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Abstract:

Embodiments of the present invention provide compounds (such as Formula
(I) compounds, Formula (II) compounds, and various embodiments thereof).
Compositions comprising those compounds are also provided. Methods for
their preparation are included. Also, uses of the compounds are included,
such as administering and treating diseases (e.g., cancer and
infections).

Claims:

1. A compound of formula (I) ##STR00044## wherein R1 and R2
can be the same or different and are selected from the group consisting
of: --OH, alkoxy, benzyloxy, --OCH2--CH═CH2, and
--OCH2OCH3; A is a bivalent, substituted or unsubstituted,
branched or unbranched alkane or alkene; and R3 is a substituted or
unsubstituted four-, five-, six-, or seven-member ring that optionally
includes one or more heteroatoms in the ring; with the proviso that the
compound is not ##STR00045##

2. The compound of claim 1, wherein R1 and R2 are both --OH;
R1 and R2 are both --OCH2CH═CH2; R1 and
R2 are both --OCH2OCH3; or R1 is --OCH2OCH3
and R2 is --OH.

3. The compound of claim 1, wherein A is --CH2--CH2--,
--CH═CH-- or --CH═CH--CH═CH--.

4. The compound of claim 1, wherein R3 is selected from the group
consisting of: ##STR00046##

5. The compound of claim 1, wherein the compound can produce an SI of at
least about 10 where the SI is a ratio of an IC50 of an undesired
cell to an IC50 of a desired cell, the undesired cell is a parasite
or a mycobacteria, and the desired cell is a VERO cell or a macrophage.

6. The compound of claim 5, wherein the compound can produce an SI of at
least about 10 for at least one of an undesired cell that is a L.
amazonensis and a desired cell that is a macrophage, or an undesired cell
that is a T. cruzi and a desired cell that is a VERO cell.

7. The compound of claim 1, wherein the compound can produce a reduction
in parasite burden of at least about 25% in an animal.

8. A composition comprising the compound of claim 1.

9. The composition of claim 8, wherein the composition is in the form
selected from the group consisting of: a solution, an emulsion, a tablet,
a capsule, a pill, a gel, an ointment, a cream, a lotion, a food, and a
drink.

10. The composition of claim 8, wherein the composition comprises from
about 25% to about 75% of the compound.

11. The composition of claim 8, wherein the composition is a
pharmaceutical composition.

12. A method for synthesizing a compound of claim 1 comprising subjecting
##STR00047## to a condensation reaction, and recovering ##STR00048##
wherein R1 and R2 can be the same or different and are selected
from the group consisting of: --OH, alkoxy, benzyloxy,
--OCH2--CH═CH2, and --OCH2OCH3; A is a bivalent,
substituted or unsubstituted, branched or unbranched alkane or alkene;
and R3 is a substituted or unsubstituted four-, five-, six-, or
seven-member ring that may include one or more heteroatoms in the ring.

13. The method of claim 12, wherein R1 and R2 are both --OH;
R1 and R2 are both --OCH2CH═CH2; R1 and
R2 are both --OCH2OCH3; or R1 is --OCH2OCH3
and R2 is --OH.

14. The method of claim 12, wherein A is --CH2--CH2--,
--CH═CH--, or --CH═CH--CH═CH--.

15. The method of claim 12, wherein R3 is selected from the group
consisting of: ##STR00049##

16. The method of claim 12, wherein the compound is not ##STR00050##

17. The method of claim 12, wherein the condensation reaction is a
Claisen-Schmidt aldol condensation reaction.

18. The method of claim 12, further comprising a deprotection reaction
after the condensation reaction and before the recovering.

19. The method of claim 18, wherein the deprotection reaction is a
dealkylation reaction.

20. The method of claim 18, wherein the deprotection reaction comprises
combining the product of the condensation reaction with
Pd(PPh3)4 in K2CO3 and MeOH.

21. A method comprising administering a composition comprising a compound
to at least one cell wherein the compound is ##STR00051## R1 and
R2 can be the same or different and are selected from the group
consisting of: --OH, alkoxy, benzyloxy, --OCH2--CH═CH2, and
--OCH2OCH3; A is a bivalent, substituted or unsubstituted,
branched or unbranched alkane or alkene; and R3 is a substituted or
unsubstituted four-, five-, six-, or seven-member ring that may include
one or more heteroatoms in the ring.

22. The method of claim 21, wherein R1 and R2 are both --OH;
R1 and R2 are both --OCH2CH═CH2; R1 and
R2 are both --OCH2OCH3; or R1 is --OCH2OCH3
and R2 is --OH.

23. The method of claim 21, wherein A is --CH2--CH2--,
--CH═CH--, or --CH═CH--CH═CH--.

24. The method of claim 21, wherein R3 is selected from the group
consisting of: ##STR00052##

25. The method of claim 21, wherein the compound can produce an SI of at
least about 10 where the SI is a ratio of an IC50 of an undesired
cell to an IC50 of a desired cell, the undesired cell is a parasite
or a mycobacteria, and the desired cell is a VERO cell or a macrophage.

26. The method of claim 21, wherein the compound can produce an SI of at
least about 10 for at least one of an undesirable cell that is a L.
amazonensis and a desirable cell that is a macrophage, or an undesirable
cell that is a T. cruzi and a desirable cell that is a VERO cell.

27. The method of claim 21, wherein the compound can produce a reduction
in parasite burden of at least about 25% in an animal.

28. The method of claim 21, wherein the at least one cell is at least one
animal cell.

29. The method of claim 21, wherein the administering is part of a
treatment of a mammal.

30. The method of claim 29, wherein the route of administration is
selected from the group consisting of: an oral route, a parenteral route,
a cutaneous route, a nasal route, a rectal route, a vaginal route, and an
ocular route.

31. The method of claim 29, wherein the mammal is being treated for an
infectious disease or for a cancer.

32. The method of claim 29, wherein the mammal is being treated for a
parasitic infection or for a microbial infection.

33. A compound of formula (II) ##STR00053## wherein R4 and R5
can be the same or different and are selected from the group consisting
of: H, methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, t-butyl, and
substituted or unsubstituted four-, five-, six-, or seven-member ring
that may include one or more heteroatoms in the ring; if R4 is a
ring then R4 may optionally be linked by a bivalent, substituted or
unsubstituted, branched or unbranched alkane or alkene; R4 and
R5 are not both H; and R4 and R5 are selected according to
either (i) R4=methyl and R5=phenyl; R.sup.4.dbd.R5=methyl;
R.sup.4.dbd.R5=ethyl; or R.sup.4.dbd.R5=phenyl, or (ii) at
least one of R4 and R5 is selected from the group consisting
of: ##STR00054##

34. A compound of claim 33, wherein the compound is selected from the
group consisting of: ##STR00055## ##STR00056## ##STR00057##

35. A compound of 33, wherein the compound is selected from the group
consisting of: ##STR00058##

36. The compound of claim 33, wherein the compound can produce an SI of
at least about 10 for an undesired cell that is a parasite or
mycobacteria and a desired cell that is a VERO cell or a macrophage.

37. The compound of claim 33, wherein the compound can produce an SI of
at least about 10 for either an undesired cell that is L. amazonensis,
and a desired cell that is a macrophage, or an undesired cell that is T.
cruzi, and a desired cell that is a VERO cell.

38. The compound of claim 33, wherein the compound can produce a
reduction in parasite burden of at least about 25% in an animal.

39. A composition comprising the compound of claim 33.

40. The composition of claim 39, wherein the composition is in the form
selected from the group consisting of: a solution, an emulsion, a tablet,
a capsule, a pill, a gel, an ointment, a cream, a lotion, a food, and a
drink.

41. The composition of claim 39, wherein the composition comprises from
about 25% to about 75% of the compound.

42. The composition of claim 39, wherein the composition is a
pharmaceutical composition.

43. A method for synthesizing the compound of claim 33 comprising
subjecting ##STR00059## to a condensation reaction, and recovering the
compound of claim 1.

44. The method of claim 43, wherein the compound is selected from the
group consisting of: ##STR00060##

45. The method of claim 43, wherein the condensation reaction comprises
refluxing in ethanol or methanol for from about 2 hours to about 24
hours.

46. A method comprising administering a compound to at least one animal
cell wherein the compound is ##STR00061## R4 and R5 can be
the same or different and are selected from the group consisting of: H,
methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, t-butyl, and
substituted or unsubstituted four-, five-, six-, or seven-member ring
that may include one or more heteroatoms in the ring; if R4 is a
ring then R4 may optionally be linked by a bivalent, substituted or
unsubstituted, branched or unbranched alkane or alkene; and with the
proviso that the compound is not ##STR00062##

47. The method of claim 46, wherein R4 and R5 can be the same
or different and are selected from the group consisting of: ##STR00063##
##STR00064## and methyl.

48. The method of claim 46, wherein the compound is selected from the
group consisting of: ##STR00065## ##STR00066## ##STR00067##

49. The method of claim 46, wherein the compound is selected from the
group consisting of: ##STR00068##

50. The method of claim 46, wherein the compound can produce an SI of at
least about 10 where the SI is a ratio of an IC50 of an undesired
cell to an IC50 of a desired cell and the undesired cell is a
parasite or mycobacteria and the desired cell is a VERO cell or a
macrophage.

51. The method of claim 46, wherein the compound can produce an SI of at
least about 10 for at least one of (a) the undesired cell is L.
amazonensis and the desired cell is a macrophage; or (b) the undesired
cell is T. cruzi and the desired cell is a VERO cell.

52. The method of claim 46, wherein the compound can produce a reduction
in parasite burden of at least about 25% in an animal.

53. The method of claim 46, wherein the administering is part of a
treatment of a mammal.

54. The method of claim 53, wherein the route of administration is
selected from the group consisting of: an oral route, a parenteral route,
a cutaneous route, a nasal route, a rectal route, a vaginal route, and an
ocular route.

55. The method of claim 53, wherein the mammal is being treated for an
infectious disease or for a cancer.

56. The method of claim 53, wherein the mammal is being treated for a
parasitic or for a microbial infection.

Description:

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit to U.S. Provisional Application No.
61/089,231, filed Aug. 15, 2008, which is incorporated by reference in
its entirety.

BACKGROUND

[0002] Embodiments of the present invention provide compounds (e.g.,
Formula (I) compounds and Formula (II) compounds, which include compounds
related to chalcones and compounds related to benzothienopyrimidines
(BTPs)), their preparations, and their uses (e.g., treating disease).

[0003] There is a need to find drugs to provide aid (such as treatment or
cure) to diseased animals that will have one or more positive-outcome
effects, such as anticancer, anti-inflammatory, immunomodulatory,
antibacterial, immunosuppressive, and antiprotozoan activity, including,
for example, trypanocidal, leishmanicidal and antimalarial activity. For
instance, Chagas' disease or American trypanosomiasis--caused by the
vector-borne flagellate protozoan parasite Trypanosoma cruzi--is an
endemic tropical disease that has infected 20 million people in Central
and South America and approximately between 50,000 and 100,000 people in
the United States. Accordingly, some embodiments of the present invention
provide compounds to treat or cure diseased animals.

SUMMARY

[0004] Some embodiments of the present invention include a compound of
formula (I)

##STR00001##

[0005] where R1 and R2 can be the same or different and are
selected from the group consisting of: --OH, alkoxy, benzyloxy,
--OCH2--CH═CH2, and --OCH2OCH3; A is a bivalent,
substituted or unsubstituted, branched or unbranched alkane or alkene;
and R3 is a substituted or unsubstituted four-, five-, six-, or
seven-member ring that optionally includes one or more heteroatoms in the
ring. In some instances, the compound is not

##STR00002##

[0006] The formula (I) compounds can be part of a composition. In another
embodiment, a method for synthesizing a compound of claim 1 comprises:
subjecting

##STR00003## [0007] to a condensation reaction, and recovering

##STR00004##

[0007] where R' and R2 can be the same or different and are selected
from the group consisting of: --OH, alkoxy, benzyloxy,
--OCH2--CH═CH2, and--OCH2OCH3; A is a bivalent,
substituted or unsubstituted, branched or unbranched alkane or alkene;
and R3 is a substituted or unsubstituted four-, five-, six-, or
seven-member ring that may include one or more heteroatoms in the ring.

[0008] In still other embodiments, a method comprises administering a
composition comprising a compound to at least one cell, where the
compound is

##STR00005##

[0009] where R1 and R2 can be the same or different and are
selected from the group consisting of: --OH, alkoxy, benzyloxy,
--OCH2--CH═CH2, and --OCH2OCH3; A is a bivalent,
substituted or unsubstituted, branched or unbranched alkane or alkene;
and R3 is a substituted or unsubstituted four-, five-, six-, or
seven-member ring that may include one or more heteroatoms in the ring.

[0010] Some embodiments of the invention include a compound of formula
(II)

##STR00006##

[0011] where R4 and R5 can be the same or different and are
selected from the group consisting of: H, methyl, ethyl, propyl,
isopropyl, butyl, sec-butyl, t-butyl, and substituted or unsubstituted
four-, five-, six-, or seven-member ring that may include one or more
heteroatoms in the ring. In some instances, if R4 is a ring then
R4 may optionally be linked by a bivalent, substituted or
unsubstituted, branched or unbranched alkane or alkene. In still other
instances, R4 and R5 are not both H. In some embodiments,
R4 and R5 can be selected according to either (i)
R4=methyl and R5=phenyl; R4═R5=methyl;
R4═R5=ethyl; or R4═R5=phenyl, or (ii) at
least one of R4 and R5 is selected from the group consisting
of:

##STR00007##

[0012] Further embodiments of the invention include compositions
comprising the formula (II) compound or embodiments, as described above.
Embodiments also include methods for synthesizing compounds of formula
(II) (and the various embodiments thereof) comprising:

subjecting

##STR00008##

to a condensation reaction, and recovering the formula (II) compounds (or
the embodiment thereof).

[0013] Further embodiments include, methods comprising administering a
compound to at least one animal cell, where the compound is

##STR00009##

[0014] and R4 and R5 can be the same or different and are
selected from the group consisting of: H, methyl, ethyl, propyl,
isopropyl, butyl, sec-butyl, t-butyl, and substituted or unsubstituted
four-, five-, six-, or seven-member ring that may include one or more
heteroatoms in the ring. In some instances, if R4 is a ring then
R4 may optionally be linked by a bivalent, substituted or
unsubstituted, branched or unbranched alkane or alkene. In some
embodiments the compound is not

##STR00010##

[0015] Other embodiments of the present invention will be apparent in
light of the description of the invention herein.

DETAILED DESCRIPTION

[0016] The compounds of the present invention can include

##STR00011##

In some discussions below, the ring with R1 and R2 is referred
to as Ring A and the R3 system is referred to as Ring B. In some
embodiments, R1 and R2 can be the same or different and
selected from --OH, alkoxy, benzyloxy, --OCH2--CH═CH2
(allyloxy), or --OCH2OCH3. For example, one of R1 or
R2 can be --OH or neither can be --OH. The alkoxy can, for example,
be methoxy, ethoxy, or propoxy. In some embodiments, R1 is
--OCH3 and R2 is --OH; R1 and R2 are both --OH;
R1 and R2 are both --OCH2CH═CH2; R1 and
R2 are both --OCH2OCH3; or R1 is --OCH2OCH3
and R2 is --OH.

[0017] A can be a bivalent, substituted or unsubstituted, branched or
unbranched alkane or alkene. Alkene is defined as a hydrocarbon with one
or more double bonds. In some instances, A is a mixture of saturated and
unsaturated hydrocarbon groups. In some embodiments, A can be,

##STR00012##

[0018] where n can be 1, 2, 3, or 4. In some instances, A is
--CH2--CH2--, --CH2CH2CH2CH2--,
--CH═CH--, or --CH═CH--CH═CH--.

[0019] R3 can be a substituted or unsubstituted four-, five-, six-,
or seven-member ring that may include one or more heteroatoms in the
ring. Embodiments include rings that have, for example, one, two, three,
four, five, or six substitutions. The ring can be conjugated, aromatic,
unsaturated, or saturated. When the rings include heteroatoms, these
heterocycles can have 1, 2, 3, or 4 heteroatoms (such as N, S, or O),
which can be the same or different for a given ring. In some embodiments,
R3 can be a substituted or unsubstituted phenyl, naphthyl, furan,
pyridine, or pyrrole. Substitutions can include, but are not limited to
alkoxy (such as methoxy, ethoxy, propoxy), hydroxyl, amine, amide,
halogens (e.g., F, Cl, Br), nitro, allyloxy, alkyloxyalkyl (such as
methoxymethyl), alkyl (such as methyl, ethyl, propyl), substituted alkyl
(e.g., tri-halogenated methyl, trifluoromethyl). R3 can also be
substituted with moieties that are attached at two ring positions to
create a fused ring system such as alkylenedioxy (e.g., methylenedioxy
(i.e., --OCH2O)), naphthyl (e.g., 1-naphthyl or 2-naphthyl),
benzofuranyl, indolyl, or quinolyl. In some embodiments, R3 can be

[0022] In some embodiments, Formula (I) excludes at least one of the
following compounds:

##STR00023##

In some embodiments, the synthesis of the Formula (I) compounds can
comprise subjecting

##STR00024## [0023] to a condensation reaction, and recovering

[0023] ##STR00025## [0024] from the reaction mixture.

[0025] In some embodiments, the synthesis of the Formula (I) compounds
starts with the preparation of related acetophenones, as exemplified in
Scheme A.

##STR00026##

[0026] In Scheme A, step (a) can be an alkylation reaction, which can
occur, for example, by combining a substituted acetophenone with
(CH3)2SO4, K2CO3, and (CH3)2CO at
about 65° C. for about 6 h. Step (b) can be a dealkylation
reaction, which can occur, for example, by combining the product of (a)
with AlCl3 and benzene, and refluxing for about 1 hour. Step (c) can
be a protection (allylation) reaction, which can occur by combining the
product of step (b) allyl bromide, K2CO3 and DMF at room
temperature (e.g., about 25° C.) overnight. Steps (d), (d'), and
(d'') can be condensation reactions (e.g., Claisen-Schmidt aldol
condensation) and can have the same or different conditions (using for
example different hydroxyl bases). For example, the products of (a), (c),
or (h) can be combined with an aromatic aldehyde, KOH, H2O,
CH3OH at room temperature (e.g., about 25° C.) for from about
1 h to about 48 h. Step (e) can be a deprotection (dealkylation) reaction
to provide a protection group removal step, which can occur by combining
the product of (d') with catalytic Pd(PPh3)4 in K2CO3
and MeOH at about 60° C. for about 1 h. Steps (f) and (f') can be
reducing steps and can be the same or different. For example, the product
of (d) or (e) can be combined with catalytic Pd/C 5%, H2 gas at
about 250 psi and EtOAc at room temperature (e.g., about 25° C.)
for about 1.5 h. Steps (g) and (g') can be a deprotection or dealkylation
reaction and can be the same or different. For example, the product of
(d'') can be refluxed with about 3N HCl in methanol for about 30 min.
Step (h) can be an alkylation or protection reaction. For example, the
product of step (b) can be refluxed with K2CO3,
(CH3)2CO, CH3OCH2Cl for about 3 hours. Products of
steps (d), (d'), (d''), (e), (f), (f'), (g), and (g') can include
compounds of formula (I). Of course, conditions (such temperatures,
times, solvents, etc.) can be varied to provide optimized or varied
outcomes (such as product yields).

[0027] In some embodiments Scheme A can include reaction sequences as
follows: Initially, 2,4,6-trihydroxyacetophenone is transformed into
2,4-dihydroxy-6-methoxyacetophenone using dimethyl sulphate as
methylating agent. Then, the methyl group on the para-methoxy position of
the acetophenone is cleaved using AlCl3, to obtain
2,4-dihydroxy-6-methoxyacetophenone, which is protected using allyl
bromide to give 2,4-allyloxy-6-methoxylacetophenone. Claisen-Schmidt
aldol condensation of acetophenone with the corresponding aromatic
aldehyde in the presence of aqueous KOH, gives a chalcone product. After
a mild deprotection procedure to remove the allyl-protecting groups,
using Pd(PPh3)4 and K2CO3, the resulting
2'4'-dihydroxy-6-methoxy chalcones were finally reduced to produce the
corresponding dihydrochalcones.

[0028] In Scheme A and when discussed below, 2',4'-AC include some
2',4'-diallyloxy-6'-methoxychalcones; 2',4'-HC include some
2',4'-dihydroxy-6'-methoxychalcones; 2',4'-HDC include some
2',4'-dihydroxy-6'-methoxy-dihydrochalcones; 4',6'-MC include some
4',6'-dimethoxy-2'-hydroxychalcones; 4',6'-MDC include some
4',6'-dimethoxy-2'-hydroxy-dihydrochalcones; 4',6'-TC include some
2'-hydroxy-4'-methoxymethyl-6'-methoxychalcones; and 4',6'-OC include
some 2',4'-dimethoxymethyl-6'-methoxychalcones.

##STR00027##

[0029] Scheme B demonstrates another strategy for synthesizing compounds
of formula (I). This one pot synthesis can include combining the reactant
with K2CO3, MOMCl (methoxymethyl chloride), and methanol at
room temperature (e.g., about 25° C.) for about 2 hours. An
aldehyde is added to the solution and combined for about 2 hours at room
temperature (e.g., about 25° C.). A protonation workup, using an
acid solution, occurs for from about 2 to about 4 hours, to remove the
methoxymethyl protecting group. Of course, conditions (such temperatures,
times, solvents, etc.) can be varied to provide optimized or varied
outcomes (such as product yields).

[0030] The compounds of the present invention can include Formula (II)
compounds such as

[0033] R4 and R5 can be a substituted or unsubstituted four-,
five-, six-, or seven-member ring that may include one or more
heteroatoms in the ring. Embodiments include rings that have, for
example, one, two, three, four, five, or six substitutions. The ring can
be conjugated, aromatic, unsaturated, or saturated. When the rings
include heteroatoms, these heterocycles can have 1, 2, 3, or 4
heteroatoms (such as N, S, or O), which can be the same or different for
a given ring. In some embodiments, R4 or R5 can be a
substituted or unsubstituted phenyl, naphthyl, furan, pyridine, or
pyrrole. Substitutions can include, but are not limited to alkoxy (such
as methoxy, ethoxy, or propoxy), hydroxyl, amine, amide, halogens (e.g.,
F, Cl, or Br), nitro, allyloxy, alkyloxyalkyl (such as methoxymethyl),
alkyl (such as methyl, ethyl, or propyl), substituted alkyl (e.g.,
tri-halogenated methyl or trifluoromethyl).

[0034] In some embodiments, R4 and R5 also include the rings (as
just described) and a link to the core structure. The link can be a
bivalent, substituted or unsubstituted, branched or unbranched alkane or
alkene. In some instances, the link is a mixture of saturated and
unsaturated hydrocarbon groups. In some embodiments, the link can be,

##STR00029##

[0035] where m can be 1, 2, 3, or 4. In some instances, the link is
--CH2CH2--, --CH2CH2CH2CH2--, --CHCH--, or
--CHCHCHCH--. In some embodiments, R4 is a cinnamyl group.

[0036] R4 or R5 can also be substituted with moieties that are
attached at two ring positions to create a fused ring system such as
alkylenedioxy (e.g., methylenedioxy (i.e., --OCH2O)), naphthyl
(e.g., 1-naphthyl or 2-naphthyl), benzofuranyl, indolyl, or quinolyl.

[0037] In some instances, R5, R4, or both, are hydrogen.

[0038] R4 and R5 can be the same or different and can be:

##STR00030## ##STR00031##

In some embodiments, R4 and R5 can be selected according to (i)
or (ii), as follows:

[0039] (i) R4=methyl and R5=phenyl; R4═R5=methyl;
R4═R5=ethyl; or R4═R5=phenyl, or

[0040] (ii) at least one of R4 and R5 is selected from the group
consisting of:

##STR00032##

In some embodiments, Formula (II) can be (with molecular formulas and
calculated molecular weights):

[0041] Some compounds of Formula (II) can be synthesized, in some
embodiments, as summarized in Scheme C, below. In step (a), a Gewald
reaction can be used to prepare the thiol ethyl ester by, for example,
combining the reactants (and in some instance refluxing) with S8, a
cyclic or alkyl ketone, Et2NH (or any other secondary amine), and
ethanol for about 30 minutes to about 2 hours. In step (b), the product
of step (a) is treated using a condensation reaction. For example, the
step (a) product is combined with a formamide to make the corresponding
pyrimidinone. In step (c), the product of step (b) is treated to provide
a leaving group for step (d)'s nucleophilic displacement reaction. For
example, step (c) can be a chlorination reaction such as refluxing with
POCl3 for about 3 hours to about 6 hours. In step (d), the
nucleophilic displacement reaction can occur by refluxing with hydrazine
and ethanol for about 5 hours. For step (e), the product of step (d) can
be reacted (via a condensation reaction) to form a Schiff base with the
corresponding ketone or aldehyde by, for example, refluxing the step (d)
product with methanol or ethanol and the corresponding ketone or aldehyde
for about 2 hours to about 24 hours (e.g., about 4 hours). Of course,
conditions (such temperatures, times, solvents, etc. . . . ) can be
varied to provide optimized or varied outcomes (such as product yields).

##STR00040##

[0042] The compounds of Formula (I) and Formula (II) can be administered
to animals by any number of administration routes or formulations. The
compounds can also be used to treat animals for a variety of diseases.
Animals include but are not limited to canine, bovine, porcine, avian,
mammalian, and human.

[0043] Diseases that can be treated or cured using the compounds include,
but are not limited to Leishmaniasis, Chagas disease, Cancer, diseases
related to Helicobacter (e.g., gastric ulcer), tuberculosis, Malaria,
helminth infectious diseases, African sleeping sickness, onchocerciasis,
blinding trachoma, buruli ulcer, Cholera, Dengue, Dracunculiasis (guinea
worm disease), Fascioliasis, Leprosy, yaws, lymphatic filariasis, and
schistosomiasis. Moreover, the compounds can have many disease
state-effects including but not limited to antimicrobial,
anti-inflammatory, antibacterial, antiprotozoan, antifungal, anti-cancer,
antiviral, immunomodulatory, immunosuppressive, and antineoplastic
effects. In some instances, the compounds can modulate (e.g., disrupt)
membrane dynamics of the Golgi apparatus of the parasite. In some
instances, the compounds can be used to inhibit an infecting organism's
enzymatic pathway and the animal being treated is minimally affected
because it does not have the inhibited enzymatic pathway.

[0044] The route of administration of the compounds may be of any suitable
route such as that which provides a concentration in the blood
corresponding to a therapeutic concentration. Administration routes that
can be used, but are not limited to the oral route, the parenteral route,
the cutaneous route, the nasal route, the rectal route, the vaginal route
and the ocular route. The choice of administration route can depend on
the compound identity, such as the physical chemical properties of the
compound, as well as the age and weight of the animal, the particular
disease, and the severity of the disease. For example, Leishmaniasis
treatment can use a cream, ointment, or oil. Treatment for Tuberculosis
can include administration by intravenous delivery, by pill or by
cutaneous injection. Of course, combinations of administration routes can
be administered, as desired.

[0045] The compounds of Formula (I) and Formula (II) can be part of a
pharmaceutical composition and can be in an amount from about 1% to about
95% by weight of the total composition (or from about 10% to about 90%,
or from about 25% to about 75%). The composition can be presented in a
dosage form which is suitable for the oral, parenteral, rectal,
cutaneous, nasal, vaginal, or ocular administration route. The
composition can be of the form of, for example, tablets, capsules, pills,
powders granulates, suspensions, emulsions, solutions, gels (including
hydrogels), pastes, ointments, creams, plasters, drenches, delivery
devices, suppositories, enemas, injectables, implants, sprays, aerosols
or other suitable forms.

[0046] Pharmaceutical compositions can be formulated to release the active
compound substantially immediately upon the administration or any
substantially predetermined time or time after administration. Such
formulations can include, for example, controlled release formulations
such as various controlled release compositions and coatings. Such
formulations also include pro-drug principles, such as converting the
active drug substance into an inactive derivative; when the pro-drug is
administered to the organism, the organism converts the pro-drug to the
active drug (e.g., by an enzymatic or non-enzymatic process) so the
active drug can exert its therapeutic effect.

[0047] Other formulations include those incorporating the drug (or control
release formulation) into food, food stuffs, feed, or drink.

[0048] The compounds of Formula (I) and Formula (II) can be in the form of
salts, optical and geometric isomers, and salts of isomers. Also, the
compounds can be in various forms, such as uncharged molecules,
components of molecular complexes, or non-irritating pharmacologically
acceptable salts, e.g. the hydrochloride, hydrobromide, sulphate,
phosphate, nitrate, borate, acetate, maleate, tartrate, salicylate, etc.
For acidic compounds, salts include metals, amines, or organic cations
(e.g. quaternary ammonium). Furthermore, simple derivatives of the
compounds (such as ethers, esters, amides, etc.) which have desirable
retention and release characteristics but which are easily hydrolyzed by
body pH, enzymes, etc., can be employed.

[0049] In some embodiments, certain properties of the compounds may help
determine their desirability and may influence experimental or
therapeutic properties. For example, the molecular weight of the
compounds can be from about 175 to about 1000. For Formula (I) compounds
the range of molecular weights can be from about 250 to about 750. For
Formula (II) compounds the range of molecular weights can be from about
200 to about 600.

[0050] Other properties include the ability of the compound to selectively
inhibit an undesired microorganism compared to a desired animal cell, as
provided by the Selectivity Index (SI) (defined as that ratio of
IC50 for a desired cell (e.g. animal cell) to the IC50 for an
undesired cell (e.g., microorganism)). SI can be about 2, about 5, about
10, about 20, about 50, about 100, about 150, about 200, about 250, about
300, about 400, and about 500. SI can, for example, be from about 2 to
about 30, from about 5 to about 25, from about 15 to about 25, from about
2 to about 250, from about 10 to about 250, from about 2 to about 500,
from about 10 to about 500, from about 15 to about 250, from about 10 to
about 100, or from about 15 to about 100. The parasitic cells can be a
pathogenic microorganism (e.g., Prokaryotes, Eukaryotes, or Protists)
including, for example, bacteria, mycobacterium, parasite, virus, worms,
or fungi. The animal cells can be, for example, macrophage cells, VERO
cells or any animal tissue cell (e.g., skin, heart, intestine cell). In
some instances, the pathogenic microorganism is a parasite (e.g.,
Trypanosoma cruzi or Leishmania amazonensis) and the animal cell is a
VERO cell or a macrophage cell. Of course, the parasites may be in the
promastigote or amastigote morphological form. In some instances, the
pathogenic microorganism is a mycobacterium (e.g., M. tuberculosis) and
the animal cell is a VERO cell or a macrophage cell.

[0051] Other properties include the ability of the compound to reduce the
parasite burden of an animal. The parasites can be, for example,
Trypanosoma cruzi or Leishmania amazonensis. The animal can be a mammal,
such as a mouse or a human. The reduction in parasite burden can be the
result of daily treatments (e.g., 3, 4, 5, 6, 7, or 8 treatments) over
many weeks (e.g., 4, 5, 6, 7, 8, 9, or 10 weeks) at a fixed or variable
dosage of the compound (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg/kg/day).
The percent reduction in parasite burden can be about 10%, about 20%,
about 50%, about 60%, about 75%, about 85%, about 90%, about 95%, or
about 100%. The percent reduction in parasite burden can be at least
about 5%, at least about 10%, or at least about 25%. The percent
reduction in parasite burden can be from about 5% to about 95%, from
about 10% to about 95%, from about 25% to about 95%, from about 5% to
about 75%, from about 10% to about 75%, or from about 25% to about 75%.

[0052] Variations of the structural components of the compounds can
influence the activity (e.g., on cell growth in vitro), selectivity
(e.g., as provided by the Selectivity Index (SI), which is defined as
that ratio of IC50 for a desired animal cell to the IC50 for an
undesired microorganism), or therapeutic effect of the compound. For
example, cytotoxicities of Formula (I) compounds with the α,β
unsaturated carbonyl group can be higher than their dihydro counterparts
lacking the α,β unsaturated carbonyl group; the latter can
sometimes react with nucleophiles such as glutathione (GSH). In some
embodiments, the activity or selectivity of Formula (I) compounds can be
influenced by the planarity of the Formula (I) compound's phenyl ring
relative to the adjacent ketone; this planarity can be affected by
substituents on the phenyl ring, such as in the ortho positions. In other
embodiments, the electron withdrawing or donating influence of R3
can also alter the activity and selectivity. In some instances, an
electron withdrawing R3 can provide high activity. In some
instances, an electron donating R3 can provide a high selectivity
index (e.g., at least 5 or at least 7 or at least 9 or at least 12). The
inclusion of two or more double bonds in A can produce a loss of
activity. Variation in the heterocyclic nature (e.g., choice of which
hetero atoms are in the heterocycle) of R3 can also produce changes
in the activity and selectivity index.

[0053] Other properties that could be useful include those calculated for
the ADME study such as the topological polar surface area and the number
of Lipinski's rule violations. A computational study can be used to
predict ADME (absorption, distribution, metabolism, and elimination)
which can aid in drug discovery. In such an analysis, topological polar
surface area (TPSA) can be a good indicator of compound absorbance in the
intestines, Caco-2 monolayers penetration, and blood-brain barrier
crossing. TPSA can be used to calculate the percentage of absorption (%
ABS) according to the equation: % ABS=109-0.345×TPSA, as reported
by Zhao et al. (Zhao et al., Rate-limited steps of human oral absorption
and QSAR studies, Pharm. Res. 2002, Vol. 19, pp. 1446-1457). In addition,
the number of rotatable bonds (n-ROTB), and Lipinski's rule of five, can
also be calculated. From one or more of these parameters, accurate
predictions can sometimes be made regarding the potential utility to
compounds for therapy.

[0056] Example syntheses of some Formula (I) compounds, such as
acetophenone-related compounds. To a refluxing solution of
2,4,6-trihydroacetophenone-monohydrate (10 g, 53.7 mmol) and
K2CO3 (15 g, 108.7 mmol) in acetone (150 mL),
(CH3)2SO4 was added at three hour intervals (3×3.5
mL, 40.0 mmol). The solution was filtered and the solvent was evaporated
to afford 2,4-dimethoxy-6-hydroxyacetophenone as a yellow solid (98%). To
obtain 2,4-dihydroxy-6-methoxyacetophenone, anhydrous AlCl3 (11.0 g,
82.5 mmol) and 2,4-dimethoxy-6-hydroxyacetophenone (11.0 g, 56.1 mmol)
were suspended in chlorobenzene (133 mL) and refluxed for 1 h. After
cooling and evaporating of the solvent, an ice-cold H2O--HCl (1:1,
290 mL) solution was added to the residue and sonicated until the white
precipitate seemed homogeneous. The solution was filtered and the solid
was dissolved in EtOAc (200 mL) and extracted with an aqueous solution of
NaOH (10%, 3×200 mL). The aqueous portions were mixed and
neutralized with HCl (conc., 40 mL) to finally be extracted with EtOAc
(2×250 mL) and recrystallized from the same solvent (44.1%).
2,4-diallyloxy-6-methoxyacetophenone, was prepared by mixing
2,4-dihydroxy-6-methoxyacetophenone (5.8 g, 10.0 mmol), K2CO3
(21.8 g, 50.0 mmol) and allylbromide (11.0 mL, 40 mmol) in DMF (100 mL).
After stiffing for 18 hours, the mixture was dissolved in deionized water
(100 mL) and extracted with diethyl ether (3×75 mL). The organic
layers were pooled and extracted with deionized water (3×50 mL).
Finally, the organic phases were combined and dried to be subjected to
column chromatography using hexanes-EtOAc step gradient (40:1 to 5:1,
colorless oil, 85.0%).

[0057] Example syntheses of some Formula (I) compounds, such as chalcone-
and dihydrochalcone-related compounds. To prepare chalcone-related
compounds, the corresponding acetophenone (1.2 mmol), aromatic aldehyde
(1.4 mmol), KOH (1.5 g, 26.7 mmol), H2O (1.5 mL) and CH3OH (3.0
mL) were stirred at room temperature (e.g., 25° C.) for 1 to 48
hours. Deionized water (50 mL) was added and the solution was extracted
with EtOAc (2×30 mL), the organic layer was dried over MgSO4
and evaporated. The crude extract was subjected to column chromatography
using hexanes-EtOAc gradient (10:1 to 1:1). To obtain
2,4-dihydroxy-6-methoxychalcone, the appropriate
2,4-diallyloxy-6-methoxychalcone (0.25 mmol) and Pd(Ph3P)4 (1
mmol %) were dissolved in CH3OH (3 mL), after 1 min of sonication,
K2CO3 (6 eq) was added to the mixture and flushed with argon
gas for 3 min. The solution was stirred for 1 h at 60° C. and then
it was poured over a solution of HCl (2N, 20 mL). The aqueous solution
was extracted with EtOAc (2×20 mL) and the organic phase was dried
over MgSO4 and evaporated. The orange residue was purified by column
chromatography using Hexanes-EtOAc step gradients (6:1 to 1:2).
2,4-dihydroxy-6-methoxydihydrochalcones, were obtained by mixing the
appropriate chalcone (1.5 mmol) with Pd/C 5% (0.1 eq) in EtOAc (10 mL)
and stiffing the solution in a Parr flask under 250 psi of H2 gas at
room temperature (e.g., 25° C.) for 1.5 hour. Then the solvent was
evaporated and the residue purified by column chromatography using
Hexanes-EtOAc step gradients (20:1 to 4:1).

[0058] Compound data: 1H and 13C NMR spectra were recorded at
500 and 125 MHz respectively, using CDCl3 or CDCl3/CD3OD
as a solvent on a Varian Inova 500. The chemical shifts are reported in
ppm values relative to CHCl3 (7.27 ppm for 1H NMR and 77.0 ppm
for 13C NMR). Coupling constants (J) are reported in hertz (Hz).
Melting points were measured on a Thomas Hoover capillary melting point
apparatus and are uncorrected. All air and/or moisture sensitive
reactions were carried out under argon atmosphere. Elemental analysis was
performed at Atlantic Microlab, Inc., Norcross, Ga. Column chromatography
was carried out over Silicycle silica gel (230-400 mesh). Reactions and
fractions obtained from column chromatography were monitored on Merck
silica gel 60 F254 aluminum sheets. TLC spots were visualized by
inspection of plates under UV light (254 and 365 nm) and after submersion
in 5% sulphuric acid or in 4% phosphomolybdic acid and heating
(110° C.). All commercial reagents were obtained either from
Aldrich, Acros or Alfa Aesar and used without any further purification.
3,5-Bisallyloxy-4-bromobenzaldehyde was available from our laboratory.

[0109] Some Formula (II) compounds can be synthesized, in some
embodiments, as summarized in Scheme 2. This Scheme begins with the
preparation of the
2-amino-4,5,6,7-tetrahydrobenzo[b]thienophene-3-carboxylic acid ethyl
ester by using the Gewald reaction and then preparing the cyclic
pyrimidinone compound F, by reacting the 2-aminothiophene carboxylic
ester with excess of formamide. Compound F undergoes chlorination using
phosphorus oxychloride (POCl3) yielding the suitable electrophile
for the nucleophilic displacement using aqueous hydrazine generating the
corresponding aromatic hydrazine derivative compound G (which is also
referred to as PC for precursor). The Scheme concludes with the synthesis
of the Formula (II) compounds by forming the Schiff base upon reaction of
compound G with the corresponding aromatic aldehyde.

##STR00042##

Examples Syntheses of Formula (II) Including Yields of Some Intermediates

[0112] The cytotoxicity of compounds was assayed as follows. Growth
inhibition was evaluated by preparing serial dilutions of each fraction
or compound (up to a maximum concentration of 62.5 μg/mL) and
incubating the cells in 96-well plates in the presence or absence of
these fractions for 48 h at 37° C. Appropriate solvent controls
were tested for comparison. The percent inhibition of cell growth
relative to the control was evaluated colorimetrically using a
sulforhodamine B dye by comparison to the control. The calorimetric
procedure followed that published in Skehan et al., J. Nat. Cancer Inst.,
Vol. 4, pp. 1107-1112 (1990) and Boyd & Paull, Drug Dev. Res., Vol. 34,
pp. 91-109 (1995). The GI50 value is defined as the concentration of
test sample resulting in a 50% reduction of absorbance as compared with
untreated controls that received a serial dilution of the solvent in
which the test samples were dissolved, and was determined by linear
regression analysis. The cell lines are: 3T3 cells are BALB/3T3 clone A31
embryonic mouse fibroblast cells; H460 cells are human large cell lung
cancer cells; DU145 cells are human prostate carcinoma cells; MCF-7 cells
are human breast adenocarcinoma cells; M-14 cells are human melanoma
cells; HT-29 cells are human colon adenocarcinoma cells; PC3 cells are
human prostate adenocarcinoma cells; K562 cells are human chronic
myelogenous leukemia cells; and VERO cells are African Green Monkey
kidney epithelial cells.

[0113] Trypanosoma cruzi (Tulahuen C4) transfected with
β-galactosidase (Lac Z) gene was obtained from Instituto de
Investigaciones Cientificas Avanzadas y servicios de Alta
Tecnologia--Panama (AIP). This transfected T. cruzi can be made using,
for example, the procedures provided in Buckner et al., Efficient
technique for screening drugs for activity against Trypanosoma cruzi
using parasites expressing β-galactosidase, Antimicrob. Agents
Chemother. 1996, Vol. 40, pp. 2592-2597. This strain permits high
throughput screening of compounds using a colorimetric enzyme assay.
Compounds that inhibit the growth of T. cruzi (Tulahuen C4) will have no
or little color while those that do not inhibit growth will permit the
strain to grow as determined by a purple color change. The strain was
maintained in monolayer VERO cells (African Green Monkey kidney
epithelial cells) in complete RPMI 1640 medium without phenol red (Sigma
company, St. Louis Mo.), supplemented with 10% heat inactivated fetal
bovine serum. All cultures and assays were conducted at 37° C.
under an atmosphere of 5% CO2, 95% air mixture.

[0114] The antitrypanoside activity of compounds was evaluated by the
colorimetric method based on the reduction of the substrate chlorophenol
red β-D-galactopyranoside (CPRG) by β-galactosidase resulting
from the expression of the gene for T. cruzi Tulahuen C4. The assay was
performed in 96 well plates containing monolayer VERO cells which were
infected with 5×104 trypomastigotes (Tulahuen C4) per well, 24
hours later 10 μg/mL of each compound were added and incubated at
37° C. After 120 hours 25 μl of 900 μM CPRG substrate
(Roche) solution were added to each well to determine the antitrypanoside
activity of the compound. Then they were incubated at 37° C., for
4-5 hours until color developed. The compounds that had antitrypanoside
activity (<50% growing inhibition) were then assayed to determine the
inhibitory concentration for 50% growth of the parasites (IC50).
These compounds were evaluated at 10, 2, 0.4, 0.8, and 0.16 μg/mL.
Each compound and concentration was made in duplicate. The intensity of
color resulting from the cleavage of CPRG by β-galactosidase was
measured at 570 nm using a VersaMax Micro® microplate reader. The
IC50 is of the compounds were calculated by logarithmic regression
of the OD values obtained, compared with the untreated control. All
active compounds also went through an evaluation of the cytotoxicity
using Thiozol Blue (MTT; 3-[4.5 dimethylthiazol-2-yl]-2,5-diphenyl
tetrazolium bromide) (Aldrich, St. Louis Mo.). This can be important
because in some instances compounds that may inhibit the parasite may
also be toxic, and thus may factor against a drug candidate. This
reaction was measured at 570 nm using a VersaMax Micro® microplate
reader. Nifurtimox (Bayer) was used as positive control at concentrations
of 0.1, 1, and 10 βg/mL. The negative control comprised a media
containing 0.1% DMSO. IC50 is the concentration that produces a 50%
inhibitory effect. VERO are normal African green monkey kidney epithelial
cells. SI is the Selectivity index which is IC50, VERO/IC50, T.
cruzi. ND means the data was not determined.

[0115] Table 2 (IC50 reported in μg/mL)) is an expanded data set
of Table 3 (IC50 reported in μM).

[0116] In general, compound cytotoxicities were higher than their dihydro
counterparts lacking the α,β unsaturated carbonyl group, which
could react with nucleophiles such as glutathione (GSH). I-05 and I-06
were more active than I-01, I-02, I-10, and I-19. The lower cytotoxic
activity of I-01, I-02, I-10, and I-19 might be explained by the
substitutions on the ortho position of the ring, which could affect the
planarity of the molecule. Compound I-10 is slightly less active than its
structurally related I-01, this might indicate that the cytotoxicity
decreases as the size of the ortho substituent on the ring increases;
however, it might also demonstrate that some variations are possible in
the substitution pattern before the activity is lost. Although active, it
can be observed that compounds bearing electron withdrawing groups on the
R3 ring (e.g., I-12, I-13, I-14, I-15, I-16, I-23, I-25, I-26, and
I-27) could be highly toxic to VERO cells. On the other hand, compounds
having electron donating groups on the R3 ring (e.g., I-11, I-17,
I-20, I-24, and I-28) could have selectivity indexes of at least 7. In
these data, the addition of an extra double bond produces the loss of
activity when compared with compounds I-07 and I-21. The high activity of
pyridinium R3 compounds (I-26 and I-27) could not be maintained by
the pyrrole analog I-22. However, when pyrrole was replaced by furan
(I-28), we obtained the highest selectivity index of the series.

[0118] For the preparation of the inoculum, a suspension of M.
tuberculosis was made by mixing growth from slants (20-30 days old) with
100 μL of Tween 80 into 0.2% bovine serum albumin (Sigma Chemical Co.,
St. Louis, Mo.). Turbidity of the suspension was then adjusted to a
McFarland standard No. 1 (3×107 CFU/mL) by adding Tween 80 and
bovine serum albumin. The bacterial suspension (300 μL) was further
transferred to 7.2 mL of 7H9GC broth (4.7 g of Middlebrook 7H9 broth base
(Difco, Detroit, Mich.), 20 mL of 10% glycerol, 1 g of Bacto Casitone
(Difco), 880 mL of distilled water, 100 mL OADC (oleic acid, albumin,
dextrose, catalase) (Remel, Lenexa, Kans.). For the bioassay, pure
compounds were resuspended in DMSO at a concentration of 4 mg/mL (stock
solution). These stock solutions were further diluted with appropriate
volumes of 7H9GC broth to yield final concentrations of 0.4 to 25
μg/mL. Final compound concentration ranges of standard antibiotics
used as positive controls were 0.125 to 32 μg/mL for isoniazid and
0.063 to 16 μg/mL for rifampin. The compound (100 μL) was mixed in
the wells with 100 μL of bacterial inoculum, resulting in a final
bacterial concentration of approx. 1.2×106 CFU/mL. The wells
in column 11 served as inoculum-only controls. Solvent (DMSO) was
included in every experiment as a negative control. The plates were
sealed in plastic bags and then incubated at 37° C. for 5 days. On
day 5, 50 μL of the tetrazolium-Tween 80 mixture was added to the
wells and the plate was incubated at 37° C. for 24 h. The
tetrazolium-Tween 80 mixture was 1.5 mL of tetrazolium
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (Aldrich
Chemical Co., Milwaukee, Wis.) at a dilution of 1 mg/mL in absolute
ethanol and 1.5 mL of 10% Tween 80. After the incubation period, the
growth of the microorganism was visualized by the change in color of the
dye from yellow to purple. The tests were carried out in triplicate. MIC
is defined as the lowest compound that prevents the aforementioned change
in color. `ND` means not determined.

[0119] Table 6 displays results from in vitro low oxygen recovery assays
(LORA) and conventional aerobic (MABA) culture assays. Prior to use,
cultures were thawed, diluted in Middlebrook 7H12 broth (Middlebrook 7H9
broth containing 1 mg/ml casitone, 5.6 μg/ml palmitic acid, 5 mg/ml
bovine serum albumin, and 4 μg/ml catalase, filter-sterilized), and
sonicated for 15 s. Cultures were diluted to obtain an A570 of
0.03-0.05 and 3000-7000 relative light units (RLU) per 100 μl. This
corresponds to 5×105 to ˜2×106 cfu/ml.
Two-fold serial dilutions of the antimicrobial agents were prepared in a
volume 100 μL in black 96 well microtiter plates and 100 μl of the
cell suspension was added. For LORA, the microplate cultures were placed
under anaerobic conditions (oxygen less than 0.16%) using an Anoxomat
Model WS-8080 (MART Microbiology) using three cycles of evacuation and
filling with a mixture of 10% H2, 5% CO2, and the balance
N2. An anaerobic indicator strip was placed inside the chamber to
visually confirm the removal of oxygen. Plates are incubated at
37° C. for 10 days and then transferred to an ambient gaseous
condition (5% CO2-enriched air) incubator for a 28 hour "recovery".
Colony forming units (determined by subculture onto 7H11 agar) during the
day incubation did not increase and remained essentially unchanged. On
day 11 (after the 28 hr aerobic recovery) 1000 culture were transferred
to white 96-well microtiter plates for determination of luminescence. For
conventional assay, the microplate cultures were placed under ambient
gaseous condition (5% CO2-enriched air) incubator for 7 days and 100
μl culture was transferred to white 96-well microtiter plates for
determination of luminescence. A 10% solution of n-decanal aldehyde
(Sigma) in ethanol was freshly diluted ten-fold in PBS and 1000 was added
to each well with an auto injector. Luminescence was measured in a
Victor2 multilabel reader (PerkinElmer Life Sciences) using a reading
time of 1 second. The MIC was defined as the lowest drug concentration
effecting an inhibition of >90% relative to drug-free controls. MIC
values were numerically extrapolated from transformed
inhibition-concentration plots as previously described.

[0120] The fatty acid elongation system FAS-II is involved in the
biosynthesis of mycolic acids, which are major and specific long-chain
fatty acids of the cell envelope of Mycobacterium tuberculosis and other
mycobacteria, including Mycobacterium smegmatis. The protein MabA, also
named FabG1, may be part of FAS-II and may catalyze the NADPH-specific
reduction of long chain β-ketoacyl derivatives. This activity could
correspond to the second step of an FAS-II elongation round. FAS-II may
be inhibited by the antituberculous drug isoniazid through the inhibition
of the 2-trans-enoyl-acyl carrier protein reductase InhA. Thus,
inhibition of MabA can be used in the bioassay to measure the
anti-tuberculosis activity of a given compound.

[0121] Luminescence-based low oxygen recovery assay (LORA) is a bioassay
developed to screen antimicrobial agents against the physiological state
of non-replicating persistence Mycobacterium tuberculosis (NRP-TB)
responsible for antimicrobial tolerance in many bacterial infections.

Anti-Leishmania amazonensis Assays--Tables 7, 8a, and 8b

[0122] To test for anti-Leishmaniasis, the compounds were first tested
against the parasite (i.e., amastigotes), if they exhibit a high
activity, then the compounds were tested in macrophages. Experiments were
conducted on promastigotes and axenic amastigotes of L. amazonensis
(strain MHOM/BR/76/LTB-012).

[0123] Promastigotes were maintained at 25° C.±1° C. by
weekly sub-passages in RPMI 1640 medium with 25 mM HEPES and 2 mM
NaHCO3 (pH 7.2) and supplemented with 20% heat-inactivated fetal
bovine serum in 25 cm2 tissue culture flasks. Axenically grown
amastigotes were maintained by weekly sub-passages in MAA/20 medium at
32° C.±1° C. in 25 cm2 tissue culture flasks.
Cultures were initiated with 5×105 promastigotes or
amastigotes/mL in 25 cm2 tissue culture flasks with 5 mL of medium.
To determine the activity of the compounds, the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
micromethod was used. Briefly, 100 μL of axenically grown
promastigotes or amastigotes, from late log phage of growth, was seeded
in 96-well flat bottom microtiter plates. Compounds, dissolved in DMSO,
were added at final concentrations ranging from 100 to 1 μg/mL. The
final DMSO concentration was not greater than 0.1%. After 72 h of
incubation, 10 μL of MTT (10 mg/mL) was added to each well and plates
were further incubated for 4 h. The enzymatic reaction was then stopped
with 100 μL of 50% isopropanol-10% sodium dodecyl sulphate and
incubated for an additional 30 min under agitation at room temperature
(e.g., 25° C.). Finally, the optical density (OD) was read at 570
nm with a 96-well scanner (Bio-Rad).

[0124] Peritoneal macrophages were prepared as follows. Non-inflammatory
macrophages (106) were collected from each BALB/C mouse. The cells
adhered at 37° C. after 2 h under 5% CO2 in a CO2
incubator. The plates were then rinsed two to three times with 0.5 mL of
RPMI+buffer MOPS without fetal calf serum to eliminate non-adhering
cells. The supernatant on the plates was replaced by fresh medium
(RPMI+glutamine+FCS+antibiotics), 0.5 mL of medium per well. To determine
the toxicity of the compounds to macrophages, dilutions of compounds were
added on a part of the macrophages prepared above. The contact between
the compounds and macrophages alone was 48 h at 37° C. in 5%
CO2. The medium was then removed and 1 μL of sterile Trypan Blue
was added. Cover glasses N° 1 were left where cells were plated
and then the number of macrophages in a microscope was determined with a
haemocytometer. All experiments were performed in triplicate.

[0125] As shown in Table 7, eight of the most active compounds (I-5, I-6,
I-27, I-26, and I-42 to I-46) showed a selectivity index (SI) greater the
10. Among the two largest series of compounds
(2',4'-diallyloxy-6'-methoxychalcones, 2',4'-AC and
2',4'-dihydroxy-6'-methoxychalcones, 2',4'-HC), it was the 2',4'-HC
series, which showed the a better selectivity against the axenic
amastigotes of L. amazonensis, even though they sometimes do not have a
higher IC50 as compared to those in the 2',4'-AC series. Among
compounds I-1, I-5, I-10, and I-44, which differ only on the substitution
pattern on ring A, can be observed that having a methoxymethyl
substituent on the 4'-position, not only maintains the activity of the
molecules, but also greatly enhances its selectivity against the
parasite, when compared to its allyoxy and hydroxy analogs. This
observation suggests that there exists considerable tolerance for the
size and substitution pattern on ring A. In the same way, the presence of
the 2',4'-diallyloxy moieties on the ring A seem to dramatically decrease
the antileishmanial character and SI of the chalcones when comparing
compounds I-9, I-12, I-17, I-18, I-24, I-25, and I-28, with their
corresponding 2',4'-dihydroxy substituted counterparts. These results
appear to be in agreement with the results reported by Liu et al. Bioorg.
Med. Chem. Vol. 11 p. 2729 (2003). When comparing the rest of active
2',4'-AC (I-8, I-10, I-11, I-14, I-15, I-16, I-19, I-20, and I-23)
against their corresponding 2',4'-HC (I-5, I-6, I-29, I-31, I-33, I-34,
I-35, I-38, and I-40) all of them bearing electron donating or electron
withdrawing groups on the ring B; it appears that the anti-parasitic
activity is independent of the substitution pattern on the ring A, since
the antileishmanial activity is conserved for each analog pair. This
appreciation is also applicable for not-as-active compounds such I-21 and
I-39, in which can also be seen that the enlargement of the double bond
on the α-β unsaturated bridge results in a loss of
bioactivity.

[0126] In Tables 8a and 8b, the IC50 data are reported in units of
μM.

[0130] Infection of macrophages by amastigotes was determined as follows.
To assess intracellular antileismanial activity, medium in the wells
containing the macrophages was replaced by the suspension of amastigotes
using an infection ratio of 3/1 amastigotes/macrophages according to
Sauvain et al., 1993 (Sauvain et al., Phytotherapy Research, 7, 167-171
(1993)). Twelve hours after infection, a solution of the compounds to be
tested was added to the cultures at various concentrations and maintained
at 37° C. in 5% CO2 for a further forty-eight hours more. The
plates were fixed with methanol and stained with 10% Giemsa stain. The
percentage of infected macrophages was determined microscopically at 100
times magnification. The number of intracellular amastigotes was
determined in 300 cells. Following Delorenzi et al. Antimicrobial Agents
and Chemotherapy, 45, 1349-1354 (2001)) the percentage of infection rate
(% IR) of each culture was calculated as follows:

% IR=100-(infection rate of the treated culture/infection rate of the
untreated culture)×100.

[0131] IC50 was also calculated as the dose capable of a 50%
reduction in the number of infected cells (calculated using the Excel
trend formula). All experiments were performed in triplicate. ANOVA was
used to test for statistical significance of differences (Epi-Info,
Statview student program). The total parasite burden was calculated as a
mean number of amastigotes per cells multiplied by the number of infected
macrophages.

[0132] Table 9 shows the bioactivities of the nine compounds which showed
the highest SI on the axenic amastigotes assay. From this
macrophage-infected model, it can be observed that in general, L.
peruviana was the species of parasites which showed the strongest
resistance towards all chalcones, while L. braziliensis and L.
amazonensis seemed to respond differently depending on the type of
chalcone administrated. Compound I-27, containing a pyridinyl moiety
seemed to exert the highest bioactivities with values ranging from 0.9 to
4.0 μM. Compound I-44 showed an interesting selectivity against L.
braziliensis, as well as compound I-36 towards L. amazonensis. Finally,
compounds I-5, I-27, I-42, I-43, and I-45, exhibited higher bioactivity
in the macrophage-infected model than against free axenic amastigotes of
L. amazonensis.

[0133] One in vivo test was used in the study of the antileishmanial
activity of the compounds. The in vivo assay is performed by infecting
sensitive BALB/c mice with amastigotes of L. amazonensis in the posterior
feet (Sauvain et al., Phytotherapy Research, 7, 167-171 (1993)).
Treatment started one week after the infection and consisted of
intralesional injections three times each week during six weeks. Growth
of cutaneous lesions of the mice is observed during seven consecutive
weeks, by measuring the thickening of the posterior legs and measuring
the parasitic load by fluorescence (Jackson et al., Science, 227, 435-438
(1985), Barreca et al., Diagn. Microbiol. Infect. Dis., 37, 247-251
(2000)) in the foot tissue. The results are compared with those obtained
with an established usual antileishmanial drug (antimonial organic
salts).

[0134] A nodule extracted from a BALB/c mouse, infected six weeks before
with the LV79 strain (MPRO/BR/72/M1841), was homogenized in a sterile
Potter crusher and taken up in PBS medium (Sigma, USA) in a dilution that
gave 200 000 amastigotes in 10 μL volume; the two posterior feet of
group of each ten mice are infected. Seven days after infection, the
products to be tested and the reference substance were injected into the
lesion three times a week during six weeks in the right posterior foot of
each mouse. A solvent control was prepared in the same manner.

[0135] The thickness of each leg was measured with a Schnell-tester to
evaluate the cutaneous fold one week after the infection and during the
following seven weeks which is the time required for the development of a
leishmanial nodule before ulceration.

[0136] The load of parasites was measured two times (after four and seven
weeks of treatment respectively) by a method using fluorescence. In every
time of evaluation the infected nodules of both later feet were
extracted. The infected nodules were weighed and homogenized in a sterile
Potter crusher with 20% SFB M199 medium (Sigma, USA). An aliquot of 50 uL
was extracted and serial dilutions were realised with the Ethidium
reagent diacetate-Orange acridin (EB-FDAmod) (Sigma, USA). Intralesional
alive (green) and died (red) amastigotes was placed in an hemocytometer
(Bright-line, Sigma) to quantify the viability and the differentiation of
the amastigote stage by means of the identification of the nucleus and
kinetoplast of green colour. The parasitic load has been expressed by the
number of amastigotes (×106)/mg of infected nodule. The
student t-test which was used to compare the averages of the parasitic
load between two treatments; the significance was defined as p<0.05.

[0137] None of the five tested compounds appeared to be as effective as
the positive control (Glucantime) to reduce the lesion diameter. However,
because this result depends on a number of parameters such, immune
response, inflammation process, and parasite virulence (which are not
proportional to the parasite load or the parasite burden), we completed
the measurement of the mice's footpad with kaliper by counting the L.
amazonensis amastigotes in the foot tissue using a fluorescent probe.

[0138] From the count, compounds I-45 and I-46 (administrated to the
infected mice in a concentration almost seven times lower than the
positive control) showed a high reduction of the parasite burden (with
P=0.0004 and P=0.0019, respectively) after the initial four weeks of
treatment, providing a reduction of the parasite burden of 92% and 74%,
respectively. These results were further confirmed by the experiment at
the seventh week in which compounds I-45 and I-46 showed a 35% and 41%
reduction of parasite burden, respectively. Compounds I-45 and I-46 did
not exhibit any cutaneous toxicity at the tested doses and they may not
exhibit a reduction of the lesion diameter, perhaps due to their lack of
anti-inflammatory activity.

Studies of GI50, TGI, and LC50 for Compound II-21 for Several
Cell Lines--Table 12

[0140] For Table 12, GI50, TGI, and LC50 are determined as
follows. GI50 is the concentration of test compound where
100×(T-T0)/(C-T0)=50. See Boyd et al. in Cytotoxic Anticancer Drugs
Models and Concepts for Drug Discovery and Development; Vleriote et al.
Eds.; Kluwer Academic: Hingham, Mass., 1992; pp 11-34 and Monks et al.
JNCI, J. Natl. Cancer Inst. 1991, Vol. 83, pp. 757-766. The optical
density of the test well after a 48-h period of exposure to test drug is
T, the optical density at time zero is T0, and the control optical
density is C. The GI50 measures the growth inhibitory power of the
test compound. The TGI is the concentration of test drug where
100×(T-T0)/(C-T0)=0. Thus, the TGI can signify a cytostatic effect.
The LC50, which can signify a cytotoxic effect, is the concentration
of drug where 100×(T-T0)/T0=-50. The control optical density is not
used in the calculation of LC50.

[0141] A computational study for prediction of ADME was used to predict
the properties of the compounds and is presented in Tables 13 and 14.
Topological polar surface area (TPSA) can be a good indicator of compound
absorbance in the intestines, Caco-2 monolayers penetration, and
blood-brain barrier crossing. TPSA was used to calculate the percentage
of absorption (% ABS) according to the equation: %
ABS=109-0.345×TPSA, as discussed above. In addition, the number of
rotatable bonds (n-ROTB), and Lipinski's rule of five, was also
calculated. From all these parameters it can be observed that although
the oral bioavailability of compounds with selectivity indexes of at
least 12, (e.g., I-20, I-24, I-25, and I-28) could be affected (e.g.,
making them less bioavailable inside the microorganism) (n-ROTB ranged
from 10 to 16) they exhibited a great % ABS ranging from 84 to 94%.
Furthermore, I-20, I-24, and I-28 violate one or none of Lipinski's
parameters, making them potentially promising agents for antitrypanosomal
therapy.

[0142] Polar surface area (TPSA), miLogP, number of rotatable bonds and
violations of Lipinski's rule of five, were calculated using
Molinspiration online property calculation toolkit according to
previously reported literature--see (Molinspiration Cheminformatics,
Bratislava, Slovak Republic,
<<http://www.molinspiration.com/services/properties.html>>).
Every LogP refers the logarithm of compound partition coefficient between
n-octanol and water; variations in this parameter as tabulated below are
given by the software used in the calculation.

[0143] Having described the invention in detail and by reference to
specific embodiments thereof, it will be apparent that modifications and
variations are possible without departing from the scope of the invention
defined in the appended claims. More specifically, although some aspects
of the present invention are identified herein as preferred or
advantageous, it is contemplated that the present invention is not
necessarily limited to these preferred or advantageous aspects of the
invention.

Patent applications by Gerald B. Hammond, Louisville, KY US

Patent applications in class Tricyclo ring system having 1,3-diazine as one of the cyclos

Patent applications in all subclasses Tricyclo ring system having 1,3-diazine as one of the cyclos