH.sub.3),
38.66 (CH.sub.2), 40.42 (CH), 67.19 (CH-OH), 77.15 (CH-O), 77.67
(C-O), 104.04 (C.sub.4a), 106.36 (C.sub.8a and C.sub.12a), 110.14
(C.sub.3), 116.51 (C8), 126.97 (C.sub.7), 151.14 (C.sub.4b), 153.10
(C.sub.8b), 154.50 (C.sub.12b), 158.88 (C.sub.4), 160.42 (C=O);
CIMS: 371 (100, M+1), 370 (23.6,M.sup.+), 353 (66.2, M-OH); 1R:
3611 (w) and 3426 (m, broad, OH), 1734 (vs. C=O), 1643 (m), 1606
(m) and 1587 (vs) cm.sup.-1; UV .lambda..sub.max (methanol): 204
(32,100), 228 (23,200), 283 (22,200), 325 (12,700) nm; Anal. calcd.
for C.sub.22H.sub.26o.sub.5.1/4H.sub.2O: C, 70.47; H, 7.12; Found:
C, 70.64; H, 7.12.
[0194] (-)-Calanolide A (1): mp 47-50.degree.
C.;[.alpha.].sup.25.sub.D75.- 6.degree. (CHCl.sub.3, c 0.7)
Lit..sup.17[.alpha.].sup.25.sub.D=-66.6.degr- ee. (CHCl.sub.3, c
0.5); .sup.1H NMR (CDCl.sub.3) .delta.1.03 (3H, t, J=7.4 Hz,
CH.sub.3), 1.15 (3H, d, J=6.8 Hz, CH.sub.3), 1.46 (3H, d, J=6.3 Hz,
CH.sub.3), 1.47 (3H, s, CH.sub.3), 1.51 (3H, s, CH.sub.3), 1.66
(2H, m, CH.sub.2), 1.93 (1H, m, H.sub.11), 2.89 (2H, m, CH.sub.2),
3.50 (1H, d, J=2.9 Hz, OH), 3.92 (1H, m, H.sub.10), 4.72 (1H, dd,
J=7.8 Hz, J=2.7 Hz, H.sub.12), 5.54 (1H, d, J=10.0 Hz, H.sub.7),
5.94 (1H, s, H.sub.3), 6.62 (1H, d, J=10.0 Hz, H8); .sup.13C NMR
(CDCl.sub.3) .delta.13.99 (CH.sub.3), 15.10 (CH.sub.3), 18.93
(CH.sub.3), 23.36 (CH.sub.2), 27.38 (CH.sub.3), 28.02 (CH.sub.3),
38.66 (CH.sub.2), 40.42 (CH), 67.19 (CH-OH), 77.15 (CH-O), 77.67
(C-O), 104.04 (C.sub.4a), 106.36 (C.sub.8a and C.sub.12a), 110.14
(C.sub.3), 116.51 (C.sub.8), 126.97 (C.sub.7), 151.14 (C.sub.4b),
153.11 (C.sub.8b), 154.50 (C.sub.12b), 158.90 (C.sub.4), 160.44
(C.dbd.O); CIMS: 371 (95.2, M+1), 370 (41.8,M.sup.+), 353 (100,
M-OH); IR: 3443 (m, broad, OH), 1732 (vs, C=O), 1643 (m), 1606 (m)
and 1584 (vs) cm.sup.-1; UV .lambda..sub.max (methanol): 200
(20,500), 230 (19,400), 283 (22,500), 326 (12,500) nm; Anal. calcd.
for (C.sub.22 H26O.sub.5.1/4H.sub.2O: C, 70.47; H, 7.12; Found: C,
70.27; H, 7.21.
EXAMPLE 14
[0195] Enzymatic Resolution of (.+-.)-Calanolide A
[0196] To a magnetically stirred suspension of (.+-.)-calanolide A,
prepared by the method of the present invention, and vinyl butyrate
(0.1 mL) in hexane (0.5 mL) at ambient temperature was added 1 mg
of lipase PS-13 (Pseudomonas Species) (Sigma Corporations, St.
Louis, Mo., USA). The reaction mixture was stirred and monitored by
conventional means such as TLC analysis. At 10 days, an additional
1 mg of lipase PS-13 was added. After stirring for a total of 20
days, the reaction was stopped because there was no obvious
increase in ester formation. The enzyme was filtered out and the
filtrate was concentrated to dryness. The residue was analyzed by
HPLC (see Example 13), which showed that 21% of (-)-calanolide A
had been converted into its butyrate ester form. The enriched
(+)-calanolide A and the butyrate ester of (-)-calanolide A can be
easily separated by conventional means such as column
chromatography. The enriched (+)-calanolide A may be repeatedly
treated with vinyl butyrate and lipase PS-13 as described above so
as to obtain high e.e. of (+)-calanolide A.
EXAMPLE 15
[0197] Aldol Reaction (Scheme III) of Chromene 4 in the Presence of
LDA
[0198] To a stirring solution of chromene 4 (1.0 g, 2.9 mmol) in
THF (15 mL) at -78.degree. C. under N.sub.2 was added 2 M LDA in
THF (3.2 mL, 6.4 mrnol). After 1 h at the same temperature,
acetaldehyde (1.0 mL, 17.5 mmol) was added via syringe. The
reaction was monitored by TLC analysis. After 1 h, the reaction
mixture was quenched with a precooled 2 N HCl in methanol (15 mL)
and extracted with ethyl acetate (30 mL.times.3). The combined
extracts were washed with brine and dried over Na.sub.2SO.sub.4.
Removal of solvents in vacuo afforded a reddish oil, which was
purified by silica gel column chromatography eluting with a
discontinuous gradient of 5%, 10%, 15%, 25% and 30% of ethyl
acetate in hexane to obtain 780 mg (70% yield) of a mixture of
(.+-.)-8a and (.+-.)-8b in a ratio of 1:1, as indicated by .sup.1H
NMR. Pure samples of (.+-.)-8a and (.+-.)-8b were obtained by
carefully collecting the front fractions and later fractions from
column chromatography, analytical data of which were described
below:
[0199]
6,6-Dimethyl-9-hydroxy-10-[2(S*)-methyl-3(R*)-hydroxybutyro-]4-prop-
yl-2H,6H-benzo[1,2-b:3,4b']dipyran-2-one [syn-(.+-.)8a]. m.p. 66-67
.degree. C.; .sup.1H NMR (CDCl.sub.3): 1.05 (3H, t, J=7.3 Hz,
CH.sub.3), 1.30 (3H, d, J=6.0 Hz, CH.sub.3), 1.33 (3H, d, J=6.6 Hz,
CH.sub.3), 1.54 (6H, s, 2 CH.sub.3), 1.67 (2H, m, CH.sub.2), 2.62
(1H, broad-s, OH), 2.91 (2H, t, J=7.7 Hz, CH.sub.2), 3.98 (1H, dq,
J=2.7 Hz, J=7.0 Hz, H.sub.2'), 4.29 (1H, m, H.sub.3'), 5.59 (1H, d,
J=10.0 Hz, H.sub.7), 6.01 (1H, s, H.sub.3), 6.73 (1H, d, J=10.0 Hz,
H.sub.8), 14.11 (1H, s, OH); 1H NMR (DMSO-d.sub.6): 1.00 (3H, t,
J=7.3 Hz, CH.sub.3), 1.13 (3H, d, J-6.6 Hz, CH.sub.3), 1.16 (3H, d,
J=6.8 Hz, CH.sub.3), 1.49 (3H, s, CH.sub.3), 1.50 (3H, s,
CH.sub.3), 1.60 (2H, apparent sextet, J-7.6 Hz, CH.sub.2), 2.88
(2H, apparent dd, J=6.3 Hz, J=9.0 Hz, CH.sub.2), 3.39 (1H, broad-s,
OH), 3.68 (1H, dq, J=5.2 Hz, J=6.7 Hz, H.sub.2'), 3.97 (1H,
apparent quintet, J=5.8 Hz, H.sub.3'), 5.78 (1H, d, J=10.1 Hz,
H.sub.7), 6.11 (1H, s, H.sub.3), 6.63 (1H, d, J=10.1 Hz, H.sub.8),
13.25 (1H, s, OH); MS (CI): 388 (36.5, M+2), 387 (100, M+1), 386
(6.6, M.sup.+), 369 (21.6, M-OH), 343 (50.7, M-C.sub.3H.sub.7); UV
.lambda..sub.max (methanol) nm: 199 (41,000), 270 (25,700), 306
(21,900); IR (KBr) cmn.sup.-1: 3395 (broad, m, OH), 1734 (s) and
1707 (vs) (C=O), 1644 (m), 1608 (vs), 1578 (vs) and 1547 (vs);
Alial. Calcd. for C.sub.22H.sub.26O.sub.6. 1/3H.sub.2O: C, 67.33;
H, 6.84; Found: C, 67.43; H, 6.93.
[0200]
6,6-Dimethyl-9-hydroxy-10-12(S*)-methyl-3(S*)-hydroxybutyro]4-propy-
l-2H,6H-benzo[1,2-b:3,4-b']dipyran-2-one [anti-(.+-.)-8b]. m.p.
115.degree. C.; .sup.1H NMR (CDCl.sub.3): 1.05 (3H, t, J=7.4 Hz,
CH.sub.3), 1.25 (3H, d, J=6.4 Hz, CH.sub.3), 1.29 (3H, d, J=6.9 Hz,
CH.sub.3), 1.54 (6H, s, 2 CH.sub.3), 1.66 (2H, apparent sextet,
J=7.6 Hz, CH.sub.2), 2.92 (2H, t, J=7.8 Hz, CH.sub.2), 2.95 (1H, d,
J=5.5 Hz, OH), 3.98 (1H, dq, J=6.1 Hz, J=6.8 Hz, H.sub.2), 4.22
(1H, apparent sextet, J=6.2 Hz, H.sub.3), 5.59 (1H, d, J=10.1 Hz,
H.sub.7), 6.03 (1H, s, H.sub.3), 6.73 (1H, d, J=10.1 Hz, H.sub.8),
14.25 (1H, s, OH); .sup.1H NMR (DMSO-d.sub.6): 1.00 (3H, t, J=7.3
Hz, CH.sub.3), 1.11 (6H, d, J=6.7 Hz, 2 CH.sub.3), 1.49 (3H, s,
CH.sub.3), 1.50 (3H, s, CH.sub.3), 1.60 (2H, apparent sextet, J=7.3
Hz, CH.sub.2), 2.85, 2.90 (2H, t-AB type, J=7.7 Hz, J.sub.AB=21.4
Hz, CH.sub.2'), 3.59 (1H, apparent quintet, J=7.1 Hz, H.sub.2),
3.96 (1H, apparent quintet, J=7.0 Hz, H3'), 4.97 (1H, broad-s, OH),
5.78 (1H, d, J=10.1 Hz, H.sub.7), 6.10 (1H, s, H.sub.3), 6.63 (1H,
d, J=10.0 Hz, H.sub.8), 12.69 (1H, s, OH); MS (EI): 387 (2.8, M+1),
386 (9.4, M.sup.+), 371 (5.3, M-CH.sub.3), 369 (1.5, M-OH), 353
(54.0, M-CH.sub.3-H.sub.2O), 342 (22.5, M-C.sub.3H.sub.7-1), 327
(100, M-C.sub.3H.sub.7-OH+1); UV .lambda..sub.max (methanol) nm:
199 (41,000), 270 (25,700), 306 (21,900); IR (KBr) cm.sup.-1: 3478
(broad, m, OH), 1736 (vs) and 1707 (vs) (C=O), 1645 (m), 1603 (vs),
1584 (vs, sh); Anal. Calcd. for
C.sub.22H.sub.26O.sub.6.1/3H.sub.2O: C, 67.33; H, 6.84; Found: C,
67.34; H, 6.45.
EXAMPLE 16
[0201] Aldol Reaction (Scheme III) of Chromene 4 in the Presence of
LDA/TiCL.sub.4
[0202] In this Example, two procedures are provided for effecting
the Aldol reaction. Procedure B was found to be more suitable for
scale-up because of simplification of temperature control.
[0203] Procedure A. To a stirring solution of chromene 4 (200 mg,
0.58 mmol) in dry methylene chloride (10 mL) at -78.degree. C.
under N.sub.2 was added 2M solution of LDA in heptane/THF/ethyl
benzene (0.64 mL, 1.28 mmol). The reaction mixture was stirred at
-78.degree. C. for 30 min and then TiCI.sub.4 (0.13 mL, 1.17 mmol)
was added. The resulting yellow solution was warmed to -40.degree.
C. and stirred for 45 min. The mixture was recooled to -78.degree.
C., and acetaldehyde (150 mg, 3.5 mmol) was added via syringe.
After 4 h, the reaction was quenched by slow addition of pre-cooled
saturated NH.sub.4Cl (10 mL). Water (3 mL) was added to dissolve
the oily solid. The mixture was extracted with ethyl acetate (50
mL.times.3). The combined extracts were washed with brine (100 mL)
and dried over MgSO.sub.4. The crude product obtained by
evaporation was purified by silica gel column chromatography,
eluting with hexane/ethyl acetate (5:1) to afford unreacted
chromene 4 (30 mg, 15% yield) and syn-(.+-.)-8a (140 mg, 61%
yield), which contained 7% of anti-(.+-.)-8b as shown by HPLC.
[0204] Procedure B. To a stirring solution of chromene 4 (20 g,
58.4 nmuol) in dry methylene chloride (300 mL) at -40.degree. C.
under N.sub.2 was added TiCl.sub.4 (19 mL, 175 mmol). The mixture
was then cooled to -78.degree. C., followed by slow addition of 2M
solution of LDA in heptane/THF/ethyl benzene (64 mL, 128 mmol).
After 30 min at the same temperature, acetaldehyde (9 mL, 175 mmol)
was added via syringe. The reaction mixture was stirred at
-78.degree. C. for 2 h. TLC analysis (hexane/ethyl acetate, 5:1)
indicated that approximately 90% chromene 4 had been converted. The
mixture was then poured into pre-cooled saturated NH.sub.4Cl (240
mnL). Water (120 mL) was added to dissolve the oily solid and the
mixture was stirred for 20 min. Layers were separated and the
aqueous solution was extracted with ethyl acetate (600 mL.times.3).
The combined extracts were washed with brine (600 mL) and dried
over MgSO.sub.4. Removal of solvents in vacuo afforded a reddish
oil (23 g), which was taken up into ether (250 mL). The undissolved
residue was filtered and the etheral solution was concentrated to
half volume and then slowly added into rapidly stirring hexane
cooled at -78.degree. C. Precipitates thus formed were collected by
filtration to afford syn-(.+-.)-8a (11.1 g, 49% yield), which
contained 4% of (.+-.)-8b as shown by HPLC.
EXAMPLE 17
[0205] Enzymatic Resolution of syn-(.+-.)-8a (Scheme III)
[0206] Into a stirring solution of syn-(.+-.)-8a (7.6 g, 19.7 mmol)
in tert-butyl methyl ether (130 mL) at ambient temperature under
N.sub.2 were added successively vinyl acetate (33 mL), 4 A
molecular sieves (17 g) and Lipase PS-30 (3.8 g) (Amano Enzyme
U.S.A. Co., Ltd., Troy, Va.). The resulting mixture was vigorously
stirred at ambient temperature for 4 days, whereupon it was
filtered through celite and the celite was washed with ethyl
acetate (20 mL). The crude product obtained from evaporation was
subjected to silica gel column chromatography eluting with a
discontinuous gradient of 5%, 10%, 15%, 25%, 30% and 40% of ethyl
acetate in hexane to afford 4.8 g (63% yield) of the acetate (9),
which was contaminated by over-acylation product of (+)-8a, and 2.8
g (37% yield) of pure syn-(+)-8a.
[0207] 6,6-Dimethyl-9-hydroxy-10-[2(R)-methyl-3(S)-hydroxybutyro
]4-propyl-2H,6H-benzo[1,2-b:3,4-b']dipyran-2-one [syn-(+)-8a]. m.p.
82-85.degree. C.; [.alpha.].sup.5.sub.D=0.degree. (CHCl.sub.3, c
0.7; [.alpha.].sup.25.sub.D=0.degree. (CHCl.sub.3, c 0.35); .sup.1H
NMR (CDCl.sub.3): 1.05 (3H, t, J=7.4 Hz, CH.sub.3), 1.31 (3H, d,
J=5.6 HZ, CH.sub.3), 1.33 (3H, d, J=6.9 Hz, CH.sub.3), 1.54 (6H, s,
2 CH.sub.3), 1.67 (2H, apparent sextet, J=7.6 Hz, CH.sub.2), 2.75
(1H, broad-s, OH), 2.91 (2H, t, J=7.8 Hz, CH.sub.2), 3.98 (1H, dq,
J=2.7 Hz, J=7.0 Hz, H.sub.2'), 4.30 (1H, dq, J=2.7 Hz, J=6.5 Hz,
H.sub.3'), 5.59 (1H, d, J=10.2 Hz, H.sub.7), 6.01 (1H, s, H.sub.3),
6.72 (1H, d, J=10.3 Hz, H.sub.8), 14.10 (1H, s, OH); .sup.13C NMR
(CDCl.sub.3); 10.42 (CH.sub.3), 14.00 (CH.sub.3), 20.61 (CH.sub.3),
23.32 (CH.sub.2), 28.31 (2 CH.sub.3), 39.05 (CH.sub.2), 50.93
(CHCO), 68.03 (CH-O), 79.92 (C-O), 102.95 (C.sub.8a), 103.69
(C.sub.4a), 106.12 (C.sub.10), 110.60 (C.sub.3), 115.80 (C.sub.8),
126.51 (C.sub.7), 157.03 and 157.11 (C.sub.9 and C.sub.10), 158.58
(C.sub.4b), 159.01 (C.sub.4), 163.13 (CO.sub.2), 210.61 (C=O); MS
(CI): 388 (33.4, M+2), 387 (100, M+1), 386 (8.5, M.sup.+), 369
(36.3, M-OH), 343 (97.2, M-C.sub.3H.sub.7); Anal. calcd. for
C.sub.22H.sub.26O.sub.6: C, 68.38; H, 6.78; Found: C, 68.02; H,
6.62.
EXAMPLE 18
[0208]
10(R),11(R)-trans-Dihydro6,6,10,11-tetramethyl-4-propyl-2H,6H,12H-b-
enzo [1,2-b:3,4-b':5,6-b"]tripyran-2,12-dione [Scheme III,
(+)-7]
[0209] Into a stirring solution of syn-(+)-8a (2.0 g, 5.2 mmol) in
THF (50 mL) were added triphenylphosphine (1.9 g, 7.2 mmol) and
diethyl azodicarboxylate (DEAD, 1.2 mL, 7.6 mmol). The resulting
reddish solution was stirred at ambient temperature under N.sub.2
for 5 h, after which the reaction mixture was quenched with
saturated aqueous NH.sub.4Cl (20 mL) and extracted with ethyl
acetate (50 mL.times.3). The combined extracts were washed with
brine (50 mL) and dried over Na.sub.2SO.sub.4. The crude product
(5.8 g) obtained by evaporation was purified by column
chromatography on silica gel eluting with a discontinuous gradient
of 10%, 20%, 30% and 40% of ethyl acetate in hexane to afford 1.2 g
(63% yield) of pure (+)-7. mp 171-175.degree. C.;
[.alpha.].sup.25.sub.D=+37.9- 0 (CHCl.sub.3, c 0.73); .sup.1H NMR
[CDCl.sub.3/CD.sub.3OD (3:1)]: 1.06 (3H, t, J=7.3 Hz, CH.sub.3),
1.22 (3H, d, J=7.0 Hz, CH.sub.3), 1.54 (3H, s, CH.sub.3), 1.57 (3H,
d, J=6.0 Hz, CH.sub.3), 1.58 (3H, s, CH.sub.3), 1.67 (2H, apparent
sextet, J=7.6 Hz, CH.sub.2), 2.59 (1H, dq, J=6.9 Hz, J=11.1 Hz,
H.sub.11), 2.92 (2H, t, J=7.8 Hz, CH.sub.2), 4.37 (1H, dq, J=6.3
Hz, J=11.1 Hz, H.sub.10), 5.66 (1H, d, J=10.1 Hz, H.sub.7), 6.05
(1H, s, H.sub.3), 6.67 (1H, d, J=10.1 Hz, H,); .sup.13C NMR
[CDCl.sub.3/CD.sub.3OD (3:1)]: .delta.9.87 (CH.sub.3), 13.34
(CH.sub.3), 18.97 (CH.sub.3), 22.85 (CH.sub.2), 27.40 and 27.73 (2
CH.sub.3), 38.38 (CH.sub.2), 46.82 (CHCO), 79.17 (CH-O and C-O),
102.91 (C.sub.8a), 104.11 (C.sub.4a), 105.46 (C.sub.12a), 111.09
(C.sub.3), 115.21 (C.sub.8), 126.90 (C.sub.7), 154.83 and 155.86
(C.sub.8b and C.sub.12b), 157.89 (C.sub.4b), 158.99 (C.sub.4),
160.27 (CO.sub.2), 190.50 (C=O); MS (CI): 370 (49.0, M+2), 369
(100, M+1), 368 (17.2, M.sup.+); Anal. Calcd. for
C.sub.22H.sub.24O.sub.5C, 71.72; H, 6.57; Found: C, 71.46; H,
6.60.
[0210] (+)-Calanolide A: To a stirring solution of (+)-7 (660 mg,
1.79 mmol) in ethanol (18 mL) were added CeCl.sub.3(H.sub.2O).sub.7
(2.7 g, 7.17 mmol) and triphenylphosphine oxide (2.0 g, 7.17 mmol).
The mixture was stirred for 1 h at ambient temperature under
N.sub.2 and then cooled to -30.degree. C. with an ethylene
glycol/H.sub.2O (1:2 w/w) dry ice bath. After the temperature was
equilibrated to -30.degree. C., NaBH.sub.4 (271 mg, 7.17 mmol) was
added and stirred at the same temperature for 5.5 h, at which time
the reaction was quenched with saturated NH.sub.4Cl (20 mL) and
extracted with ethyl acetate (30 mL.times.3). The combined extracts
were washed with brine (50 mL) and dried over Na.sub.2SO.sub.4. The
crude product obtained by removal of solvent under reduced pressure
was purified by column chromatography on silica gel eluting with
20% of ethyl acetate in hexane to afford 520 mg (78% yield) of a
mixture containing 90% of (+)-calanolide A [(+)-1] and 10% of
(+)-calanolide B. (+)-Calanolide A [(+)-1] was further separated
from (+)-calanolide B by normal phase HPLC and was identical with
an authentic sample.
EXAMPLE 19
[0211] Enzymatic Resolution (Scheme IV) of anti-(.+-.)-8b
[0212] Into a stirring solution of anti-(.+-.)-8b (3.0 g, 7.8 mmol)
in tert-butyl methyl ether (78 mL) at ambient temperature under
N.sub.2 were added successively vinyl acetate (26 mL), 4 A
molecular sieves (3.0 g) and Lipase PS-30 (1.5 g) (Amano Enzyme
U.S.A. Co., Ltd., Troy, Va.). The resulting mixture was vigorously
stirred at ambient temperature for 41 h, whereupon it was filtered
through the celite and the celite was washed with ethyl acetate (20
mL). The crude yellowish solid product (3.2 g) obtained from
evaporation was purified by silica gel column chromatography
eluting with a discontinuous gradient of 5%, 10%, 15%, 25%, 30% and
40% of ethyl acetate in hexane to afford 1.68 g (50% yield) of the
acetate (10) and 1.37 g (46% yield) of anti-(+)-8b.
[0213] 6,6-Dimethyl-9-hydroxy-10-[2(S)-methyl-3(S)-hydroxybutyro
]-4-propyl-2H,6H-benz[1,2-b:3,4-b']dipyran-2-one [anti-(+)-8b].
m.p. 131-134.degree. C.; [.alpha.].sup.25.sub.D=+45.3.degree.
(CHCl.sub.3, c 0.72); .sup.1H NMR (CDCl.sub.3): 1.06 (3H, t, J=7.3
Hz, CH.sub.3), 1.25 (3H, d, J=6.6 Hz, CH.sub.3), 1.29 (3H, d, J=6.7
Hz, CH.sub.3), 1.55 (6H, s, 2 CH.sub.3), 1.67 (2H, apparent sextet,
J=7.6 Hz, CH.sub.2), 2.92 (2H, t, J=7.8 Hz, CH.sub.2), 2.96 (1H, d,
J=7.1 Hz, OH), 3.98 (1H, apparent quintet, J=6.1 Hz, H.sub.2'),
4.22 (1H, apparent sextet, J=6.0 Hz, H.sub.3'), 5.60 (1H, d, J=10.1
Hz, H.sub.7), 6.03 (1H, s, H.sub.3), 6.73 (1H, d, J=10.1 Hz,
H.sub.8), 14.25 (1H, s, OH); MS (CI): 388 (41.4, M+2), 387 (100,
M+1), 386 (13.0, M.sup.+), 369 (42.8, M-OH), 343 (63.8,
M-C.sub.3H.sub.9); Anal. calcd. for C.sub.22H.sub.26O.sub.6: C,
68.38; H, 6.78; Found: C, 68.50; H, 6.91.
[0214] 6,6Dimethyl-9-hydroxy-10-[2(R)-methyl-3(R)-acetoxybutyro
]-4-propyl-2H,6H-benzo-[1,2-b:3,4-b']dipyran-2-one [anti-(+)-10].
m.p. 61-64.degree. C.; [.alpha.].sup.25.sub.D=+30.0.degree.
(CHCl.sub.3, c 0.73); .sup.1H NMR (CDCl.sub.3): 1.06 (3H, t, J=7.2
Hz, CH.sub.3), 1.29 (3H, d, J=6.2 Hz, CH.sub.3), 1.32 (3H, d, J=6.7
Hz, CH.sub.3), 1.54 (6H, s, 2 CH.sub.3), 1.67 (2H, apparent sextet,
J=7.6 Hz, CH.sub.2), 1.93 (3H, s, CH.sub.3CO), 2.91 (2H, m,
CH.sub.2), 4.18 (1H, dq, J=8.3 Hz, J=6.9 Hz, H.sub.2'), 5.34 (1H,
dq, J=8.2 Hz, J=6.4 Hz, H.sub.3'), 5.59 (1H, d, J=10.1 Hz,
H.sub.7), 6.02 (1H, s, H.sub.3), 6.73 (1H, d, J=10.1 Hz, H.sub.8),
14.02 (1H, s, OH); MS (CI): 430 (37.1, M+2), 429 (95.2, M+1), 428
(7.2, M.sup.+), 369 (100, M-AcO); Anal. calcd. for
C.sub.24H.sub.28O.sub.7: C, 67.28; H, 6.59; Found: C, 67.75: H,
6.90.
EXAMPLE 20
[0215] 5,7-Dihydroxy-4-trifluoromethylcoumarin (Scheme V, 11a,
R.sub.1=CF.sub.3, R.sub.2=H)
[0216] Into a mixture of anhydrous phloroglucinol (8 g, 63.0 mmol)
and ethyl 4,4,4-trifluoroacetoacetate (12 g, 65.0 mmol) was added
concentrated H.sub.2SO.sub.4 (11 mL). The resulting mixture was
heated at 100.degree. C. and stirred for 2 h, whereupon the
reaction mixture was cooled to room temperature. Ice (100 g) and
H.sub.20 (150 mL) were then added while cooling with ice bath. The
precipitated product was collected and dissolved in AcOEt (100 mL),
which was washed with H.sub.2O and dried over Na.sub.2SO.sub.4. The
crude product (16 g) obtained by evaporation under vacuum was
chromatographed in methylene chloride-ethanol (95:5) to furnish 11a
(6 g, 39% yield) along with another unidentified product. lha: mp
250-252.degree. C. after recrystallization from methylene
chloride-hexane. .sup.1H NMR (DMSO-d.sub.6): 6.30 (1H, s, H.sub.3),
6.33 and 6.54 (2H, 2 s, H.sub.7 and H.sub.8), 10.68 and 10.99 (2H,
2 s, 2 OH); MS (CI) m/z: 246 (100, M.sup.+), 226 (14.6, M-HF), 218
(10.0, M-CO), 198 (59.6, M-HF-CO); IR (KBr) cm.sup.-1: 3537(m, sh)
and 3384 (s, broad, OH), 1709 (s, C.dbd.O), 1618 (s,
C.dbd.C--C.dbd.O), 1154 (s, C-F); Anal. Calcd. for
C.sub.10,h.sub.5,O.sub.4: C, 48.80; H, 2.05; Found, C, 48.83; H,
2.10.
EXAMPLE 21
[0217] 5,7-Dihydroxy-8isobutyryl4ipropylcoumarin (Scheme V, 12a,
R.sub.1=n-Pr, R.sub.2=H, R.sub.3=R.sub.4=Me)
[0218] Into a flame-dried 500 mL 3-necked round-bottom flask was
placed 5,7-dihydroxy-4-propylcoumarin (2, 10.0 g, 48.1 mmol) and
AlCl.sub.3 (12.0 g, 90 mmol) under N.sub.2. Dichloroethane (120 mL)
was then added, and the solution warmed to 75.degree. C. with a
water bath with mechanical stirring. After stirring 15 min at
75.degree. C., a homogenous solution was obtained. To this solution
was added a mixture of isobutyric anhydride (7.61 g, 48.1 mmol) and
AlCl.sub.3 (12.0 g) in dichloroethane (60 mL) dropwise over 1 h.
After addition was completed, the solution was stirred for an
additional 1 h at 75.degree. C., then cooled to room temperature.
The solution was poured into a mixture of crushed ice (100 g) and 2
N HCl (100 mL), at which point a white precipitate formed. The
mixture was diluted with ethyl acetate (1.8 L), and the organic
layer separated. The organic solution was washed sequentially with
1 N HCl (500 mL) and saturated brine (500 mL), dried over magnesium
sulfate, filtered and evaporated to provide an orange powder. The
powder was triturated with acetone (80 mL), collected on a Buichner
fimel, rinsed with diethyl ether (80 mL) and dried to provide a
cream colored solid (4.22 g). The product was further purified via
recrystallization from ethanol (200 mL) to give colorless plates
(3.63 g, 26.0%); mp 263-265.degree. C., with softening at
250.degree. C. (Lit. .sup.5 272-273.degree. C.); .sup.1H NMR
(DMSO-d.sub.6): 0.95 (3H, t, J=7.4 Hz, CH.sub.3), 1.08 (6H, d,
J=6.9 Hz, 2 CH.sub.3), 1.59 (2H, sextet, J=7.4 Hz, CH.sub.2), 2.87
(2H, t, J=7.4 Hz, CH.sub.2), 3.24 (1H, heptet, J=6.9 Hz, CH), 5.93
(1H, s, H.sub.3), 6.37 (1H, s, H6), 11.16 and 11.44 (2H, 2 s, 2
OH); EIMS: 290 (23.2, M.sup.+), 247 (100, M-C.sub.3H.sub.7), 219
(11.1, M-C.sub.3H.sub.7CO); IR (KBr) cm.sup.-1: 3216 (s, OH), 1684
(s, C.dbd.O); Anal. calcd. for C.sub.16H.sub.18O.sub.5: C, 66.20;
H, 6.25. Found: C, 66.15; H, 6.21.
EXAMPLE 22
[0219] 6,6-Dimethyl-9-hydroxy-10-isobutyryl-4-propyl-2H,6H-benzo
[1,2-b:3,4-b']dipyran-2-one (Scheme V, 13a, R.sub.1=n-Pr,
R.sub.2=R.sub.7=H, R.sub.3=R.sub.4=R.sub.5=Me)
[0220] To a solution of 12a (2.90 g, 10.0 mmol) in pyridine (5 mL)
was added 4,4-dimethoxy-2-methylbutan-2-ol (1.49 g, 10.1 mmol), and
the solution heated to reflux. After heating for 40 h, TLC
indicated complete consumption of starting material. The reaction
was cooled to room temperature and the pyridine removed in vacuo.
The dark colored residue was dissolved in ethyl acetate (50 mL) and
washed sequentially with 2 N HCl (50 mL.times.2), 5% NaHCO.sub.3
(50 mL) and saturated brine (50 mL). The solution was dried over
magnesium sulfate, filtered and evaporated to provide a dark orange
solid, which was chromatographed on a silica gel column (125 g) and
eluted with ethyl acetate/hexane (1:4) to afford the pure product
as a bright orange crystalline solid (2.51 g, 70.5%/); mp
70-72.degree. C.; .sup.1H NMR (CDCl.sub.3): 1.05 (3H, t, J=7.3 Hz,
CH.sub.3), 1.26 (6H, d, J=6.7 Hz, 2 CH.sub.3), 1.54 (6H, s, 2
CH.sub.3), 1.66 (2H, sextet, J=7.7 Hz, CH.sub.2), 2.91 (2H, t,
J=7.7 Hz, CH.sub.2), 4.06 (1H, heptet, J=6.7 Hz, CH), 5.58 (1H, d,
J=9.9 Hz, H.sub.7), 6.01 (1H, s, H.sub.3), 6.73 (1H, d, J=9.9 Hz,
H.sub.8), 14.45 (1H, s, OH); EIMS: 356 (48.0, M.sup.+), 341 (100,
M-CH.sub.3), 313 (65.0, M-C.sub.3H.sub.7); IR (KBr) cm.sup.-1:
1732; Anal. calcd. for C.sub.21H.sub.24O.sub.5: C, 70.77; H, 6.79.
Found: C, 70.73; H, 6.78.
EXAMPLE 23
[0221]
(.+-.)-6,6-Dimethyl-10-(2,2-dimethyl-3-hydroxybutyro)-9-hydroxy-4-p-
ropyl-2H,6H-benzo[1,2-b:3,4-b']dipyran-2-one (Scheme V, 14a,
R.sub.1=n-Pr, R.sub.2=R.sub.7=R.sub.8=H,
R.sub.3=R.sub.4=R.sub.5=R.sub.6=R.sub.9=Me)
[0222] To a solution of 13a (1.25 g, 3.51 nunol) in anhydrous THF
(20 mL) under N.sub.2 at -78.degree. C. was added LDA (2.0 M in
heptane/THF/ethyl benzene, 4.39 mL, 8.78 mmol) dropwise, and the
resulting red solution stirred for 1 h. A solution of acetaldehyde
(1.54 g, 35.1 mmol) in THF (6 mL) was added dropwise, and the
reaction mixture stirred at -78.degree. C. for 3 hours whereupon
the reaction was quenched by slowly adding 2.5 M ethanolic HCl (10
mL), and the solution then allowed to warm to room temperature. The
solvent was evaporated in vacuo and the residue partitioned between
ethyl acetate (100 mL) and saturated NaHCO.sub.3 (100 mL). The
organic layer was collected and washed with saturated brine (100
mL), dried over magnesium sulfate, filtered and evaporated to
provide a brown solid. The product was triturated with ethyl
acetate/hexane (1:1, 15 mL), collected on a Buichner funnel, rinsed
with fresh solvent and air dried to give the desired product as a
white powder (654 mg, 46.6%). An analytical sample was obtained via
recrystallization from ethyl acetate/hexane (1:1); mp
190-191.degree. C.; .sup.1H NMR (CDCl.sub.3): 1.04 (3H, t, J=7.4
Hz, CH.sub.3), 1.25 (3H, s, CH.sub.3), 1.29 (3H, d, J=6.4 Hz,
CH.sub.3), 1.33 (3H, s, CH.sub.3), 1.48 (3H, s, CH.sub.3), 1.52
(3H, s, CH.sub.3), 1.66 (2H, sextet, J=7.5 Hz, CH.sub.2), 2.39 (1H,
broad-s, OH), 2.88 (m, 2H, CH.sub.2), 4.47 (1H, q, J=6.4 Hz, CH),
5.56 (1H, d, J=10.0 Hz, H.sub.7), 5.92 (1H, s, H.sub.3), 6.64 (1H,
d, J=10.0 Hz, H.sub.8), 8.99 (1H, s, OH): EIMS: 400 (1.1, M.sup.+),
356 (37.5, M- C.sub.2H.sub.4O), 341 (100,
M-CH.sub.3-C.sub.2H.sub.4O), 313 (68.2,
M-C.sub.3H.sub.7-C.sub.240); IR (KBr) cm.sup.-1: 3246 (broad-s,
OH), 1686 (s, C=O); Anal. calcd. for C.sub.23H.sub.280.sub.6: C,
68.98; H, 7.05. Found. C, 69.03; H, 6.99.
EXAMPLE 24
[0223]
(.+-.)-6.6-Dimethyl-10-(2,3-dimethyl-3-hydroxybutyro)-9-hydroxy4-pr-
opyl-2H,6H-benzo[1,2-b:3,4-b']dipyran-2-one (Scheme V, 14b,
R.sub.1=n-Pr, R.sub.2=R.sub.3=R.sub.7=H,
R.sub.4=R.sub.5=R.sub.6=R.sub.8=R.sub.9=Me)
[0224] To a suspension of 4 (1.2 g, 3.50 mmol) in THF (16 mL) at
-78.degree. C. was added a solution of LDA in heptane/THF/ethyl
benzene (2 M, 5.0 mL, 10.0 mmol) dropwise under N.sub.2. The
solution was stirred at -78.degree. C. for 1 h and acetone (2.0 mL,
27.2 mmol) was added quickly via syringe. The solution was stirred
at -78 .degree. C. for 3 h, quenched with methanolic HCl (2M, 15
mL) at -78.degree. C., then allowed to warm to room temperature.
The reaction mixture was concentrated and partitioned between ethyl
acetate (150 mL) and saturated NaHCO.sub.3 (100 mL). The organic
layer was collected and washed with saturated brine (50 mL), dried
over magnesium sulfate, filtered and concentrated to provide a red
oil (1.36 g), an analytical sample of which was obtained via silica
gel column chromatography (ethyl acetate/hexane, 1:4) as an
off-white solid: mp 99-102.degree. C.; .sup.1H NMR (CDCl.sub.3):
1.05 (3H, t, J=7.3 Hz, CH.sub.3), 1.29 (3H, s, CH.sub.3), 1.32 (3H,
s, CH.sub.3), 1.39 (3H, d, J=6.8 Hz, CH.sub.3), 1.55 (6H, s, 2
CH.sub.3), 1.67 (2H, sextet, J=7.7 Hz, CH.sub.2), 2.91 (2H, t,
J=7.7 Hz, CH.sub.2), 3.52 (1H, broad-s, OH), 4.03 (1H, q, J=6.8 Hz,
CH), 5.60 (1H, d, J=9.9 Hz, H.sub.7), 6.03 (1H, s, H.sub.3), 6.73
(1H, d, J=10.1 Hz, H.sub.8), 13.81 (1H, s, OH); EIMS: 401 (5.1,
M+1), 400 (21.5, M.sup.+), 385 (6.2, M-CH.sub.3), 342 (38.9,
M-C.sub.3H.sub.7O+1), 327 (100, M-CH.sub.3-C.sub.3H.sub.7O+1); IR
(KBr) cm.sup.-1 : 3547 (w, OH), 3449 (vw, broad, OH), 1734 (vs,
C.dbd.O); Anal. calcd. for C.sub.23H.sub.28O.sub.6: C, 68.98; H,
7.04. Found: C, 68.98; H, 7.04.
EXAMPLE 25
[0225] (.+-.)-syn and
(.+-.)-anti-6,6-Dimethyl-9-hydroxy-10-(2-methyl-3-hy-
droxypentanoyl)-4propyl-2H,6H-benzo [1,2-b:3,4-b']dipyran-2-one
(Scheme V, 14c, R.sub.1=n=]Pr, R.sub.2=R.sub.3=R.sub.7=R.sub.8=H,
R.sub.4=R.sub.5=R.sub.6=Me, R.sub.9=Et)
[0226] To a solution of 4 (1.75 g, 5.11 mmol) in THF (27.0 mL) at
-78.degree. C. was added dropwise a solution of LDA in
heptane/THF/ethyl benzene (2 M, 7.0 mL, 14.0 mmol) under N.sub.2.
The solution was stirred at -78.degree. C. for 1 h, and
propionaldehyde (2.2 mL, 31.2 mmol) was added quickly via syringe.
The solution was stirred at -78.degree. C. for 3 h, quenched with
methanolic HCl (2 M, 25 mL) at -78.degree. C., then warmed to room
temperature. The mixture was extracted with ethyl acetate (350 mL),
washed sequentially with 150 mL each of saturated NaHCO.sub.3 and
saturated brine, dried over magnesium sulfate, filtered and
concentrated to provide a diastereomeric mixture of the product as
a red oil (2.44 g, 100%), which was not further purified and used
for the next step.
EXAMPLE 26
[0227]
(.+-.)-10,11-Dihydro-6,6,10,11,11-pentamethyl-4-propyl-2H,6H,
12H-benzo[1,2-b:3,4-b':5,6-b"]tripyran-2,12-dione (Scheme V, 15a,
R.sub.1=n-Pr, R.sub.2=R.sub.7=R.sub.8=H,
R.sub.3=R.sub.4=R.sub.5=R.sub.6=- R.sub.9=Me)
[0228] To a solution of 14a (0.5 g, 1.25 mmol) and
triphenylphosphine (492 mg, 1.88 mmol) in THF (10 mL) was added a
solution of diethyl azodicarboxylate (327 mg, 1.88 mmol) in THF (2
mL) dropwise under N.sub.2. The reaction mixture was stirred for
2.5 h, after which it was poured into saturated NH.sub.4Cl (100
mL). The solution was extracted with ethyl acetate (100 mL), and
the separated organic layer washed sequentially with H.sub.2O (100
mL) and saturated brine (100 mL). After drying over magnesium
sulfate, the solution was filtered and concentrated in vacuo to
provide a yellow oil. Column chromatography through 75 g silica gel
(ethyl acetate/hexane, 1:2) provided the desired product as a white
crystalline solid (449 mg, 94.0%). An analytical sample was
obtained via recrystallization from ethyl acetate/hexane (2:1): mp
157.degree. C.; .sup.1H NMR (CDCl.sub.3): 1.03 (3H, t, J=7.3 Hz,
CH.sub.3), 1.09 (3H, s, CH.sub.3), 1.19 (3H, s, CH.sub.3), 1.43
(3H, d, J=6.5 Hz, CH.sub.3), 1.53 (3H, s, CH.sub.3), 1.55 (3H, s,
CH.sub.3), 1.64 (2H, sextet, J=7.7 Hz, CH.sub.2), 2.88 (2H, t,
J=7.7 Hz, CH.sub.2), 4.34 (1H, q, J=6.4 Hz, H.sub.10), 5.60 (1H, d,
J=10.0 Hz, H.sub.7), 6.04 (1H, s, H.sub.3), 6.66 (1H, d, J=10.0 Hz,
H.sub.8); EIMS: 382 (60.8, M.sup.+), 367 (100, M-CH.sub.3), 312
(50.3 (M-C.sub.5H.sub.10), 297 (74.5, M-CH.sub.3-C.sub.5H.sub.10);
IR (KBr) cm.sup.-1: 1730 (vs, C.dbd.O); Anal. calcd. for
C.sub.23H.sub.26O,: C, 72.23; H, 6.85. Found: C, 72.35; H,
6.90.
EXAMPLE 27
[0229] (.+-.)-10,11-Dihydro-6,6,10,10,11-pentamethyl-4-propyl-2H,
6H, 12H-benzo[1,2-b:3,4-b':5,6-b"]tripyran-2,12-dione (Scheme V,
15b, R.sub.1=n-Pr, R.sub.2=R.sub.3=R.sub.7=H,
R.sub.4=R.sub.5=R.sub.6=R.sub.8=- R.sub.9=RgMe)
[0230] To a solution of crude 14b (980 mg, 2.19 mmol) and
triphenylphosphine (859.0 mg, 3.28 mmol) in TBF (15 mL) was slowly
added diethyl azodicarboxylate (DEAD, 0.50 mL, 3.17 mmol) under
N.sub.2. The red solution was stirred for 2.5 h at room
temperature, then quenched with saturated NH.sub.4Cl (10 mL). The
solution was extracted with ethyl acetate (200 mL), washed
sequentially with 50 mL each of H.sub.2O and saturated brine, dried
over magnesium sulfate, filtered and concentrated to provide a
yellow residue (2.37 g). Purification by silica gel column
chromatography (ethyl acetate/hexane, 1:10) provided, after
overnight drying under high vacuum in the presence of
P.sub.2O.sub.5, the desired product as an off-white solid (373.7
mg, 44.6%): mp 140-141.degree. C.; .sup.1H NMR (CDCl.sub.3): 1.03
(3H, t, J=7.3 Hz, CH.sub.3), 1.19 (3H, d, J=7.0 Hz, CH.sub.3), 1.34
(3H, s, CH.sub.3), 1.53 (6H, s, 2 CH.sub.3), 1.55 (3H, s,
CH.sub.3), 1.65 (2H, sextet, J=7.8 Hz, CH.sub.2), 2.72 (1H, q,
J=7.0 Hz, H.sub.11), 2.85-2.91 (2H, m, CH.sub.2), 5.60 (1H, d,
J=10.1 Hz, H.sub.7), 6.03 (1H, s, H.sub.3), 6.65 (1H, d, J=10.0 Hz,
H.sub.8); EIMS: 382 (61.2, M.sup.+), 367 (82.0, M-CH.sub.3), 312
(46.0, M-C.sub.5H.sub.10), 297 (100, M-CH.sub.3-C.sub.5H.sub.10);
IR (KBr) cm.sup.-1: 1728 (vs, C=O); Anal. calcd. for
C.sub.23H.sub.2601: C, 72.23; H, 6.85. Found: C, 71.95; H,
6.88.
EXAMPLE 28
[0231]
(.+-.)-10,11-trans-10,11-Dihydro-10-ethyl-4-propyl-6,6,11-trimethyl-
-2H,6H,12H-benzo[1,2-b:3,4-b "]tripyran-2,12-dione (15c) and
(.+-.)-10,11-cis-10,11-dihydro-10-ethyl-4-propyl-6,6,11-trimethyl-2H,6H,1-
2H-benzo [1,2-b:3,4-b':5,6-b"]tripyran-2,12-dione (15d, Scheme
V)
[0232] To a solution of 14c (2.44 g, 5.11 mmol) and
triphenylphosphine (1.96 mg, 7.48 mmol) in THF (30.0 mL) was slowly
added diethyl azodicarboxylate (DEAD, 1.16 mL, 7.37 mmol) under
N.sub.2. The red solution was stirred for 2.5 h at room
temperature, then quenched with saturated NH.sub.4Cl (22 mL). The
solution was warmed to room temperature and extracted with ethyl
acetate (400 mL), washed with H.sub.2O (100 mL) and brine (100 mL)
and dried over magnesium sulfate. After filtration, the solution
was concentrated in vacuo to provide a yellow residue (5.75 g). The
crude product was purified by repetitive silica gel column
chromatography (3X) using ethyl acetate/hexane (1:4.5) as eluent.
The desired fractions were combined, concentrated in vacuo and
dried under high vacuum overnight in the presence of P.sub.2O.sub.5
to afford 15c (765.4 mg, 39.2%) and 15d (350.4 mg, 17.9%).
[0233] 15c (R.sub.1=n-Pr, R.sub.2=R.sub.4=R.sub.7=R.sub.8=H,
R.sub.3=R.sub.5=R.sub.6=Me, R.sub.9=Et): mp 155-158.degree. C.;
.sup.1H NMR (CDCl.sub.3): 1.03 (3H, t, J=7.4 Hz, CH.sub.3), 1.13
(3H, t, J=7.4 Hz, CH.sub.3), 1.22 (3H, d, J=6.9 Hz, CH.sub.3), 1.53
(3H, s, CH.sub.3), 1.56 (3H, s, CH.sub.3), 1.64 (2H, sextet, J=7.6
Hz, CH.sub.2), 1.78-1.95 (2H, m, CH.sub.2), 2.62 (1H, dq, J=10.4
Hz, J=7.0 Hz, H.sub.11), 2.88 (2H, t, J=7.7 Hz,CH.sub.2), 4.14 (1H,
ddd, J=3.5 Hz, J=7.8 Hz, J=10.7 Hz, H.sub.10), 5.61 (1H, d, J=10.0
Hz, H.sub.7), 6.04 (1H, s, H.sub.3), 6.66 (1H, d, J=10.0 Hz,
H.sub.8); EIMS: 382 (37.2, M), 367 (100, M-CH.sub.3), 297 (47.2,
M-CH.sub.3-C.sub.5H.sub.10); IR (KBr) cm.sup.-1: 1738 (vs,
C.dbd.O); Anal. calcd. for C.sub.23H.sub.26O.sub.5: C, 72.23; H,
6.85. Found: C, 71.75; H, 7.02.
[0234] 15d (R.sub.1=n-Pr, R.sub.2=R.sub.3=R.sub.7=R.sub.8=H,
R.sub.4=R.sub.5=R.sub.6=Me, R.sub.9=Et): mp 100-102.degree. C.;
.sup.1H NMR (CDCl.sub.3): 1.03 (3H, t, J=7.3 Hz, CH.sub.3), 1.07
(3H, t, J=7.4 Hz, CH.sub.3), 1.14 (3H, d, J=7.3 Hz, CH.sub.3), 1.54
(3H, s, CH.sub.3), 1.55 (3H, CH.sub.3), 1.65 (2H, sextet, J=7.6 Hz,
CH.sub.2), 1.83-1.98 (2H, m, CH.sub.2), 2.70 (1H, dq, J=3.2 Hz,
J=7.3 Hz, H.sub.11), 2.88 (2H, t, J=7.6 Hz, CH.sub.2), 4.39 (1H,
ddd, J=3.4 Hz, J=5.3 Hz, J=8.8 Hz, H.sub.10), 5.60 (1H, d, J=10.0
Hz, H.sub.7), 6.05 (1H, s, H.sub.3), 6.66 (1H, d, J=10.0 Hz,
H.sub.8); EIMS: 382 (55.0, M.sup.+), 367 (100, M-CH.sub.3), 297
(52.7, M-CH.sub.3-C.sub.5H.sub.10); IR (KBr) cm.sup.-1: 1732 (vs,
C.dbd.O); Anal. calcd. for C.sub.23H.sub.26O.sub.5: C, 72.23; H,
6.85. Found: C, 71.80; H, 6.97.
EXAMPLE 29
[0235]
(.+-.)-10,12-cis-10,11-Dihydro-12-hydroxy-6,6,10,11,11-pentamethyl--
4propyl-2H, 6H, 12H-benzo [1,2-b:3,4-b':5,6-b"]tripyran-2-one (16a)
and
(.+-.)-10,12-trans-10,11-dihydro-12-hydroxy-6,6,10,11,11-pentamethyl-4-pr-
opyl-2H,6H,12H-benzo [1,2-b:3,4-b':5,6-b"]tripyran-2-one (16b,
Scheme V)
[0236] To a solution of 15a (252 mg, 0.661 mmol) in ethanol/THF
(1:1, 8 mL) was added sodium borohydride (25.1 mg, 0.661 mmol) and
the solution stirred at room temperature for 30 minutes. The
reaction was quenched by the addition of water (1 mL), and the
solvent then removed in vacuo. The residue was partitioned between
20 mL each of ethyl acetate and 1 M HCl, and the organic phase
separated and washed sequentially with 5% NaHCO.sub.3 and saturated
brine. After drying over magnesium sulfate, the solution was
evaporated to give the product as a paleyellow foam. TLC analysis
(ethyl acetate/hexane, 1:2) showed the two epimeric alcohols 16a
and 16b at R.sub.f 0.30 and 0.25, as well as a minor impurity at
R.sub.f 0.55. Separation via column chromatography (75 g silica
gel, ethyl acetate/hexane, 1:2) provided 16a (127.7 mg, 50.3%) and
16b (18.8 mg, 7.4%) as a white foam and an off-white waxy solid,
respectively.
[0237] 16a (R.sub.1=n-Pr, R.sub.2=R.sub.7=R.sub.8=H,
R.sub.3=R.sub.4=R.sub.5=R.sub.6=R.sub.9=Me): .sup.1H NMR
(CDCl.sub.3): 1.04 (3H, t, J=7.3 Hz, CH.sub.3), 1.06 (6H, s, 2
CH.sub.3), 1.40 (3H, d, J=6.7 Hz, CH.sub.3), 1.47 (3H, s,
CH.sub.3), 1.50 (3H, s, CH.sub.3), 1.66 (2H, sextet, J=7.3 Hz,
CH.sub.2), 2.80-2.99 (2H, m, CH.sub.2), 3.39 (1H, d, J=3.2 Hz, OH),
3.99 (1H, q, J=6.7 Hz, H.sub.10), 4.70 (1H, d, J=3.2 Hz, H.sub.12),
5.54 (1H, d, J=9.9 Hz, H.sub.7), 5.94 (1H, s, H.sub.3), 6.63 (1H,
d, J=9.9 Hz, H.sub.8); EIMS: 384 (59.0, M.sup.+), 369 (100,
M-CH.sub.3), 314 (44.7, M-C.sub.5H.sub.10), 299 (88.8,
M-CH.sub.3-C.sub.5H.sub.10); IR (KBr) cm.sup.-1: 3432 (broad-s,
OH), 1734 (vs, C.dbd.O); Anal. calcd. for C.sub.23H.sub.28O.sub.5:
C, 71.85; H, 7.34. Found: C, 71.74; H, 7.43.
[0238] 16b (R.sub.1=n-Pr, R.sub.2=R.sub.7=R.sub.8=H,
R.sub.3=R.sub.4=R.sub.5=R.sub.6=R.sub.9=Me): .sup.1H NMR
(CDCl.sub.3): 0.78 (3H, s, CH.sub.3), 1.04 (3H, t, J=7.3 Hz,
CH.sub.3), 1.11 (3H, s, CH.sub.3), 1.36 (3H, d, J=6.5 Hz,
CH.sub.3), 1.49 (6H, s, 2 CH.sub.3), 1.64 (2H, m, CH.sub.2), 2.47
(1H, broad-s, OH), 2.89 (2H, m, CH.sub.2), 4.35 (1H, q, J=6.5 Hz,
H.sub.10), 4.63 (1H, broad-s, H.sub.12), 5.54 (1H, d, J=9.8 Hz,
H7), 5.96 (1H, s, H.sub.3), 6.65 (1H, d, J=9.8 Hz, H.sub.8); EIMS:
384 (40.7, M.sup.+), 369 (100, M-CH.sub.3), 314 (13.5,
M-C.sub.5H.sub.10), 299 (48.4, M-CH.sub.3-C.sub.5H.sub.10); Anal.
calcd. for C.sub.23H.sub.28O.sub.5: C, 71.85; H, 7.34. Found: C,
71.79; H, 7.49.
EXAMPLE 30
[0239]
(.+-.)-11,12-cis-10,11-Dihydro-12-hydroxy-6,6,10,10,11-pentamethyl--
4-propyl-2H, 6H, 12H-benzo [1,2-b:3,4-b':5,6-b"]tripyran-2-one
(16c) and
(.+-.)-11,12-trans-10,11-dihydro-12-hydroxy-6,6,10,10,11-pentamethyl-4-pr-
opyl-2H,6H,12H-benzo [1,2-b:3,4-b':5,6-b"]tripyran-2-one (16d,
Scheme V)
[0240] To a solution of 15b (289.7 mg, 0.75 mmol),
triphenylphosphine oxide (927.0 mg, 3.33 mmol) and
CeCl.sub.3(H.sub.2O).sub.7 (842.0 mg, 2.25 mmol) in ethanol (15 mL)
at 0.degree. C. was slowly added NaBH.sub.4 (195.0 mg, 5.15 mmol)
under N.sub.2. The suspension was stirred for 1 h at room
temperature, then quenched with saturated NH.sub.4Cl (30 mL). The
solution was extracted with ethyl acetate (200 mL), washed with
brine (50 mL), dried over magnesium sulfate, filtered and
concentrated to afford a pink crystalline solid (1.38 g). Silica
gel column chromatography (ethyl acetate/hexane, 1:5) provided 16c
(100.0 mg, 34.3%) as off-white foam and 16d which was fuirther
purified by preparative TLC (silica gel, diethyl ether/hexane, 2:1)
as off-white foam (56.0 mg, 19.2%).
[0241] 16c (R.sub.1=n-Pr, R.sub.2=R.sub.3=R.sub.7=H,
R.sub.4=R.sub.4=R.sub.6=R.sub.8=R.sub.9=Me): mp 44-45.degree. C.;
.sup.1H NMR (CDCl.sub.3): 1.04 (3H, t, J=7.3 Hz, CH.sub.3), 1.24
(3H, d, J=7.1 Hz, CH.sub.3), 1.38 (3H, s, CH.sub.3), 1.45 (3H, s,
CH.sub.3), 1.47 (3H, s, CH.sub.3), 1.51 (3H, s, CH.sub.3), 1.66
(2H, sextet, J=7.3 Hz, CH.sub.2), 1.96-2.04 (1H, m, H.sub.11),
2.8-3.0 (2H, m, CH.sub.2), 3.02 (1H, d, J=4.0 Hz, OH), 4.94 (1H, t,
J=4.2 Hz, H,.sub.2), 5.53 (1H, d, J=10.0 Hz, H.sub.7), 5.94 (1H, s,
H.sub.3), 6.65 (1H, d, J=9.9 Hz, H.sub.8); EIMS: 385 (22.1, M+1),
384 (61.8, M.sup.+), 369 (71.1, M-CH.sub.3), 351 (29.5,
M-CH.sub.3-H.sub.2O), 299 (100, M-CH.sub.3-C.sub.5l H.sub.10); IR
(KBr) cm.sup.-1: 3451 (broad-m, OH), 1709 (s, C.dbd.O); Anal.
calcd. for C.sub.23H.sub.28O.sub.5l : C, 71.85; H, 7.33. Found: C,
71.63; H, 7.64.
[0242] 16d (R.sub.1=n-Pr, R.sub.2=R.sub.4=R.sub.7=H,
R.sub.3=R.sub.5=R.sub.6=R.sub.8=R.sub.9=Me): mp 40-42.degree. C.;
.sup.1H NMR (CDCl.sub.3): 1.04 (3H, t, J=7.3 Hz, CH.sub.3), 1.13
(3H, d, J=7.0 Hz, CH.sub.3), 1.21 (3H, s, CH.sub.3), 1.46 (3H, s,
CH.sub.3), 1.48 (3H, s, CH.sub.3), 1.52 (3H, s, CH.sub.3), 1.67
(2H, sextet, J=7.6 Hz, CH.sub.2), 2.03 (1H, quintet, J=7.2 Hz,
H.sub.11), 2.8-3.0 (2H, m, CH.sub.2), 3.66 (1H, s, OH), 4.69 (1H,
d, J=7.4 Hz, H.sub.12), 5.54 (1H, d, J=10.0 Hz, H.sub.7), 5.94 (1H,
s, H.sub.3), 6.63 (1H, d, J=9.9 Hz, H.sub.8); EIMS: 385 (8.7, M+1),
384 (36.0, M.sup.+), 369 (65.8, M-CH.sub.3), 351 (17.6,
M-CH.sub.3-H.sub.2O), 299 (100, M-CH.sub.3-C.sub.5H.sub.11); IR
(KBr) cm.sup.-1: 3437 (w, OH), 1734 (s, C.dbd.O); Anal. calcd. for
C.sub.23H.sub.28O.sub.5: C, 71.85; H, 7.33. Found: C, 71.70; H,
7.56.
EXAMPLE 31
[0243]
(.+-.)-10,11-trans-11,12-cis-10,11-Dihydro-10-ethyl-12-hydroxy-4-pr-
opyl-6,6,11-trimethyl-2H,6H,12H-benzo
[1,2-b:3,4-b':5,6-b"]tripyran-2-one (16e) and
(.+-.)-10,11-qrans-11,12-trans-10,11-dihydro-10-ethyl-12-hydrox-
y4-propyl-6,6,11-trimethyl-2H,6H,12H-benzo
[1,2-b:3,4-b':5,6-b"]tripyran-2- -one (16f, Scheme V)
[0244] To a suspension of 15c (454.7 mg, 1.19 mmol),
triphenylphosphine oxide (1.38 g, 4.96 mmol) and
CeCl.sub.3(H.sub.2O).sub.7 (1.21 g, 3.25 mmol) in ethanol (10 mL)
at 0.degree. C. was slowly added NaBH.sub.4 (312.0 mg, 8.25 mmol)
under N.sub.2. The suspenion was stirred for 3 h at room
temperature. The reaction mixture was quenched with saturated
NH.sub.4Cl (15 mL), extracted with ethyl acetate (100 mL.times.3),
washed with brine (50 mL), dried over magnesium sulfate, filtered
and concentrated to provide pink crystals (1.97 g). Silica gel
column chromatography (ethyl acetate/hexane, 1:4) afforded a yellow
oil, which consisted of mixture of 16e and 16f (261.0 mg). The
compounds were separated using preparative HPLC (normal phase,
ethyl acetate/hexane, 3:7). The desired fractions were combined and
concentrated in vacuo and dried overnight under high vacuum in the
presence of P.sub.2O.sub.5 to afford 16e (yellow oil, 46.5 mg,
10.1%) and 16f (white solid, 137.6 mg, 30.1%).
[0245] 16e (R.sub.1=n-Pr, R.sub.2=R.sub.4=R.sub.7=R.sub.8=H,
R.sub.3=R.sub.5=R.sub.6=Me, R.sub.9=EI): .sup.1H NMR (CDCl.sub.3):
1.03 (3H, t, J=7.3 Hz, CH.sub.3), 1.10 (3H, t, J=7.6 Hz, CH.sub.3).
1.13 (3H, d, J=6.8 Hz, CH.sub.3), 1.48 (3H, s, CH.sub.3), 1.49 (3H,
s, CH.sub.3), 1.65 (2H, sextet, J=7.4 Hz, CH.sub.2), 1.76-1.98 (3H,
m, CH.sub.2+H.sub.11), 2.80-2.92 (3H, m, CH.sub.2+OH), 4.10 (1H,
ddd, J=2.9 Hz, J=7.9 Hz, J=10.7 Hz, H.sub.10), 4.98 (1H, d, J=3.3
Hz, H.sub.12), 5.54 (1H, d, J=9.9 Hz, H.sub.7), 5.94 (1H, s,
H.sub.3), 6.63 (1H, d, J=9.9 Hz, H.sub.8); EIMS: 385(10.5, M+1),
384 (35.8,M.sup.+), 369 (78.4, M-CH.sub.3), 366 (43.1, M-H.sub.2l
O), 351 (39.0, M-CH.sub.3-H.sub.2O), 337 (100,
M-H.sub.2O-C.sub.2H.sub.5), 299 (37.7, M-CH.sub.3-C.sub.5H.sub.-
10); IR (neat, thin film) cm.sup.-1: 3432 (w, OH), 1709 (s,
C.dbd.O); Anal. calcd. for C.sub.23H.sub.28O.sub.5.1/4 H.sub.2O: C,
71.02; H, 7.38. Found: C, 71.10; H, 7.40.
[0246] 16f (R.sub.1=n-Pr, R.sub.2=R.sub.4=R.sub.7=R.sub.8=H,
R.sub.3=R.sub.5=R.sub.6=Me, R.sub.9=Et): mp 103-105.degree. C.;
.sup.1H NMR (CDCl.sub.3): 1.04 (3H, t, J=7.3 Hz, CH.sub.3), 1.07
(3H, t, J=7.4 Hz, CH.sub.3), 1.13 (3H, d, J=6.9 Hz, CH.sub.3), 1.47
(3H, s, CH.sub.3), 1.51 (3H, s, CH.sub.3), 1.66 (2H, sextet, J=7.6
Hz, CH.sub.2), 1.79-1.90 (2H, m, CH.sub.2), 2.05 (1H, m, H.sub.11),
2.90 (2H, m, CH.sub.2), 3.53 (1H, s, OH), 3.78 (1H, dt, J=4.1 Hz,
J=8.1 Hz, Hlo), 4.73 (1H, d, J=6.7 Hz, H.sub.12), 5.54 (1H, d,
J=10.0 Hz, H.sub.7), 5.95 (1H, s, H.sub.3), 6.63 (1H, d, J=9.9 Hz,
H.sub.8); EIMS: 385 (7.6, M+1), 384 (31.1, M.sup.+), 369 (100,
M-CH.sub.3), 351 (9.5, M-CH.sub.3-H.sub.2O), 337 (11.5,
M-H.sub.2O-C.sub.2H.sub.5), 299 (36.9, M-CH.sub.3-C.sub.5H.sub.10)-
; IR (KBr) cm.sup.-1: 3493, 3435 and 3250 (w, OH), 1699 (s, C=O);
Anal. calcd. for C.sub.23H.sub.28O.sub.5: C, 71.85; H, 7.33. Found:
C, 71.46; H, 7.34.
EXAMPLE 32
[0247]
(.+-.)-10,11-cis-11,12-trans-10,11-Dihydro-10-ethyl-12-hydroxy-4-pr-
opyl-6,6,11-trimethyl-2H,6H,12H-benzo
[1,2-b:3,4-b':5,6-b"]tripyran-2-one (16g) and
(.+-.)-10,11,12-cis-10,11-dihydro10-ethyl-12-hydroxy4-propyl-6,-
6,11-trimethyl-2H,6H,12H-benzo [1,2-b:3,4-b':5,6-b"]tripyran-2-one
(16h, Scheme V)
[0248] To a solution of 15d (290.5 mg,0.76 mmol) in ethanol (15 mL)
at 25.degree. C. was added NaBH.sub.4 (269.0 mg, 7.11 mmol)
portionwise under N.sub.2. The suspension was stirred for 1 h at
room temperature, then quenched with saturated NH.sub.4Cl (6 mL).
The solution was extracted with ethyl acetate (200 mL), washed with
brine (80 mL), dried over magnesium sulfate, filtered and
concentrated to provide a pink residue (455.8 mg). The crude
product was purified by silica gel preparative TLC (ethyl
acetate/hexane, 2:1). The desired bands were scraped, combined,
extracted, concentrated in vacuo and dried under high vacuum
overnight in the presence of P.sub.2O.sub.5 to afford the desired
products 16g (229 mg, 78% yield) with 95% purity as indicated by
HPLC) and 16h (55.9 mg, 19% yield) with 92% purity. The analytical
samples were obtained by further purification via preparative HPLC
(normal phase, ethyl acetate/hexane, 3:7).
[0249] 16g (R.sub.1=n-Pr, R.sub.2=R.sub.3=R.sub.7=R.sub.8=H,
R.sub.4=R.sub.5=R.sub.6=Me, R.sub.9=Et): .sup.1H NMR (CDCl.sub.3):
1.03 (3H, t, J=7.4 Hz, CH.sub.3), 1.04 (3H, t, J=7.3 Hz,
CH.sub.3),1.12 (3H, d, J=7.1 Hz, CH.sub.3), 1.49 (3H, s, CH.sub.3),
1.66 (2H, sextet, J=7.3 Hz, CH.sub.2), 1.8-2.0 (2H, m, CH.sub.2),
2.3-2.4 (1H, m, H.sub.11), 2.8-3.0 (2H, m, CH.sub.2), 3.30 (1H, s,
OH), 4.06 (1H, dt, J=10.1 Hz, J=3.3 Hz, H.sub.10), 5.10 (1H, d,
J=5.2 Hz, H,.sub.2), 5.55 (1H, d, J=10.0 Hz, H.sub.7), 5.94 (1H, s,
H.sub.3), 6.63 (1H, d, J=10.0 Hz, H.sub.8); EIMS: 385 (6.3, M+1),
384 (27.3, M.sup.+), 369 (100, M-CH.sub.3), 351 (6.8,
M-CH.sub.3-H.sub.2O), 337 (4.2, M-H.sub.2O-C.sub.2H.sub.5), 299
(34.7, M-CH.sub.3-C.sub.5H.sub.10); IR (KBr) cm.sup.-1: 3449 (m,
OH), 1734 (vs, C.dbd.O); Anal. calcd. for C.sub.23H.sub.28O.sub.5:
C, 71.85; H, 7.33. Found: C, 71.79; H, 7.39.
[0250] 16h (R.sub.1=n-Pr, R.sub.2=R.sub.3=R.sub.7=R.sub.8=H,
R.sub.4=R.sub.5=R.sub.6=Me, R.sub.9=Et): .sup.1H NMR (CDCl.sub.3):
0.79 (3H, d, J=7.3 Hz, CH.sub.3), 1.04 (3H, t, J=7.3 Hz, CH.sub.3),
1.11 (3H, t, J=7.3 Hz, CH.sub.3), 1.49 (3H, s, CH.sub.3), 1.51 (3H,
s, CH.sub.3), 1.67 (2H, sextet, J=7.4 Hz, CH.sub.2), 1.92 (2H, m,
CH.sub.2), 2.10 (1H, tq, J=2.0 Hz, J=7.3 Hz, H.sub.11), 2.79 (1H,
s, OH), 2.81-2.90 (2H, m, CH.sub.2), 4.23 (1H, ddd, J=1.9 Hz, J=5.4
Hz, J=8.7 Hz, H.sub.10) 4.87 (1H, d, J=1.9 Hz, H.sub.12), 5.54 (1H,
d, J=10.0 Hz, H.sub.7), 5.96 (1H, s, H.sub.3), 6.66 (1H, d, J=9.9
Hz, H.sub.8); EIMS: 385 (6.1, M+1), 384 (26.0, M.sup.+), 369 (100,
M-CH.sub.3), 351 (9.8, M-CH.sub.3-H.sub.2O), 337 (8.2,
M-H.sub.2O-C.sub.2H.sub.5), 299 (17.6, M-CH.sub.3-C.sub.5H.sub.-
10); IR (neat, thin film) cm.sup.-1: 3410 (w, OH), 1732 (s,
C.dbd.O).
EXAMPLE 33
[0251]
(.+-.)-10,11-trans4-Propyl-7,8,10,11-tetrahydro-6,6,10,11-tetrameth-
yl-2H,6H,12H-benzo [1,2-b:3,4-b'5,6-b"]tripyran-2,12-dione (Scheme
VI, 18a, R,=n-Pr, R.sub.2=R.sub.4=R.sub.7=R.sub.8=H,
R.sub.3=R.sub.5=R.sub.6=- R.sub.9=Me)
[0252] To a solution of (.+-.)-7 (534 mg, 1.45 mmol) in
ethanol/methylene chloride (1:1, 50 mL, Parr apparatus) under
N.sub.2 was added 10% palladium/carbon (53.4 mg) at ambient
temperature. The mixture was shaken under hydrogen (2 atm) for 1 h,
then gravity filtered through Whatmann filter paper. The solvent
was evaporated to give a white crystalline solid which was filtered
through a short plug of silica gel, eluting with methylene
chloride/methanol (97:3). The pure compound (.+-.)-18a (441 mg,
82.2%) was obtained as white plates by recrystallization from ethyl
acetate: mp 165.degree. C.; .sup.1H NMR (CDCl.sub.3): 1.01 (3H, t,
J=7.3 Hz, CH.sub.3), 1.21 (3H, d, J=6.8 Hz, CH.sub.3), 1.42 (3H, s,
CH.sub.3), 1.44 (3H, s, CH.sub.3), 1.53 (3H, d, J=6.2 Hz,
CH.sub.3), 1.61 (2H, sextet, J=7.5 Hz, CH.sub.2), 1.84 (2H,
apparent dt, J=2.4 Hz, J=6.7 Hz, CH.sub.2), 2.53 (1H, dq, J=11.2
Hz, J=6.8 Hz, H.sub.11), 2.69 (2H, apparent dt, J=3.4 Hz, J=6.7 Hz,
CH.sub.2), 2.88 (2H, t, J=7.5 Hz, CH.sub.2), 4.28 (1H, dq, J=11.2
Hz, J=6.2 Hz, H.sub.10), 6.02 (1H, s, H.sub.3); EIMS: 371 (40.8,
M+1); 370 (100, M+), 314 (99.3, M-C.sub.4H.sub.8), 299 (21.6,
M-C.sub.5H.sub.10), 286 (65.0, M-CH.sub.3-C.sub.5H.sub.10+1), 271
(20.5, M-CH.sub.3-C.sub.5H.sub.8O), 259 (47.5,
M-C.sub.4H.sub.8-C.sub.3H.sub.4O+1); IR (KBr) cmn.sup.-1: 1740 (vs,
C.dbd.O); Anal. calcd. for C.sub.22H.sub.26O.sub.5: C, 71.33; H,
7.07. Found: C, 71.00; H, 7.22.
EXAMPLE 34
[0253]
(.+-.)10,11-trans-10,11-Dihydro4-propyl-6,6,10,11-tetramethyl-2H,
6H, 12H-benzo [1,2-b:3,4-b':5,6-b"]tripyran-2,12-dione-12-oxime
(Scheme VI, 19a, R.sub.1=n-Pr, R.sub.2=R.sub.4=R.sub.7=R.sub.8=H,
R.sub.5=R.sub.6R.sub.9=Me, R.sub.10=H)
[0254] Into a 100 mL one-necked round-bottom flask was placed
(.+-.)-7 (1.47 g, 4.00 mmol) and NH.sub.2OHHCl (1.39 g, 20.0 mmol).
To this mixture was added methanol (60 mL), and the solution heated
to reflux with magnetic stirring until the ketone dissolved. Solid
K.sub.2CO.sub.3 powder (1.38 g, 10.0 mmol) was then carefully
added, and the reaction allowed to stir at reflux for 4 hours. The
solution was cooled at room temperature, filtered to remove the
K.sub.2CO.sub.3 and evaporated in vacuo, to provide a yellow solid.
The residue was partitioned between 150 mL each of H.sub.2O and
ethyl acetate. The organic phase was collected and washed
sequentially with 1 N HCl and saturated brine, then dried over
magnesium sulfate, filtered and evaporated to afford a thick yellow
syrup, which was purified via silica gel column chromatography (75
g), eluting with methylene chloride/methanol (97:3) to afford the
desired product as a white solid (657 mg, 43%). An analytical
sample was obtained via recrystallization from acetone/hexane (1:3)
as colorless prisms; mp 200-201 .degree. C; .sup.1H NMR
(CDCl.sub.3): 1.04 (3H, t, J=7.4 Hz, CH.sub.3), 1.23 (3H, d, J=7.0
Hz, CH.sub.3), 1.33 (3H, d, J=6.5 Hz, CH.sub.3), 1.51 (3H, s,
CH.sub.3), 1.54 (3H, s, CH.sub.3), 1.67 (2H, sextet, J=7.4 Hz,
CH.sub.2), 2.82-3.01 (2H, m, CH.sub.2), 3.79 (1H, dq, J=2.0 Hz,
J=7.0 Hz, H.sub.11), 4.46 (1H, dq, J=2.0 Hz, J=6.5 Hz, H.sub.10),
5.57 (1H, d, J=9.9 Hz, H.sub.7), 6.02 (1H, s, H.sub.3), 6.67 (1H,
d, J=9.9 Hz, Hg), 9.46 (1H, broad-s, OH); EIMS: 384 (12.9,M+1), 383
(49.22, Me), 368 (100, M-CH.sub.3), 366 (21.1, M-OH), 352 (15.2,
M-NOH); IR (KBr) cm.sup.-1: 3223 (broad, OH), 1740 (C.dbd.O); Anal.
calcd. for C.sub.22H.sub.25NO.sub.5.1/4 H.sub.2O): C, 68.1 1; H,
6.63; N, 3.61. Found: C, 68.40; H, 6.59; N,3.58.
EXAMPLE 35
[0255] (.+-.)-10,11-trans-10,11
-Dihydro4-propyl-6,6,10,11-tetramethyl-2H, 6H, 12H-benzo
[1,2-b:3,4-b':5,6-b "]tripyran-2,12-dione-12-methoxime (Scheme VI,
19b, R.sub.1=n-Pr, R.sub.2=R.sub.4=R.sub.7=R.sub.8=R.sub.8=H,
R.sub.3=R.sub.5=R.sub.6=R.sub.9=Me, R.sub.10=Me)
[0256] Into a one-necked 100 mL round-bottom flask was placed
(.+-.)-7 (1.47 g, 4.00 mmol) and NH.sub.2OCH.sub.3HCl (1.67 g, 20.0
mmol). To this mixture was added methanol (60 mL), and the solution
heated to reflux with magnetic stirring until the ketone dissolved.
Solid K.sub.2CO.sub.3 powder (1.38 g, 10.0 mmol) was then carefully
added, and the reaction allowed to stir at reflux for 4 hours. The
solution was cooled to room temperature, filtered to remove the
K.sub.2CO.sub.3 and evaporated in vacuo, to provide a yellow oil.
The residue was partitioned between 150 mnL H.sub.2O and 150 mL
ethyl acetate. The organic phase was collected and washed
sequentially with 1 N HCl and saturated brine, then dried over
magnesium sulfate, filtered and evaporated to afford a thick yellow
syrup. The product was purified via silica gel column
chromatography (75 g), eluting with ethyl acetate/hexane (1:3) to
afford the desired product as a faintly yellow oil which, upon
standing, formed a white solid (598 mg, 38%). An analytical sample
was obtained via recrystallization from acetone/hexane (1:3) as
white plates; mp 143-144.degree. C.; .sup.1H NMR (CDCl.sub.3): 1.01
(3H, t, J=7.3 Hz, CH.sub.3), 1.16 (3H, d, J=7.0 Hz, CH.sub.3), 1.28
(3H, d, J=6.4 Hz, CH.sub.3), 1.49 (3H, s, CH.sub.3), 1.50 (3H, s,
CH.sub.3), 1.64 (2H, sextet, J=7.3 Hz, CH.sub.2), 2.79-2.99 (2H, m,
CH.sub.2), 3.57 (H, dq, J=1.9 Hz, J=7.0 Hz, H.sub.11), 4.06 (3H, s,
OCH.sub.3), 4.37 (1H, dq, J=1.9 Hz, J=6.4 Hz, H.sub.10), 5.54 (1H,
d, J=10.0 Hz, H.sub.7), 6.00 (1H, s, H.sub.3), 6.62 (1H, d, J=10.0
Hz, H.sub.8); EIMS: 397 (61.2, M.sup.+), 382 (100, M-CH.sub.3), 366
(12.9, M-OCH.sub.3); IR (KBr) cm.sup.-1: 1728 (vs, C.dbd.O); Anal.
calcd. for C.sub.23H.sub.27NO.sub.5: C, 69.50; H, 6.85; N, 3.52.
Found: C, 69.39; H, 6.90; N, 3.59.
EXAMPLE 36
[0257] Conversion of (-)-Calanolide A into (-)-Calanolide B
[0258] To a solution of (-)-calanolide A (341 mg, 0.922 mmol) in
anhydrous methylene chloride (5 mL) at -78.degree. C. under N.sub.2
was added a solution of diethylarnidosulfur trifluoride (DAST, 178
mg, 1.11 nmmol) in methylene chloride (1 mL) and the resulting
yellow solution stirred at -78.degree. C. for 4 hours. The reaction
was quenched with 0.5 mL methanol, then allowed to warm to room
temperature. The solution was diluted with methylene chloride (20
mL), then washed with water (50 mL) and saturated brine (50 mL).
After drying over magnesium sulfate, the solution was filtered and
evaporated to provide a light yellow solid. TLC analysis (silica
gel, 3% methanol in methylene chloride) showed two components, one
fast-moving and one slow. The material was chromatographed through
80 g silica gel, eluting with 1% methanol in CH.sub.2Cl.sub.2, and
the fractions containing the respective components combined and
evaporated to afford 198 mg (61% yield) of compound 22 and 75.3 mg
(22%) of (-)-calanolide B.
[0259] 10(S)4-propyl-6,6,10,11-tetramethyl-2H,6H,10H-benzo
[1,2-b:3,4-b':5,6-b"]tripyran-2-one (22): .sup.1H NMR (CDCl.sub.3):
1.03 (3H, t, J=7.4 Hz, CH.sub.3), 1.39 (3H, d, J=6.6 Hz, CH.sub.3),
1.47 (3H, s, CH.sub.3). 1.51 (3H, s, CH.sub.3), 1.66 (2H, sextet,
J=7.4 Hz, CH.sub.2), 1.85 (3H, s, CH.sub.3), 2.88 (2H, m,
CH.sub.2), 4.89 (1H, q, J=6.6 Hz, H.sub.10), 5.55 (1H, d, J=10.0
Hz, H.sub.7), 5.93 (1H, s, H.sub.3), 6.62 (1H, d, J=10.0 Hz,
H.sub.8), 6.64 (1H, s, H.sub.12); EIMS: 353 (15.5, M+1), 352 (53.2,
M.sup.+), 337 (100, M-CH.sub.3). IR (KBr) cm.sup.-1: 1724 (s,
C.dbd.O); Anal. calcd. for C.sub.22H.sub.24O.sub.4: C, 74.98; H,
6.86. Found: C, 74.87; H, 7.00.
[0260] (-)-Calanolide B: .sup.1H NMR (CDCl.sub.3): 1.03 (3H, t,
J=7.3 Hz, CH.sub.3), 1.14 (3H, d, J=7.0 Hz, CH.sub.3), 1.43 (3H, d,
J=6.4 Hz, CH.sub.3), 1.48 (3H, s, CH.sub.3), 1.49 (3H, CH.sub.3),
1.66 (2H, sextet, J=7.6 Hz, CH.sub.2), 1.72-1.79 (1H, m, H.sub.11),
2.60 (1H, d, J=3.8 Hz, OH), 2.89 (2H, m, CH.sub.2), 4.26 (1H, dq,
J=10.7 Hz, 6.3 Hz, H.sub.10), 4.97 (1H, J=3.8 Hz, H.sub.12), 5.53
(1H, d, J=10.0 Hz, H.sub.7), 5.95 (1H, s, H.sub.3), 6.63 (1H, d,
J=10.0 Hz, H8); EIMS: 370 (31.1, M.sup.+), 355 (100, M-CH.sub.3),
299 (29.7, M-CH.sub.3-C.sub.4H.sub.8); IR (KBr) cm.sup.-1: 3478 (s,
sharp, OH), 1703 (s, C.dbd.O).
EXAMPLE 37
[0261] In Vitro evaluation of (+)-, (+- and (-)-calanolide A
[0262] This example illustrates the anti-HIV viral activity of the
synthetic (.+-.) -calanolide A and its pure enantiomers,
(+)-calanolide A and (-)-calanolide A, which were evaluated using
the published MTT-tetrazolium method.sup.18. Retroviral agents AZT
and DDC were used as controls for comparison purposes.
[0263] The cells used for screening were the MT-2 and the human
T4-lymphoblastoid cell line, CEM-SS, and were grown in RPMI 1640
medium supplemented with 10% fetal (v/v) heat-inactivated fetal
calf serum and also containing 100 units/mL penicillin, 100
.mu.g/mL streptomycin, 25 mM HEPES and 20 .mu.g/mL gentamicin. The
medium used for dilution of drugs and maintenance of cultures
during the assay was the same as above. The HTLV-IllB and HTLV-RF
were propagated in CEM-SS. The appropriate amounts of the pure
compounds for anti-HIV evaluations were dissolved in DMSO, then
diluted in medium to the desired initial concentration. The
concentrations (.mu.g drug/mL medium) employed were 0.0032
.mu.g/mL; 0.001 .mu.g/mL; 0.0032 .mu.g/mL; 0.01 .mu.g/mL; 0.032
.mu.g/mL; 0.1 .mu.g/mL; 0.32 .mu.g/mL; 1 .mu.g/mL; 3.2 .mu.g/mL; 10
.mu.g/mL; 32 .mu.g/mL; and 100 .mu.g/mL. Each dilution was added to
plates in the amount of 100 .mu.L/well. Drugs were tested in
triplicate wells per dilution with infected cells while in
duplicate wells per dilution with uninfected cells for evaluation
of cytotoxicity. On day 6 (CEM-SS cells) and day 7 (MT-2 cells)
post-infection, the viable cells were measured with a tetrazolium
salt, MTT (5 mg/mL), added to the test plates. A solution of 20%
SDS in 0.001 N HCl is used to dissolve the MTT formazan produced.
The optical density value was a function of the amount of formazan
produced which was proportional to the number of viable cells. The
percent inhibition of CPE per drug concentration was measured as a
test over control and expressed in percent (T/C%). The data is
summarized in FIGS. 1(a-e), 2(a-e), 3(a-e), 4(a-d), and 5(a-d).
[0264] FIGS. 1(a) to 1(e) illustrate in vitro MTT assay results
using an isolate, G910-6 HIV viral strain.sup.19, which is
AZT-resistant. The data shows that (-)-calanolide A was relatively
non-toxic at concentrations of 1 .mu.g/mL but exhibited very little
antiviral effect. Moreover, (.+-.)-calanolide A and (+)-calanolide
A were effective in reducing viral CPE. As expected, AZT had little
to no effect in reducing viral CPE and enhancing cell
viability.
[0265] FIGS. 2(a) to 2(e) illustrate in vitro MTT assay results
using H112-2 HIV viral strain.sup.19 which was not pre-treated with
AZT. As expected, the viral strain was sensitive to AZT. The data
also showed that (-)-calanolide A was relatively non-toxic at
concentrations of 1 g/mL but exhibited very little antiviral
effect. (.+-.)-Calanolide A was nearly as effective as
(+)-calanolide A in reducing viral CPE.
[0266] FIGS. 3(a) to 3(e) illustrate in vitro MTT assay results
using A-17 HIV viral strain.sup.20 which is resistant to to
non-nucleoside inhibitors such as TIBO but is sensitive to AZT. The
results here parallel those shown in FIGS. 2(a)-2(e).
[0267] FIGS. 4(a)-(d) and 5(a)-(d) illustrate in vitro MTT assay
results using lab cultivated HIV viral strains IIIB and RF,
respectively. The results here also parallel those shown in FIGS.
2(a)-2(e).
EXAMPLE 38
[0268] IN VITRO EVALUATION OF CALANOLIDE ANALOGUES
[0269] Selected calanolide A intermediates and analogues, prepared
as described above, were evaluated using the in vitro
MTT-tetrazoliurn assay described in Example 37. As shown in Table I
below, compounds (.+-.)-7, (+)-7, (.+-.)-8a, (.+-.)-8b, (+)-10,
(.+-.)-16b, (.+-.)-16d, (.+-.)-16e, and (.+-.)-16f were highly
efficacious in protecting cells against HIV infection.
Table I. In Vitro Anti-HIV-1 Activity of Analoguesa
[0270]
1TABLE I In Vitro Anti-HIV-1 Activity of Analognes.sup.a Maximum
EC.sub.50 IC.sub.50 Compound Protection (%) (.mu.M) (.mu.M)
TI.sup.b (.+-.)-7 90 1.18 19.10 16 (+)-7 90 0.68 72.80 107
(.+-.)-7a 55 2.82 12.00 4 (.+-.)-8a 84 6.16 23.8 4 (.+-.)-8b 81
2.28 21.50 9 (+)-10 88 0.89 9.27 11 (.+-.)-14a c c 37.00 c
(.+-.)-14b c c 27.10 c (.+-.)-15a c c 19.30 c (.+-.)-15b c c 21.00
c (.+-.)-15c 47 c 6.80 c (.+-.)-16a c c 20.30 c (.+-.)-16b 78 2.36
16.90 7 (.+-.)-16c c c 21.30 c (.+-.)-16d 88 5.66 21.00 4
(.+-.)-16e 88 1.67 14.00 8 (.+-.)-16f 86 1.97 17.70 9 (.+-.)-16g c
c 11.60 c (.+-.)-16h c c 20.90 c (.+-.)-18a 60 2.47 9.19 4
(.+-.)-19a c c 15.80 c (.+-.)-19b c >100 >100 c 22 c c 24.70
c a. CEM-SS MTT assay b. IC.sub.50/EC.sub.50 c. not measurable
EXAMPLE 39
[0271] ANTIVIRAL ACTIVITIES OF CALANOLIDE ANALOGUES AGAINST VIRUSES
OTHER THAN HIV
[0272] Selected calanolide analogues, prepared as described above,
were evaluated against hepatitis B virus, herpes viruses (HSV-1,
HSV-2, HCMV, VZV, and EBV), and respiratory viruses (influenza A,
influenza B, parainfluenza, adenovirus, measles, and respiratory
syncytial virus). Laboratory procedures for determining antiviral
efficacy and toxicity, as well as test design, are described more
fully below. Several compounds were found to be active against
various viruses and the results are summarized in Table II below.
Compound (+)-7 in particular was very potent against HCMV and EBV,
and potent against HBV and measles.
[0273] TESTING DESIGN DETERMINING IN VITRO ACTIVITY AND TOMCITY OF
POTENTIAL ANTIVIRAL DRUGS FOR HERPES VIRUS INFECTION
2 A. Primary Screening System - Human Foreskin Fibroblast Cells 1.
Antiviral HSV-1 or 2 Semi-automated CPE-inhibition assay (HSV-1
E-377 strain; HSV-2 MS strain) CMV Semi-automated CPE-inhibition
assay (AD169 strain) VZV Plaque reduction assay (Ellen strain) EBV
Superinfection of Raji or Daudi cells with P3HR-1; assay for early
antigen (EA) and viral capsid antigen (VCA) production 2. Toxicity
Neutral red uptake - stationary cells Cell proliferation assay -
rapidly growing cells B. Confirmatory Assay Systems - Human
Foreskin Fibroblast Cells 1. Antiviral HSV-1 or 2 Plaque reduction
assay - liquid overlay CMV Plaque reduction assay - liquid overlay
VZV Plaque reduction assay or yield reduction assay EBV P3HR-1
infection of other B-lymphocyte cell lines. Inhibition of EBV DNA
synthesis - Hybridization assay 2. Toxicity MTT assay for
cytotoxicity - stationary cells. C. Additional Follow-up Studies 1.
Antiviral Determine activity in cell lines from other species, i.e.
mice, rabbits, guinea pigs Test sensitivity of other virus strains
and clinical isolates Determine activity against ACV and GCV
resistant mutants Determine mechanism of action 2. Toxicity Bone
marrow assays - Human CFU-GM and BFU-E clonogenic assays II.
Description of Virus Isolates Used For Antiviral Evaluation A.
Herpes simplex virus type 1 (HSV-I) 1. E-377 - laboratory passaged
standard strain 2. E-115 - laboratory passaged standard strain 3.
HL-3 - low passaged clinical isolate from herpes labialis 4. HL-34
- low passaged clinical isolate from herpes labialis 5. 4E -
clinical isolate from herpes encephalitis 6. SC16 - ACV sensitive,
TK positive 7. SC 16-SI - ACV resistant, TK altered 8. DM 2.1 - ACV
resistant, TK deficient 9. PAAr - PAA and PFA resistant, polymerase
mutant 10. 11893 - ACV resistant, TK altered 11. 11359 - ACV
resistant, TK deficient 12. 11360 - ACV resistant, TK deficient 13.
B-2006 - ACV resistant, TK deficient B. Herpes simplex virus type 2
(HSV-2) 1. MS - laboratory passaged standard strain 2. X-79 -
laboratory passaged standard strain 3. Jensen - low passaged
clinical isolate from herpes genitalis 4. Heeter - low passaged
clinical isolates from herpes genitalis 5. SR - recent clinical
isolate from neonatal herpes 6. 8705 - ACV sensitive, TK positive
7. 8707 - ACV resistant, TK altered 8. 11680 - ACV resistant, TK
altered 9. 12247 - ACV resistant, TK altered 10. 11575 - ACV-
resistant, TK partial (low producers) 11. 11572 - ACV resistant, TK
partial (low producers) 12. 11785 - ACV resistant, TK partial (low
producers) 13. 8711 - ACV resistant, TK deficient 14. 11361 - ACV
resistant, TK deficient 15. AG-3 - ACV resistant, TK deficient C.
Human cytomegalovirus (HCMV) 1. AD109 - standard laboratory strain
2. Davis - standard laboratory strain 3. Towne - standard
laboratory strain 4. EC - recent low passaged clinical isolate 5.
LA - recent low passaged clinical isolate 6. CH - recent low
passaged clinical isolate 7. Mann - recent low passaged clinical
isolate 8. Coffman - recent low passaged clinical isolate 9.
C8708/17-1-1 - clinical isolate 10. C9207 3-3-1 - ganciclovir
sensitive 11. C8704 9-4-1 - ganciclovir resistant 12. C9208 5-4-2 -
ganciclovir sensitive 13. C9209 1-4-4 - ganciclovir resistant 14.
C8912-3 - ganciclovir sensitive 15. C8914-6 - ganciclovir resistant
16. C8805 37-1-1 - ganciclovir resistant 17. C8706 13-1-1 -
ganciclovir resistant 18. AD169 177.sup.R - ganciclovir resistant
and HPMPC resistant D. Murine Cytomegalovirus (MCMV) 1. Smith
strain - standard laboratory strain 2. JS strain E. Varicella
Zoster Virus (VZV) 1. Ellen - standard laboratory strain 2. Oka -
varicella vaccine strain 3. GLM - recent clinical isolate 4. DKG -
recent clinical isolate 5. KS 1027 - recent clinical isolate 6.
V8907 - clinical isolate 7. V8908 - Acyclovir resistant mutant of
V8907 8. V8602 5-1-1 - clinical isolate 9. V8602 7-1-3 - ACV
resistant. TK deficient 10. V8602 24-3-1 - ACV resistant,
polymerase mutant 11. 40 A.sup.2 - ACV resistant F. Epstein-Barr
Virus (EBV) 1. P3HR-1 - standard laboratory strain
[0274] Laboratory Procedures for Determining Antiviral Efficacy and
Toxicity
[0275] A. Preparation of Human Foreskin Fibroblast Cells
[0276] Newborn human foreskins were obtained as soon as possible
after circumcisions were performed and placed in minimal essential
medium containing vancomycin, fingizone, penicillin, and
gentamycin, at the usual concentrations, for four hours. The medium
was then removed, the foreskin minced into small pieces and washed
repeatedly until red cells were no longer present. The tissue was
then trypsinized using trypsin at 0.25% with continuous stirring
for 15 minutes at 37.degree. C. in a CO.sub.2 incubator. At the end
of each 15 minute period the tissue was allowed to settle to the
bottom of the flask. The supernatant containing cells was poured
through sterile cheesecloth into a flask containing MEM and 10%
fetal bovine serum. The flask containing the medium was kept on ice
throughout the trypsinizing procedure. After each addition of
cells, the cheesecloth was washed with a small amount of MEM
containing serum. Fresh trypsin was added each time to the foreskin
pieces and the procedure repeated until no more cells became
available. The cell-containing medium was then centrifuged at 1000
RPM at 4.degree. C. for ten minutes. The supernatant liquid was
discarded and the cells resuspended in a small amount of MEM with
10% FBS. The cells were then placed in an appropriate number of 25
cm.sup.2 tissue culture flasks. As cells became confluent and
needed trypsinization, they were gradually expanded into larger
flasks. The cells were kept on vancomycin and fungizone to passage
four.
[0277] B. Cytopathic Effect Inhibition Assay--HSV, HCMV, VZV
[0278] Low passage human foreskin fibroblast cells were seeded into
96-well tissue culture plates 24 h prior to use at a cell
concentration of 2.5 x 10.sup.4 cells per mL in 0.1 mL of minimal
essential medium (MEM) supplemented with 10% fetal bovine serum
(FBS). The cells were then incubated for 24 h at 37.degree. C. in a
CO.sub.2 incubator. After incubation, the medium was removed and
100 .mu.l of MEM containing 2% FBS was added to all but the first
row. In the first row, 125 .mu.L of experimental drug was added in
triplicate wells. Medium alone was added to both cell and virus
control wells. The drug in the first row of wells was then diluted
serially 1:5 throughout the remaining wells by transferring 25
.mu.L using the Cetus Liquid Handling Machine. After dilution of
drug, 100 .mu.L of the appropriate virus concentration was added to
each well, excluding cell control wells, which received 100 .mu.L
of MEM. For HSV-1 and HSV-2 assays, the virus concentration
utilized was 1000 PFU's per well. For CMV and VZV assays, the virus
concentration added was 2500 PFU per well. The plates were then
incubated at 37.degree. C. in a CO.sub.2 incubator for three days
for HSV-1 and HSV-2, 10 days for VZV, or 14 days for CMV. After the
incubation period, media was aspirated and the cells stained with a
0.1% crystal violet solution for 30 minutes. The stain was then
removed and the plates rinsed using tap water until all excess
stain was removed. The plates were allowed to dry for 24 h and then
read on a Skatron Plate Reader at 620 nm.
[0279] C. Plaque Reduction Assay for HSV-l and HSV-2 using
Semi-Solid Overlay
[0280] Two days prior to use, HFF cells are plated into six-well
plates and incubated at 37.degree. C. with 5% CO.sub.2 and 90%
humidity. On the date of assay, the drug is made up at twice the
desired concentration in 2x MEM and then serially diluted 1:5 in 2x
MEM using six concentrations of drug. The initial starting
concentration is usually 200 gg/mL down to 0.06 .mu.g/mL. The virus
to be used is diluted in MEM containing 10% FBS to a desired
concentration which will give 20-30 plaques per well. The media is
then aspirated from the wells and 0.2 mL of virus is added to each
well in duplicate with 0.2 mL of media being added to drug toxicity
wells. The plates are then incubated for one hour with shaking
every fifteen minutes. After the incubation period, an equal amount
of 1% agarose was added to an equal volume of each drug dilution.
This will give final drug concentrations beginning with 100
.mu.g/mL and ending with 0.03 .mu.g/mL and a final agarose overlay
concentration of 0.5%. The drug agarose mixture is applied to each
well in 2 mL volume and the plates then incubated for three days,
after which the cells were stained with a 1.5% solution of neutral
red. At the end of 4-6hr incubation period, the stain is aspirated,
and plaques counted using a stereomicroscope at lOx
magnification.
[0281] EC.sub.50 (50% effective concentration) is the concentration
required to inhibit viral cytopathogenicity by 50%
[0282] IC.sub.50 (50% inhibitory concentration) is the
concentration required to inhibit cell proliferation by 50%
[0283] Selective Index (S. I.) IC.sub.50/EC.sub.50
[0284] D. VZV Plaque Reduction Assay--Semi-Solid overlay.
[0285] The procedure is essentially the same as for the HSV plaque
assay described above with two exceptions:
[0286] 1. After addition of the drug, the plates are incubated for
ten days.
[0287] 2. On days three and six, an additional 1 mL overlay with
equal amounts of 2x MEM and 1% agarose are added.
[0288] E. CMV Plaque Assay--Semi-Solid Overlay
[0289] The procedure again is nearly the same as for HSV with a few
minor changes. The agarose used for both the initial overlay and
the two subsequent overlays is 0.8% rather than 1%. The assay is
incubated for 14 days with the additional 1 mL overlays being
applied on days four and eight.
[0290] F. Plaque Reduction Assays Using Liquid Medium Overlay
[0291] The procedure for the liquid overlay plaque assay is similar
to that using the agarose overlay. The procedure for adding the
virus is the same as for the regular plaque assay. The drugs are
made up in a concentration to be used in MEM with 2% FBS. The drugs
are not made up at 2x concentration as in the previous assays but
are made up at the desired concentration. For HSV-1 and HSV-2
assays, an antibody preparation obtained from Baxter Health Care
Corporation is diluted 1:500 and added to the media that the drug
is diluted in. For CMV and VZV, no antibody in the overlay is
utilized. For the CMV assay, additional medium without new drug is
added on day five and allowed to incubate for a total of 10 days.
For VZV, additional medium is added on day five and incubated for a
total of 10 days. At the end of the incubation period for all of
the assays, 2 mL of 1:10 dilution of stock neutral is added to each
well incubated for six hours. The liquid is then aspirated off and
plaques enumerated using a stereomicroscope.
[0292] G. Screening and Confirmation Assays for EBV
[0293] 1. Virus
[0294] There are two prototypes of infectious EBV. One is
exemplified by the virus derived from supernatant fluids of the
P3HR-1cell line. This cell line produces nontransforming virus that
causes the production of early antigen (EA) after primary infection
or superinfection of B cell lines. The other prototype is
exemplified by the B-95-8 virus. This virus immortalized cord blood
lymphocytes and induced tumors in marmosets.
[0295] It does not, however, induce an abortive productive
infection even in cell lines harboring EBV genome copies. The virus
used in our assays is P3HR-1.
[0296] 2. Cell Lines
[0297] Ramos is an exceptional B cell line derived from Burkitt's
lymphoma tumor but containing no detectable EBV genome copies and
is EBNA negative. Ramos/AW was obtained by in vitro infection of
Ramos with the P3HR-1 virus and contains one resident EBV genome
copy/cell. Raji is a Burkitt's lymphoma cell line containing 60 EBV
genomes/cell, and will be the primary cell used for screening
antiviral activity against EBV EA expression. Daudi is a low level
producer that contains 152 EBV genome copies/cell. It spontaneously
expresses EBV EA in 0.25%-0.5% of the cells. It will be used in
follow-up studies to confirm activity. These cell lines respond to
superinfection by EBV by expressing EA(D), EA(R), and VCA. All cell
lines are maintained in RPMI-1640 medium supplemented by 10% FCS,
L-glutamine and 100 .mu.g/mL gentamicin. The cultures are fed twice
weekly and the cell concentration adjusted to 3.times.10.sup.5/mL.
The cells are kept at 37.degree. C. in a humidified atmosphere with
5% CO.sub.2.
[0298] 3. Immunofluorescence Assays with Monoclonal Antibodies
[0299] Cells are infected with the P3HR-1 strain of EBV and the
drugs to be tested are added after adsorption (45 minutes at
37.degree. C.) and washing of the cell cultures. The cultures are
incubated for two days in complete medium to allow viral gene
expression. Following the 48 hr incubation period, the number of
cells of each sample are counted and smears made. Monoclonal
antibodies to the different EA components and VCA are then added to
the cells incubated and washed. This is followed by a fluorescein
conjugated rabbit anti-mouse Ig antibody; and the number of
fluorescence positive cells in the smears are counted. The total
number of cells in the cultures positive for EA or VCA are then
calculated and compared.
[0300] H. Cell Proliferation Assay--Toxicity
[0301] Twenty-four hours prior to assay, HFF cells are seeded in
6-well plates at a concentration of 2.5.times.10.sup.4 cells per
well in MEM containing 10% FBS. On the day of the assay, drugs are
diluted serially in MEM containing 10% FBS at increments of 1:5
covering a range from 100 .mu.g/mL to 0.03 gg/mL. For drugs that
have to be solubilized in DMSO, control wells receive MEM
containing 10% DMSO. The media from the wells is then aspirated and
2 mL of each drug concentration is then added to each well. The
cells are then incubated in a CO.sub.2 incubator at 37.degree. C.
for 72 h. At the end of this time, the media-drug solution is
removed and the cells washed. One mL of 0.25% trypsin is added to
each well and incubated until the cells start to come off of the
plate. The cell-media mixture is then pipetted up and down
vigorously to break up the cell suspension, and 0.2 mL of the
mixture is added to 9.8 mL of Isoton III and counted using a
Coulter Counter. Each sample is counted three times with three
replicate wells per sample.
[0302] I. MTT Assay for Cell Cytotoxicity
[0303] Twenty-four hours prior to assay, HFF cells are plated into
96-well plates at a concentration of 2.5.times.10.sup.4 cells per
well. After 24 h, the media is aspirated and 125 mL of drug is
added to the first row of wells and then diluted serially 1:5 using
the automated Cetus Liquid Handling System in a manner similar to
that used in the CPE assay. The plates are then incubated in a
CO.sub.2 incubator at 37.degree. C. for seven days. At this time,
each well receives 50 mL of I ig/mL solution of MTT in Dulbeccop's
Phosphate Buffered Saline. The plates are then incubated for an
additional four hours. At this time, the media is removed and
replaced with 100 .mu.L of 0.04N hydrochloric acid in isopropanol.
After shaking briefly, the plates are then read on a plate reader
at 550 nm.
[0304] J. Neutral Red Uptake Assay--Toxicity
[0305] The procedure for plating cells and adding drug is the same
as for the MTT Assay.
[0306] After drug addition, the plates are incubated for seven days
in a CO.sub.2 incubator at 37.degree. C. At this time the
media/drug is aspirated and 200 RL/well of 0.01% neutral red in
DPBS is added. This is incubated in the CO.sub.2 incubator for one
hour. The dye is aspirated and the cells are washed using a Nunc
Plate Washer. After removing the DPBS wash, 200 .mu.g/well of 50%
EtOH/1% glacial acetic acid (in H.sub.2O) is added. The plates are
rotated for 15 minutes and the optical densities are read at 550 nm
on a plate reader.
[0307] ASSAY METHODS OF HBV & INFLUENZA VIRUS: ANALYSIS OF
POTENTIAL ANTIVIRAL AGENTS AGAINST HBV REPLICATION IN CULTURES OF
2.2.15 CELLS
[0308] ANTIVIRAL ASSAYS
[0309]
[0310] The protocol for assaying anti-HBV compounds in cultures of
2.2.15 cells can be briefly summarized as follows (Korba and
Milman, 1991, Antiviral Res. 217:217). Chronically HBV-producing
human liver cells (Acs, et al., 1987, PNAS 84:4641) are seeded into
24-well tissue culture plates and grown to confluence. Test
compounds are then added daily for a continuous 9 day period.
Culture medium (changed daily during the treatment period) is
collected and stored for analysis of extracellular (virion) HBV DNA
after 0, 3, 6, and 9 days of treatment. Treated cells are lysed 24
hours following day 9 of treatment for the analysis of
intracellular HBV genomic forms. HBV DVA is then analyzed in a
quantitative and qualitative manner for overall levels of HBV DNA
(both extracellular and intracellular DNA) and the relative rate of
HBV replication (intracellular DNA).
[0311] TOXICITY ASSAYS
[0312] The protocol for determining toxicity of compounds in
cultures of 2.2.15 cells can be briefly summarized as follows.
Cells of 2.2.15 were grown to confluence in 96-well flat-bottomed
tissue culture plates and treated with compounds (in 0.2 mL culture
medium/well) as described above. Four concentrations of each
compound were assayed, each in triplicate cultures, in 3- to
10-fold steps. Untreated control cultures were maintained on each
96-well plate. On each 96-well plate, wells containing no cells
were used to correct for light scattering. Toxicity was determined
by the inhibition of the uptake of neutral red dye, determined by
absorbance at 510 nm relative to untreated cells (Finter et al.,
1969, J. Med. Chem 5:419), 24 hours following day 9 of
treatment.
[0313] ASSAY PARAMETERS
[0314] Both intracellular and extracellular HBV DNA are analyzed in
order to (i) allow for verification of compound efficacy and (ii)
provide possible data on the target site in the HBV replication
pathway for the compound from examination of the pattern of viral
replicative forms. The culture medium is changed daily during the
treatment period to (i) prevent the buildup of potentially toxic
metabolites derived from test compounds and (ii) provide an
analysis of HBV virion production during discrete 24-hour intervals
which enables a quantitative comparison of any effect on virion
production.
[0315] The analysis of HBV DNA is performed using blot
hybridization techniques (Southern and slot blot) and
[.sup.32P]-labeled HBV-specific probes. HBV DNA levels are measured
by comparison to known amounts of HBV DNA standards applied to
every nitrocellulose membrane (gel or slot blot). An AMBIS beta
scanner, which measures the radioactive decay of the hybridized
probes directly from the nitrocellulose membranes, is used for the
quantitative analysis. Standard curves, generated by multiple
analyses, are used to correlate CPM measurements made by the beta
scanner with relative levels of target HBV DNA. The levels of HBV
virion DNA released into the culture medium are analyzed by a slot
blot hybridization procedure. HBV DNA levels are then compared to
those at Day 0 to determine the effect of drug treatment.
[0316] A typical pattern of intracellular HBV DNA is displayed in
the figure below (panel A, lanes 1 and 2). The levels of HBV DNA in
each of three classes of viral genomic forms are individually
quantitated in order to evaluate the replication status of the
virus: episomal monomers, DNA replication intermediates [RI], and
integrated HBV DNA.
[0317] The levels of RI and episomal monomers are used as an
indicator of the relative level of HBV replication. Integrated HBV
DNA is used to normalize the relative amounts of DNA in each lane
because the levels of this class of HBV DNA would be expected to
remain constant on a per cell basis. The type of changes in the
intracellular HBV DNA patterns which are indicative of a decline in
HBV replication are shown in lanes 3 and 4 of the figure.
Inhibition of HBV DNA replication is indicated by the loss of RI
without changes in the level of integrated HBV DNA.
[0318] ASSAYS FOR ANTIVIRAL ACTIVITY AGAINST RESPIRATORY
VIRUSES
[0319] 1. Viruses used in primary screen
[0320] A. Influenza A and B
[0321] Virus strains: A/Texas/36191 (H1N1) (Source: Center for
Disease Control and Prevention [CDC]), A/Beijing/2/92 (H3N2)
(Source: CDC), B/Panama/45/90 (Source: CDC), A/NWS/33 (H1N1)
(Source: American Type Culture Collection [ATCC]). (All but
A/NWS/33 are tested in the presence of trypsin). Cell lines: Madin
Darby canine kidney (MDCK) cells.
[0322] B. Respiratory syncytial virus
[0323] Virus strain: Utah 89 (source: Utah State Diagnostic
Laboratory) Cell line: African green monkey kidney (MA-104)
cells.
[0324] C. Parainfluenza type 3 virus
[0325] Virus strain: C243 (Source ATCC) Cell line: African green
monkey kidney (MA-104) cells.
[0326] D. Measles virus
[0327] Virus strain: CC (Source: Pennsylvania State University)
Cell line: African green monkey kidney (BSC-1) cells.
[0328] E. Adenovirus type 5
[0329] Virus strain: Adenoid 75 (Source ATCC) Cell line: Human lung
carcinoma (A549) cells.
[0330] 2. Methods for assay of antiviral activity
[0331] A. Inhibition of Viral Cytopathic Effect (CPE)
[0332] This test, run in 96-well flat-bottomed microplates, is used
for the initial antiviral evaluation of all new test compounds. In
this CPE inhibition test, seven one-half loglo dilutions of each
test compound will be added to 4 cups containing the cell
monolayer; within 5 minutes, the virus is then added and the plate
sealed, incubated at 37.degree. C and CPE read microscopically when
untreated infected controls develop a 3 to 4+ CPE (approximately 72
hr). A known positive control drug is evaluated in parallel with
test drugs in each test. This drug is ribavirin for influenza,
measles, respiratory syncytial and parainfluenza viruses, and HPMPA
for adenovirus. The data are expressed as 50% effective
(virus-inhibitory) concentrations (EC.sub.50).
[0333] B. Increase in Neutral Red (NR) Dye Uptake
[0334] This test is run to validate the CPE inhibition seen in the
initial test, and utilizes the same 96-well microplates after the
CPE has been read.
[0335] Neutral red is added to the medium; cells not damaged by
virus take up a greater amount of dye, which is read on a
computerized microplate autoreader. An EC.sub.50 is determined from
this dye uptake.
[0336] C. Decrease in Virus Yield
[0337] Compounds considered active by CPE inhibition and by NR dye
uptake will be retested using both CPE inhibition, and, using the
same plate, effect on reduction of virus yield by assaying frozen
and thawed eluates from each cup for virus titer by serial dilution
onto monolayers of susceptible cells. Development of CPE in these
cells is the indication of presence of infectious virus. As in the
initial tests, a known active drug is run in parallel as a positive
control. The 90% effective concentration (EC.sub.90), which is that
test drug concentration that inhibits virus yield by 1 log.sub.10,
is determined from these data.
[0338] 3. Methods for assay of cytotoxicity
[0339] A. Visual Observation
[0340] In the CPE inhibition tests, two wells of uninfected cells
treated with each concentration of test compound are run in
parallel with the infected, treated wells. At the time CPE is
determined microscopically, the toxicity control cells are also
examined microscopically for any changes in cell appearance
compared to normal control cells run in the same plate. These
changes may be enlargement, granularity, cells with ragged edges, a
filmy appearance, rounding, detachment from the surface of the
well, or other changes. These changes are given a designation of T
(100% toxic), PVH (partially toxic-very heavy-80%), P. (partially
toxic-heavy-60%), P (partially toxic 40%), Ps, (partially
toxic-slight-20%), or 0 (no toxicity-0%), confonning to the degree
of cytotoxicity seen. A 50% cell inhibitory (cytotoxic)
concentration (IC.sub.50) is determined by regression analysis of
these data.
[0341] B. Neutral Red Uptake
[0342] In the neutral red dye uptake phase of the antiviral test
described above, the two toxicity control wells also receive
neutral red and the degree of color intensity is determined
spectrophotometrically. A neutral red IC.sub.50 (NR IC.sub.50) is
subsequently determined.
[0343] C. Viable Cell Count
[0344] Compounds considered to have significant antiviral activity
in the initial CPE and NR tests are retested for their effects on
cell growth. In this test, 12-well tissue culture plates are seeded
with cells (sufficient to be approximately 20% confluent in the
well) and exposed to varying concentrations of the test drug while
the cells are dividing rapidly. The plates are then incubated in a
CO.sub.2 incubator at 37.degree. C. for 72 hr, at which time the
media-drug solution is removed and the cells washed. Trypsin is
added to remove the cells, which are then counted using a Coulter
cell counter. An IC.sub.50 is then determined using the average of
three separate counts at each drug dilution.
[0345] 4. Data analysis
[0346] Each test compound's antiviral activity is expressed as a
selectivity index (SI), which is the IC.sub.50 or IC.sub.90 divided
by the EC.sub.50. Generally, an SI of 10 or greater is indicative
of positive antiviral activity, although other factors, such as a
low SI for the positive control, are also taken into
consideration.
3TABLE II Antiviral Activities of Calanolide Analogues against
Viruses Other Than HIV HBV.sup.a HCMV.sup.b EBV.sup.b EC.sub.50
IC.sub.50 SI.sup.c EC.sub.50 IC.sub.50 SI.sup.c EC.sub.50 IC.sub.50
SI.sup.c (.+-.)-7 (+)-7 7.2 57 8 0.11 47.9 435 0.94 >50 >53
(-)-7 >10 327 <32 >20 23.7 <1.2 >20 23.7 <1
(.+-.)-7a 44 32 1 0.15 19.8 132 >50 >50 1 (+)-7a 2.5 382
153.sup.d >4.0 11.6 <3 >4.0 11.9 <3 (-)-7a >10 86
<8.6 >4.0 11.9 <3 (.+-.)-18a >10 88 <9 >4.0 19.5
<5 24.5 >50 >2 (+)-18a >10 88 1 >100 >100 <1
>50 >50 <1 5 >10 2085 <209 >100 >100 <1
>50 >50 <1 (.+-.)-15c >10 152 <15 >20 77.6 <4
>50 >50 <1 (+)-15e >10 86 <8.6 >20 75.3 <4
>50 >50 <1 (.+-.)-15b >10 307 <31 >20 52.3 <3
>50 >50 <1 (.+-.)-15f 0.67 270 400.sup.e >100 >100
<1 >50 >50 <1 (.+-.)-15a 2.0 1986 993.sup.f >100
>100 <1 (-)-calanolide B >10 1000 <100 2.6 78.4 30
>50 >50 <1 (-)-23 3.5 340 97 >4.0 18.8 <5 (-)-24
>10 151 <15 >4.0 14.4 <4 >50 >50 <1 (+)-1
>2.6 175 <67 >20 86.2 <4 >50 >50 <1 (-)-1
>30 173 <6 >20 80.3 <4 >100 >100 <1 VZV.sup.b
Others.sup.a EC.sub.50 IC.sub.50 SI.sup.c EC.sub.50 IC.sub.50
SI.sup.c (.+-.)-7 (+)-7 55.9 >100 1.8 3.sup.g 10.sup.g 33.sup.g
(-)-7 (.+-.)-7a 13.7 33.3 2.4 13.7.sup.g 333.sup.g 2.4.sup.g (+)-7a
(-)-7a (.+-.)-18a 15.4 33.3 2.2 (+)-18a >100 >100 <1 5
>20 52.2 <3 (.+-.)-15c >20 68.0 <3 (.+-.)-15e >100
>100 <1 (.+-.)-15b >20 66.7 <3 (.+-.)-15f >20 60.0
<3 (.+-.)-15a (-)-calanolide B >4.0 20.0 <5 (-)-23 >4.0
16.0 <4 20.sup.g >100.sup.h >5.sup.h (-)-24 >4.0 12.0
<3 57.sup.i >100.sup.i >18.sup.i (+)-1 (-)-1 .sup.aThe
unit for EC.sub.50 and IC.sub.50 .mu.M; .sup.bThe unit for
EC.sub.50 and IC.sub.50 is .mu.g/mL; .sup.cSelectivity index is
expressed as IC.sub.50/EC.sub.50; .sup.dEC.sub.90 = 11 .mu.M, and
IC.sub.50/EC.sub.90 = 35; .sup.eEC.sub.90 = 4.2 .mu.M, and
IC.sub.50/EC.sub.90 = 64; .sup.fEC.sub.90 = 16 .mu.M, and
IC.sub.50/EC.sub.90 =124; .sup.gagainst measles; .sup.hagainst
adenovirus type-1; .sup.iagainst influenza A(H1N1).
EXAMPLE 40
[0347] ANTI-CMV ACTIVITIES OF CALANOLIDE ANALOGUES AGAINST VARIOUS
ISOLATES IN PLAQUE REDUCTION ASSAY
[0348] Five calanolide analogues, prepared as described above, were
further evaluated for their anti-HCMV activity in plaque reduction
assay against various laboratory and clinical isolates (Towne, AD
169, E. Mann, Coffinan, and C9208). Laboratory procedures were as
described in Example 39. The results are summarized in Table III
below. All compounds tested exhibited activity against all isolates
tested.
4TABLE III Anti-CMV Activities of Calanolide Analogues against
Various Isolates in Plaque Reduction Assay (+)-7.sup.a
(.+-.)-7.sup.a (.+-.)-7a.sup.a Isolate EC.sub.50 MTC.sup.b TI.sup.c
EC.sub.50 MTC.sup.b TI.sup.c EC.sub.50 MTC.sup.b TI.sup.c Towne
5.02 >100 >20 3.34 >100 >30 3.08 10 3.2 AD169 3.16 100
32 3.16 100 32 3.16 10 3.2 E. Mann 4.65 100 22 3.16 100 32 3.16 10
3.2 Coffman 4.78 >100 >21 3.34 >100 >30 3.16 10 3.2
C9208 4.91 >100 >20 7.16 100 14 2.60 10 3.8 (-)-calanolide
B.sup.a (-)-23.sup.a Isolate EC.sub.50 MTC.sup.b TI.sup.c EC.sub.50
MTC.sup.b TI.sup.c Towne 3.20 <100 <31 3.16 10 3.2 AD169 3.16
10 3.2 3.16 10 3.2 E. Mann 3.16 10 3.2 3.16 10 3.2 Coffman 3.18
<100 <31 3.16 <100 <32 C9208 2.01 10 3.3 2.99 10 3.3
.sup.aThe unit for EC.sub.50 and MTC is .mu.M; .sup.bThe minimum
drug concentration that reduced cell viability by 25%; .sup.cTI is
MTC/EC.sub.50.
EXAMPLE 41
[0349] ANTI-HBV ACTIVITIES OF CALANOLIDE ANALOGUES IN SECONDARY
ASSAY
[0350] Compounds (.+-.)-15f and (.+-.)-15a, prepared as described
above, were investigated in a secondary assay against HBV.
Laboratory procedures were as described in Example 39. The results
are summarized in Table IV below. Compound (.+-.)-15a was active in
primary assay (HBV virion), and compound (+)-15f was active in both
primary assay and secondary assay (HBV RI).
5TABLE IV Anti-HBV Activities of Calanolide Analogues in Secondary
Assay HBV Virion.sup.a HBV RI.sup.a Compound EC.sub.50 EC.sub.90
IC.sub.50 SI.sup.b EC.sub.50 EC.sub.90 IC.sub.50 SI.sup.b (+)-15f
0.81 5.0 303 61 5.7 15 303 20 (.+-.)-15a 2.8 20 1789 89 >30
>30 1789 <60 .sup.aThe unit for EC.sub.50, EC.sub.90 and
IC.sub.50 is .mu.M; .sup.bSI is IC.sub.50/EC.sub.90.
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