U.S. patent application number 14/013842 was filed with the patent office on 2015-01-15 for compositions and methods for drug-sensitization or inhibition of a cancer cell.
The applicant listed for this patent is Dwight Baker, Steven A. Maxwell, James Sacchettini, Deeann Wallis, Nian Zhou. Invention is credited to Dwight Baker, Steven A. Maxwell, James Sacchettini, Deeann Wallis, Nian Zhou.
Application Number | 20150017673 14/013842 |
Document ID | / |
Family ID | 49170892 |
Filed Date | 2015-01-15 |
United States Patent
Application |
20150017673 |
Kind Code |
A1 |
Sacchettini; James ; et
al. |
January 15, 2015 |
COMPOSITIONS AND METHODS FOR DRUG-SENSITIZATION OR INHIBITION OF A
CANCER CELL
Abstract
The disclosure provides rifamycin and rifamycin derivative
compositions, including rifabutin and rifabutin derivative
compositions able to cause drug-sensitization in a cancer cell or
inhibition of a cancer cell. The disclosure also provides methods
of administering such compositions to cancer cells to sensitize
them to drugs, such as chemotherapeutics, or directly inhibit them.
The disclosure also provides methods of administering such
compositions to increase reactive oxygen species (ROS),
particularly superoxides, in cancer cells. The disclosure further
provides methods of determining whether a cancer will respond to
chemotherapeutics and whether to administer rifamycin or a
rifamycin derivative based on ROS levels in cancer cells of a
patient.
Inventors: |
Sacchettini; James; (College
Station, TX) ; Zhou; Nian; (College Station, TX)
; Baker; Dwight; (College Station, TX) ; Maxwell;
Steven A.; (College Station, TX) ; Wallis;
Deeann; (College Station, TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Sacchettini; James
Zhou; Nian
Baker; Dwight
Maxwell; Steven A.
Wallis; Deeann |
College Station
College Station
College Station
College Station
College Station |
TX
TX
TX
TX
TX |
US
US
US
US
US |
|
|
Family ID: |
49170892 |
Appl. No.: |
14/013842 |
Filed: |
August 29, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61695041 |
Aug 30, 2012 |
|
|
|
61784416 |
Mar 14, 2013 |
|
|
|
Current U.S.
Class: |
435/29 ;
546/17 |
Current CPC
Class: |
G01N 33/84 20130101;
A61K 31/337 20130101; A61K 31/573 20130101; A61K 31/675 20130101;
C07D 498/22 20130101; A61K 38/08 20130101; A61K 31/475 20130101;
A61K 31/69 20130101; G01N 2800/52 20130101; A61K 31/122 20130101;
A61K 31/4745 20130101; A61K 31/395 20130101; A61K 31/437 20130101;
A61P 35/00 20180101; A61K 31/7068 20130101; A61K 31/435 20130101;
A61K 33/24 20130101; A61K 45/06 20130101; A61K 31/704 20130101;
A61K 31/5377 20130101; G01N 33/582 20130101; A61K 31/136 20130101;
A61K 31/435 20130101; A61K 2300/00 20130101; A61K 31/395 20130101;
A61K 2300/00 20130101 |
Class at
Publication: |
435/29 ;
546/17 |
International
Class: |
G01N 33/84 20060101
G01N033/84; G01N 33/58 20060101 G01N033/58; A61K 31/435 20060101
A61K031/435 |
Claims
1.-38. (canceled)
39. A method of determining whether to administer rifamycin or
rifamycin derivative to a patient with cancer comprising: obtaining
a cancer cell sample from the patient; measuring the reactive
oxygen species (ROS) amount in the sample; and determining if the
ROS amount is abnormally low for the cancer cell type, wherein an
abnormally low ROS level indicates administration of rifamycin or
rifamycin derivative to the patient.
40. The method of claim 39, wherein the ROS comprises a
superoxide.
41. The method of claim 39, further comprising additionally
determining whether to administer a drug to the patient in addition
to rifamycin or a rifamycin derivative.
42. The method of claim 41, wherein the drug comprises a
chemotherapeutic.
43. The method of claim 42, wherein the cancer cell comprises a
cancer cell resistant to the drug.
44. The method of claim 42, wherein the chemotherapeutic drug
comprises an alkylating agent, an antimetabolite, an anti-tumor
antibiotic, a hormonal agent, a targeted therapy, or a
differentiating agent.
45. The method of claim 39, wherein rifamycin or a rifamycin
derivative has been previously administered to the patient, and
determining comprises determining whether to administer rifamycin
or a rifamycin derivative to the patient a second or greater
time.
46. The method of claim 39, wherein the cancer cell is a carcinoma,
a sarcoma, a leukemia, a lymphoma, or a glioma.
47. The method of claim 39, wherein the cancer cell is a metastatic
cancer cell.
48. The method of claim 39, wherein the ROS comprises a
superoxide.
49. The method of claim 39, wherein the rifamycin derivative
comprises a rifabutin derivative.
50. The method of claim 49, wherein the rifabutin derivative has
the following formula: ##STR00087## wherein R is an alkyl, aryl, or
hetero-aryl group.
51. The method of claim 49, wherein the rifabutin derivative has
the following formula: ##STR00088## wherein R is an alkyl, aryl, or
hetero-aryl group.
52. The method of claim 51, wherein R comprises one of the
following structures: ##STR00089##
53. The method of claim 52, wherein R comprises one of the
following structures: ##STR00090##
54. The method of claim 49, wherein the rifabutin derivative has
the following formula: ##STR00091## wherein X is a C, O, or N and R
is an alkyl, aryl, or hetero-aryl group.
55. The composition of claim 54, wherein R comprises one of the
following structures: ##STR00092##
56. The method of claim 49, wherein the rifabutin derivative has
the following formula: ##STR00093## wherein X is a C, O, or N and R
is an alkyl, aryl, or hetero-aryl group.
57. The method of claim 56, wherein R comprises one of the
following structures: ##STR00094##
58. The method of claim 49, wherein the rifabutin derivative has
the following formula: ##STR00095## wherein R comprises one of the
following structures: ##STR00096##
59. The method of claim 49, wherein the rifabutin derivative has
the following formula: ##STR00097## wherein R comprises
##STR00098##
60. The method of claim 49, wherein the rifabutin derivative has
the following formula: ##STR00099## wherein X is a C, O, or N and R
is an alkyl, aryl, or hetero-aryl group.
61. The method of claim 60, wherein R comprises one of the
following structures: ##STR00100##
62. The method of claim 49, wherein the rifamycin or rifamycin
derivative further comprises a pharmaceutically acceptable carrier,
a salt, a buffer, a preservative, or a solubility enhancer.
Description
PRIORITY CLAIM
[0001] The present application claims priority under 35 U.S.C.
.sctn.119 to U.S. Provisional Patent Application Ser. No.
61/695,041 filed Aug. 30, 2012, titled "COMPOSITIONS AND METHODS
FOR DRUG-SENSITIZATION OR INHIBITION OF A CANCER CELL" and to
Provisional Patent Application Ser. No. 61/784,416,filed Mar. 14,
2013, titled "COMPOSITIONS AND METHODS FOR DRUG-SENSITIZATION OR
INHIBITION OF A CANCER CELL." Both provisional applications are
incorporated by reference herein in their entirety.
TECHNICAL FIELD
[0002] The present disclosure relates to compositions for
drug-sensitization of cancer cells. In particular, it relates to
compositions including rifamycin or a rifamycin derivative, such as
rifabutin or a rifabutin derivative. The present disclosure also
relates to methods of sensitizing a cancer cell to another drug or
combination of drugs by applying rifamycin or a rifamycin
derivative, such as rifabutin or a rifabutin derivative to the
cancer cell. The present disclosure additionally relates to methods
of damaging cancer cells by applying rifamycin or a rifamycin
derivative.
BACKGROUND
Cancer Therapeutics
[0003] Effective cancer treatment is frequently inhibited by the
inability of the patient to withstand an effective dose of a
therapeutic drug, by the development of resistance to therapeutic
drugs by cancer cells, or both. These problems are exhibited across
a wide range of cancers and therapeutic drugs. Physicians and
researchers have attempted to address these problems through
various approaches, such as administering multiple therapeutic
drugs at once or in series, but these solutions are not optimal
because they frequently pose additional risks to the patient, such
as increased rates of relapse, increased chances of opportunistic
infections due to increased length of treatment, and increased
chances of adverse drug reactions due to exposure to more
drugs.
[0004] Many of these problems could be avoided or lessened by
rendering the cancer cells more sensitive to one or more
therapeutic drugs. However, safe and effective methods for
sensitizing cancer cells in such a manner are lacking
Rifamycin and Rifabutin
[0005] Rifabutin is a member of the rifamycin class of antibiotics.
Rifabutin was approved for use as an antibiotic in the United
States in 1992. Although rifabutin has been tested for other
antibiotic and anti-inflammatory uses, its most common use remains
the treatment of tuberculosis and other Mycobacterium infections.
Rifampicin, another member of the rifamycin class of antibiotics,
was introduced in 1967 and is also used to treat tuberculosis and
similar infections.
SUMMARY
[0006] The present disclosure, in one embodiment, relates to a
composition including a rifamycin derivative or a pharmaceutically
acceptable salt, hydrate, or prodrug thereof. The derivative is
operable to induce drug-sensitization in a cancer cell. The
derivative is also operable to inhibit a cancer cell.
[0007] According to another embodiment, the disclosure provides a
method of sensitizing a cancer cell to a drug by administering
rifamycin or a rifamycin derivative to the cancer cell in an amount
and for a time sufficient to sensitize the cancer cell to the
drug.
[0008] According to a third embodiment, the disclosure provides a
method of increasing the amount of a chemotherapeutic in a cancer
cell by administering rifamycin or a rifamycin derivative in an
amount and for a time sufficient to decrease activity of or inhibit
a p-glycoprotein (P-gp) efflux pump in the cell by.
[0009] According to a fourth embodiment, the disclosure provides a
method of increasing reactive oxygen species (ROS) in a cancer cell
by administering rifamycin or a rifamycin derivative to the cancer
cell in an amount and for a time sufficient to increase ROS in the
cancer cell.
[0010] According to a fifth embodiment, the disclosure provides a
method of inhibiting a cancer cell with a drug by administering
rifamycin or a rifamycin derivative to the cancer cell in an amount
and for a time sufficient to sensitize the cancer cell to the drug
and administering the drug to the cancer cell in an amount and for
a time sufficient to inhibit the cancer cell. The amount or time of
administration with respect to the drug are less than that required
to achieve the same inhibition in the absence of rifamycin or a
rifamycin derivative with respect to a given cancer cell type.
[0011] A sixth embodiment of the disclosure relates to a method of
increasing susceptibility of a cancer cell to a drug by
administering rifamycin or a rifamycin derivative to the cancer
cell in an amount and for a time sufficient to increase reactive
oxygen species (ROS) in the cancer cell and administering the drug
to the cancer cell in an amount and for a time sufficient to
inhibit the cancer cell. The amount or time of administration with
respect to the drug is less than that required to achieve the same
inhibition in the absence of increased ROS.
[0012] A seventh embodiment of the disclosure provides a method of
inhibiting a cancer cell by administering rifamycin or a rifamycin
derivative to the cancer cell in an amount and for a time
sufficient to inhibit the cell.
[0013] According to an eighth embodiment, the disclosure provides a
method of increasing susceptibility of a cancer cell to a drug by
administering rifamycin or a rifamycin derivative to a cancer cell
in an amount and for a time sufficient to increase the amount of
the drug in the cancer cell as compared to the amount of the drug
that would be present in the absence of the rifamycin or rifamycin
derivative and administering the drug to the cancer cell in an
amount and for a time sufficient to inhibit the cancer cell.
[0014] According to a ninth embodiment, the disclosure provides a
method of increasing susceptibility of a cancer cell to a drug by
administering rifamycin or a rifamycin derivative to the cancer
cell in an amount and for a time sufficient to inhibit a
p-glycoprotein (P-gp) efflux pump in the cell and administering the
drug to the cancer cell in an amount and for a time sufficient to
inhibit the cancer cell, wherein the amount or time are less than
that required to achieve the same inhibition in the absence of
inhibition of the P-gp pump.
[0015] A tenth embodiment of the disclosure provides a method of
determining whether to administer rifamycin or a rifamycin
derivative to a patient with cancer. The method includes obtaining
a cancer cell sample from the patient, measuring the reactive
oxygen species (ROS) amount in the sample, and determining if the
ROS amount is low for the cancer cell type. A low ROS level
indicates that administration of rifamycin or a rifamycin
derivative to the patient may be beneficial
[0016] The following abbreviations are used throughout the
specification:
CHOP--cyclophosphamide, doxorubicin, vincristine, prednisone
NHL--non-Hodgkin's lymphoma ROS--reactive oxygen species
RTI-x--designates a rifamycin derivative in which "x" is replaced
by an identification number used in the present specification to
designate a particular composition. DOX--doxorubicin.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0018] A more complete understanding of the present embodiments and
advantages thereof may be acquired by referring to the following
description taken in conjunction with the accompanying drawings,
which depict embodiments of the present disclosure, and in which
like numbers refer to similar components, and in which:
[0019] FIG. 1 illustrates a cellular network via which rifabutin or
a rifabutin derivative may cause drug-sensitization and an example
drug-sensitization effect in CHOP-resistant DLBCL cells;
[0020] FIG. 2A illustrates the effects of rifabutin on growth of
CHOP-sensitive (CRL2631) NHL cells and CHOP-resistant (G3) NHL
cells in the presence or absence of CHOP as demonstrated by
resazurin fluorescence;
[0021] FIG. 2B illustrates the effects of rifabutin on growth of
CHOP-resistant (G3) NHL cells in the absence of CHOP (top panel) as
compared to a control drug as demonstrated by resazurin
fluorescence and in the presence of varying dilutions of CHOP for
24 hrs (bottom panel);
[0022] FIG. 2C illustrates the effects of rifabutin on growth of
another CHOP-resistant NHL cell line (SUDHL10-R) and the parental
CHOP-sensitive NHL cell line (SUDHL10-S) after 24 hrs (top panel)
and 48 hrs (bottom panel) of treatment as demonstrated by resazurin
fluorescence;
[0023] FIG. 3A illustrates the effects of rifabutin or rifabutin
derivatives RTI-79 and RTI-176 on cell growth of primary human
dermal fibroblasts both with and without 2 uM Dox;
[0024] FIG. 3B illustrates the effects of doxorubicin and rifabutin
on cell growth of primary human dermal fibroblasts;
[0025] FIG. 4 illustrates the effects of rifabutin on growth of
CHOP-resistant lymphoma cells obtained by aspiration from a dog as
demonstrated by resazurin fluorescence;
[0026] FIG. 5 illustrates the effects of rifabutin in combination
with CHOP or CHOP alone on tumor burden in mm.sup.3 over time in
SCID mice injected with CHOP-resistant (G3) NHL cells;
[0027] FIG. 6 illustrates the effects of CHOP or control solution
with no CHOP on tumor burden in mm.sup.3 over time in SCID mice
injected with CHOP-sensitive (CRL2631) NHL cells;
[0028] FIG. 7 illustrates the effects of reduced dosages of
CHOP+rifabutin or control solution with no CHOP or rifabutin on
tumor burden in mm.sup.3 over time in SCID mice injected with
CHOP-resistant (G3) NHL cells;
[0029] FIG. 8 illustrates a Kaplan-Meier curve showing average life
span for SCID mice injected with CHOP-resistant (G3) cells when
treated with either doxorubicin alone (DOX) or
doxorubicin+rifabutin derivative RTI-81 (DOX+NZ);
[0030] FIG. 9 illustrates the average tumor volume of
chemo-resistant SK-OV-3 xenografts in mice after control treatment
with saline, treatment with 3.3 mg/kg DOX, and treatment with 3.3
mg/kg DOX+25 mg/kg rifabutin over time.
[0031] FIG. 10 illustrates the average tumor volume of multi-drug
resistant cancer cell line (NCI/ADR-RES) xenografts in mice after
control treatment with saline, treatment with 7 mg/kg DOXIL.RTM.
and treatment with 7 mg/kg DOXIL.RTM.+25 mg/kg RTI-79 over
time.
[0032] FIG. 11 illustrates the average tumor volume of multi-drug
resistant cancer cell line (NCI/ADR-RES) xenografts in mice with
multiple, large tumors after control treatment with saline,
treatment with 7 mg/kg DOXIL.RTM., and treatment with 7 mg/kg
DOXIL.RTM.+25 mg/kg RTI-79 over time.
[0033] FIG. 12 illustrates the effects of rifabutin or RTI-79 on
growth of CHOP-resistant (G3) NHL cells;
[0034] FIG. 13 illustrates the effects of rifabutin or RTI-176 on
growth of CHOP-resistant (G3) NHL cells;
[0035] FIG. 14 illustrates the effects of rifabutin or RTI-81 on
growth of CHOP-resistant (G3) NHL cells;
[0036] FIG. 15 illustrates the interaction of rifabutin and
doxorubicin on CHOP-sensitive (CRL2631) NHL cells;
[0037] FIG. 16 illustrates the interaction of RTI-79 and
doxorubicin on CHOP-sensitive (CRL2631) NHL cells.
[0038] FIG. 17 illustrates the effects of rifabutin or RTI-82 on
multidrug-resistant breast cancer (MDA-MB-231) cells;
[0039] FIG. 18 illustrates the interaction of rifabutin with
actinomycin D on multi-drug resistant sarcoma (MES-SA-Dx5)
cells;
[0040] FIG. 19 illustrates the interaction of rifabutin with
menadione on dexamethasone resistant multiple myeloma (MM.1R)
cells;
[0041] FIG. 20 illustrates the interaction of rifabutin and RTI-79
with and without doxorubicin at an 8:1 rifabutin or
RTI-79:doxorubicin molar ratio on multi-drug resistant cancer cell
line (NCI/ADR-RES) cells;
[0042] FIG. 21 illustrates the interaction of RTI-79 and
doxorubicin on multi-drug resistant T lymphoblastoid leukemia
(MOLT-4) cells;
[0043] FIG. 22 illustrates the effects of rifabutin and RTI-79 with
and without doxorubicin at an 8:1 rifabutin or RTI-79:doxorubicin
molar ratio on ovarian carcinoma (OVCAR8) cells;
[0044] FIG. 23 illustrates the effects of rifabutin and actinomycin
D on multi-drug resistant sarcoma (MES-SA-Dx5) cells.
[0045] FIG. 24 illustrates the effects of rifabutin and menadione
on dexamethasone resistant multiple myeloma (MM.1R) cells;
[0046] FIG. 25 illustrates the interaction of rifabutin and
mitoxantrone on osteosarcoma (U-2 OS) cells;
[0047] FIG. 26 illustrates the interaction of rifabutin with
gemcitabine on multi-drug resistant breast cancer (MDA-MB-231)
cells;
[0048] FIG. 27 illustrates the interaction of rifabutin with
paclitaxel on myeloid leukemia cells (HL-60) cells;
[0049] FIG. 28 illustrates the interaction of rifabutin and
camptothecin on ovarian cancer (OVCAR-8) cells;
[0050] FIG. 29 illustrates the number of viable cells present after
re-exposure to CHOP of CHOP-sensitive (CRL2631) cells to a full or
half dose of CHOP in the presence or absence of rifabutin;
[0051] FIG. 30A illustrates a Western blot for phosphorylated Akt
(pAkt) Akt, 14-3-3.zeta., and an actin control in CHOP-sensitive
(CRL2631) and CHOP-resistant (G3) cells. FIG. 30B illustrates the
effect of varying amounts of Akt Inhibitor VIII on growth of G3
cells as demonstrated by resazurin fluorescence. FIG. 30C
illustrates a Western blot for phosphorylated Akt (pAkt) Akt,
14-3-3.zeta., and a Vimentin control in G3 cells exposed or not
exposed to Akt Inhibitor VIII;
[0052] FIG. 31 illustrates the amount of ROS in CHOP-sensitive
(CRL2631) or CHOP-resistant (G3) cells before and after 101 ng/ml
CHOP treatment (cyclophosphamide=240 ng/ml [0.83 uM];
Doxorubicin=33 ng/ml [0.057 uM]; Vincristine=0.93 ng/ml [0.0045
uM]; Prednisone=67 ng/ml [0.828 uM].
[0053] FIG. 32 illustrates the ROS levels in distinct populations
of cells in CHOP-sensitive (CRL2631) cells purified by flow
cytometry;
[0054] FIG. 33 illustrates the number of viable cells present after
treatment of low-ROS CRL2631 cells and high-ROS CRL2631 cells with
101 ng/ml CHOP treatment (cyclophosphamide=240 ng/ml [0.83 uM];
Doxorubicin=33 ng/ml [0.057 uM]; Vincristine=0.93 ng/ml [0.0045
uM]; Prednisone=67 ng/ml [0.828 uM];
[0055] FIG. 34 illustrates the effect on cell growth of varying
amounts of CHOP in the presence or absence of 10 uM rifabutin on
low-ROS CRL2631 cells as demonstrated by resazurin
fluorescence;
[0056] FIG. 35 illustrates the effect of 10 uM rifabutin on ROS in
CHOP-resistant (G3) cells over time;
[0057] FIG. 36A provides a western-blot showing different ABCB1
protein levels in si-ABCB1 and si-NC1 (control si-RNA) treated
ADR-RES cells, as well as the untreated ADR-RES cells and its
parental drug-sensitive strain OVCAR8;
[0058] FIG. 36B shows the effects of rifabutin (RBT) on calcein-AM
efflux in OVCAR8 than in ADR-RES cells.
[0059] FIG. 36C shows the effects of 5 .mu.M rifabutin (RBT),
RTI-79, and rifampin (RMP) on calcein-AM efflux and the further
effects of ABCB1 RNA-silencing;
[0060] FIG. 37A shows dose-response curves of various RTIs on
calcein-AM efflux.;
[0061] FIG. 37B shows the effects of various RTIs on 1 uM
doxorubicin's toxicity in G3 cells;
[0062] FIG. 37C shows the correlation between efflux inhibition
effect and drug sensitizing ability for various RTI-x rifamycin
derivatives;
[0063] FIG. 37D shows the comparison of doxorubicin fluorescence
intensity in the NCI/ADR-RES cells with rifabutin treatment or
dimethyl sulfoxide (DMSO) control.
[0064] FIG. 38A shows the effects of MDR/P-gp inhibitors and two
control drugs (carboxin, nifazoxinide) on ROS in
doxorubicin-sensitive OVCAR8 cells;
[0065] FIG. 38B shows the effects of MDR/P-gp inhibitors and two
control drugs (carboxin, nifazoxinide) on ROS in
doxorubicin-resistant ADR-RES cells;
[0066] FIG. 38C shows the effects of MDR/P-gp inhibitors and two
control drugs (carboxin, nifazoxinide) on ROS in
doxorubicin-resistant G3 cells;
[0067] FIG. 39 shows staining of ADR-RES cells treated with RTI-79;
ADR-RES cells were infected 24 hrs with a baculovirus expressing a
recombinant GFP protein fused with a mitochondrial localization
signal (green); cells were stained with CellROX to detect ROS (red)
or DAPI to detect nuclei (blue);
[0068] FIG. 40 shows the effects of cell-permeable calcium
modulators (BAPTA, Verapamil) and a Complex I inhibitor (Rotenone)
on ROS in G3 cells;
[0069] FIG. 41A shows the effects of P-gp inhibitors (Reserpine,
Elacridar) on ROS levels in ADR and OVCAR8 cells
[0070] FIG. 41B shows the effects of RTI-79 on ROS and calcium
mobilization in doxorubicin-sensitive lymphoma (CRL2631, 10S, WSU)
and ovarian carcinoma (OVCAR8) cells and doxorubicin-resistant
lymphoma (G3R,10R, WSUR) cells;
[0071] FIG. 41C shows the levels of ROS and calcium mobilization in
more CHOP-sensitive lymphoma (CRL2631, 10S, WSU) compared to the
more resistant derivative cell lines (G3, 10R, WSU-R), and in the
more doxorubicin-sensitive OVCAR8 versus the more resistant
derivative cell line ADR.
[0072] FIG. 42 shows a time course of RTI-79 induction of ROS and
calcium mobilization in G3 cells; and
[0073] FIG. 43 shows the effects of siRNA knockdown of P-gp on
induction of ROS and mobilization of calcium.
[0074] FIG. 44 shows the effects of rifabutin on G3 and CRL2631
cells in a collagen invasion 3D assay.
[0075] FIG. 45 shows the effects of rifabutin on G3 and CRL2631
cells in a modified Boyden chamber assay.
DETAILED DESCRIPTION
[0076] The present disclosure relates to compositions and methods
for drug-sensitization of a cancer cell or for inhibiting a cancer
cell, as well as methods for diagnosis of whether a cancer cell may
respond to a chemotherapeutic or to a composition described herein.
These compositions and methods are described in further detail
below.
[0077] Unless otherwise indicated by the specific context of this
specification, a cancer cell may include a cell of any type of
cancer. Furthermore, it may include a cancer cell in a patient,
either in a cancerous growth, such as a tumor, or in isolation from
other cancer cells, such as during metastasis. The patient may be
any animal. In particular, the patient may be a mammal, such as a
human, a pet mammal such as a dog or cat, an agricultural mammal,
such as a horse, cow, pig, sheep, or goat, or a zoo mammal.
Although many embodiments herein are expressed in terms of a cancer
cell, the same or similar effects may be seen in groups of cancer
cells in a patient.
[0078] Drug-sensitization, unless otherwise indicated by the
specific context of this specification, may include increased
sensitivity to a drug, decreased resistance to a drug, or
potentiation of a drug's activity or efficacy. Any effect may be
measured using any methods accepted in the art. In a specific
embodiment, drug-sensitization may be determined by an increased
ability of the drug to inhibit a cell. Cellular inhibition may
include killing the cell, such as via apoptisis or necrosis,
reducing the growth of the cell, thus reducing the growth of the
cancer containing the cell, rendering the cell more susceptible to
the immune system, preventing or reducing metastasis, reducing the
size of a tumor containing the cell, or otherwise negatively
affecting a cancer cell. An increased ability of the drug to
inhibit a cancer cell may be demonstrated by an ability to inhibit
the cell with a reduced amount of drug or in a shorter period of
time than in the absence of drug-sensitization. In the case of
drug-resistant cancer cells, which include cells with inherent or
acquired resistance, drug-sensitization may result in a renewed or
newly acquired ability of the drug to inhibit a cancer cell or type
of cancer cell.
Compositions
[0079] The present disclosure includes drug-sensitization
compositions, such as chemosensitizer compositions, including
rifamycin and rifamycin derivatives, such as rifabutin or rifabutin
derivatives or rifampicin and rifampicin derivatives. The present
disclosure also includes compositions for inhibition of cancer
cells including rifamycin and rifamycin derivatives, such as
rifabutin or rifabutin derivatives or rifampicin (also called
rifampin) and rifampicin derivatives. Other rifamycin derivates
include rifapentine and rifalazil.
[0080] In certain embodiments, the present disclosure provides
derivatives of rifabutin according to one of the following general
structures:
##STR00001## ##STR00002##
in which R may be an alkyl, aryl, or hetero aryl group.
[0081] In other embodiments, the present disclosure provides
enantiomers of the general structures. In particular embodiments,
it provides enantiomers with the following general chrial
structures:
##STR00003## ##STR00004##
in which R may be an alkyl, aryl, or hetero aryl group.
[0082] In certain embodiments having general structures I or II or
general chiral structures Ia or IIa, R may be one of the following
structures:
##STR00005##
[0083] In certain embodiments, the present disclosure provides
derivatives of rifabutin according to the following formula:
##STR00006##
[0084] In certain embodiments, the present disclosure provides
derivatives of rifabutin according to the following formula:
##STR00007##
[0085] In certain embodiments, the present disclosure provides
derivatives of rifabutin according to the following formula:
##STR00008##
where X and R may include the following combinations:
##STR00009##
[0086] The structure with the general formula above may also be the
following enantiomer:
##STR00010##
[0087] In certain embodiments, the present disclosure provides
derivatives of rifabutin according to the following formula:
##STR00011##
[0088] In certain embodiments having general structures III or IV
or general chiral structures IIIa or IVa, R may be one of the
following structures:
##STR00012##
wherein X is a C, O, or N and R is an alkyl, aryl, or hetero-aryl
group.
[0089] In another embodiment, the present disclosure provides
derivatives of rifabutin according to the following formula:
##STR00013##
wherein X is a C, O, or N and R is an alkyl, aryl, or hetero-aryl
group or wherein X and R are as follows:
##STR00014##
[0090] In one embodiment, a composition of the general formula
above may be the following enantiomer:
##STR00015##
[0091] In certain embodiments, the present disclosure provides
derivatives of rifabutin according to the following formula:
##STR00016##
wherein X is a C, O, or N and R may include the structures listed
below:
##STR00017##
[0092] In certain embodiments, the present disclosure provides
derivatives of rifabutin according to the following formula,
wherein X is a C, O, or N:
##STR00018##
[0093] In certain embodiments, a composition with the general
formula above may be the following enantiomer:
##STR00019##
[0094] In certain embodiments, the present disclosure provides
derivatives of rifabutin according to the following formula:
##STR00020##
[0095] In certain embodiments, the present disclosure provides
derivatives of rifabutin according to the following formula:
##STR00021##
wherein X is a C, O, or N and R is an alkyl, aryl, or hetero-aryl
group or wherein X and R are as follows:
##STR00022##
[0096] In certain embodiments, the present disclosure provides
derivatives of rifabutin according to the following formula:
##STR00023##
[0097] In other embodiments, the present disclosure provides a
drug-sensitization composition including a series of
3,4-cyclo-rifamycin derivatives. Examples of such compositions are
as follows:
##STR00024##
or the following enantiomer:
##STR00025##
[0098] In certain embodiments X may be CH, S, SO, SO.sub.2 or N.Y.
may be H or an acetyl group. R1 may be hydrogen. R2 may be a
hydroxyl or an amino (--NH.sub.2) group. R1 and R2 together may be
an oxo or imine group. R3 may be one of the following groups:
hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and
heterocycloalkyl groups that may be additionally substituted with
from zero to four substituents chosen independently from halogen,
hydroxy, alkoxy-alkyl, --CN, nitro, --S-alkyl, amino, alkylamino,
dialkylamino, dialkylaminoalkyl, carboxy, carboalkoxy, acyl,
carboxamido, alkylsulfoxide, acylamino, phenyl, benzyl, phenoxy,
and benzyloxy. In certain embodiments, R3 may be --C(.dbd.O)--R4,
--C(.dbd.O)--O--R4 and --C(.dbd.O)--NH--R4 where R4 is
independently selected from alkyl, alkenyl, alkynyl, cycloalkyl,
aryl, heteroaryl and heterocycloalkyl groups that may be
additionally substituted with from zero to four substituents chosen
independently from halogen, hydroxy, alkoxy-alkyl, --CN, nitro,
--S-alkyl, amino, alkylamino, dialkylamino, dialkylaminoalkyl,
carboxy, carboalkoxy, acyl, carboxamido, alkylsulfoxide, acylamino,
phenyl, benzyl, phenoxy and benzyloxy.
[0099] In other embodiments, the present invention provides
compositions of the following structure:
##STR00026##
or the following enantiomer:
##STR00027##
wherein Y is H or an acetyl group and R4 may be selected from
alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and
heterocycloalkyl groups that may be additionally substituted with
from zero to four substituents chosen independently from halogen,
hydroxy, alkoxy-alkyl, --CN, nitro, --S-alkyl, amino, alkylamino,
dialkylamino, dialkylaminoalkyl, carboxy, carboalkoxy, acyl,
carboxamido, alkylsulfoxide, acylamino, phenyl, benzyl, phenoxy and
benzyloxy.
[0100] In certain embodiments, the present invention provides
compositions with the following structure:
##STR00028##
or the following enantiomer:
##STR00029##
wherein Y is H, or acetyl group; Z is carbon, oxygen or nitrogen
atom; and R4 is independently selected from alkyl, alkenyl,
alkynyl, cycloalkyl, aryl, heteroaryl and heterocycloalkyl groups
that may be additionally substituted with from zero to four
substituents chosen independently from halogen, hydroxy,
alkoxy-alkyl, --CN, nitro, --S-alkyl, amino, alkylamino,
dialkylamino, dialkylaminoalkyl, carboxy, carboalkoxy, acyl,
carboxamido, alkylsulfoxide, acylamino, phenyl, benzyl, phenoxy and
benzyloxy.
[0101] Examples of drug-sensitization compositions in accordance
with certain aspects of the present disclosure may include those
listed in Table 1. Compositions of Table 1 are designated by like
names throughout this specification.
TABLE-US-00001 TABLE 1 Rifamycin Derivatives RTI- General x
structure R Name 33 I ##STR00030##
11-deoxy-11-imino-4-deoxy-3,4[2-spino-[1-(t-
butyloxycarbonyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5-
dihydro)rifamycin S 44 I --H
11-deoxy-11-imino-4-deoxy-3,4[2-spiro-[piperidin-4-yl]]-(1H)-
imidazo-(2,5-dihydro)rifamycin S 49 I ##STR00031##
11-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-(benzyl)-piperidin-
4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycin S 51 I ##STR00032##
11-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-(2-methoxyethyl)-
piperidin-4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycin S 53 I
##STR00033## 11-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-(2-
morpholinoethyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5-
dihydro)rifamycin S 57 I ##STR00034##
11-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-(cyclobutylmethyl)-
piperidin-4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycin S 59 I
##STR00035## 11-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-
(cyclopropylmethyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5-
dihydro)rifamycin S 60 I ##STR00036##
11-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-(isopropyl)-
piperidin-4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycin S 61 I
##STR00037## 11-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-(t-
ethyloxycarbonyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5-
dihydro)rifamycin S 63 I ##STR00038##
11-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-(acetyl)-piperidin-4-
yl]]-(1H)-imidazo-(2,5-dihydro)rifamycin S 64 I ##STR00039##
11-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-(n-propyl)-
piperidin-4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycin S 65 I
##STR00040##
11-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-(cyclopropyl)-
piperidin-4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycin S 66 I
##STR00041##
11-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-(ethyl)-piperidin-4-
yl]]-(1H)-imidazo-(2,5-dihydro)rifamycin S 67 I ##STR00042##
11-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-(beRTIoyl)-
piperidin-4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycin S 68 I
##STR00043## 11-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-
(benzyloxycarbonyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5-
dihydro)rifamycin S 69 I --CH.sub.3
11-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-(methyl)-piperidin-
4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycin S 70 I ##STR00044##
11-deoxy-11-imino-4-deoxy-3,4[2-spino-[1-(2-methylpropyl)-
piperidin-4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycin S 74 I
##STR00045## 11-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-
(phenylaminocarbonyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5-
dihydro)rifamycin S 75 II ##STR00046##
4-deoxy-3,4[2-spiro-[1-(t-butyloxycarbonyl)-piperidin-4-yl]]-
(1H)-imidazo-(2,5-dihydro)rifamycin S 76 II ##STR00047##
4-deoxy-3,4[2-spiro-[1-(ethyloxycarbonyl)-piperidin-4-yl]]-
(1H)-imidazo-(2,5-dihydro)rifamycin S 77 I ##STR00048##
11-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-(ethyloxycarbonyl)-
piperidin-4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycin S 78 II
##STR00049##
4-deoxy-3,4[2-spiro-[1-(n-propyloxycarbonyl)-piperidin-4-yl]]-
(1H)-imidazo-(2,5-dihydro)rifamycin S 79 II ##STR00050##
4-deoxy-3,4[2-spiro-[1-(isobutyloxycarbonyl)-piperidin-4-yl]]-
(1H)-imidazo-(2,5-dihydro)rifamycin S 80 II ##STR00051##
4-deoxy-3,4[2-spiro-[1-(benzyloxycarbonyl)-piperidin-4-yl]]-
(1H)-imidazo-(2,5-dihydro)rifamycin S 81 I ##STR00052##
11-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-
(isobutyloxycarbonyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5-
dihydro)rifamycin S 82 I ##STR00053##
11-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-
(ethylaminocarbonyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5-
dihydro)rifamycin S 83 II ##STR00054##
4-deoxy-3,4[2-spiro-[1-(ethylaminocarbonyl)-piperidin-4-yl]]-
(1H)-imidazo-(2,5-dihydro)rifamycin S 84 I ##STR00055##
11-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-
(isopropyloxycarbonyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5-
dihydro)rifamycin S 85 II ##STR00056##
4-deoxy-3,4[2-spiro-[1-(isopropyloxycarbonyl)-piperidin-4-yl]]-
(1H)-imidazo-(2,5-dihydro)rifamycin S 86 II ##STR00057##
4-deoxy-3,4[2-spiro-[1-(phenylaminocarbonyl)-piperidin-4-yl]]-
(1H)-imidazo-(2,5-dihydro)rifamycin S 87 II ##STR00058##
4-deoxy-3,4[2-spiro-[1-(acetyl)-piperidin-4-yl]]-(1H)-imidazo-
(2,5-dihydro)rifamycin S 88 II ##STR00059##
4-deoxy-3,4[2-spiro-[1-(beRTIoyl)-piperidin-4-yl]]-(1H)-
imidazo-(2,5-dihydro)rifamycin S 89 II ##STR00060##
4-deoxy-3,4[2-spiro-[1-(3,3-dimethylbutanoyl)-piperidin-4-yl]]-
(1H)-imidazo-(2,5-dihydro)rifamycin S 91 I ##STR00061##
11-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-(3,3-
dimethylbutanoyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5-
dihydro)rifamycin S 94 I ##STR00062##
11-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-(n-pentanoyl)-
piperidin-4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycin S 97 I
##STR00063## 11-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-(2-
methylpropanoyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5-
dihydro)rifamycin S 98 I ##STR00064##
11-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-(3-methylbutanoyl)-
piperidin-4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycin S 101 II
##STR00065##
4-deoxy-3,4[2-spiro-[1-(dimethylaminocarbonyl)-piperidin-4-
yl]]-(1H)-imidazo-(2,5-dihydro)rifamycin S 102 II ##STR00066##
4-deoxy-3,4[2-spiro-[1-(isobutylaminocarbonyl)-piperidin-4-
yl]]-(1H)-imidazo-(2,5-dihydro)rifamycin S 103 II ##STR00067##
4-deoxy-3,4[2-spiro-[1-(isopropylaminocarbonyl)-piperidin-4-
yl]]-(1H)-imidazo-(2,5-dihydro)rifamycin S 104 II ##STR00068##
4-deoxy-3,4[2-spiro-[1-((1-methylpropyl) aminocarbonyl)-
piperidin-4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycin S 105 II
##STR00069##
4-deoxy-3,4[2-spiro-[1-(t-butylaminocarbonyl)-piperidin-4-yl]]-
(1H)-imidazo-(2,5-dihydro)rifamycin S 174 IV ##STR00070##
11-deoxy-11-hydroxy-4-deoxy-3,4[2-spiro-[1-(2-
methylpropyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5- dihydro)rifamycin
S 175 IV ##STR00071## 11-deoxy-11-hydroxy-4-deoxy-3,4[2-spiro-[1-
(isobutyloxycarbonyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5-
dihydro)rifamycin S 176 III ##STR00072##
11-deoxy-11-amino-4-deoxy-3,4[2-spiro-[1-
(isobutyloxycarbonyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5-
dihydro)rifamycin S 181 III ##STR00073##
11-deoxy-11-amino-4-deoxy-3,4[2-spiro-[1-(2-methylpropyl)-
piperidin-4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycin S 182 I
##STR00074## 11-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-
(isobutylaminocarbonyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5-
dihydro)rifamycin S 183 III ##STR00075##
11-deoxy-11-amino-4-deoxy-3,4[2-spiro-[1-
(isobutylaminocarbonyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5-
dihydro)rifamycin S 197 V ##STR00076##
11-deoxy-11-hydroxyimino-4-deoxy-3,4[2-spiro-[1-
(isobutyloxycarbonyl)-piperidin-4-yl]]-(1H)-imidazo-(2,5-
dihydro)rifamycin S 217 V ##STR00077##
11-deoxy-11-hydroxyimino-4-deoxy-3,4[2-spiro-[1-
(isobutylaminocarbonyl)-piperidin-4-yl]](1H)-imidazo-(2,5-
dihydro)rifamycin S
[0102] Modification of the rifamycin structure in locations
corresponding to the 21-OH, 23-OH or 25-O--Ac sites of the
rifabutin structures I, II, III, IV and V do not generally affect
drug-sensitization activity and thus variations with modifications
at these sites or even elimination of these sites are encompassed
herein. Such variations may be used to improve synthesis yields,
control costs, increase water solubility, or improve pharmaceutical
properties of the composition. Sites 21, 23 and 25 are located as
follows:
##STR00078## ##STR00079##
[0103] The present disclosure also includes pharmaceutically
acceptable salts, hydrates, prodrugs, and mixtures of any of the
above compositions. The term "pharmaceutically acceptable salt"
refers to salts whose counter ion derives from pharmaceutically
acceptable non-toxic acids and bases.
[0104] The 3,4-cyclo-rifamycin derivatives which contain a basic
moiety, such as, but not limited to an amine or a pyridine or
imidazole ring, may form salts with a variety of organic and
inorganic acids. Suitable pharmaceutically acceptable (i.e.,
non-toxic, physiologically acceptable) base addition salts for the
compounds of the present invention include inorganic acids and
organic acids. Examples include acetate, adipate, alginates,
ascorbates, aspartates, benzenesulfonate (besylate), benzoate,
bicarbonate, bisulfate, borates, butyrates, carbonate,
camphorsulfonate, citrate, digluconates, dodecylsulfates,
ethanesulfonate, fumarate, gluconate, glutamate, glycerophosphates,
hemisulfates, heptanoates, hexanoates, hydrobromides,
hydrochloride, hydroiodides, 2-hydroxyethanesulfonates,
isethionate, lactate, maleate, malate, mandelate, methanesulfonate,
2-naphthalenesulfonates, nicotinates, mucate, nitrate, oxalates,
pectinates, persulfates, 3-phenylpropionates, picrates, pivalates,
propionates, pamoate, pantothenate, phosphate, salicylates,
succinate, sulfate, sulfonates, tartrate, p-toluenesulfonate, and
the like.
[0105] The 3,4-cyclo-rifamycin derivatives which contain an acidic
moiety, such as, but not limited to a carboxylic acid, may form
salts with variety of organic and inorganic bases. Suitable
pharmaceutically acceptable base addition salts for the compounds
of the present invention include, but are not limited to, ammonium
salts, metallic salts made from calcium, lithium, magnesium,
potassium, sodium and zinc or organic salts made from lysine,
N,N-dialkyl amino acid derivatives (e.g. N,N-dimethylglycine,
piperidine-1-acetic acid and morpholine-4-acetic acid),
N,N'-dibenzylethylenediamine, chloroprocaine, choline,
diethanolamine, ethylenediamine, meglumine (N-methylglucamine),
t-butylamine, dicyclohexylamine, hydrabamine, and procaine.
[0106] The 3,4-cyclo-rifamycin derivatives, and salts thereof, may
exist in their tautomeric form (for example, as an amide or imino
ether). All such tautomeric forms are contemplated herein as part
of the present invention.
[0107] The compounds described herein may contain asymmetric
centers and may thus give rise to enantiomers, diastereomers, and
other stereoisomeric forms. Each chiral center may be defined, in
terms of absolute stereochemistry, as (R)- or (S)-. The present
invention is meant to include all such possible isomers, as well
as, their racemic and optically pure forms. Optically active (R)-
and (S)-, or (D)- and (L)-isomers may be prepared using chiral
synthons or chiral reagents, or resolved using conventional
techniques. When the compounds described herein contain olefinic
double bonds or other centers of geometric asymmetry, and unless
specified otherwise, it is intended that the compounds include both
E and Z geometric isomers.
[0108] The configuration of any carbon-carbon double bond appearing
herein is selected for convenience only and unless explicitly
stated, is not intended to designate a particular configuration.
Thus the carbon-carbon double bond depicted arbitrarily above as E
may be Z, E, or a mixture of the two in any proportion.
[0109] Abbreviations as used herein have the meanings known by one
skilled in the art. Specifically, Ac represent acetyl group, Boc
represents t-butoxycarbonyl group, Bn represents benzyl group, DCM
represents dichloromethane, DMF represents N,N-dimethylformamide,
DMSO represents dimethyl sulfoxide, Et represents ethyl group,
EtOAc represents ethylacetate, Me represents methyl group, Ph
represents phenyl group, TEA represents triethylamine, TFA
represents trifluoroacetic acid, THF represents tetrahydrofuran,
and TMS is trimethylsilane group.
[0110] Compositions of the present disclosure may also include a
pharmaceutically acceptable carrier, in particular a carrier
suitable for the intended mode of administration, or salts,
buffers, or preservatives. Rifamycin and many of its derivatives,
such as rifabutin and rifabutin derivatives are poorly soluble in
water. Accordingly, aqueous compositions of the present disclosure
may include solubility enhancers. Compositions for oral use may
include components to enhance intestinal absorption. The overall
formulation of the compositions may be based on the intended mode
of administration. For instance, the composition may be formulated
as a pill or capsule for oral ingestion. In other examples, the
composition may be encapsulated, such as in a liposome or
nanoparticle. In particular, it may be encapsulated with the drug
to sensitize the cancer cell, such as encapsulated in a liposome
with doxorubicin. It may also be administered with a liposomal or
nanoparticle drug, such as DOXIL.RTM. (doxorubicin HCl liposome
injection) (Centocor Ortho Biotech Products, LP, Raritan, N.J.),
whether encapsulated with the drug or not. It may also be
separately encapsulated.
[0111] Compositions of the present disclosure may contain a
sufficient amount of rifamycin or rifamycin derivative to cause
drug-sensitization or other inhibition of a cancer cell to occur
when the composition is administered to a cancer cell. The amount
of rifamycin or rifamycin derivative, such as rifabutin or
rifabutin derivative may vary depending on other components of the
composition and their effects on drug availability in a patient,
the type of drug or drugs to which the cancer cell is sensitized,
the amount of drug otherwise required to inhibit the cancer cell,
the intended mode of administration, the intended schedule for
administration, any drug toxicity concerns, drug-drug interactions,
such as interactions with other medications used by the patient, or
the individual response of a patient. Many compositions may contain
an amount of rifamycin or rifamycin derivative, such as rifabutin
or rifabutin derivative, well below levels at which toxicity to
normal cells or to the patient overall becomes a concern.
[0112] Compositions of the present disclosure may also contain one
or more drugs for which the rifamycin or rifamycin derivative, such
as rifabutin or rifabutin derivative, causes drug-sensitization.
Example drugs are described in the current specification. In
another embodiment, compositions of the present disclosure may
contain one or more other drugs commonly used in combination with
the drug for which sensitization occurs. For example, a composition
may include rifabutin or a rifabutin derivative with any CHOP drug,
regardless of whether rifabutin causes drug-sensitization for that
drug. In still another embodiment, the composition may contain
another drug that also causes drug sensitization, such as a drug
that affects the amount or ROS, particularly superoxide, in a cell.
For example it may contain superoxide dismutase inhibitors. In
still another embodiment, the composition may contain another drug
that affects drug resistance or a property causing drug resistance
in cancer cells. For example, it may contain drugs affecting the
apoptotic pathway, such as the apoptotic pathway inhibitors for
Bc1-XL or mimetics for BH3 proteins.
[0113] Compositions of the present disclosure may further include
other therapeutic agents. For example, they may include any one or
more of the chemotherapeutic agents listed herein, particularly
those described below in connection with Drug Sensitization
Methods. The amounts of those chemotherapeutic agents in
compositions of the present disclosure may be reduced as compared
to normal doses of such agents administered in a similar
fashion.
[0114] The amount of rifamycin or rifamycin derivative, such as
rifabutin or a rifabutin derivative, present in a compostion may be
measured in any of a number of ways. The amount may, for example,
express concentration or total amount. Concentration may be for
example, weight/weight, weight/volume, moles/weight, or
moles/volume. Total amount may be total weight, total volume, or
total moles. Typically, the amount of rifamycin or rifamycin
derivative may be expressed in a manner standard for the type of
formulation or dosing regimen used.
[0115] The present disclosure further includes methods of
identifying whether a rifamycin derivative, such as a rifabutin
derivative is able to sensitize a cancer cell or inhibit a cancer
cell. Such methods include preparing or obtaining such a
derivative, applying it to a cancer cell, and identifying that the
derivative renders the cancer cell more susceptible to a
chemotherapeutic in any manner described herein.
Drug-Sensitization Methods
[0116] The present disclosure also includes drug-sensitization
methods in which a rifamycin or rifamycin derivative, such as
rifabutin or rifabutin derivative, composition is administered to a
cancer cell in order to sensitize the cancer cell to another drug.
The composition may be any composition described above. In a
specific embodiment, the composition may be administered with any
other drug which may alternatively be present in a pharmaceutical
composition as described herein. For example the other drug may
include DOXIL.RTM..
[0117] The drug may be any drug for which rifamycin or a rifamycin
derivative, such as rifabutin or a rifabutin derivative, increases
drug-sensitization. In a specific embodiment, the drug may be a
chemotherapeutic. Example types of chemotherapeutics include
alkylating agents, antimetabolites, anti-tumor antibiotics,
hormonal agents, targeted therapies, differentiating agents and
other drugs.
[0118] Example alkylating agents include nitrogen mustards such as
mechlorethamine (nitrogen mustard), chlorambucil, cyclophosphamide,
ifosfamide, and melphalen. Example alkylating agents further
include nitrosoureas, such as streptozocin, carmustine (BCNU), and
lomustine. Example alkylating agents further include alkyl
sulfonates such as busulfan, triazines, such as procarbazine and
dacarbazine (DTIC) and temozolomide, and ethylenimines, such as
thiotepa and altretamine (hexamethylmelamine). Example alkylating
agents further include platinum drugs, such as cisplatin,
carboplatin, and oxalaplatin.
[0119] Example antimetabolites include purine antagonists such as
mercaptopurine (6-MP), thioguanine (6-TG), fludarabine phosphate,
clofarabine, cladribine, and pentostatin. Example antimetabolites
also include pyrimidine antagonists such as fluorouracil (5-FU),
floxuridine, capecitabine, cytarabine, gemcitabine and azacitidine.
Example antimetabolites further include plant alkaloids. Some plant
alkaloids include topoisomerase inhibitors such as topoisomerase I
inhibitors such as camptothecin, topotecan and irinotecan, or
topoisomerase II inhibitors such as amsacrine, etoposide, and
teniposide. Other plant alkyloids include mitotic inhibitors such
as taxanes, including paclitaxel and docetaxel, epothilones,
including ixabepilone, vinca alkaloids, including vinblastine,
vincristine, and vinorelbine, as well as estramustine. Example
antimetabolites further include folate antimetabolites such as
methotrexate and pemetrexed. Other antimetabolites include
hydroxyurea.
[0120] Example anti-tumor antibiotics include anthracyclines or
anthracycline analogs such as daunorubicin, doxorubicin,
epirubicin, mitoxantrone, and idarubicin. Other anti-tumor
antibiotics include dactinomycin, plicamycin, mitomycin, bleomycin,
apicidin, and actinomycin.
[0121] Example hormonal agents include gonadotropin-releasing
hormone agonists such as leuprolide and goserelin. Other example
hormonal agents include aromatase inhibitors such as
aminoglutethimide, exemestane, letrozole and anastrozole. Other
hormonal agents include tamoxifen and flutamide. Still other
example hormonal agents include anti-estrogens such as fulvestrant,
tamoxifen, and toremifene or anti-androgens such as bicalutamide,
flutamide, and nilutamde. Example hormonal agents further include
progestins such as megestrol acetate, and estrogens.
[0122] Example targeted therapies include antibodies or other
therapeutics that act on a molecular level such as imatinib,
gefitinib, sunitinib, and bortezomib.
[0123] Example differentiating agents include retinoids such as
tretinoin, bexarotene, and arsenic trioxide.
[0124] Other chemotherapeutics include L-asparaginase, phenoxodiol,
rapamycin, and menadione.
[0125] In methods of the current disclosure, the cancer cell may be
sensitized to a drug already known to inhibit the cancer cell, or
it may be sensitized to a drug not previously used with that type
of cancer cell. If the cancer cell is a drug-resistant cancer cell
that has acquired resistance, it may be sensitized to a drug that
previously exhibited a decreased ability to inhibit the cancer cell
or cancer cells of the same type.
[0126] In another embodiment, the composition may directly inhibit
the cancer cell instead of or in addition to causing
drug-sensitization.
[0127] The cancer cell that undergoes drug-sensitization or
inhibition may be any type of cancer cell. It may, for instance, be
a carcinoma, a sarcoma, a leukemia, a lymphoma, or a glioma. It may
also be a soft cancer or a hard cancer. It may also be a cancer
affecting a particular organ or tissue, such as: an
immunological-related cancer such as leukemia, lymphoma, including
Non-Hodgkin's lymphoma, or Hodgkin's disease, myeloma, including
multiple myeloma, sarcoma, lung cancer, breast cancer, ovarian
cancer, uterine cancer, including endometrial cancer, testicular
cancer, intestinal cancer, including colon cancer, rectal cancer,
and small intestinal cancers, stomach cancer, esophageal cancer,
oral cancer, pancreatic cancer, liver cancer, prostate cancer,
glandular cancers such as adrenal gland cancer and pituitary tumor,
bone cancer, bladder cancer, brain and other nervous tissue
cancers, including glioma, eye cancer, including retinoblasoma,
skin cancer, including basal cell carcinoma and melanoma, and
kidney cancer.
[0128] The composition may be delivered to the cancer cell in a
patient by delivering the composition to the patient. The mode of
delivery may be selected based on a number of factors, including
metabolism of the rifamycin or rifamycin derivative, such as the
rifabutin or rifabutin derivative, or another drug in the
composition, mode of administration of other drugs to the patient,
such as the drug to which the cancer cell is sensitized, the
location and type of cancer cell to be drug-sensitized, health of
the patient, ability or inability to use particular dosing forms or
schedules with the patient, preferred dosing schedule, including
any adjustment to dosing schedules due to side effects of
chemotherapeutics, and ease of administration. In specific
embodiments, the mode of administration may be enteral, such as
orally or by introduction into a feeding tube. In other specific
embodiments, the mode of administration may be parenteral, such as
intravenously.
[0129] The dosage amounts of the rifamycin or rifamycin derivative,
such as rifabutin or rifabutin derivative and administration
schedule may vary depending on other components of the composition
and their effects on drug availability in a patient, the type of
drug or drugs to which the cancer cell is sensitized, the intended
mode of administration, the intended schedule for administration,
when other drugs are administered, any drug toxicity concerns, and
the patient's response to the drug. In a specific embodiment, the
amount and frequency of rifamycin or rifamycin derivative such as
rifabutin or rifabutin derivative delivered may be such that levels
in the patient remain well below levels at which toxicity to normal
cells or to the patient becomes a concern. However the amount and
frequency may also be such that the rifamycin or rifamycin
derivative, such as rifabutin and rifabutin derivative, levels in
the cancer cell remain continuously at a level sufficient to induce
drug-sensitization or are at a level sufficient to induce-drug
sensitization when or shortly after the drug to which the cancer
cell is sensitized is delivered to it. Accordingly, the rifabutin
or rifabutin derivative composition may be taken on a regular basis
during treatment with the drug to which the cancer cell is
sensitized or it may be taken only a set time before, at the same
time, or a set time after the drug to which the cancer cell is
sensitized.
Cancer Inhibition Methods
[0130] In some specific embodiments, the disclosure provides
methods of inhibiting a cancer cell using a drug to which the
cancer cell is resistant by administering rifamycin or a rifamycin
derivative, such as rifabutin or a rifabutin derivative, to the
cancer cell.
[0131] In other specific embodiments, the disclosure provides
methods of reducing the amount of a drug administered to a patient
by also administering rifamycin or a rifamycin derivative, such as
rifabutin or a rifabutin derivative. Such methods may, in
particular, be employed with drugs that have other harmful effects.
For example, use of certain alkylating agents, such as
topoisomerase inhibitors, increases the later chances of leukemia
in the patient. The chance of this adverse effect may be lessened
if lower doses of the alkylating agent may be administered with the
same therapeutic effect. Similarly, methods of the present
disclosure may be used to reduce the amount of mitotic inhibitors
administered, reducing the chance or amount of resulting peripheral
nerve damage, or the methods may be used to reduce the amount of
anti-tumor antibiotics administered, reducing the chance or amount
of resulting hearing damage. In the case of anti-tumor antibiotics
for which there is a total lifetime dosage limit, methods of the
present disclosure may allow a patient to be treated with the drug
for a longer time, increasing life expectancy or improving quality
of life. Methods of the present disclosure may also allow amounts
of some chemotherapeutics administered to remain sufficiently low
as to allow the patient to have children after cancer treatment.
Methods of the present disclosure may further allow amounts of the
chemotherapeutics administered to be lowered into a range where a
drug approved for use in adults might also be used in children.
[0132] In an alternative embodiment in which the rifamycin or
rifamycin derivative, such as rifabutin or rifabutin derivative
directly inhibits a cancer cell alone or in addition to causing
drug-sensitization, the dosage and administration may be adequate
to allow this inhibition. In an example embodiment, it may consist
of regular administration of an amount of the rifamycin or
rifamycin derivative, such as rifabutin or rifabutin derivative, to
maintain a certain level in the patient, the blood, a tissue, or a
tumor. However, dosage amounts and the administration schedule may
be adjusted based on other components of the composition and their
effects on drug availability in a patient, the intended mode of
administration, the intended schedule for administration, when
other drugs are administered, any drug toxicity concerns, and the
patient's response to the drug.
[0133] Without limiting the compositions and methods of
administration described herein, in one embodiment, rifamycin or a
rifamycin derivative, such as rifabutin or a rifabutin derivative,
may exhibit its drug-sensitization effect on a cancer cell by
directly or indirectly inhibiting an efflux pump, such as the
ATP-binding cassette sub-family B member 1 (ABCB1) pump. This
glycoprotein is found in the cell membrane and actively transports
certain chemotherapeutics, such as doxorubicin, out of cancer
cells, reducing efficacy of the drug. By inhibiting this pump, the
amount of chemotherapeutic present in a cancer cell can be
increased and thus the killing effect on the cancer cell may be
increased.
[0134] According to one embodiment of the present disclosure,
rifabutin and rifabutin derivatives suppress ABCB1 activity,
increasing the effective amount of a chemotherapeutic within a
cancer cell.
[0135] Also, without limiting the compositions and methods of
administration described herein, in one embodiment, rifamycin or a
rifamycin derivative, such as rifabutin or a rifabutin derivative,
may exhibit its drug-sensitization effect on a cancer cell by
acting on the Akt (protein kinase B)/14-3-3.zeta./mitochondrial
electron transport chain (ETC)/reactive oxygen species (ROS)
signaling network within a cell. An example of how the rifamycin or
rifamycin derivative can effect this pathway in a drug-resistant
cancer cell is shown in FIG. 1. In this example, a CHOP-resistant
cancer cell, such as a CHOP-resistant diffuse large B-cell lymphoma
(DLBCL) cell, undergoes cellular changes such that Akt is
constitutively activated. This constitutively activated Akt
phosphorylates mitochondrial GSK-3.beta.. This phosphorylated
GSK-3.beta. then binds the 14-3-3.zeta. protein, rendering the
GSK-3.beta. unavailable to bind to mitochondrial ETC Complex 1.
GSK-3.beta. binding to ETC Complex 1 inhibits the complex activity,
so the overall result of constitutive Akt activation is that ETC
Complex 1 is not inhibited when it otherwise should be.
Downregulation of Complex I activity by GSK-3.beta. can lead to
increased electron leakage from the ETC, resulting in increases in
ROS.
[0136] ETC Complex 1 acts to reduce the amount of electron spillage
from the ETC during mitochondrial activity. Electrons spilled in
such a manner react with oxygen to produce reactive oxygen species
(ROS). Thus, increased ETC Complex 1 activity and the resultant
reduction in electron leakage decrease the amount of ROS in the
cell. Low levels of ROS may lead to an intracellular environment
that inhibits the ability of chemotherapeutics such as CHOP to
induce cancer cell death by apoptosis. Thus, one effect of
constitutive Akt activation is a decrease in ROS, making the cancer
cell harder to kill.
[0137] According to one embodiment of the present disclosure,
rifamycin or a rifamycin derivative, such as rifabutin and
rifabutin derivative suppress ETC Complex 1 activity, restoring it
to a more normal level. As a result, more ROS are present in the
cell and the cellular environment is restored to one in which CHOP
may once again induce cell death via apoptosis.
[0138] A similar effect may be seen with other chemotherapeutics or
other drugs whose efficacy relies on a cellular environment with
minimum amount of ROS or other factors (such as other intracellular
chemicals, proteins, or conditions) resulting from a minimum amount
of ROS in the cell.
[0139] As a result of this effect on the Akt/14-3-3.zeta./ETC/ROS
network, the present disclosure also includes methods of inducing
drug-sensitization in a cancer cell by administering an amount of
rifabutin or rifabutin derivative sufficient to decrease activity
of ETC Complex 1 or increase cellular levels of ROS. In particular,
the disclosure includes methods of administering an amount of
rifabutin or rifabutin derivative sufficient to increase cellular
levels of ROS to an amount sufficient to allow a drug to which a
cancer cell is sensitized to kill, reduce the growth of, or
negatively affect the cancer cell.
[0140] Although the above example relates to cancer cells that have
become resistant to a drug due to abnormal Akt activity, the same
methods are applicable to cancer cells that exhibit low ROS levels
for other reasons. Furthermore, the same methods may be used for
drug-sensitization in cancer cells that have no ROS abnormality by
increasing ROS to an abnormal level if the cancer cells then become
sensitive to the drug at the abnormal ROS level.
[0141] Effects mediated by ROS described above may, in particular,
be mediated by superoxide species and superoxide species may be the
particular form of ROS affected.
[0142] Although some drug-sensitization or cancer cell inhibition
effects may be mediated by the ROS pathway, compositions and
methods of the present disclosure may act via other cellular
pathways alternatively to or in addition to the
Akt/14-3-3.zeta./ETC/ROS network. This may be particularly true
with respect to drug-sensitization to chemotherapeutics that
operate in a different manner than CHOP. For example, ROS may
affect the mitochondrial-directed Bcl-2 apoptosis pathway as well.
Furthermore, the effect of rifabutin on ROS induction has been
shown to be very rapid, whereas the effect on Akt has been shown to
take at least 18 hours. Accordingly, it appears likely that an
initial ROS induction event may occur, followed by a secondary
downstream effect downregulating Akt. In CHOP-resistant cells, Akt
is constitutively active thereby increasing Complex I activity
resulting in decreases in ROS. Induction of ROS by compositions and
methods of the present disclosure will further promote drug
sensitivity in the resistant cancer cell by downregulating the Akt
pathway.
[0143] Again without limiting the compositions and methods of
administration described herein, in one embodiment, rifamycin or a
rifamycin derivative, such as rifabutin or a rifabutin derivative,
may exhibit its drug-sensitization effect on a cancer cell by
mobilizing calcium within the cell. Increased calcium mobilization
correlates with increased ROS amounts. Drug-sensitive cells often
exhibit both increased levels of calcium and increased ROS levels
as compared to drug-resistant cells. Typically, ROS levels rise
first in such cells, followed by calcium mobilization. Accordingly,
rifamycin or a rifamycin derivative, such as rifabutin or a
rifabutin derivative, may directly inhibit efflux pump activity,
which then causes a burst of ROS followed by calcium
mobilization.
[0144] According to one embodiment of the present disclosure,
rifamycin or a rifamycin derivative, such as rifabutin or a
rifabutin derivative, may inhibit a cancer cell through more than
one activity. For instance, it may both decrease efflux pump
activity and increase ROS. In certain embodiments, these multiple
activities may have synergistic effects.
[0145] Further without limiting the compositions and methods of
administration described herein, the compositions and methods may
prevent or reduce metastasis. Metastasis from solid tumors is a
complex, multistep process whereby cancer cells must breach the
basement membrane and migrate away from the primary tumor
environment to invade the surrounding stroma and enter the
vasculature directly or via the lymphatics. The cancer cells must
then also invade another area of the body. Rifamycin or a rifamycin
derivative, such as rifabutin or a rifabutin derivative, may
prevent or reduce metastais by preventing or reducing any of these
movements or activities of the cancer cells. Rifamycin or a
rifamycin derivative, such as rifabutin or a rifabutin derivative,
may also decrease the levels of metastasis-associated cellular
factors in or around cancer cells. Such factors include matrix
metalloproteinase (MMP) 2 or other MMP family members and vascular
endothelial growth factor (VEGF). MMP family members are involved
in the breakdown of extracellular matrix in disease processes such
as metastasis. VEGF is an important signaling protein involved in
both vasculogenesis (the formation of the circulatory system) and
angiogenesis (the growth of blood vessels from pre-existing
vasculature).
Determining Appropriate Cancer Targets
[0146] The present disclosure also provides a method of determining
whether a cancer cell is likely to be resistant to
chemotherapeutics or experience an increase in ROS or
drug-sensitization in response to rifamycin or a rifamycin
derivative, such as rifabutin or a rifabutin derivative, or if
treatment with such a composition is having an effect on a cancer
cell. In such a method, ROS, such as superoxide species, may be
measured in cancer cells of the same type. If ROS is abnormally low
compared to ROS levels previously measured in cancer cells of the
same type (e.g. 3-10 fold lower), then the cancer cell may be more
likely to not respond to a chemotherapeutic than cancer cells with
higher ROS levels. The cancer cell may also exhibit an increase in
ROS or be sensitized to a drug in response to rifamycin or a
rifamycin derivative, such as rifabutin or a rifabutin derivative,
so such a composition may be administered to the cancer cell along
with a drug to which a cancer cell is sensitized to inhibit the
cancer cell. If the patient has been treated with rifamycin or a
rifamycin derivative, such as rifabutin or a rifabutin derivative,
and ROS levels are normal for that type of cancer cell, higher than
in previous measurements from that patient, or higher than normal
for the type of cell from which the cancer is derived, then the
treatment is likely successful and should be continued.
[0147] Alternatively, rather than measure ROS directly, an
indicator of ROS levels may be measured. In a specific embodiment,
ROS may be measured using ROS stains.
[0148] In another embodiment, the amount of ROS (or indicator of
ROS levels) in cancer cells of a certain type may be measured and,
if below a certain threshold, rifamycin or a rifamycin derivative,
such as rifabutin or a rifabutin derivative, may be administered to
a cancer cell of the same type along with a drug to which a cancer
cell is sensitized to inhibit the cancer cell.
[0149] In specific embodiments, ROS-related measurements may be
made by any conventional methods. For example, ROS-related
measurements may be made on a biopsy, resection or aspirant of a
tumor or cancer-bearing tissue, a blood sample, or cancer cells
isolated by other means. Measurements are compatible with presently
known methods of obtaining cancer cells from patients and are
expected to be similarly compatible with additional methods
developed in the future.
EXAMPLES
[0150] The following examples are provided to further illustrate
specific embodiments of the disclosure. They are not intended to
disclose or describe each and every aspect of the disclosure in
complete detail and should be not be so interpreted. Unless
otherwise specified, designations of cells lines and compositions
are used consistently throughout these examples.
Example 1
Drug-Sensitization of CHOP-Resistant NHL Cell Lines
[0151] Several human cell lines were utilized as in vitro models of
NHL, including the CRL2631 line obtained from the American Type
Culture Collection (ATCC). CRL2631 was established from peripheral
blood leukocytes (PBL) of a patient with DLBCL. CHOP-resistant NHL
cell lines (designated G3) were generated by repeated cycles of
on-off treatments with CHOP, a treatment protocol that is similar
to clinical regimens.
[0152] The effects of rifabutin on cell growth of both
CHOP-sensitive (CRL2631) and CHOP-resistant (G3) cells in the
presence or absence of CHOP are shown in FIG. 2A. A reduction in
cell growth is demonstrated by a reduction in fluorescence emitted
by the cell growth indicator dye, resazurin.
[0153] Rifabutin was confirmed to have drug-sensitization activity
in clinically derived CHOP resistant cell lines. As shown in FIG.
2A, CHOP inhibited growth of CHOP-sensitive (CRL2631) cells but had
little effect on G3 cells. Rifabutin did not affect the growth of
cells in the absence of CHOP, indicating low toxicities (FIG. 2A).
Rifabutin enhanced the sensitivity of CHOP-sensitive cells to CHOP
as shown in FIG. 2B, relative to a control drug.
[0154] FIG. 2C shows similar effects in another CHOP-resistant NHL
line.
Example 2
Toxicity
[0155] Doxorubicin and rifabutin or its derivatives RTI-79 and
RTI-176 were applied to primary human fibroblasts to determine
comparative cytotoxicity. Results are presented in FIG. 3A and FIG.
3B and demonstrate that rifabutin and the analogs are not toxic to
normal cells.
[0156] To further test safety of rifabutin and its derivatives,
rifabutin and rifabutin derivates RTI-79 and RTI-81 were
administered as an adjunct therapy to doxorubicin (DOX).
[0157] Swiss mice were dosed with levels equal to and exceeding
that of intended doses. Swiss mice were given repeated weekly oral
doses of rifabutin at 180 mg/kg, RTI-79 at 250 mg/kg or RTI-81 at
30 mg/kg in conjunction with intravenous 3.3 mg/kg DOX. No overt
toxicity or weight loss was seen over several weeks time. Further,
no significant differences between mice treated with RTI-79 with or
without DOX were observed after both histological analysis of heart
tissue by hematoxylin and eocin (H&E) and analysis of blood and
serum for complete blood count and manual differential. Intravenous
rifabutin or RTI-81 were also given repeatedly both at 75 mg/kg in
conjunction with intravenously administered 3.3 mg/kg DOX and no
overt toxicity or weight loss was seen over several weeks time.
Further data in Example 4 below shows treatment efficacy using less
than one-fifth the above oral dose of 33 mg/kg rifabutin with
intravenous 3.3 mg/kg doxorubicin.
Example 3
Drug-Sensitization of CHOP-Resistant Lymphoma Cells from Dog
Model
[0158] A single lymphoma aspirate from a dog with CHOP-resistant
lymphoma was tested for responsiveness to CHOP in the presence or
absence of rifabutin. CHOP-responsiveness was measured by a
decrease in fluorescence signal generated by resazurin. FIG. 4
shows that growth of aspirated lymphoma cells was resistant to CHOP
at doses up to 640 ng/ml, but significant growth inhibition was
observed at a dose of 1280 ng/ml CHOP. The inclusion of 5 .mu.M
rifabutin significantly enhanced the sensitivity of the aspirated
lymphoma cells to CHOP such that significant growth inhibition was
observed at 320 and 640 ng/ml CHOP. Rifabutin had no effect on cell
viability in the absence of CHOP.
Example 4
Drug-Sensitization In Vivo
[0159] In a first efficacy study, 6-8 week old female SCID mice (7
mice per treatment arm) were injected subcutaneously on both flanks
with 1.times.10.sup.7 G3 CHOP-resistant NHL cells. Once palpable
tumors (about 50-100 cc size) appeared, therapies (CHOP or
CHOP+rifabutin) were started. CHOP was administered at the maximum
tolerated dose (cyclophosphamide, 40 mg/kg i.v.; doxorubicin, 3.3
mg/kg i.v.; vincristine, 0.5 mg/kg i.v.; and prednisone, 0.2 mg/kg
orally daily for 5 d) weekly for 3 weeks. Rifabutin in an amount of
100 mg/kg was administered on the day of each CHOP treatment and
24-hours later by gavage. Mouse body weight and tumor size were
monitored every two days and tumor size measured by caliper. The
tumor volume formula (L*W*W)/2 was used to calculate tumor
mass.
[0160] The overall tumor burden per mouse was much lower in mice
that received CHOP+rifabutin than for those receiving CHOP only
treatments. CHOP treatment alone of the SCID mice harboring
subcutaneous G3 lymphomas resulted in relatively fast tumor growth,
as compared to tumors in CHOP+rifabutin treated mice (FIG. 5). The
dosage of rifabutin administered had little or not toxicity in the
mice. Control mice injected with CRL2631 cells, in contrast,
exhibited a marked decrease in tumor growth in response to CHOP
alone (FIG. 6).
[0161] A second efficacy study was conducted where mice were
treated before the appearance of palpable tumors. In that
experiment, one week after transplantation of CHOP-resistant G3
cells, one group (7 mice) was treated with CHOP-only and a second
group (8 mice) was treated with CHOP+rifabutin. One week later,
mice received a second treatment and tumors began to appear in the
CHOP-only group. The two treatment groups differed not only in the
tumor size but also in the number of tumors developed. More tumors
appeared and grew at a significantly higher rate in CHOP-only mice
compared to CHOP+rifabutin mice. The CHOP only treatment group
developed tumors at 12 of 14 (85.7%) injection sites. The
CHOP+rifabutin treatment group developed fewer tumors at only 6 of
16 (37.5%) injection sites. In a separate experiment, SCID mice
developed G3 tumors at 35 of 42 (83.3%) injection sites when
receiving no treatment; this is similar to the CHOP only treatment
group. Significance was analyzed by the T test yielding a highly
significant difference between the means of the tumor burdens of
the two groups (p<0.01) at Day 7. Thus, rifabutin actually
reduces the tumor take rate which could translate into more
complete responses when humans are treated early with this
combination.
[0162] A third study was conducted in which mice injected with
CHOP-resistant G3 cells received reduced dosages of CHOP in
combination with 33 mg/kg rifabutin. CHOP+rifabutin was
administered weekly beginning one week post-inoculation. Control
mice were given no CHOP or rifabutin. Tumor load was significantly
less in mice that received even reduced CHOP dosages as compared to
untreated mice, demonstrating that rifabutin may allow the use of
lower dosages of CHOP without a significant decrease in therapeutic
effect (FIG. 7).
[0163] A fourth efficacy study was conducted using DOX in
combination with the rifabutin derivative RTI-81. SCID mice were
injected with CHOP-resistant G3 cells in the same manner as the
first efficacy study above. Treatments began 2 weeks
post-inoculation and were administered twice weekly. DOX was given
at 3.3 mg/kg iv and RTI-81 was given at 10 mg/kg by gavage. A
statistically significant difference in average life-span is seen
when mice were treated with doxorubicin and RTI-81 as compared to
DOX alone. Mice receiving doxorubicin+RTI-81 lived 27% longer than
those receiving doxorubicin only (X.sup.2=8.6 p=0.00336 (dof=1))
(FIG. 8). Respective mean and median lifespans for each group were:
42.6, 42 and 34.6, 33. Mice treated with doxorubicin only were
10.37 times as likely to die before those treated with
doxorubicin+RTI-81. Cox proportional hazard ratio was 0.0964 with a
likelihood ratio of 7.24 (p=0.00714 (dof=1, n=15).
[0164] In a fifth efficacy study, we generated xenografts of the
human ovarian cancer cell line SK-OV-3, a cell line considered
doxorubicin-resistant, by bilateral subcutaneous (s.c.) injection
of 1.times.10.sup.7 tumor cells to establish localized tumors in
6-8 week old female SCID mice. Using rifabutin co-administered with
DOX, in vivo efficacy was assessed. Once tumor volumes were at
least 75 mm.sup.3 and showed consistent growth rates, therapies
(DOX only 3.3 mg/kg i.v. or DOX 3.3 mg/kg i.v.+rifabutin 25 mg/kg
oral) were started. Cycles of Dox or Dox+rifabutin were given once
a week for 4 cycles. This cyclical dosing scheme of mouse models
has precedent in the literature and is intended to mimic the cycles
of DOXIL.RTM. (Centocor Ortho Biotech Products, LP, Raritan, N.J.)
given in the clinic. Rifabutin was administered on the day of each
DOX treatment and by gavage. Mouse body weight and tumor size were
monitored. As shown in FIG. 9, after 19 days treatment, average
tumor volumes were 587 mm.sup.3 for the DOX-only treatment group,
and 348 mm.sup.3 for the DOX+rifabutin group. This is a 40%
reduction in tumor size for the DOX+rifabutin group.
[0165] In a sixth efficacy study, we generated xenografts of
multi-drug resistant ovarian cancer cell line (NCI/ADR-RES) by
implantation of NCI/ADR-RES cell xenografts in the left and right
flanks of nude mice, resulting in two tumors per mice. In vivo
efficacy of RTI-79 was assessed by co-administration with
DOXIL.RTM.. Once tumor volumes were at least 90 mm.sup.3 and showed
consistent growth rate, therapies (DOXIL.RTM. only 7 mg/kg i.v. or
DOXIL.RTM.7 mg/kg i.v.+RTI-79 25 mg/kg oral) were started. Cycles
of DOXIL.RTM. or DOXIL.RTM.+RTI-79 were given every week for six
cycles. RTI-79 was administered by oral gavage 24 and 48 hours
after each DOXIL.RTM. administration. Tumor size was monitored. As
shown in FIG. 10, after 41 days the tumor volume in the
RTI-79-treated mice was 66% lower than in mice receiving only
DOXIL.RTM..
[0166] In a seventh efficacy study, we generated xenografts of
multi-drug resistant ovarian cancer cell line (NCI/ADR-RES) by
implantation of NCI/ADR-RES cells xenografts in the left and right
flanks of nude mice, resulting in two tumors per mouse. In vivo
efficacy of RTI-79 was assessed by co-administration with
DOXIL.RTM.. Therapies were DOXIL.RTM. only 7 mg/kg i.v. or
DOXIL.RTM. 7 mg/kg i.v.+RTI-79 25 mg/kg oral. Cycles of DOXIL.RTM.
or DOXIL.RTM.+RTI-79 were given every week for six cycles. RTI-79
was administered by oral gavage 24 and 48 hours after each
DOXIL.RTM. administration. Tumor size was monitored. As shown in
FIG. 11, after 46 days the tumor volume in the RTI-79-treated mice
was 55% lower than in mice receiving only DOXIL.RTM.. Furthermore,
tumor volume in RTI-79-treated mice was reduced by 50% during the
course of the study.
Example 5
Sensitization to CHOP Using Other Rifabutin Derivatives
[0167] Several compositions of the present disclosure were tested
and their effects on cell growth were measured. A reduction in cell
growth is demonstrated by a reduction in fluorescence emitted by
the cell growth indicator dye, resazurin. Compositions were tested
on CHOP-resistant G3 NHL cells that had been cultivated in RPMI
medium for five days. Prior to assay, the cells were counted by
haemocytometer and cell concentration standardized to 625,000
cells/ml. Test drugs were solubilized in 100% DMSO and then diluted
to final assay concentration with 0.1M phosphate buffered saline
(PBS) and a final DMSO concentration of 0.5%. Cells were added to
assay plates containing the test drugs (rifabutin+148 ng/ml, 74
ng/ml, 37 ng/ml, or 0 ng/ml doxorubicin) and allowed to incubate
for 96 hours at 37.degree. C. and 5% CO.sub.2. The metabolic dye
rezasurin was added to the wells of the assay plate at a final
concentration of 20 .mu.g/ml and the plates were incubated for an
additional 24 hours. The plates were then read in a BMG Polarstar
plate reader at wavelength (573-605) and the data plotted as OD
versus increasing dilutions (i.e. decreasing total amounts) of
rifabutin derivative concentration.
[0168] Results of tests were performed on G3 cells to compare the
effects of rifabutin and certain rifabutin derivatives on cell
growth in the presence or absence of 1 .mu.M doxorubicin (DOX) are
presented in Table 2, which indicates the IC.sub.50s for selected
rifamycin analogs on lymphoma cell line G3.
TABLE-US-00002 TABLE 2 IC.sub.50s for selected rifamycin analogs on
lymphoma cell line G3. IC.sub.50 (.mu.M) Fold increase with
doxorubicin in potency Analog IC.sub.50 (.mu.M) (1 .mu.M) over DOX
alone Doxorubicin 2.36 NA NA Rifabutin >64 .25 9.4 RTI-51 >64
3.3 0.7 RTI-53 >64 11.8 0.2 RTI-78 58 0.08 29.5 RTI-79 43 1.14
2.1 RTI-81 >64 0.3 7.9 RTI-82 >64 3.5 0.7 RTI-102 >64 0.95
2.5 RTI-174 51 0.64 3.7 RTI-175 >64 1.06 2.2 RTI-176 >64 0.45
5.2 RTI-181 52 0.43 5.5 RTI-182 62 1.2 2.0 RTI-183 >64 5.19
0.5
[0169] Example data for RTI-79 and rifabutin is shown in FIG. 12.
Example data for RTI-176 and rifabutin is shown in FIG. 13. Example
data for RTI-81 and rifabutin is shown in FIG. 14. Example data for
interaction of rifabutin and doxorubicin on CRL2631 cells is shown
in FIG. 15. Example data for interaction of RTI-79 and doxorubicin
on CRL2631 cells is shown in FIG. 16. These results establish that
a variety of rifabutin derivatives are similarly effective at
restoring doxorubicin sensitivity to CHOP-resistant cells.
Example 6
Drug-Sensitization of Multiple Cell Lines
[0170] The ability of rifabutin and rifabutin derivatives to cause
drug-sensitization to doxorubicin in multiple types of cancer cells
was investigated by performing experiments similar to those
described above. In these experiments, the following cell lines
were used: CHOP-resistant NHL cell line G3, CHOP-sensitive NHL cell
line CRL2631, the multi-drug resistant sarcoma cell line
MES-SA-Dx5; multi-drug-resistant breast cancer cell line
MDA-MB-231, multi-drug resistant ovarian carcinoma cell line
SK-OV3, multi-drug resistant ovarian cancer cell line NCI/ADR-RES,
drug-sensitive ovarian cancer cell line OVCAR-5, and multi-drug
resistant ovarian cancer cell line OVCAR-3. Results are presented
in Table 3.
TABLE-US-00003 TABLE 3 Magnitude of potentiation observed with
rifamycin analogs in combination with doxorubicin Cancer RTI- RTI-
RTI- RTI- Type Tissue Cell Line RBT 51 53 79 81 Lymphoma B cells G3
+++ ++ +++ Lymphoma B cells CRL CRL2631 Sarcoma Uterus MES-SA-Dx5
++ ++ ++ Carcinoma Breast MDA-MB-231 +++ + Carcinoma Ovarian SK-OV3
+ + + +++ +++ Carcinoma Ovarian OVCAR-3 + + + Carcinoma Ovarian
OVCAR-5 + Carcinoma Ovarian NCI/ADR-RES ++ + Cancer RTI- RTI- RTI-
RTI- Type Tissue Cell Line RBT 82 102 174 175 Lymphoma B cells G3
+++ ++ ++ ++ Lymphoma B cells CRL CRL2631 Sarcoma Uterus MES-SA-Dx5
++ + Carcinoma Breast MDA-MB-231 +++ +++ Carcinoma Ovarian SK-OV3 +
+++ + +++ Carcinoma Ovarian OVCAR-3 + + Carcinoma Ovarian OVCAR-5
++ Carcinoma Ovarian NCI/ADR-RES + ++ Cancer RTI- RTI- RTI- RTI-
Type Tissue Cell Line RBT 176 181 182 183 Lymphoma B cells G3 +++
+++ +++ + Lymphoma B cells CRL CRL2631 Sarcoma Uterus MES-SA-Dx5 ++
Carcinoma Breast MDA-MB-231 +++ ++ Carcinoma Ovarian SK-OV3 + +++
+++ ++ Carcinoma Ovarian OVCAR-3 + + + Carcinoma Ovarian OVCAR-5 +
Carcinoma Ovarian NCI/ADR-RES RBT = rifabutin; + potentiation
between 1.2 to 2.0 fold increase; ++ potentiation between 2.1 to 5
fold increase; +++ potentiation greater than 5 fold increase
[0171] Example data for rifabutin or RTI-82 on MDA-MB-231 cells is
presented in FIG. 17. Example data for rifabutin or RTI-79 with or
without doxorubicin on SK-OV3 cells is presented in FIG. 18.
Example data for rifabutin or RTI-81 on MES-SA-Dx5 cells is
presented in FIG. 19. Example data for interaction of rifabutin or
RTI-79 and doxorubicin on ADR-RES cells is shown in FIG. 20.
Example data for interaction of RTI-79 and doxorubicin on MOLT-4
cells is shown in FIG. 21. Example data for the interation of
rifabutin or RTI-79 and doxorubicin on ovarian carcinoma OVCAR-8
cells is shown in FIG. 22. These results establish that rifabutin
and rifabutin analogs are able to induce drug-sensitization for a
variety of types of cancer.
Example 7
Sensitization to Various Chemotherapeutics Using Rifabutin and
Rifabutin Derivatives
[0172] Similar tests were performed to compare the effects of
rifabutin and certain rifabutin derivatives on cell growth in the
presence or absence of various chemotherapeutics on various cell
lines. Chemotherapeutics include: the targeted therapy bortezomib
(Velcade.RTM.), the pyrimidine antagonist gemcitabine, the platinum
drug cis-platin, the anti-tumor antibiotic actinomycin D, the
anti-tumor antibiotic apicidin, the topoisomerase I inhibitor
camptothecin, the anti-tumor antibiotic doxorubicin, the mitotic
inhibitor vinblastine, the nitrogen mustard alkylating agent
melphalen, the hormonal agent tamoxifen, the folate antimetabolite
methotrexate, the toposimerase II inhibitor etoposide, phenoxodiol,
the antibiotic rapamycin, and menadione. Additional cell lines used
include: ovarian cancer OVCAR-8, T lymphoblastoid leukemia MOLT-4,
dexamethasone-resistant multiple myeloma MM.1R, myeloid leukemia
cells HL-60, osteosarcoma cells U-2 OS, and myeloma RPMI 8226.
Results are shown in Table 4.
TABLE-US-00004 TABLE 4 IC.sub.50s for selected cancer cell lines
and clinically relevant cancer therapeutics in interaction with
Rifabutin IC.sub.50 (.mu.M) Fold IC.sub.50 with increase in Cancer
type Cell line Therapeutic drug (.mu.M) Rifabutin potency Diffuse
large B G3 Doxorubicin 2.36 0.25 9.4 cell lymphoma Diffuse large B
G3 Vinblastine 8.00 1.00 8.0 cell lymphoma Diffuse large B G3
Mitoxantrone 0.46 0.04 11.5 cell lymphoma Diffuse large B CRL2631
Doxorubicin 0.35 0.12 2.9 cell lymphoma Ovarian carcinoma OVCAR-3
Menadione >32 10.88 >2.9 Ovarian carcinoma OVCAR-5 Velcade
0.17 0.08 2.1 Ovarian carcinoma OVCAR-8 Mitoxantrone 14.0 3.0 4.7
Ovarian carcinoma SK-OV3 Mitoxantrone >32 12.28 >2.6 Ovarian
carcinoma ADR-RES Doxorubicin >32 6.59 >4.9 Leukemia MOLT-4
Doxorubicin 0.03 0.01 3 Leukemia MOLT-4 Actinomycin D 0.04
<0.008 >5 Breast Cancer MDA-MB-231 Gemcitabine >32 4.83
>6.6 Multiple myeloma MM.1R Camptothecin 1.13 0.3 3.8 Multiple
myeloma MM.1R Menadione 4 2 2 Myeloid leukemia HL-60 Paclitaxel 0.4
0.2 2 Uterine Sarcoma MES-SA-Dx5 Actinomycin D 0.03 0.01 3
Osteosarcoma U-2OS Mitoxantrone 0.14 0.06 2.3 Myeloma RPMI 8226
Paclitaxel 1.0 0.89 1.1
[0173] Example data for interaction of rifabutin with actinomycin D
on MES-SA-Dx5 cells is shown in FIG. 23. Example data for
interaction of rifabutin with menadione on MM.1R cells is shown in
FIG. 24. Example data for interaction of rifabutin and mitoxantrone
on U-2 OS cells is shown in FIG. 25. Example data for interaction
of rifabutin andgemcitabine on MDA-MB-231 cells is shown in FIG.
26. Example data for interaction of rifabutin with paclitaxel on
HL-60 cells is shown in FIG. 27. Example data for interaction of
rifabutin and camptothecin on OVCAR-8 cells is shown in FIG. 28.
These results demonstrate the ability of rifabutin and rifabutin
derivatives to induce drug-sensitivity for a wide variety of
chemotherapeutics in a wide variety of cancers.
Example 8
Prevention of the Emergence of CHOP Resistance
[0174] The ability of rifabutin to prevent the emergence of
CHOP-resistance was determined by treating CHOP-sensitive CRL2631
cells with either CHOP alone or CHOP+rifabutin for one week.
Following treatment, the cells were grown in the absence of CHOP,
then their sensitivity to CHOP was assayed by retreatment with
CHOP, followed by counting of viable cells. Results are shown in
FIG. 29. Rifabutin was able to significantly repress the emergence
of CHOP-resistant cells at both half (0.5.times.) and full
(1.times.) doses of CHOP. A 1.times.CHOP dose in this experiment
corresponds to final concentrations of the following components:
0.83 .mu.M 4-hydroxycyclophosphamide [4HC, a pre-activated form of
cyclophosphamide], 0.057 .mu.M doxorubicin, 0.01 .mu.M vincristine,
and 0.186 .mu.M prednisone.
Example 9
Effects of Rifabutin and Rifabutin Derivatives on ROS
[0175] A Western blot of CHOP-sensitive (CRL2631) or CHOP-resistant
(G3) lymphoma cells revealed that Akt, phosphorylated Akt, and
14-3-3.zeta. levels were consistent with the model proposed in FIG.
1 (FIG. 30A) in that Akt was markedly more active in CHOP-resistant
G3 cells than in CRL2631. The model was further confirmed by
treatment of CHOP-resistant (G3) cells with Akt Inhibitor VIII,
which caused a dose-dependent reversal of CHOP resistance (FIG.
30B). The inhibitory effect of Akt inhibitor VIII on the expression
of phosphorylated Akt and total 14-3-3.zeta. protein was confirmed
by Western blot (FIG. 30C).
[0176] Additional studies further confirmed the model of FIG. 1 by
demonstrating that CHOP-sensitive (CRL2631) cells make more ROS
than do CHOP-resistant (G3 cells) (FIG. 31). Furthermore, CHOP
increased ROS in CHOP-sensitive (CRL2631) cells, but not in
CHOP-resistant (G3) cells (FIG. 31).
[0177] Examination of CHOP-sensitive CRL2631 cells revealed that
these cells include two distinct populations, a low-ROS population
and high-ROS population (FIG. 32). When these populations were
separated, the low-ROS population proved more resistant to CHOP
than high-ROS population (FIG. 33). However, this low-ROS cell
population was sensitized to CHOP by rifabutin (FIG. 34). Rifabutin
also rapidly induces ROS in CHOP-resistant (G3) cells (FIG.
35).
[0178] Overall, these results demonstrate that, at least in the
CRL2631 lymphoma cell line and cell lines derived therefrom,
CHOP-resistance is mediated by ROS levels and that rifabutin and
rifabutin derivatives decrease CHOP-resistance by increasing
ROS.
Example 10
Rifabutin and RTI-79 Decrease Drug Efflux and Mobilize Calcium
[0179] Rifabutin and its derivatives, such as RTI-79, showed clear
inhibition of efflux pumps in NCI/ADR-RES and G3 cells when tested
in calcein-AM assays. This inhibitory effect was unambiguously due
to inhibition of ABCB1 pumps. The difference in pump activity
between ADR-RES cells and its drug-sensitive parental strain,
OVCAR-8, may be seen in FIGS. 34A and 34B
[0180] It is known that mitigation of ABCB1 activity will lead to
more effective accumulation of doxorubicin in cells (Shen, F., Chu,
S., Bence, A. K., Bailey, B., Xue, X., Erickson, P. A., Montrose,
M. H., Beck, W. T., and Erickson, L.C. (2008). Quantitation of
doxorubicin uptake, efflux, and modulation of multidrug resistance
(MDR) in MDR human cancer cells. J Pharmacol Exp Ther 324,
95-102.). Thus the inhibition on ABCB1 by RTI-79 directly
contributes to its potentiating doxorubicin toxicity on these
drug-resistant cells. This was confirmed by testing with additional
rifabutin derivatives. As shown in FIG. 36, the stronger inhibitors
of ABCB1, also better re-sensitized drug-resistant cells. RTI-79
was the strongest inhibitor as well as best re-sensitizer.
[0181] Doxorubicin-sensitive (OVCAR8 ovarian) and
Doxorubicin-resistant (G3 lymphoma; ADR-RES ovarian) cells were
treated for 2 hrs with 10 uM RTI-79, p-glycoprotein (P-gp)
inhibitors (reserpine, elacridar), or control drugs (DMSO,
carboxin, nifazoxidine). Cells were then stained with the
fluorescent ROS indicator, CellROX, and subjected to flow cytometry
to quantitate total intracellular ROS. As FIG. 38 shows, RTI-79
induced ROS in ovarian carcinoma and lymphoma cell lines, as did
the MDR/P-gp inhibitors, reserpine, elacridar. This suggests that
RTI's ability to induce ROS was the result of inhibition of efflux
pumps. The degree of ROS induced by RTI-79 and P-gp inhibitors was
much greater in the doxorubicin-resistant ADR-RES and G3 cell lines
than in the doxorubicin-sensitive OVCAR8 cell line. Control drugs
established that this effect is specific to MDR/P-gp inhibitors and
RTI-79.
[0182] The intracellular origin of RTI-induced ROS was determined
by staining ADR-RES cells with the red fluorescent ROS indicator,
CellROX (Invitrogen), and visualizing where the ROS was
concentrated by confocal microscopy. Results are presented in FIG.
39. Mitochondria were localized by infecting cells for 24 hrs with
the BacMAM mitotracker baculovirus, which expresses a GFP fused to
a mitochondria localization signal. Nuclei were stained with the
blue DAPI stain. There was a good co-localization of red CellROX
staining with the green GFP mitotracker, indicating that the ROS
were originating from the mitochondria.
[0183] The electron transport chain (ETC) is known to be a primary
generator of ROS in the cell. Most of the ROS is generated by
Complexes I and III of the ETC Inhibition of Complex I results in
electrons piling up and leaking to react with oxygen to produce
ROS. The effects of a Complex I inhibitor (rotenone) and a Complex
III inhibitor (antimycin A) on ROS levels in the cell were tested
and results are shown in FIG. 40. Specifically, Dox-resistant G3
lymphoma cells were treated 10 uM RTI-79, BAPTA-AM (cell permeable
calcium chelator), verapamil (a calcium channel blocker and P-gp
inhibitor), a Complex I inhibitor (Rotenone), a Complex III
inhibitor (antimycin A), or control drugs (oxaloacetate, carboxin,
nifazoxinide). Rotenone, but not antimycin A, induced ROS,
suggesting that RTI-79-induced and efflux pump inhibitor-induced
ROS originate at Complex I of the ETC.
[0184] MDR/P-gp activity is closely associated with calcium status
in the cell, so calcium modulators were tested for effects on ROS.
As shown in FIG. 40, both a cell-permeable calcium chelator
(BAPTA-AM) and a calcium channel blocker (and efflux pump
inhibitor) induced ROS in G3 cells. As shown in FIGS. 41A and 39B,
P-gp inhibitors (Reserpine, Elacridar) induced ROS relative to
control drugs (Carboxin, Nifazoxinide). As shown in FIG. 41B, a
P-gp inhibitor (Elacridar) induced calcium in a similar manner as
RTI-79. indicating connections between calcium, ROS, and efflux
pump activity in the mechanism of action of RTIs. Because calcium
modulators induced ROS, testing was performed to investigate
whether RTI-induced ROS was associated with calcium mobilization in
doxorubicin-sensitive and doxorubicin-resistant cells and in
resistant cells treated with RTI-79. Relatively Dox-sensitive
lymphoma (CRL2631, 10S, WSU) and ovarian carcinoma (OVCAR8) and
more Dox-resistant lymphoma (G3R,10R, WSUR) and ovarian carcinoma
(ADR) were treated with 10 uM DMSO for 2 hrs. Dox-resistant cells
were also treated with 10 uM RTI-79 for 2 hrs (G3R+RTI79;
10R+RTI-79, WSUR+RTI-79). Cells were co-stained with the
cell-permeable red fluorescent ROS indicator, CellROX, and
cell-permeable green fluorescent calcium indicator, Fluo-4AM, and
then subjected to flow cytometry to quantitate changes in ROS and
calcium levels. As shown in FIG. 41C, levels of both ROS and
calcium in doxorubicin-sensitive cells were much higher than in the
resistant lines, and RTI-79 induced both ROS and calcium
mobilization in resistant cells. Thus, the ability of RTI-79 to
sensitize doxorubicin-resistant cells was closely correlated with
the inhibition of efflux pumps, induction of ROS, and mobilization
of calcium.
[0185] To determine whether increases in ROS led to calcium
mobilization or calcium mobilization resulted in ROS induction, a
time course of RTI-79 treatment of G3 cells monitoring ROS and
calcium was conducted. Cells were co-stained with the red
fluorescent ROS indicator, CellROX, and the green fluorescent
calcium indicator, Fluo-4AM for 30 minutes and treated with 10 uM
RTI-79 for 0 to 30 minutes. All samples were analyzed at the same
time in flow cytometry. As shown in FIG. 42, increases in ROS were
seen as soon as 1 minute after exposure of cells to RTI-79 and
gradually increase to 4 minute, level off to 5 minute, and then
decrease after 6 minute, followed by increases up to 15 minute. In
contrast, calcium mobilization did not occur until after 15 minutes
of RTI-79 treatment, thus indicating that ROS levels increased
first followed by calcium mobilization.
[0186] RTI-79 might inhibit MDR/P-gp by inducing ROS, which then
increase calcium mobilization that then inhibits efflux pump
activity. Alternatively, RTI-79 may first directly inhibit efflux
pump activity, which then causes a burst of ROS followed by calcium
mobilization. To determine which mechanism most likely involved,
ADR-RES (Dox-resistant) and OVCAR8 (Dox-sensitive) ovarian
carcinoma cells were transfected with siRNA to knockdown efflux
pumps to determine the effect on ROS and calcium. Cells were then
co-stained with CellROX and Fluo-4AM for 1 hour. Some cells were
treated with RTI-79 for 1 hour and controls (no RTI-79) were
treated with DMSO. As shown in FIG. 43, knockdown of P-gp in
ADR-RES cells led to increases in both ROS and calcium
mobilization, and greatly enhanced the ability of RTI-79 to
increase ROS and calcium mobilization. As expected, the effect of
downregulating efflux pump activity on ROS and calcium in OVCAR8
was much less than in ADR-RES, due to the lower efflux pump
activity in OVCAR8 cells. However, the degree of induction of ROS
and calcium mobilization by RTI-79 in P-gp knockdown cells (greater
than 90% repression of P-gp expression) is much greater than what
would be expected if the P-gp was the sole mechanism involved in
RTI-79-induced upregulation of ROS. Thus, is it likely that RTI-79
acts not only to induce ROS and calcium mobilization through
inhibition of ROS, but also acts at a second target, namely Complex
I, to induce ROS.
Example 11
Preventing or Reducing Metastasis
[0187] The effects of rifabutin on cell invasion was assessed in a
collagen invasion 3D assay. Increased interest in the use of 3D
culture systems has been motivated by accumulating evidence that 3D
models better reflect the microenvironment of tumors and metastases
and more accurately predict therapeutic response in vivo compared
with conventional 2D assays. A collagen invasion 3D assay allows
the rapid and quantitative assessment of invasiveness and a means
to screen for drugs which alter the invasive phenotype of tumor
cells. Malignant cell lines with high metastatic potential in vivo
show a higher rate of invasion than non-metastatic tumor cells and
normal cells showed little or no ability to penetrate the
barrier.
[0188] The CHOP-resistant G3 cell line is much more invasive in a
collage invasion 3D assay than its CHOP-sensitive parent cell line
(CRL2631). Collagen matrices (1 mg/ml) were prepared as previously
described in Su, S. C., et al., Molecular profile of endothelial
invasion of three-dimensional collagen matrices: insights into
angiogenic sprout induction in wound healing. Am. J. Physiol. Cell
Physiol., 295(5): C1215-29 (2008), incorporated in material part by
reference herein, with the inclusion of either DMSO control or 10
uM Rifabutin (Rif). Cells were allowed to invade for 24 hours.
Culture medium was removed and collagen gels containing invading
cells were fixed in 3% glutaraldehyde in PBS for 30 minutes. Gels
were stained with 0.1% toluidine blue in 30% methanol for 10
minutes prior to destaining with water. Cell invasion density was
quantified by counting fixed cultures under transmitted light using
an Olympus CK2 inverted microscope equipped with eyepieces
displaying a 10.times.10 ocular grid. For each condition, four
random fields were selected and the number of invading cells per
high power field (HPF) was counted manually at 10.times.
magnification, corresponding to 1 mm.sup.2 area.
[0189] Data are reported as mean number of invading cells per HPF
(.+-.S.D.) in FIG. 44. G3 cells were more invasive than CRL2631
cells. The inclusion of rifabutin in the collagen matrix reduced
the amount of G3 invasion by up to 30%. Less of this effect was
observed for CRL2631 cells.
[0190] A modified Boyden chamber assay was used as an independent
method to evaluate rifabutin's ability to suppress
invasion/metastasis. G3 and CRL2631 cells were grown in the
presence of 10 .mu.M rifabutin or dose volume equivalent DMSO for
24 hours at 37.degree. C. Cell invasion was assessed with a
Chemicon QCM Collagen Invasion Assay (Millipore). The assay is a
96-well plate assay wherein each well is equipped with a suspended
insert. Inserts contain an 8-micron membrane coated with a thin
layer of polymerized collagen. Invading cells migrate through the
collagen layer and attach to the bottom of the membrane. Cells were
detached from the membrane and lysed prior to detection via CyQuant
dye. Fluorescence intensity is proportional to number of invading
cells. As shown in FIG. 45, the presence of rifabutin resulted in
decreased relative fluorescence from 170,374 to 114,395 RLU in G3
cells. In CRL2631 cells RLU decreases from 39,356 to 27,432 RLU in
the presence of rifabutin (p<0.05).
[0191] The effect of RTI-79 treatment on the secretion of MMP2 and
VEGF was also analyzed. Treatment with RTI-79 resulted in
statistically significant decreases in both MMP2 and VEGF in U2-OS
osteosarcoma cells in commercially available ELISA based assays. In
U2-OS cells, MMP2 was reduced from 22.4 ng/million cells to 10.5
ng/million cells (p<0.01) with the addition of 5 uM RTI-79. When
evaluating the effects of RTI-79 on VEGF, a decrease from 998 to
436 pg/million cells (p<0.01) was observed.
Example 12
Rifamycin Derivative Synthesis
[0192] The 3,4-cyclo-rifamycin (rifabutin) derivatives of the
current disclosure made be prepared as shown in the schemes listed
below.
[0193] Scheme 1 illustrates the general preparation of
11-deoxo-11-imino-3,4-spiro-piperidyl-rifamycins (1c) and
11-deoxo-11-amino-3,4-spiro-piperidyl-rifamycins (1d). The
compounds of (1c) are synthesized by condensation of
3-amino-4-deoxy-4-imino-rifamycin S (1a) with a substituted
piperidone or hexanon-type of ketone (1b) at a temperature range
from 10.degree. C. to 70.degree. C. in organic solvent, such as THF
or ethanol, in the presence of an excess of ammonium salt, such as
ammonium acetate, in a sealed reaction tube. Reduction of
11-imino-rifamycin (1c) with reducing reagent, such as NaBH.sub.4,
in organic solvent, such as THF and EtOH at a temperature range
from 0.degree. C. to room temperature produces 11-amino-rifamycin
(1d). When the compound is RTI-35, the thioether could be oxidized
to sulfoxide (--SO--) or sulfone (--SO2-) depending upon the ratio
of compound 1c and oxidizing agents. When the compound is RTI-44,
product is obtained by de-protection of Boc-propected-piperidine or
Fmoc-protected-piperidine.
##STR00080## ##STR00081##
[0194] Scheme 2 illustrates the general preparation of
3,4-spiro-piperidyl-rifamycins (2c) and
11-deoxo-11-hydroxy-3,4-spiro-piperidyl-rifamycins (2d). The
compounds of (2c) are synthesized by condensation of
3-amino-4-deoxy-4-imino-rifamycin S (1a) with a substituted
piperidone or hexanon-type of ketone (1b) at a temperature range
from 10.degree. C. to 70.degree. C. in organic solvent, such as THF
or ethanol, in the presence or absence of a catalyst, such as Zinc.
Reduction of 11-oxo of rifamycin (2c) with reducing reagent, such
as NaBH.sub.4, in organic solvent, such as THF and EtOH at a
temperature range from 0.degree. C. to room temperature produce
11-hydroxy-rifamycin (2d).
##STR00082## ##STR00083##
[0195] The intermediate of (1a) is commercially available or may be
obtained from the rifamycin S. The hexanon-type of ketone or
4-substituted piperidone (1b or 2b: Z.dbd.C, or O) is either
commercially available or may be prepared by known procedures. The
4-oxo-piperidine-1-carboxamide (2b: X.dbd.NH) is prepared by
reacting 4-oxo-piperidine-1-carbonyl chloride.
[0196] Scheme 3 illustrates the general preparation of
11-deoxo-1'-hydroxyimino-3,4-spiro-piperidyl-rifamycins (3c). The
compounds of (3c) are synthesized from the reaction of
11-oxy-rifamycin compound (2c) with hydroxylamine (or its HCl salt)
at a temperature range from 10.degree. C. to 70.degree. C. in
organic solvent, such as THF or methanol, in the presence or
absence of base, such as pyridine.
##STR00084##
[0197] The above syntheses schemes are preferred schemes for the
preparation of the indicated types of compounds It is apparent to
one skilled in art that other sequences of the reactions, and
alternative reagents can be used for the synthesis of the rifamycin
derivatives of the present disclosure. These alternatives for the
synthesis of the derivatives are within the scope of this
invention.
[0198] The following examples provide synthesis schemes for
specific rifabutin derivative compositions. All starting material
used in these examples are either purchased from commercial sources
or prepared according to published procedures. Reagents were
purchased from commercial sources and used without further
purification. Reactions with moisture-sensitive reagents were
performed under a nitrogen atmosphere. Concentration of solutions
was performed by reduced pressure (in vacuum) rotary evaporation.
Column flash chromatography was performed using silica gel 60 as
stationary phase. The preparative thin-layer chromatography (TLC)
was performed using glass plates (20.times.20 cm) of silica gel (60
F254, thickness 1 mm or 2 mm).
[0199] Proton nuclear magnetic resonance (1H-NMR) spectra were
recorded on a Varian Inova 300, or 500 MHz magnetic resonance
spectrometer. 1H-NMR refers to proton nuclear magnetic resonance
spectroscopy with chemical shifts reported in ppm (parts per
million) downfield from tetramethylsilane or referred to a residue
signal of solvent (CHCl.sub.3=7.27). 13C-NMR spectra were recorded
on Varian Inova 500 MHz spectrometer operating at 125 MHz and
Chemical shifts were reported in ppm and referenced to residual
solvent signals (CHCl.sub.3=d 77.23 for carbon)
[0200] The high resolution mass spectra (HRMS) were carried out in
a Bruker-micrOTOF-QII spectrometer, using electro spray ionization
positive (ESI+) method and reported as M+H or M+Na, referring to
protonated molecular ion or its sodium complex.
[0201] The following examples are for illustration purposes and are
not intended to limit the scope of the invention. It will be
apparent to one skilled in the art that the compounds of current
invention can be prepared by a variety of synthetic routes,
including but not limited to substitution of appropriate reagents,
solvents or catalyst, change of reaction sequence, and variation of
protecting groups.
[0202] General Procedure (A) for Synthesis of Compounds (1c in
Scheme 1):
[0203] In a sealed reaction tube, a reaction mixture of
3-amino-4-imino-rifamycin S (1a) (0.1 mmol), piperidone or
hexanon-type of ketone (1b) (0.2-0.3 mmol), and ammonium acetate (1
mmol) in THF (3 ml) was stirred at 60.degree. C. overnight under
nitrogen. The reaction mixture was allowed to cool to room
temperature and diluted with DCM (20 ml) and water (20 ml). The
aqueous phase was extracted with DCM (2.times.20 ml). The combined
organic phase was washed with water (20 ml) and brine. The organic
phase was dried over anhydrous sodium sulphate, filtered and
concentrated under vacuum. The residue was purified either by
silica gel column chromatography or by silica gel preparative
thin-layer chromatography with methanol in DCM as eluent to give
the product as purple solid.
[0204] General Procedure (B) for Synthesis of Compounds (2c in
Scheme 1):
[0205] In a round bottom flask with condenser, a reaction mixture
of 3-amino-4-imino-rifamycin S (1a) (0.1 mmol), piperidone or
hexanon-type of ketone (1b) (0.2-0.3 mmol), and ammonium acetate
(0.2-0.3 mmol) in THF (8 ml) was stirred at 75.degree. C. overnight
under nitrogen. The reaction mixture was allowed to cool to room
temperature and diluted with DCM (20 ml) and water (20 ml). The
aqueous phase was extracted with DCM (2.times.20 ml). The combined
organic phase was washed with water (20 ml) and brine. The organic
phase was dried over anhydrous sodium sulphate, filtered and
concentrated under vacuum. The residue was purified either by
silica gel column chromatography or by silica gel preparative
thin-layer chromatography with methanol in DCM as eluent to give
the product as purple solid.
[0206] General Procedure (C) for Synthesis of Compounds (1d in
Scheme 1 and 2d in Scheme 2):
[0207] To a solution of rifamycin 11-imine or 11-oxo-compound (1c
or 2c) (0.1 mmol) in THF (4 ml) was added a suspension of NaBH4
(0.2 mmol) in ethanol (4 ml) at room temperature. The reaction
mixture stirred at room temperature for 1.5 hours and diluted with
ethyl acetate (20 ml) and water (20 ml). The aqueous phase was
extracted with ethyl acetate (2.times.20 ml). The combined organic
phase was washed with water and brine. The organic phase was dried
over anhydrous sodium sulphate, filtered and concentrated under
vacuum. The residue was purified either by silica gel column
chromatography or by silica gel preparative thin-layer
chromatography with methanol in DCM as eluent to give the product
as purple solid.
Preparation of RTI-33
11-deoxy-11-imino-4-deoxy-3,4[2-spiro[1-(t-butyloxycarbonyl)-piperidin-4--
yl]]-(1H)-imidazo-(2,5-dihydro)rifamycin S
[0208] Following the general procedure (A), the title compound was
obtained as a pure solid. HRMS (ESI.sup.+): 890.4570 (M+H).sup.+;
calculated for (M+H).sup.+: 890.4553; 1H-NMR (300 MHz, CDCl.sub.3)
.delta. -0.09 (d, J=7 Hz, 3H), 0.61 (d, J=7 Hz, 3H), 0.84 (d, J=7
Hz, 3H), 1.04 (d, J=7 Hz, 3H), 1.44 (m, 1H), 1.50 (s, 9H), 1.6-1.85
(m, 4H), 1.88 (s, 3H), 1.9-2.15 (m, 2H), 2.02 (s, 3H), 2.05 (s,
3H), 2.35 (s, 3H), 2.40 (m, 1H), 3.00 (m, 1H), 3.09 (s, 3H), 3.33
(m, 1H), 3.49 (s, 1H), 3.60 (d, J=5 Hz, 1H), 3.68 (d, J=10 Hz, 1H),
3.6-3.8 (br, 2H), 3.95-4.1 (br, 2H), 4.72 (d, J=10 Hz, 1H), 5.07
(dd, J=12 and 7 Hz, 1H), 6.03 (dd, J=16 and 7 Hz, 1H), 6.16 (d,
J=12 Hz, 1H), 6.28 (d, J=10 Hz, 1H), 6.40 (dd, J=16 and 10 Hz, 1H),
8.26 (s, 1H), 8.71 (bs, 1H), 12.93 (s, 1H), 14.21 (s, 1H).
Preparation of RTI-35
11-deoxy-11-imino-4-deoxy-3,4[2-spiro-tetrahydrothiopyran-4-yl]-(1H)-imid-
azo-(2,5-dihydro)rifamycin S
[0209] Following the general procedure (A), the title compound was
obtained as a pure solid. HRMS (ESI.sup.+): 807.3665 (M+H).sup.+;
calculated for (M+H).sup.+: 807.3640; RTI-035A, 1H-NMR (300 MHz,
CDCl3): -0.08 (d, J=7 Hz, 3H), 0.62 (d, J=7 Hz, 3H), 0.85 (d, J=7
Hz, 3H), 1.05 (d, J=7 Hz, 3H), 1.44 (m, 1H), 1.75-1.85 (m, 2H),
1.89 (s, 3H), 2.02 (s, 3H), 2.07 (s, 3H), 1.9-2.15 (m, 4H), 2.35
(s, 3H), 2.40 (m, 1H), 2.75-2.9 (m, 2H), 3.00 (m, 1H), 3.09 (s,
3H), 3.15-3.3 (m, 2H), 3.34 (dd, J=7 and 2 Hz, 1H), 3.47 (s, 1H),
3.60 (d, J=6 Hz, 1H), 3.68 (d, J=10 Hz, 1H), 4.72 (d, J=10 Hz, 1H),
5.07 (dd, J=12 and 8 Hz, 1H), 6.03 (dd, J=15 and 6 Hz, 1H), 6.18
(d, J=12 Hz, 1H), 6.30 (d, J=10 Hz, 1H), 6.40 (dd, J=15 and 10 Hz,
1H), 8.23 (s, 1H), 8.78 (s, 1H), 12.93 (s, 1H), 14.21 (s, 1H).
Preparation of RTI-44
11-deoxy-11-imino-4-deoxy-3,4[2-spiro[piperidin-4-yl]]-(1H)-imidazo-(2,5--
dihydro)rifamycin S
[0210] Following the general procedure (A), the title compound was
obtained as a pure solid. HRMS (ESI.sup.+): 790.4078 (M+H).sup.+;
calculated for (M+H).sup.+: 790.4029; RTI-044C, 1H-NMR (300 MHz,
CDCl3): -0.08 (d, J=7 Hz, 3H), 0.61 (d, J=7 Hz, 3H), 0.85 (d, J=7
Hz, 3H), 1.05 (d, J=7 Hz, 3H), 1.44 (m, 1H), 1.75-1.85 (m, 2H),
1.89 (s, 3H), 2.02 (s, 3H), 2.07 (s, 3H), 1.85-2.15 (m, 4H), 2.35
(s, 3H), 2.40 (m, 1H), 3.00 (m, 1H), 3.09 (s, 3H), 3.15-3.3 (m,
2H), 3.3-3.45 (m, 4H), 3.50 (s, 1H), 3.45-3.65 (br, 1H), 3.69 (d,
J=10 Hz, 1H), 4.75 (d, J=10 Hz, 1H), 5.08 (dd, J=12 and 7 Hz, 1H),
6.04 (dd, J=15 and 6 Hz, 1H), 6.18 (d, J=12 Hz, 1H), 6.30 (d, J=10
Hz, 1H), 6.42 (dd, J=15 and 10 Hz, 1H), 8.24 (s, 1H), 8.82 (s, 1H),
13.00 (s, 1H), 14.28 (s, 1H).
Preparation of RTI-46
11-deoxy-11-imino-4-deoxy-3,4[2-spiro-cyclohexyl]-(1H)-imidazo-(2,5-dihyd-
ro)rifamycin S
[0211] Following the general procedure (A), the title compound was
obtained as a pure solid. HRMS (ESI.sup.+): 789.4122 (M+H).sup.+;
calculated for (M+H).sup.+: 789.4076; RTI-046C, 1H-NMR (300 MHz,
CDCl3): -0.09 (d, J=7 Hz, 3H), 0.61 (d, J=7 Hz, 3H), 0.85 (d, J=7
Hz, 3H), 1.04 (d, J=7 Hz, 3H), 1.44 (m, 1H), 1.7-1.9 (m, 10H), 1.89
(s, 3H), 2.01 (s, 3H), 2.06 (s, 3H), 1.95-2.1 (m, 2H), 2.33 (s,
3H), 2.40 (m, 1H), 3.00 (m, 1H), 3.08 (s, 3H), 3.34 (dd, J=7 and 3
Hz, 1H), 3.45 (s, 1H), 3.62 (d, J=6 Hz, 1H), 3.68 (d, J=10 Hz, 1H),
4.75 (d, J=10 Hz, 1H), 5.08 (dd, J=12 and 7 Hz, 1H), 6.03 (dd, J=15
and 6 Hz, 1H), 6.16 (d, J=12 Hz, 1H), 6.27 (d, J=10 Hz, 1H), 6.40
(dd, J=15 and 10 Hz, 1H), 8.21 (s, 1H), 8.87 (s, 1H), 13.00 (s,
1H), 14.33 (s, 1H).
Preparation of RTI-49
11-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-(benzyl)-piperidin-4-yl]]-(1H)-i-
midazo-(2,5-dihydro)rifamycin S
[0212] Following the general procedure (A), the title compound was
obtained as a pure solid. HRMS (ESI.sup.+): 880.4535 (M+H).sup.+;
calculated for (M+H).sup.+: 880.4498; RTI-049A, 1H-NMR (300 MHz,
CDCl3): -0.09 (d, J=7 Hz, 3H), 0.60 (d, J=7 Hz, 3H), 0.84 (d, J=7
Hz, 3H), 1.04 (d, J=7 Hz, 3H), 1.44 (m, 1H), 1.65-1.85 (m, 2H),
1.91 (s, 3H), 2.01 (s, 3H), 2.07 (s, 3H), 2.35 (s, 3H), 2.40 (m,
1H), 2.47 (t, J=6 Hz, 2H), 2.76 (t, J=6 Hz, 2H), 2.8-2.95 (m, 4H),
3.00 (m, 1H), 3.09 (s, 3H), 3.33 (dd, J=7 and 2 Hz, 1H), 3.46 (s,
1H), 3.60-3.72 (m, 4H), 4.74 (d, J=10 Hz, 1H), 5.08 (dd, J=12 and 7
Hz, 1H), 6.04 (dd, J=16 and 7 Hz, 1H), 6.18 (d, J=12 Hz, 1H), 6.27
(d, J=10 Hz, 1H), 6.40 (dd, J=16 and 10 Hz, 1H), 7.3-7.45 (m, 5H),
8.22 (s, 1H), 8.80 (s, 1H), 12.99 (s, 1H), 14.31 (s, 1H).
Preparation of RTI-51
11-deoxy-11-imino-4-deoxy-3,4[2-spiro[1-(2-methoxyethyl)-piperidin-4-yl]]-
-(1H)-imidazo-(2,5-dihydro)rifamycin S
[0213] Following the general procedure (A), the title compound was
obtained as a pure solid. HRMS (ESI.sup.+): 848.4487 (M+H).sup.+;
calculated for (M+H).sup.+: 848.4447; RTI-051A, 1H-NMR (300 MHz,
CDCl3): -0.09 (d, J=7 Hz, 3H), 0.61 (d, J=7 Hz, 3H), 0.85 (d, J=7
Hz, 3H), 1.04 (d, J=7 Hz, 3H), 1.44 (m, 1H), 1.65-1.85 (m, 4H),
1.90 (s, 3H), 2.02 (s, 3H), 2.07 (s, 3H), 1.85-2.15 (br, 2H), 2.35
(s, 3H), 2.40 (m, 1H), 2.79 (t, J=5 Hz, 2H), 2.85-2.95 (m, 4H),
3.00 (m, 1H), 3.09 (s, 3H), 3.33 (dd, J=7 and 2 Hz, 1H), 3.41 (s,
3H), 3.49 (s, 1H), 3.59 (t, J=5 Hz, 2H), 3.64 (d, J=6 Hz, 1H), 3.68
(d, J=10 Hz, 1H), 4.75 (d, J=10 Hz, 1H), 5.08 (dd, J=12 and 7 Hz,
1H), 6.04 (dd, J=15 and 7 Hz, 1H), 6.16 (d, J=12 Hz, 1H), 6.27 (d,
J=10 Hz, 1H), 6.41 (dd, J=15 and 10 Hz, 1H), 8.25 (s, 1H), 8.77 (s,
1H), 12.94 (s, 1H), 14.31 (s, 1H).
Preparation of RTI-53
11-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-(2-morpholinoethyl)-piperidin-4--
yl]]-(1H)-imidazo-(2,5-dihydro)rifamycin S
[0214] Following the general procedure (A), the title compound was
obtained as a pure solid. HRMS (ESI.sup.+): 903.4904 (M+H).sup.+;
calculated for (M+H).sup.+: 903.4869; RTI-053A, 1H-NMR (300 MHz,
CDCl3): -0.09 (d, J=7 Hz, 3H), 0.61 (d, J=7 Hz, 3H), 0.85 (d, J=7
Hz, 3H), 1.04 (d, J=7 Hz, 3H), 1.44 (m, 1H), 1.65-1.85 (m, 4H),
1.90 (s, 3H), 2.02 (s, 3H), 2.07 (s, 3H), 1.85-2.15 (br, 2H), 2.34
(s, 3H), 2.40 (m, 1H), 2.5-2.65 (m, 6H), 2.74 (m, 2H), 2.85-2.95
(m, 4H), 3.00 (m, 1H), 3.09 (s, 3H), 3.33 (dd, J=7 and 2 Hz, 1H),
3.49 (s, 1H), 3.64 (d, J=6 Hz, 1H), 3.68 (d, J=10 Hz, 1H), 3.74 (t,
J=5 Hz, 4H), 4.75 (d, J=10 Hz, 1H), 5.08 (dd, J=12 and 7 Hz, 1H),
6.04 (dd, J=15 and 7 Hz, 1H), 6.16 (d, J=12 Hz, 1H), 6.28 (d, J=10
Hz, 1H), 6.40 (dd, J=15 and 10 Hz, 1H), 8.25 (s, 1H), 8.77 (s, 1H),
12.94 (s, 1H), 14.29 (s, 1H).
Preparation of RTI-57
11-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-(cyclobutylmethyl)-piperidin-4-y-
l]]-(1H)-imidazo-(2,5-dihydro)rifamycin S
[0215] Following the general procedure (A), the title compound was
obtained as a pure solid. HRMS (ESI.sup.+): 858.4690 (M+H).sup.+;
calculated for (M+H).sup.+: 858.4655; RTI-057A, 1H-NMR (300 MHz,
CDCl3): -0.09 (d, J=7 Hz, 3H), 0.61 (d, J=7 Hz, 3H), 0.85 (d, J=7
Hz, 3H), 1.04 (d, J=7 Hz, 3H), 1.44 (m, 1H), 1.7-1.85 (m, 8H), 1.90
(s, 3H), 1.9-2.15 (m, 4H), 2.02 (s, 3H), 2.07 (s, 3H), 2.35 (s,
3H), 2.40 (m, 1H), 2.60 (m, 3H), 2.7-2.9 (br, 4H), 3.00 (m, 1H),
3.09 (s, 3H), 3.34 (dd, J=7 and 2 Hz, 1H), 3.46 (s, 1H), 3.63 (d,
J=6 Hz, 1H), 3.68 (d, J=10 Hz, 1H), 4.75 (d, J=10 Hz, 1H), 5.08
(dd, J=12 and 7 Hz, 1H), 6.03 (dd, J=16 and 7 Hz, 1H), 6.17 (d,
J=12 Hz, 1H), 6.28 (d, J=10 Hz, 1H), 6.40 (dd, J=16 and 10 Hz, 1H),
8.22 (s, 1H), 8.80 (s, 1H), 12.95 (s, 1H), 14.31 (s, 1H).
Preparation of RTI-59
11-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-(cyclopropylmethyl)-piperidin-4--
yl]]-(1H)-imidazo-(2,5-dihydro)rifamycin S
[0216] Following the general procedure (A), the title compound was
obtained as a pure solid. HRMS (ESI.sup.+): 844.4536 (M+H).sup.+;
calculated for (M+H).sup.+: 844.4498; RTI-059A, 1H-NMR (300 MHz,
CDCl3): -0.09 (d, J=7 Hz, 3H), 0.18 (m, 2H), 0.57 (m, 2H), 0.61 (d,
J=7 Hz, 3H), 0.84 (d, J=7 Hz, 3H), 0.93 (m, 1H), 1.04 (d, J=7 Hz,
3H), 1.44 (m, 1H), 1.7-1.85 (m, 4H), 1.90 (s, 3H), 1.95-2.15 (br,
2H), 2.02 (s, 3H), 2.07 (s, 3H), 2.35 (s, 3H), 2.40 (m, 1H), 2.46
(d, J=7 Hz, 2H), 2.8-3.05 (m, 5H), 3.09 (s, 3H), 3.35 (dd, J=7 and
2 Hz, 1H), 3.49 (s, 1H), 3.63 (d, J=6 Hz, 1H), 3.68 (d, J=10 Hz,
1H), 4.74 (d, J=10 Hz, 1H), 5.07 (dd, J=12 and 7 Hz, 1H), 6.03 (dd,
J=16 and 7 Hz, 1H), 6.17 (d, J=12 Hz, 1H), 6.28 (d, J=10 Hz, 1H),
6.40 (dd, J=16 and 10 Hz, 1H), 8.25 (s, 1H), 8.78 (s, 1H), 12.93
(s, 1H), 14.31 (s, 1H).
Preparation of RTI-60
11-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-(isopropyl)-piperidin-4-yl]]-(1H-
)-imidazo-(2,5-dihydro)rifamycin S
[0217] Following the general procedure (A), the title compound was
obtained as a pure solid. HRMS (ESI.sup.+): 832.4542 (M+H).sup.+;
calculated for (M+H).sup.+: 832.4498; RTI-060A, 1H-NMR (300 MHz,
CDCl3): -0.09 (d, J=7 Hz, 3H), 0.60 (d, J=7 Hz, 3H), 0.84 (d, J=7
Hz, 3H), 1.04 (d, J=7 Hz, 3H), 1.16 (d, J=6 Hz, 6H), 1.44 (m, 1H),
1.7-1.8 (m, 4H), 1.88 (s, 3H), 1.95-2.15 (br, 2H), 2.01 (s, 3H),
2.05 (s, 3H), 2.33 (s, 3H), 2.40 (m, 1H), 2.75-3.05 (m, 6H), 3.08
(s, 3H), 3.34 (dd, J=7 and 2 Hz, 1H), 3.47 (s, 1H), 3.64 (d, J=6
Hz, 1H), 3.68 (d, J=10 Hz, 1H), 4.75 (d, J=10 Hz, 1H), 5.07 (dd,
J=12 and 7 Hz, 1H), 6.03 (dd, J=16 and 7 Hz, 1H), 6.16 (d, J=12 Hz,
1H), 6.27 (d, J=10 Hz, 1H), 6.40 (dd, J=16 and 10 Hz, 1H), 8.22 (s,
1H), 8.76 (s, 1H), 12.91 (s, 1H), 14.31 (s, 1H).
Preparation of RTI-61
11-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-(t-ethyloxycarbonyl)-piperidin-4-
-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycin S
[0218] Following the general procedure (A), the title compound was
obtained as a pure solid. HRMS (ESI.sup.+): 862.4270 (M+H).sup.+;
calculated for (M+H).sup.+: 862.4240; RTI-61A, 1H-NMR (300 MHz,
CDCl3): -0.08 (d, J=7 Hz, 3H), 0.62 (d, J=7 Hz, 3H), 0.84 (d, J=7
Hz, 3H), 1.04 (d, J=7 Hz, 3H), 1.30 (t, J=7 Hz, 3H), 1.44 (m, 1H),
1.6-1.85 (m, 4H), 1.89 (s, 3H), 2.0-2.15 (m, 2H), 2.02 (s, 3H),
2.06 (s, 3H), 2.35 (s, 3H), 2.40 (m, 1H), 3.00 (m, 1H), 3.09 (s,
3H), 3.33 (m, 1H), 3.50 (s, 1H), 3.61 (d, J=5 Hz, 1H), 3.68 (d,
J=10 Hz, 1H), 3.6-3.8 (br, 2H), 4.0-4.2 (br, 2H), 4.21 (q, J=7 Hz,
2H), 4.72 (d, J=10 Hz, 1H), 5.07 (dd, J=12 and 7 Hz, 1H), 6.03 (dd,
J=16 and 7 Hz, 1H), 6.17 (d, J=12 Hz, 1H), 6.29 (d, J=10 Hz, 1H),
6.41 (dd, J=16 and 10 Hz, 1H), 8.26 (s, 1H), 8.72 (bs, 1H), 12.93
(s, 1H), 14.21 (s, 1H).
Preparation of RTI-63
11-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-(acetyl)-piperidin-4-yl]]-(1H)-i-
midazo-(2,5-dihydro)rifamycin S
[0219] Following the general procedure (A), the title compound was
obtained as a pure solid. HRMS (ESI.sup.+): 832.4181 (M+H).sup.+;
calculated for (M+H).sup.+: 832.4134. RTI-63A, 1H-NMR (300 MHz,
CDCl3): -0.06 (d, J=7 Hz, 3H), 0.62 (d, J=7 Hz, 3H), 0.85 (d, J=7
Hz, 3H), 1.04 (d, J=7 Hz, 3H), 1.45 (m, 1H), 1.6-1.85 (m, 4H), 1.89
(s, 3H), 2.03 (s, 3H), 2.06 (s, 3H), 2.0-2.2 (m, 2H), 2.20 (s, 3H),
2.35 (s, 3H), 2.40 (m, 1H), 3.00 (m, 1H), 3.10 (s, 3H), 3.33 (m,
1H), 3.47 (s, 0.4H), 3.51 (s, 0.6H), 3.55-3.70 (m, 3H), 3.90 (m,
2H), 4.48 (m, 1H), 4.73 (m, 1H), 5.07 (m, 1H), 6.03 (dd, J=16 and 6
Hz, 1H), 6.18 (d, J=12 Hz, 1H), 6.29 (d, J=10 Hz, 1H), 6.38 (m,
1H), 8.25 (s, 1H), 8.66 (s, 0.6H), 8.71 (s, 0.4H), 12.92 (s, 1H),
14.16 (s, 0.4H), 14.19 (s, 0.6H).
Preparation of RTI-64
11-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-(n-propyl)-piperidin-4-yl]]-(1H)-
-imidazo-(2,5-dihydro)rifamycin S
[0220] Following the general procedure (A), the title compound was
obtained as a pure solid. HRMS (ESI.sup.+): 832.4552 (M+H).sup.+;
calculated for (M+H).sup.+: 832.4498; RTI-064A, 1H-NMR (300 MHz,
CDCl3): -0.09 (d, J=7 Hz, 3H), 0.61 (d, J=7 Hz, 3H), 0.85 (d, J=7
Hz, 3H), 0.96 (t, J=7 Hz, 3H), 1.04 (d, J=7 Hz, 3H), 1.44 (m, 1H),
1.55-1.65 (m, 2H), 1.7-1.85 (m, 4H), 1.90 (s, 3H), 1.95-2.15 (br,
2H), 2.02 (s, 3H), 2.07 (s, 3H), 2.35 (s, 3H), 2.40 (m, 1H), 2.54
(m, 2H), 2.8-2.9 (m, 4H), 3.00 (m, 1H), 3.09 (s, 3H), 3.35 (dd, J=7
and 2 Hz, 1H), 3.46 (s, 1H), 3.62 (d, J=6 Hz, 1H), 3.67 (d, J=10
Hz, 1H), 4.75 (d, J=10 Hz, 1H), 5.07 (dd, J=12 and 7 Hz, 1H), 6.03
(dd, J=16 and 7 Hz, 1H), 6.17 (d, J=12 Hz, 1H), 6.27 (d, J=10 Hz,
1H), 6.40 (dd, J=16 and 10 Hz, 1H), 8.21 (s, 1H), 8.78 (s, 1H),
12.95 (s, 1H), 14.30 (s, 1H).
Preparation of RTI-65
11-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-(cyclopropyl)-piperidin-4-yl]]-(-
1H)-imidazo-(2,5-dihydro)rifamycin S
[0221] Following the general procedure (A), the title compound was
obtained as a pure solid. HRMS (ESI.sup.+): 830.4386 (M+H).sup.+;
calculated for (M+H).sup.+: 830.4342; RTI-065A, 1H-NMR (300 MHz,
CDCl3): -0.09 (d, J=7 Hz, 3H), 0.45-0.55 (m, 5H), 0.61 (d, J=7 Hz,
3H), 0.85 (d, J=7 Hz, 3H), 1.04 (d, J=7 Hz, 3H), 1.44 (m, 1H),
1.7-1.85 (m, 4H), 1.90 (s, 3H), 1.95-2.15 (br, 2H), 2.02 (s, 3H),
2.07 (s, 3H), 2.35 (s, 3H), 2.40 (m, 1H), 2.9-3.1 (m, 5H), 3.09 (s,
3H), 3.35 (dd, J=7 and 2 Hz, 1H), 3.46 (s, 1H), 3.63 (d, J=6 Hz,
1H), 3.67 (d, J=10 Hz, 1H), 4.75 (d, J=10 Hz, 1H), 5.08 (dd, J=12
and 7 Hz, 1H), 6.04 (dd, J=16 and 7 Hz, 1H), 6.17 (d, J=12 Hz, 1H),
6.27 (d, J=10 Hz, 1H), 6.40 (dd, J=16 and 10 Hz, 1H), 8.21 (s, 1H),
8.79 (s, 1H), 12.97 (s, 1H), 14.30 (s, 1H).
Preparation of RTI-66
11-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-(ethyl)-piperidin-4-yl]]-(1H)-im-
idazo-(2,5-dihydro)rifamycin S
[0222] Following the general procedure (A), the title compound was
obtained as a pure solid. HRMS (ESI.sup.+): 818.4388 (M+H).sup.+;
calculated for (M+H).sup.+: 818.4342; RTI-066A, 1H-NMR (300 MHz,
CDCl3): -0.08 (d, J=7 Hz, 3H), 0.61 (d, J=7 Hz, 3H), 0.85 (d, J=7
Hz, 3H), 1.04 (d, J=7 Hz, 3H), 1.18 (t, J=7 Hz, 3H), 1.44 (m, 1H),
1.7-1.85 (m, 4H), 1.90 (s, 3H), 1.95-2.15 (br, 2H), 2.02 (s, 3H),
2.07 (s, 3H), 2.35 (s, 3H), 2.40 (m, 1H), 2.64 (q, J=7 Hz, 2H),
2.8-2.95 (m, 4H), 3.00 (m, 1H), 3.09 (s, 3H), 3.35 (d, J=7 Hz, 1H),
3.46 (s, 1H), 3.63 (d, J=6 Hz, 1H), 3.67 (d, J=10 Hz, 1H), 4.75 (d,
J=10 Hz, 1H), 5.08 (dd, J=12 and 7 Hz, 1H), 6.04 (dd, J=16 and 7
Hz, 1H), 6.16 (d, J=12 Hz, 1H), 6.27 (d, J=10 Hz, 1H), 6.40 (dd,
J=16 and 10 Hz, 1H), 8.22 (s, 1H), 8.77 (s, 1H), 12.95 (s, 1H),
14.29 (s, 1H).
Preparation of RTI-67
11-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-(beRTIoyl)-piperidin-4-yl]]-(1H)-
-imidazo-(2,5-dihydro)rifamycin S
[0223] Following the general procedure (A), the title compound was
obtained as a pure solid. HRMS (ESI.sup.+): 916.4169 (M+Na).sup.+;
calculated for (M+Na).sup.+: 916.4109. RTI-67A, 1H-NMR (300 MHz,
CDCl3): -0.07 (br, 3H), 0.60 (br, 3H), 0.84 (br, 3H), 1.02 (d, J=7
Hz, 3H), 1.45 (m, 1H), 1.6-1.85 (m, 4H), 1.88 (s, 3H), 2.00 (s,
3H), 2.04 (s, 3H), 1.9-2.2 (m, 2H), 2.34 (s, 3H), 2.40 (m, 1H),
3.00 (m, 1H), 3.08 (s, 3H), 3.2-3.9 (br, 7H), 4.2 (br, 1H), 4.6
(br, 1H), 5.05 (br, 1H), 6.0 (br, 1H), 6.18 (br, 1H), 6.29 (br,
1H), 6.40 (br, 1H), 7.40 (m, 2H), 7.45 (m, 3H), 8.25 (s, 1H), 8.6
(brs, 1H), 12.93 (s, 1H), 14.16 (s, 1H).
Preparation of RTI-68
11-deoxy-11-imino-4-deoxy-3,4[2-spiro[1-(benzyloxycarbonyl)-piperidin-4-y-
l]]-(1H)-imidazo-(2,5-dihydro)rifamycin S
[0224] Following the general procedure (A), the title compound was
obtained as a pure solid. HRMS (ESI.sup.+): 924.4435 (M+H).sup.+;
calculated for (M+H).sup.+: 924.4396; RTI-68A, 1H-NMR (300 MHz,
CDCl3): -0.09 (d, J=7 Hz, 3H), 0.60 (d, J=7 Hz, 3H), 0.84 (d, J=7
Hz, 3H), 1.04 (d, J=7 Hz, 3H), 1.44 (m, 1H), 1.6-1.85 (m, 4H), 1.88
(s, 3H), 2.0-2.15 (m, 2H), 2.02 (s, 3H), 2.05 (s, 3H), 2.35 (s,
3H), 2.40 (m, 1H), 3.00 (m, 1H), 3.09 (s, 3H), 3.33 (br, 1H), 3.49
(s, 1H), 3.60 (d, J=5 Hz, 1H), 3.68 (d, J=10 Hz, 1H), 3.6-3.8 (br,
2H), 4.0-4.2 (m, 2H), 4.72 (d, J=10 Hz, 1H), 5.07 (dd, J=12 and 7
Hz, 1H), 5.20 (s, 2H), 6.03 (dd, J=16 and 7 Hz, 1H), 6.17 (d, J=12
Hz, 1H), 6.29 (d, J=10 Hz, 1H), 6.41 (dd, J=16 and 10 Hz, 1H), 7.38
(m, 5H), 8.26 (s, 1H), 8.70 (bs, 1H), 12.92 (s, 1H), 14.20 (s,
1H).
Preparation of RTI-69
11-deoxy-11-imino-4-deoxy-3,4[2-spiro[1-(methyl)-piperidin-4-yl]]-(1H)-im-
idazo-(2,5-dihydro)rifamycin S
[0225] Following the general procedure (A), the title compound was
obtained as a pure solid. HRMS (ESI.sup.+): 804.4213 (M+H).sup.+;
calculated for (M+H).sup.+: 804.4185; RTI-069A, 1H-NMR (300 MHz,
CDCl3): -0.08 (d, J=7 Hz, 3H), 0.61 (d, J=7 Hz, 3H), 0.85 (d, J=7
Hz, 3H), 1.04 (d, J=7 Hz, 3H), 1.45 (m, 1H), 1.7-1.85 (m, 4H), 1.90
(s, 3H), 1.95-2.15 (br, 2H), 2.02 (s, 3H), 2.07 (s, 3H), 2.35 (s,
3H), 2.40 (m, 1H), 2.49 (s, 3H), 2.7-2.95 (m, 4H), 3.00 (m, 1H),
3.09 (s, 3H), 3.34 (d, J=7 Hz, 1H), 3.48 (s, 1H), 3.63 (d, J=6 Hz,
1H), 3.68 (d, J=10 Hz, 1H), 4.75 (d, J=10 Hz, 1H), 5.08 (dd, J=12
and 7 Hz, 1H), 6.04 (dd, J=16 and 7 Hz, 1H), 6.17 (d, J=12 Hz, 1H),
6.27 (d, J=10 Hz, 1H), 6.40 (dd, J=16 and 10 Hz, 1H), 8.23 (s, 1H),
8.77 (s, 1H), 12.95 (s, 1H), 14.29 (s, 1H).
Preparation of RTI-70
11-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-(2-methylpropyl)-piperidin-4-yl]-
]-(1H)-imidazo-(2,5-dihydro)rifamycin S
[0226] Following the general procedure (A), the title compound was
obtained as a pure solid. HRMS (ESI.sup.+): 846.4682 (M+H).sup.+;
calculated for (M+H).sup.+: 846.4655; RTI-070A, 1H-NMR (500 MHz,
CDCl3): -0.09 (d, J=7 Hz, 3H), 0.60 (d, J=7 Hz, 3H), 0.84 (d, J=7
Hz, 3H), 0.94 (d, J=7 Hz, 6H), 1.04 (d, J=7 Hz, 3H), 1.44 (m, 1H),
1.74-1.85 (m, 3H), 1.89 (s, 3H), 1.9-2.15 (m, 4H), 2.01 (s, 3H),
2.05 (s, 3H), 2.29 (d, J=7 Hz, 2H), 2.33 (s, 3H), 2.40 (m, 1H),
2.75-2.85 (m, 4H), 3.00 (m, 1H), 3.08 (s, 3H), 3.33 (dd, J=7 and 2
Hz, 1H), 3.46 (s, 1H), 3.63 (d, J=6 Hz, 1H), 3.68 (d, J=10 Hz, 1H),
4.75 (dd, J=10 and 2 Hz, 1H), 5.07 (dd, J=12 and 7 Hz, 1H), 6.03
(dd, J=16 and 7 Hz, 1H), 6.16 (d, J=12 Hz, 1H), 6.27 (d, J=10 Hz,
1H), 6.40 (dd, J=16 and 10 Hz, 1H), 8.23 (s, 1H), 8.78 (s, 1H),
12.96 (s, 1H), 14.30 (s, 1H). .sup.13C-NMR (125 MHz, CDCl3).
Preparation of RTI-74
11-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-(phenylaminocarbonyl)-piperidin--
4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycin S
[0227] Following the general procedure (A), the title compound was
obtained as a pure solid. HRMS (ESI.sup.+): 909.4433 (M+H).sup.+;
calculated for (M+H).sup.+: 909.4400; RTI-074A, 1H-NMR (300 MHz,
CDCl3): -0.07 (d, J=7 Hz, 3H), 0.62 (d, J=7 Hz, 3H), 0.84 (d, J=7
Hz, 3H), 1.04 (d, J=7 Hz, 3H), 1.44 (m, 1H), 1.6-1.85 (m, 3H), 1.89
(s, 3H), 1.9-2.25 (m, 3H), 2.02 (s, 3H), 2.05 (s, 3H), 2.35 (s,
3H), 2.40 (m, 1H), 3.00 (m, 1H), 3.09 (s, 3H), 3.33 (m, 1H), 3.51
(s, 1H), 3.61 (d, J=6 Hz, 1H), 3.68 (d, J=10 Hz, 1H), 3.6-3.8 (br,
2H), 4.0-4.2 (br, 2H), 4.72 (d, J=10 Hz, 1H), 5.07 (dd, J=12 and 7
Hz, 1H), 6.03 (dd, J=16 and 7 Hz, 1H), 6.16 (d, J=12 Hz, 1H), 6.28
(d, J=10 Hz, 1H), 6.40 (m, 2H), 7.15 (m, 1H), 7.34 (m, 4H), 8.27
(s, 1H), 8.69 (s, 1H), 12.92 (s, 1H), 14.19 (s, 1H).
Preparation of RTI-77
11-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-(ethyloxycarbonyl)-piperidin-4-y-
l]]-(1H)-imidazo-(2,5-dihydro)rifamycin S
[0228] Following the general procedure (A), the title compound was
obtained as a pure solid. HRMS (ESI.sup.+): 876.4417 (M+H).sup.+;
calculated for (M+H).sup.+: 876.4396; RTI-77A, 1H-NMR (300 MHz,
CDCl3): -0.08 (d, J=7 Hz, 3H), 0.61 (d, J=7 Hz, 3H), 0.84 (d, J=7
Hz, 3H), 1.04 (d, J=7 Hz, 3H), 0.99 (t, J=7 Hz, 3H), 1.44 (m, 1H),
1.6-1.85 (m, 6H), 1.88 (s, 3H), 2.0-2.15 (m, 2H), 2.02 (s, 3H),
2.05 (s, 3H), 2.35 (s, 3H), 2.40 (m, 1H), 3.00 (m, 1H), 3.09 (s,
3H), 3.33 (m, 1H), 3.49 (s, 1H), 3.60 (d, J=5 Hz, 1H), 3.68 (d,
J=10 Hz, 1H), 3.6-3.8 (br, 2H), 4.0-4.2 (m, 4H), 4.72 (d, J=10 Hz,
1H), 5.07 (dd, J=12 and 7 Hz, 1H), 6.03 (dd, J=16 and 7 Hz, 1H),
6.17 (d, J=12 Hz, 1H), 6.28 (d, J=10 Hz, 1H), 6.41 (dd, J=16 and 10
Hz, 1H), 8.25 (s, 1H), 8.7 (bs, 1H), 12.93 (s, 1H), 14.20 (s,
1H).
Preparation of RTI-81
11-deoxy-11-imino-4-deoxy-3,4[2-spiro[1-(isobutyloxycarbonyl)-piperidin-4-
-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycin S
[0229] Following the general procedure (A), the title compound was
obtained as a pure solid. HRMS (ESI.sup.+): 890.4552 (M+H).sup.+;
calculated for (M+H).sup.+: 890.4553; RTI-081, 1H-NMR (300 MHz,
CDCl3): -0.09 (d, J=7 Hz, 3H), 0.61 (d, J=7 Hz, 3H), 0.84 (d, J=7
Hz, 3H), 0.98 (d, J=7 Hz, 6H), 1.04 (d, J=7 Hz, 3H), 1.44 (m, 1H),
1.6-1.85 (m, 4H), 1.88 (s, 3H), 1.9-2.15 (m, 3H), 2.01 (s, 3H),
2.05 (s, 3H), 2.35 (s, 3H), 2.40 (m, 1H), 3.00 (m, 1H), 3.09 (s,
3H), 3.33 (m, 1H), 3.49 (s, 1H), 3.60 (d, J=6 Hz, 1H), 3.68 (d,
J=10 Hz, 1H), 3.6-3.8 (br, 2H), 3.95 (m, 2H), 4.0-4.2 (br, 2H),
4.72 (d, J=10 Hz, 1H), 5.07 (dd, J=12 and 7 Hz, 1H), 6.03 (dd, J=16
and 7 Hz, 1H), 6.16 (d, J=12 Hz, 1H), 6.27 (d, J=10 Hz, 1H), 6.40
(dd, J=16 and 10 Hz, 1H), 8.25 (s, 1H), 8.7 (bs, 1H), 12.93 (s,
1H), 14.20 (s, 1H).
Preparation of RTI-82
11-deoxy-11-imino-4-deoxy-3,4[2-spiro[1-(ethylaminocarbonyl)-piperidin-4--
yl]]-(1H)-imidazo-(2,5-dihydro)rifamycin S
[0230] Following the general procedure (A), the title compound was
obtained as a pure solid. HRMS (ESI.sup.+): 883.4175 (M+Na).sup.+;
calculated for (M+Na).sup.+: 883.4218; RTI-082A, 1H-NMR (300 MHz,
CDCl3): -0.08 (d, J=7 Hz, 3H), 0.61 (d, J=7 Hz, 3H), 0.84 (d, J=7
Hz, 3H), 1.04 (d, J=7 Hz, 3H), 1.20 (t, J=7 Hz, 3H), 1.44 (m, 1H),
1.6-1.85 (m, 3H), 1.88 (s, 3H), 1.9-2.25 (m, 3H), 2.02 (s, 3H),
2.05 (s, 3H), 2.35 (s, 3H), 2.40 (m, 1H), 3.00 (m, 1H), 3.09 (s,
3H), 3.3-3.4 (m, 3H), 3.50 (s, 1H), 3.61 (d, J=6 Hz, 1H), 3.68 (d,
J=10 Hz, 1H), 3.6-3.7 (br, 2H), 3.8-4.0 (br, 2H), 4.52 (m, 1H),
4.72 (d, J=10 Hz, 1H), 5.07 (dd, J=12 and 7 Hz, 1H), 6.03 (dd, J=16
and 7 Hz, 1H), 6.16 (d, J=12 Hz, 1H), 6.28 (d, J=10 Hz, 1H), 6.40
(m, 1H), 8.25 (s, 1H), 8.69 (s, 1H), 12.92 (s, 1H), 14.20 (s,
1H).
Preparation of RTI-83
4-deoxy-3,4[2-spiro[1-(ethylaminocarbonyl)-piperidin-4-yl]]-(1H)-imidazo--
(2,5-dihydro)rifamycin S
[0231] Following the general procedure (B), the title compound was
obtained as a pure solid. HRMS (ESI.sup.+): 884.4048 (M+Na).sup.+;
calculated for (M+Na).sup.+: 884.4058; RTI-083A, 1H-NMR (300 MHz,
CDCl3): -0.04 (d, J=7 Hz, 3H), 0.61 (d, J=7 Hz, 3H), 0.84 (d, J=7
Hz, 3H), 1.04 (d, J=7 Hz, 3H), 1.20 (t, J=7 Hz, 3H), 1.4-1.6 (m,
2H), 1.65-1.85 (m, 3H), 1.74 (s, 3H), 1.95-2.2 (m, 2H), 2.02 (s,
3H), 2.04 (s, 3H), 2.35 (s, 3H), 2.40 (m, 1H), 3.00 (m, 1H), 3.09
(s, 3H), 3.3-3.4 (m, 3H), 3.43 (s, 1H), 3.56 (d, J=6 Hz, 1H), 3.68
(d, J=10 Hz, 1H), 3.7-4.0 (m, 4H), 4.50 (m, 1H), 4.72 (d, J=10 Hz,
1H), 5.13 (dd, J=12 and 7 Hz, 1H), 6.03 (dd, J=16 and 7 Hz, 1H),
6.18 (d, J=12 Hz, 1H), 6.27 (d, J=10 Hz, 1H), 6.38 (m, 1H), 8.18
(s, 1H), 8.90 (s, 1H), 14.57 (s, 1H).
Preparation of RTI-84
11-deoxy-11-imino-4-deoxy-3,4[2-spiro[1-(isopropyloxycarbonyl)-piperidin--
4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycin S
[0232] Following the general procedure (A), the title compound was
obtained as a pure solid. HRMS (ESI.sup.+): 898.4203 (M+Na).sup.+;
calculated for (M+Na).sup.+: 898.4215; RTI-084A, 1H-NMR (300 MHz,
CDCl3): -0.09 (d, J=7 Hz, 3H), 0.61 (d, J=7 Hz, 3H), 0.84 (d, J=7
Hz, 3H), 1.04 (d, J=7 Hz, 3H), 1.30 (d, J=6 Hz, 6H), 1.44 (m, 1H),
1.6-1.85 (m, 4H), 1.88 (s, 3H), 1.9-2.15 (m, 2H), 2.02 (s, 3H),
2.05 (s, 3H), 2.35 (s, 3H), 2.40 (m, 1H), 3.00 (m, 1H), 3.09 (s,
3H), 3.33 (m, 1H), 3.50 (s, 1H), 3.61 (d, J=6 Hz, 1H), 3.68 (d,
J=10 Hz, 1H), 3.6-3.8 (br, 2H), 4.0-4.2 (br, 2H), 4.72 (d, J=10 Hz,
1H), 4.99 (m, 1H), 5.07 (dd, J=12 and 7 Hz, 1H), 6.03 (dd, J=16 and
7 Hz, 1H), 6.16 (d, J=12 Hz, 1H), 6.28 (d, J=10 Hz, 1H), 6.40 (dd,
J=16 and 10 Hz, 1H), 8.27 (s, 1H), 8.7 (bs, 1H), 12.93 (s, 1H),
14.21 (s, 1H).
Preparation of RTI-86
4-deoxy-3,4[2-spiro-[1-(phenylaminocarbonyl)-piperidin-4-yl]]-(1H)-imidaz-
o-(2,5-dihydro)rifamycin S
[0233] Following the general procedure (B), the title compound was
obtained as a pure solid. HRMS (ESI.sup.+): 932.4038 (M+Na).sup.+;
calculated for (M+Na).sup.+: 932.4058; RTI-086A, 1H-NMR (300 MHz,
CDCl3): -0.02 (d, J=7 Hz, 3H), 0.62 (d, J=7 Hz, 3H), 0.84 (d, J=7
Hz, 3H), 1.04 (d, J=7 Hz, 3H), 1.4-1.6 (m, 2H), 1.65-1.85 (m, 3H),
1.75 (s, 3H), 1.95-2.2 (m, 3H), 2.02 (s, 3H), 2.05 (s, 3H), 2.35
(s, 3H), 3.00 (m, 1H), 3.09 (s, 3H), 3.3 (m, 1H), 3.45 (s, 1H),
3.58 (d, J=6 Hz, 1H), 3.67 (d, J=10 Hz, 1H), 3.8-4.2 (m, 4H), 4.72
(d, J=10 Hz, 1H), 5.13 (dd, J=12 and 7 Hz, 1H), 6.03 (dd, J=16 and
7 Hz, 1H), 6.18 (d, J=12 Hz, 1H), 6.27 (d, J=10 Hz, 1H), 6.38 (m,
1H), 6.44 (s, 1H), 7.10 (m, 1H), 7.37 (m, 4H), 8.21 (s, 1H), 8.88
(s, 1H), 14.56 (s, 1H).
Preparation of RTI-91
11-deoxy-11-imino-4-deoxy-3,4[2-spiro-1-(3,3-dimethylbutanoyl)-piperidin--
4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycin S
[0234] Following the general procedure (A), the title compound was
obtained as a pure solid. HRMS (ESI.sup.+): 910.4589 (M+Na).sup.+;
calculated for (M+Na).sup.+: 910.4579; RTI-91A, 1H-NMR (300 MHz,
CDCl3): -0.07 (d, J=7 Hz, 3H), 0.61 (d, J=7 Hz, 3H), 0.85 (d, J=7
Hz, 3H), 1.05 (m, 3H), 1.10 (s, 9H), 1.45 (m, 1H), 1.6-1.85 (m,
4H), 1.88 (s, 3H), 2.02 (s, 3H), 2.05 (s, 3H), 2.0-2.2 (m, 2H),
2.35 (s, 3H), 2.3-2.45 (m, 3H), 3.00 (m, 1H), 3.09 (s, 3H), 3.33
(m, 1H), 3.47 (s, 0.4H), 3.52 (s, 0.6H), 3.55-3.70 (m, 3H), 3.8-4.0
(m, 2H), 4.5 (m, 1H), 4.75 (m, 1H), 5.06 (m, 1H), 6.0 (m, 1H), 6.17
(m, 1H), 6.29 (d, J=10 Hz, 1H), 6.4 (m, 1H), 8.27 (s, 1H), 8.63 (s,
0.6H), 8.71 (s, 0.4H), 12.92 (s, 1H), 14.16 (s, 0.4H), 14.20 (s,
0.6H).
Preparation of RTI-94
11-deoxy-11-imino-4-deoxy-3,4[2-spiro[1-(n-pentanoyl)-piperidin-4-yl]]-(1-
H)-imidazo-(2,5-dihydro)rifamycin S
[0235] Following the general procedure (A), the title compound was
obtained as a pure solid. HRMS (ESI.sup.+): 874.4644 (M+H).sup.+;
calculated for (M+H).sup.+: 874.4604; RTI-94A, 1H-NMR (300 MHz,
CDCl3): -0.07 (d, J=7 Hz, 3H), 0.61 (d, J=7 Hz, 3H), 0.84 (d, J=7
Hz, 3H), 0.97 (t, J=7 Hz, 3H), 1.04 (m, 3H), 1.42 (m, 3H), 1.6-1.85
(m, 6H), 1.88 (s, 3H), 2.02 (s, 3H), 2.05 (s, 3H), 1.9-2.2 (m, 2H),
2.35 (s, 3H), 2.3-2.45 (m, 3H), 3.00 (m, 1H), 3.09 (s, 3H), 3.33
(m, 1H), 3.49 (s, 0.4H), 3.53 (s, 0.6H), 3.55-3.70 (m, 3H), 3.8-4.0
(m, 2H), 4.5 (m, 1H), 4.72 (m, 1H), 5.06 (m, 1H), 6.0 (m, 1H), 6.17
(m, 1H), 6.29 (d, J=10 Hz, 1H), 6.4 (m, 1H), 8.29 (s, 1H), 8.63 (s,
0.6H), 8.70 (s, 0.4H), 12.92 (s, 1H), 14.17 (s, 0.4H), 14.20 (s,
0.6H).
Preparation of RTI-97
11-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-(2-methylpropanoyl)-piperidin-4--
yl]]-(1H)-imidazo-(2,5-dihydro)rifamycin S
[0236] Following the general procedure (A), the title compound was
obtained as a pure solid. HRMS (ESI.sup.+): 860.4482 (M+H).sup.+;
calculated for (M+H).sup.+: 860.4447. RTI-97A, 1H-NMR (300 MHz,
CDCl3): -0.07 (d, J=7 Hz, 3H), 0.61 (d, J=7 Hz, 3H), 0.84 (d, J=7
Hz, 3H), 1.04 (m, 3H), 1.20 (d, J=7 Hz, 6H), 1.43 (m, 1H), 1.6-1.85
(m, 4H), 1.88 (s, 3H), 2.02 (s, 3H), 2.05 (s, 3H), 2.0-2.2 (m, 2H),
2.35 (s, 3H), 2.40 (m, 1H), 2.89 (m, 1H), 3.01 (m, 1H), 3.09 (s,
3H), 3.33 (m, 1H), 3.47 (s, 0.4H), 3.50 (s, 0.6H), 3.55-3.70 (m,
3H), 3.8-4.1 (m, 2H), 4.5 (m, 1H), 4.72 (m, 1H), 5.06 (m, 1H), 6.01
(dd, J=15 and 6 Hz, 1H), 6.18 (d, J=12 Hz, 1H), 6.29 (d, J=10 Hz,
1H), 6.39 (m, 1H), 8.25 (s, 1H), 8.67 (s, 0.6H), 8.70 (s, 0.4H),
12.93 (s, 1H), 14.16 (s, 0.4H), 14.19 (s, 0.6H).
Preparation of RTI-98
11-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-(3-methylbutanoyl)-piperidin-4-y-
l]]-(1H)-imidazo-(2,5-dihydro)rifamycin S
[0237] Following the general procedure (A), the title compound was
obtained as a pure solid. HRMS (ESI.sup.+): 874.4632 (M+H).sup.+;
calculated for (M+H).sup.+: 874.4604.RTI-98A, 1H-NMR (300 MHz,
CDCl3): -0.07 (d, J=7 Hz, 3H), 0.61 (d, J=7 Hz, 3H), 0.84 (d, J=7
Hz, 3H), 1.04 (m, 3H), 1.02 (d, J=7 Hz, 6H), 1.43 (m, 1H), 1.6-1.85
(m, 4H), 1.88 (s, 3H), 2.02 (s, 3H), 2.05 (s, 3H), 2.0-2.2 (m, 3H),
2.30 (m, 2H), 2.35 (s, 3H), 2.40 (m, 1H), 3.00 (m, 1H), 3.09 (s,
3H), 3.33 (m, 1H), 3.47 (s, 0.4H), 3.50 (s, 0.6H), 3.55-3.70 (m,
3H), 3.8-4.0 (m, 2H), 4.5 (m, 1H), 4.72 (m, 1H), 5.06 (m, 1H), 6.01
(m, 1H), 6.17 (d, J=12 Hz, 0.6H), 6.18 (d, J=12 Hz, 0.4H), 6.29 (d,
J=10 Hz, 1H), 6.40 (m, 1H), 8.24 (s, 1H), 8.65 (s, 0.6H), 8.72 (s,
0.4H), 12.92 (s, 1H), 14.16 (s, 0.4H), 14.19 (s, 0.6H).
Preparation of RTI-101
4-deoxy-3,4[2-spiro-[1-(dimethylaminocarbonyl)-piperidin-4-yl]]-(1H)-imid-
azo-(2,5-dihydro)rifamycin S
[0238] Following the general procedure (B), the title compound was
obtained as a pure solid. HRMS (ESI.sup.+): 884.4036 (M+Na).sup.+;
calculated for (M+Na).sup.+: 884.4058; RTI-101, 1H-NMR (300 MHz,
CDCl3): -0.04 (d, J=7 Hz, 3H), 0.61 (d, J=7 Hz, 3H), 0.84 (d, J=7
Hz, 3H), 1.04 (d, J=7 Hz, 3H), 1.44 (m, 1H), 1.6 (m, 1H), 1.65-1.90
(m, 3H), 1.75 (s, 3H), 1.95-2.2 (m, 2H), 2.01 (s, 3H), 2.04 (s,
3H), 2.35 (s, 3H), 2.40 (m, 1H), 2.90 (s, 6H), 3.00 (m, 1H), 3.09
(s, 3H), 3.33 (m, 1H), 3.42 (s, 1H), 3.57 (d, J=6 Hz, 1H), 3.6-3.8
(m, 5H), 4.72 (d, J=10 Hz, 1H), 5.14 (dd, J=12 and 7 Hz, 1H), 6.00
(dd, J=16 and 7 Hz, 1H), 6.18 (d, J=12 Hz, 1H), 6.27 (d, J=10 Hz,
1H), 6.37 (m, 1H), 8.19 (s, 1H), 8.96 (s, 1H), 14.62 (s, 1H).
Preparation of RTI-102
4-deoxy-3,4[2-spiro-[1-(isobutylaminocarbonyl)-piperidin-4-yl]]-(1H)-imid-
azo-(2,5-dihydro)rifamycin S
[0239] Following the general procedure (B), the title compound was
obtained as a pure solid. HRMS (ESI.sup.+): 912.4326 (M+Na).sup.+;
calculated for (M+Na).sup.+: 912.4371; RTI-102, 1H-NMR (300 MHz,
CDCl3): -0.04 (d, J=7 Hz, 3H), 0.61 (d, J=7 Hz, 3H), 0.84 (d, J=7
Hz, 3H), 0.95 (d, J=7 Hz, 6H), 1.04 (d, J=7 Hz, 3H), 1.44 (m, 1H),
1.6 (m, 1H), 1.65-1.90 (m, 4H), 1.75 (s, 3H), 1.95-2.2 (m, 2H),
2.02 (s, 3H), 2.05 (s, 3H), 2.35 (s, 3H), 2.40 (m, 1H), 3.00 (m,
1H), 3.09 (s, 3H), 3.12 (m, 2H), 3.33 (m, 1H), 3.45 (s, 1H), 3.58
(d, J=6 Hz, 1H), 3.65 (d, J=10 Hz, 1H), 3.7-4.0 (m, 4H), 4.62 (m,
1H), 4.73 (d, J=10 Hz, 1H), 5.13 (dd, J=12 and 7 Hz, 1H), 6.00 (dd,
J=16 and 7 Hz, 1H), 6.18 (d, J=12 Hz, 1H), 6.27 (d, J=10 Hz, 1H),
6.38 (m, 1H), 8.20 (s, 1H), 8.89 (s, 1H), 14.58 (s, 1H).
Preparation of RTI-103
4-deoxy-3,4[2-spiro-[1-(isopropylaminocarbonyl)-piperidin-4-yl]]-(1H)-imi-
dazo-(2,5-dihydro)rifamycin S
[0240] Following the general procedure (B), the title compound was
obtained as a pure solid. HRMS (ESI.sup.+): 898.4194 (M+Na).sup.+;
calculated for (M+Na).sup.+: 898.4215; RTI-103, 1H-NMR (300 MHz,
CDCl3): -0.04 (d, J=7 Hz, 3H), 0.61 (d, J=7 Hz, 3H), 0.84 (d, J=7
Hz, 3H), 1.04 (d, J=7 Hz, 3H), 1.21 (d, J=7 Hz, 6H), 1.44 (m, 1H),
1.55 (m, 1H), 1.65-1.90 (m, 3H), 1.75 (s, 3H), 2.0-2.15 (m, 2H),
2.02 (s, 3H), 2.05 (s, 3H), 2.35 (s, 3H), 2.40 (m, 1H), 3.00 (m,
1H), 3.09 (s, 3H), 3.33 (m, 1H), 3.45 (s, 1H), 3.58 (d, J=6 Hz,
1H), 3.66 (d, J=10 Hz, 1H), 3.7-4.0 (m, 4H), 4.03 (m, 1H), 4.33 (d,
J=7 Hz, 1H), 4.73 (d, J=10 Hz, 1H), 5.13 (dd, J=12 and 7 Hz, 1H),
6.00 (dd, J=16 and 7 Hz, 1H), 6.18 (d, J=12 Hz, 1H), 6.27 (d, J=10
Hz, 1H), 6.38 (m, 1H), 8.20 (s, 1H), 8.89 (s, 1H), 14.59 (s,
1H).
Preparation of RTI-104
4-deoxy-3,4[2-spiro-[1-methylpropyl)aminocarbonyl)-piperidin-4-yl]]-(1H)--
imidazo-(2,5-dihydro)rifamycin S
[0241] Following the general procedure (B), the title compound was
obtained as a pure solid. HRMS (ESI.sup.+): 912.4337 (M+Na).sup.+;
calculated for (M+Na).sup.+: 912.4371; RTI-104, 1H-NMR (300 MHz,
CDCl3): -0.04 (d, J=7 Hz, 3H), 0.61 (d, J=7 Hz, 3H), 0.84 (d, J=7
Hz, 3H), 0.95 (t, J=7 Hz, 3H), 1.04 (d, J=7 Hz, 3H), 1.18 (d, J=7
Hz, 3H), 1.4-1.6 (m, 4H), 1.65-1.85 (m, 3H), 1.75 (s, 3H), 2.0-2.15
(m, 2H), 2.02 (s, 3H), 2.05 (s, 3H), 2.35 (s, 3H), 2.40 (m, 1H),
3.00 (m, 1H), 3.09 (s, 3H), 3.33 (m, 1H), 3.45 (s, 1H), 3.58 (d,
J=6 Hz, 1H), 3.66 (d, J=10 Hz, 1H), 3.7-4.0 (m, 5H), 4.30 (d, J=8
Hz, 1H), 4.73 (d, J=10 Hz, 1H), 5.13 (dd, J=12 and 7 Hz, 1H), 6.00
(dd, J=16 and 7 Hz, 1H), 6.18 (d, J=12 Hz, 1H), 6.27 (d, J=10 Hz,
1H), 6.38 (m, 1H), 8.20 (s, 1H), 8.89 (s, 1H), 14.59 (s, 1H).
Preparation of RTI-105
4-deoxy-3,4[2-spiro-[1-(t-butylaminocarbonyl)-piperidin-4-yl]]-(1H)-imida-
zo-(2,5-dihydro)rifamycin S
[0242] Following the general procedure (B), the title compound was
obtained as a pure solid. HRMS (ESI.sup.+): 912.4333 (M+Na).sup.+;
calculated for (M+Na).sup.+: 912.4371; RTI-105, 1H-NMR (300 MHz,
CDCl3): -0.05 (d, J=7 Hz, 3H), 0.61 (d, J=7 Hz, 3H), 0.84 (d, J=7
Hz, 3H), 1.04 (d, J=7 Hz, 3H), 1.40 (s, 9H), 1.4-1.6 (m, 2H),
1.7-1.85 (m, 3H), 1.75 (s, 3H), 2.0-2.15 (m, 2H), 2.01 (s, 3H),
2.05 (s, 3H), 2.35 (s, 3H), 2.40 (m, 1H), 3.00 (m, 1H), 3.09 (s,
3H), 3.33 (m, 1H), 3.46 (s, 1H), 3.59 (d, J=6 Hz, 1H), 3.66 (d,
J=10 Hz, 1H), 3.7-4.0 (m, 4H), 4.43 (s, 1H), 4.73 (d, J=10 Hz, 1H),
5.13 (dd, J=12 and 7 Hz, 1H), 6.00 (dd, J=16 and 7 Hz, 1H), 6.18
(d, J=12 Hz, 1H), 6.27 (d, J=10 Hz, 1H), 6.38 (m, 1H), 8.22 (s,
1H), 8.87 (s, 1H), 14.60 (s, 1H).
Preparation of RTI-175
11-deoxy-11-hydroxy-4-deoxy-3,4[2-spiro[1-(isobutyloxycarbonyl)-piperidin-
-4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycin S
[0243] Following the general procedure (C), the title compound was
obtained as a pure solid. HRMS (ESI.sup.+): 915.4334 (M+Na).sup.+;
calculated for (M+Na).sup.+: 915.4368; RTI-175, 1H-NMR (300 MHz,
CDCl3): 0.05 (d, J=7 Hz, 3H), 0.63 (d, J=7 Hz, 3H), 0.85 (d, J=7
Hz, 3H), 0.96 (d, J=7 Hz, 6H), 1.04 (d, J=7 Hz, 3H), 1.40-1.60 (m,
2H), 1.7-2.1 (m, 6H), 1.93 (s, 3H), 2.05 (s, 3H), 2.07 (s, 3H),
2.24 (s, 3H), 2.40 (m, 1H), 3.00 (m, 1H), 3.07 (s, 3H), 3.48 (m,
1H), 3.68 (s, 1H), 3.5-3.8 (m, 2H), 3.86 (d, J=6 Hz, 2H), 3.85-4.1
(m, 4H), 4.95 (dd, J=12 and 4 Hz, 1H), 5.05 (d, J=10 Hz, 1H), 5.54
(s, 1H), 5.99 (d, J=12 Hz, 1H), 6.16 (dd, J=16 and 6 Hz, 1H), 6.27
(d, J=10 Hz, 1H), 6.44 (dd, J=16 and 10 Hz, 1H), 6.72 (s, 1H), 8.07
(s, 1H), 8.22 (bs, 1H), 13.61 (s, 1H).
Preparation of RTI-176
11-deoxy-11-amino-4-deoxy-3,4[2-spiro-[1-(isobutyloxycarbonyl)-piperidin--
4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycin S
[0244] Following the general procedure (C), the title compound was
obtained as a pure solid. HRMS (ESI.sup.+): 892.4689 (M+H).sup.+;
calculated for (M+H).sup.+: 892.4710; RTI-176 (RTI2-63B, 1H-NMR
(300 MHz) (CDCl3): -0.05 (d, J=7 Hz, 3H), 0.64 (d, J=7 Hz, 3H),
0.85 (d, J=7 Hz, 3H), 0.96 (d, J=7 Hz, 6H), 1.04 (d, J=7 Hz, 3H),
1.40-1.70 (m, 2H), 1.7-1.9 (m, 4H), 1.9-2.1 (m, 2H), 1.94 (s, 3H),
2.05 (s, 3H), 2.08 (s, 3H), 2.24 (s, 3H), 2.40 (m, 1H), 2.6-2.8
(br, 2H), 3.03 (m, 1H), 3.07 (s, 3H), 3.52 (m, 1H), 3.67 (s, 1H),
3.6-3.7 (m, 2H), 3.80 (d, J=10 Hz, 1H), 3.91 (d, J=6 Hz, 2H),
3.85-4.1 (m, 2H), 4.11 (d, J=4 Hz, 1H), 4.77 (s, 1H), 4.87 (dd,
J=12 and 4 Hz, 1H), 5.09 (d, J=10 Hz, 1H), 5.98 (d, J=12 Hz, 1H),
6.18 (dd, J=16 and 6 Hz, 1H), 6.25 (d, J=10 Hz, 1H), 6.44 (dd, J=16
and 11 Hz, 1H), 8.19 (s, 1H), 8.24 (bs, 1H), 13.93 (s, 1H).
Preparation of RTI-181
11-deoxy-11-amino-4-deoxy-3,4[2-spiro-[1-(2-methylpropyl)-piperidin-4-yl]-
](1H)-imidazo-(2,5-dihydro)rifamycin S
[0245] Following the general procedure (C), the title compound was
obtained as a pure solid. HRMS (ESI.sup.+): 848.4777 (M+H).sup.+;
calculated for (M+H).sup.+: 848.4811; RTI-181, 1H-NMR (300 MHz,
CDCl3): -0.05 (d, J=7 Hz, 3H), 0.63 (d, J=7 Hz, 3H), 0.85 (d, J=7
Hz, 3H), 0.92 (d, J=6 Hz, 6H), 1.04 (d, J=7 Hz, 3H), 1.40-1.50 (m,
1H), 1.7-2.1 (m, 9H), 1.94 (s, 3H), 2.05 (s, 3H), 2.07 (s, 3H),
2.23 (s, 3H), 2.24 (m, 2H), 2.40 (m, 1H), 2.6-2.8 (m, 4H), 3.03 (m,
1H), 3.07 (s, 3H), 3.50 (m, 1H), 3.68 (s, 1H), 3.80 (d, J=10 Hz,
1H), 4.11 (d, J=4 Hz, 1H), 4.76 (s, 1H), 4.87 (dd, J=12 and 4 Hz,
1H), 5.09 (d, J=10 Hz, 1H), 5.98 (d, J=12 Hz, 1H), 6.18 (dd, J=16
and 6 Hz, 1H), 6.25 (d, J=10 Hz, 1H), 6.44 (dd, J=16 and 11 Hz,
1H), 8.27 (s, 1H), 8.32 (s, 1H), 14.03 (s, 1H).
Preparation of RTI-182
11-deoxy-11-imino-4-deoxy-3,4[2-spiro-[1-(isobutylaminocarbonyl)-piperidi-
n-4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycin S
[0246] Following the general procedure (A), the title compound was
obtained as a pure solid. HRMS (ESI.sup.+): 889.4678 (M+H).sup.+;
calculated for (M+H).sup.+: 889.4713; RTI-182, 1H-NMR (300 MHz,
CDCl3): -0.08 (d, J=7 Hz, 3H), 0.61 (d, J=7 Hz, 3H), 0.84 (d, J=7
Hz, 3H), 0.96 (d, J=7 Hz, 6H), 1.04 (d, J=7 Hz, 3H), 1.4 (m, 1H),
1.65 (m, 1H), 1.7-1.85 (m, 4H), 1.88 (s, 3H), 1.95-2.15 (m, 2H),
2.02 (s, 3H), 2.05 (s, 3H), 2.34 (s, 3H), 2.40 (m, 1H), 3.00 (m,
1H), 3.09 (s, 3H), 3.12 (m, 2H), 3.33 (m, 1H), 3.50 (s, 1H), 3.62
(d, J=5 Hz, 1H), 3.67 (d, J=9 Hz, 1H), 3.6-3.7 (m, 2H), 3.8-4.0 (m,
2H), 4.62 (t, J=5 Hz, 1H), 4.72 (d, J=10 Hz, 1H), 5.06 (dd, J=12
and 7 Hz, 1H), 6.02 (dd, J=15 and 7 Hz, 1H), 6.16 (d, J=12 Hz, 1H),
6.29 (d, J=10 Hz, 1H), 6.38 (m, 1H), 8.27 (s, 1H), 8.67 (s, 1H),
12.92 (s, 1H), 14.58 (s, 1H).
Preparation of RTI-183
11-deoxy-11-amino-4-deoxy-3,4[2-spiro-[1-(isobutylaminocarbonyl)-piperidi-
n-4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycin S
[0247] Following the general procedure (C), the title compound was
obtained as a pure solid. HRMS (ESI.sup.+): 891.4843 (M+H).sup.+;
calculated for (M+H).sup.+: 891.4870; RTI-183, 1H-NMR (300 MHz,
CDCl3): -0.05 (d, J=7 Hz, 3H), 0.64 (d, J=7 Hz, 3H), 0.85 (d, J=7
Hz, 3H), 0.94 (d, J=7 Hz, 6H), 1.04 (d, J=7 Hz, 3H), 1.48 (m, 1H),
1.7-1.89 (m, 8H), 1.94 (s, 3H), 2.01 (m, 1H), 2.04 (s, 3H), 2.08
(s, 3H), 2.24 (s, 3H), 2.40 (m, 1H), 3.03 (m, 1H), 3.07 (s, 3H),
3.09 (m, 2H), 3.52 (m, 1H), 3.55-3.75 (m, 3H), 3.75 (s, 1H), 3.81
(d, J=10 Hz, 1H), 3.85-4.0 (m, 1H), 4.13 (d, J=4 Hz, 1H), 4.62 (t,
J=5 Hz, 1H), 4.77 (s, 1H), 4.88 (dd, J=12 and 4 Hz, 1H), 5.09 (d,
J=10 Hz, 1H), 5.98 (d, J=12 Hz, 1H), 6.18 (dd, J=16 and 6 Hz, 1H),
6.26 (d, J=10 Hz, 1H), 6.44 (dd, J=16 and 11 Hz, 1H), 8.20 (s, 1H),
8.35 (s, 1H), 13.94 (s, 1H).
Preparation of RTI-75
4-deoxy-3,4[2-spiro-[1-(t-butyloxycarbonyl)-piperidin-4-yl]]-(1H)-imidazo-
-(2,5-dihydro)rifamycin S
[0248] Following the general procedure (B), the title compound was
obtained as a pure solid. HRMS (ESI.sup.+): 913.4267 (M+Na).sup.+;
calculated for (M+Na).sup.+: 913.4211; RTI-75A, 1H-NMR (300 MHz,
CDCl3): -0.04 (d, J=7 Hz, 3H), 0.61 (d, J=7 Hz, 3H), 0.84 (d, J=7
Hz, 3H), 1.04 (d, J=7 Hz, 3H), 1.40-1.60 (m, 2H), 1.51 (s, 9H),
1.7-1.85 (m, 3H), 1.75 (s, 3H), 1.9-2.1 (m, 2H), 2.02 (s, 3H), 2.05
(s, 3H), 2.35 (s, 3H), 2.40 (m, 1H), 3.00 (m, 1H), 3.09 (s, 3H),
3.33 (m, 1H), 3.43 (s, 1H), 3.57 (d, J=5 Hz, 1H), 3.67 (d, J=10 Hz,
1H), 3.6-3.8 (br, 2H), 3.9-4.1 (br, 2H), 4.72 (d, J=10 Hz, 1H),
5.13 (dd, J=12 and 7 Hz, 1H), 6.02 (dd, J=16 and 7 Hz, 1H), 6.18
(d, J=12 Hz, 1H), 6.28 (d, J=10 Hz, 1H), 6.40 (dd, J=16 and 10 Hz,
1H), 8.19 (s, 1H), 8.93 (bs, 1H), 14.59 (s, 1H).
Preparation of RTI-76
4-deoxy-3,4[2-spiro-[1-(ethyloxycarbonyl)-piperidin-4-yl]]-(1H)-imidazo-(-
2,5-dihydro)rifamycin S
[0249] Following the general procedure (B), the title compound was
obtained as a pure solid. HRMS (ESI.sup.+): 885.3945 (M+Na).sup.+;
calculated for (M+Na).sup.+ 885.3898; RTI-76A, 1H-NMR (300 MHz,
CDCl3): -0.04 (d, J=7 Hz, 3H), 0.61 (d, J=7 Hz, 3H), 0.84 (d, J=7
Hz, 3H), 1.04 (d, J=7 Hz, 3H), 1.30 (t, J=7 Hz, 3H), 1.40-1.60 (m,
2H), 1.7-1.85 (m, 3H), 1.75 (s, 3H), 1.9-2.1 (m, 2H), 2.02 (s, 3H),
2.05 (s, 3H), 2.35 (s, 3H), 2.40 (m, 1H), 3.00 (m, 1H), 3.09 (s,
3H), 3.33 (m, 1H), 3.44 (s, 1H), 3.57 (d, J=5 Hz, 1H), 3.66 (d,
J=10 Hz, 1H), 3.7-3.9 (br, 2H), 4.0-4.2 (br, 2H), 4.21 (q, J=7 Hz,
2H), 4.72 (d, J=10 Hz, 1H), 5.13 (dd, J=12 and 7 Hz, 1H), 6.00 (dd,
J=16 and 7 Hz, 1H), 6.18 (d, J=12 Hz, 1H), 6.27 (d, J=10 Hz, 1H),
6.40 (dd, J=16 and 10 Hz, 1H), 8.20 (s, 1H), 8.92 (bs, 1H), 14.58
(s, 1H).
Preparation of RTI-78
4-deoxy-3,4[2-spiro-[1-(n-propyloxycarbonyl)-piperidin-4-yl]]-(1H)-imidaz-
o-(2,5-dihydro)rifamycin S
[0250] Following the general procedure (B), the title compound was
obtained as a pure solid. HRMS (ESI.sup.+): 899.3989 (M+Na).sup.+;
calculated for (M+Na).sup.+ 899.4054; RTI-78A, 1H-NMR (300 MHz,
CDCl3): -0.04 (d, J=7 Hz, 3H), 0.61 (d, J=7 Hz, 3H), 0.84 (d, J=7
Hz, 3H), 0.99 (t, J=7 Hz, 3H), 1.04 (d, J=7 Hz, 3H), 1.40-1.60 (m,
2H), 1.69 (m, 2H), 1.7-1.85 (m, 3H), 1.75 (s, 3H), 1.95-2.1 (m,
2H), 2.02 (s, 3H), 2.05 (s, 3H), 2.35 (s, 3H), 2.40 (m, 1H), 3.00
(m, 1H), 3.09 (s, 3H), 3.33 (m, 1H), 3.42 (s, 1H), 3.56 (d, J=5 Hz,
1H), 3.66 (d, J=10 Hz, 1H), 3.7-3.9 (br, 2H), 4.0-4.2 (br, 2H),
4.11 (t, J=7 Hz, 2H), 4.72 (d, J=10 Hz, 1H), 5.13 (dd, J=12 and 7
Hz, 1H), 6.00 (dd, J=16 and 7 Hz, 1H), 6.18 (d, J=12 Hz, 1H), 6.27
(d, J=10 Hz, 1H), 6.40 (dd, J=16 and 10 Hz, 1H), 8.17 (s, 1H), 8.92
(bs, 1H), 14.57 (s, 1H).
Preparation of RTI-79
4-deoxy-3,4[2-spiro-[1-(isobutyloxycarbonyl)-piperidin-4-yl]]-(1H)-imidaz-
o-(2,5-dihydro)rifamycin S
[0251] Following the general procedure (B), the title compound was
obtained as a pure solid. HRMS (ESI.sup.+): 913.4163 (M+Na).sup.+;
calculated for (M+Na).sup.+ 913.4211; RTI-79A, 1H-NMR (300 MHz,
CDCl3): -0.03 (d, J=7 Hz, 3H), 0.61 (d, J=7 Hz, 3H), 0.84 (d, J=7
Hz, 3H), 0.97 (d, J=7 Hz, 6H), 1.04 (d, J=7 Hz, 3H), 1.40-1.60 (m,
2H), 1.7-1.85 (m, 3H), 1.75 (s, 3H), 1.9-2.1 (m, 3H), 2.02 (s, 3H),
2.05 (s, 3H), 2.35 (s, 3H), 2.40 (m, 1H), 3.00 (m, 1H), 3.09 (s,
3H), 3.33 (m, 1H), 3.42 (s, 1H), 3.56 (d, J=5 Hz, 1H), 3.66 (d,
J=10 Hz, 1H), 3.7-3.9 (br, 2H), 3.93 (d, J=6 Hz, 2H), 4.0-4.2 (br,
2H), 4.72 (d, J=10 Hz, 1H), 5.13 (dd, J=12 and 7 Hz, 1H), 6.00 (dd,
J=16 and 7 Hz, 1H), 6.19 (d, J=12 Hz, 1H), 6.27 (d, J=10 Hz, 1H),
6.39 (dd, J=16 and 10 Hz, 1H), 8.17 (s, 1H), 8.93 (bs, 1H), 14.57
(s, 1H).
Preparation of RTI-80
4-deoxy-3,4[2-spiro-[1-(beRTIyloxycarbonyl)-piperidin-4-yl]]-(1H)-imidazo-
-(2,5-dihydro)rifamycin S
[0252] Following the general procedure (B), the title compound was
obtained as a pure solid. HRMS (ESI.sup.+): 947.3987 (M+Na).sup.+;
calculated for (M+Na).sup.+ 947.4054; RTI-80A, 1H-NMR (300 MHz,
CDCl3): -0.04 (d, J=7 Hz, 3H), 0.61 (d, J=7 Hz, 3H), 0.84 (d, J=7
Hz, 3H), 1.04 (d, J=7 Hz, 3H), 1.40-1.60 (m, 2H), 1.7-1.85 (m, 3H),
1.74 (s, 3H), 1.9-2.1 (m, 2H), 2.01 (s, 3H), 2.04 (s, 3H), 2.35 (s,
3H), 2.40 (m, 1H), 3.00 (m, 1H), 3.09 (s, 3H), 3.33 (br, 1H), 3.42
(br, 1H), 3.56 (d, J=5 Hz, 1H), 3.66 (d, J=10 Hz, 1H), 3.7-3.9 (br,
2H), 4.0-4.2 (br, 2H), 4.72 (d, J=10 Hz, 1H), 5.13 (dd, J=12 and 7
Hz, 1H), 5.20 (m, 2H), 6.00 (dd, J=16 and 7 Hz, 1H), 6.18 (d, J=12
Hz, 1H), 6.27 (d, J=10 Hz, 1H), 6.39 (dd, J=16 and 10 Hz, 1H), 7.39
(m, 5H), 8.16 (s, 1H), 8.93 (bs, 1H), 14.57 (s, 1H).
Preparation of RTI-174
11-deoxy-11-hydroxy-4-deoxy-3,4[2-spiro[1-(2-methylpropyl)-piperidin-4-yl-
]]-(1H)-imidazo-(2,5-dihydro)rifamycin S
[0253] Following the general procedure (C), the title compound was
obtained as a pure solid. HRMS (ESI.sup.+): 871.4433 (M+Na).sup.+;
calculated for (M+Na).sup.+ 871.4470.
Preparation of RTI-197
11-deoxy-11-hydroxyimino-4-deoxy-3,4[2-spiro-[1-(isobutyloxycarbonyl)-pip-
eridin-4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycin S
[0254] Following the general procedure (D), the title compound was
obtained as a solid. HRMS (ESI.sup.+): 906.4535 (M+H).sup.+;
calculated for (M+H).sup.+ 906.4535; RTI-197, 1H-NMR (300 MHz,
CDCl3): -0.03 (d, J=7 Hz, 3H), 0.62 (d, J=7 Hz, 3H), 0.84 (d, J=7
Hz, 3H), 0.97 (d, J=7 Hz, 6H), 1.04 (d, J=7 Hz, 3H), 1.35-1.40 (m,
1H), 1.7-1.8 (m, 1H), 1.85-2.1 (m, 6H), 2.00 (s, 3H), 2.04 (s, 3H),
2.13 (s, 3H), 2.33 (s, 3H), 2.40 (m, 1H), 3.00 (m, 1H), 3.10 (s,
3H), 3.34 (m, 1H), 3.42-3.50 (m, 2H), 3.67 (d, J=10 Hz, 1H),
3.8-3.9 (m, 4H), 3.93 (d, J=6 Hz, 2H), 4.60 (d, J=10 Hz, 1H), 5.23
(dd, J=12 and 8 Hz, 1H), 5.98 (dd, J=15 and 6 Hz, 1H), 6.30 (d,
J=12 Hz, 2H), 6.40 (dd, J=16 and 10 Hz, 1H), 8.35 (s, 1H), 8.92
(bs, 1H), 14.13 (s, 1H).
Preparation of RTI-217
11-deoxy-11-hydroxyimino-4-deoxy-3,4[2-spiro-1-(isobutylaminocarbonyl)-pi-
peridin-4-yl]]-(1H)-imidazo-(2,5-dihydro)rifamycin S
[0255] Following the general procedure (D), the title compound was
obtained as a solid. HRMS (ESI.sup.+): 905.4695 (M+H).sup.+;
calculated for (M+H).sup.+ 905.4662; RTI-217, 1H-NMR (300 MHz,
CDCl3): -0.03 (d, J=7 Hz, 3H), 0.62 (d, J=7 Hz, 3H), 0.84 (d, J=7
Hz, 3H), 0.95 (d, J=7 Hz, 6H), 1.04 (d, J=7 Hz, 3H), 1.35-1.40 (m,
1H), 1.7-1.8 (m, 1H), 1.85-2.1 (m, 6H), 2.00 (s, 3H), 2.04 (s, 3H),
2.13 (s, 3H), 2.33 (s, 3H), 2.40 (m, 1H), 3.00 (m, 1H), 3.10 (s,
3H), 3.08-3.14 (m, 2H), 3.34 (m, 1H), 3.45 (s, 1H), 3.47 (d, J=6
Hz, 1H), 3.65-3.8 (m, 5H), 4.60 (m, 2H), 5.23 (dd, J=12 and 8 Hz,
1H), 5.98 (dd, J=16 and 7 Hz, 1H), 6.30 (d, J=12 Hz, 2H), 6.40 (dd,
J=16 and 10 Hz, 1H), 8.34 (s, 1H), 8.89 (s, 1H), 14.14 (s, 1H).
Example 13
Preparation of a Rifabutin Derivative Modified on Alternative
Sites
[0256] Biotin-glycine-substituted rifabutin derivative RTI-173
contains a substitution at the 21-hydroxy site, yet has a similar
activity as rifabutin on G3 cells when combined with doxorubicin,
suggesting that this site may be modified without affecting
drug-sensitization or cancer inhibition effects.
Biotin-glycine-linked rifabutin derivative (RTI-173) has the
following formula:
##STR00085##
RTI-173 was prepared by the following method:
##STR00086##
[0257] A solution of Glycine-rifabutin (240 mg, 0.27 mmole) in DMF
(2 ml) was added to a solution of biotin (65 mg, 0.27 mmol), DMAP
(33 mg, 0.27 mmol) and EDCI (52 mg, 0.27 mmole) in DMF (3 ml) at
room temperature. The reaction mixture stirred at room temperature
overnight and diluted with DCM (40 ml) and washed with water and
brine. The organic phase was dried over anhydrous sodium sulphate,
filtered and concentrated under vacuum. The residue was purified by
silica gel column chromatography with methanol in DCM as eluent to
give 108 mg of the product as purple solid. HRMS (ESI.sup.+):
1152.5538 (M+Na).sup.+; calculated for (M+Na).sup.+ 1152.5304.
Example 14
Example Rifabutin and Rifabutin Derivative Compositions and Methods
of Administration to a Chemotherapeutic-Resistant Cancer
Patient
[0258] Rifamycin and rifamycin derivatives, such as rifabutin and
rifabutin derivative compositions may be prepared as described
herein. Compositions formulated in the same ways as rifabutin,
rifamycin, or related drugs typically currently formulated may be
useful for administration to cancer patients. These compositions
may contain rifamycin, a rifamycin derivative, rifabutin, or a
rifabutin derivative, such as the RTI-79 derivative described
herein.
[0259] In particular, compositions may be formulated in tablets or
capsules for oral use. These tablets or capsules may be extended
release tablets or capsules to provide a more stable and continuous
supply of the rifamycin or rifamycin derivative to the cancer cells
in the patient. Tablets or capsules may contain at least 10 mg, at
least 50 mg, at least 100 mg, at least 150 mg, or at least 200 mg
of rifamycin or rifamycin derivative. Combination tablets or
capsules with other drugs, such as chemotherapeutic drugs or other
drugs commonly administered with chemotherapy may be prepared,
particularly if the recommended dosing schedule for those drugs is
similar to that of the rifamycin or rifamycin derivative. For
example, the rifamycin or rifamycin derivative may be combined with
the prednisone portion of CHOP therapy or another steroid or other
drug that is intended to be administered daily.
[0260] Compositions may also be formulated for intravenous
injection as well. In general, the amount of rifamycin or rifamycin
derivative, such as rifabutin or a rifabutin derivative, may be
lower in a dose formulated for intravenous injection than in a dose
formulated for oral administration because intravenous injection
avoids the need for absorption through the intestines. Injectable
doses of rifamycin or rifamycin derivative, including rifabutin or
a rifabutin derivative, may be provided in multi-use containers or
in single-use containers. These containers may be compatible for
use with standard intravenous needles and syringes as well as
intravenous drip systems and more complex chemotherapeutic
administration systems. Single-use containers may contain the
entire amount of rifamycin or rifamycin derivative administered
with a round a chemotherapy to avoid the need for multiple
injections of the drug. Alternatively, they may contain amounts
appropriate for daily doses. Single-use containers may contain at
least 1 mg, at least 5 mg, at least 10 mg, at least 50 mg, at least
100 mg, or at least 150 mg of rifamycin or rifamycin derivative.
Multi-use containers may be designed to allow administration of
these same amounts of rifamycin or rifamycin derivative. Injectable
compositions may further contain other injectable chemotherapeutic
drugs or other drugs commonly administered with chemotherapy. In
one specific example, injectable compostions may contain
doxorubicin or a similar chemotherapeutic in a liposome. In such
compositions, the rifamycin or rifamycin derivate may also be in
the liposome. In general, due to improvements in delivery via
liposomes, if the rifamycin or rifamycin derivative is contained in
a liposome, the total amount in the dose may be less than if the
rifamycin or rifamycin derivative is injectable, but not in a
liposome.
[0261] Rifamycin and rifamycin derivatives, such as rifabutin and
rifabutin derivatives may be administered to patients with cancer
in the form of any compositions described in this example or
elsewhere herein or any any other form. The patients with cancer
may have a cancer that is resistant to one or more
chemotherapeutics, may be at risk for developing cancer resistant
to one or more chemotherapeutics, may benefit from administration
of reduced amounts of one or more chemotherapeutics, or may benefit
from the administration of a particular chemotherapeutic to which
rifamycin or a rifamycin derivative sensitizes the patient's cancer
cells.
[0262] In one example, the rifamycin or rifamycin derivates may be
administered orally to patients with cancer. In particular, they
may be administered in the form of tablets or capsules. The
rifamycin or rifamycin derivative may be administered such that the
patient receives at least 50 mg/adult human/week, at least 100
mg/adult human/week, at least 150 mg/adult human/week, or at least
300 mg/adult human/week. Amounts may be reduced for children. For
example, a child under age 5 might receive one quarter or less of
an adult human dose. A child age 5 to age 10 may receive one half
to one quarter the adult human dose. A child age 10 or over may
receive three quarters to one half the adult human dose. In another
embodiment, the rifamycin or rifamycin derivative may be
administered such that the patient receives at least 0.5
mg/kg/week, at least 1 mg/kg/week, at least 2 mg/kg/week, at least
5 mg/kg/week, at least 10 mg/kg/week, at least 20 mg/kg/week, at
least 30 mg/kg/week, at least 50 mg/kg/week or at least 100
mg/kg/week.
[0263] Rifamycin or a rifamycin derivative administered orally in
this fashion may be administered weekly, daily, or multiple times
per day. The dosing schedule may be adjusted so as to maintain
minimal blood concentrations for a period of time, particularly
with extended release formulations. Alternatively, maintenance of
minimal blood concentrations may not be necessary for some methods
of treatment and dosing may instead be designed to achieve a total
blood concentration for a shorter period of time, such as for four
hours or less. Although amounts are expressed as weekly totals, it
will be understood that the compositions do not have to be
administered for a full week. For example, a patient may receive a
single dose in connection with a chemotherapeutic treatment and may
not receive a further dose until much later, with another
chemotherapeutic treatment, or not at all. Furthermore, it is
possible to administer the weekly total through various
combinations of doses on various days. For example, it may be
possible to administer doses only every other day or every few
days. Doses also need not be the same each day. For example, a
patient may receive doses that increase or decrease over time,
particularly due to the schedule for administration of
chemotherapeutics. In one example, the patient may be provided with
a pack of varying-dose tablets or capsules labeled by day (e.g. Day
1, Day 2, etc.), by portions of the day (e.g. Day 1 morning, Day 1
evening, etc.), or by week (e.g. Week 1, Week 2, etc.) and
instructed to begin taking the tablets or capsules at a specified
time dictated by the schedule for administration of a
chemotherapeutic.
[0264] In general, the rifamycin or rifamycin derivative may be
administered in connection with administration of a
chemotherapeutic. In one example, it may be administered at least
weekly or at least daily the entire time the patient is receiving a
course of a chemotherapeutic, such as for several months. In
another example it may be administered only to coincide with
administration of a chemotherapeutic, such as for one day to one
week each month coinciding with a once monthly chemotherapeutic
administration.
[0265] In one specific example, the rifamycin or rifamycin
derivative may be rifabutin or RTI-79 administered orally in one to
three doses of rifabutin or RTI-79 in 100 mg to 300 mg amounts over
a period of up to 48 hours beginning within 24 hours before or
after the administration of a chemotherapeutic, such as DOXIL.RTM..
A single oral dose of 300 mg rifabutin causes a mean (.+-.SD) peak
plasma concentration (Cmax) of 375 (.+-.267) ng/mL (range 141 to
1033 ng/mL). The plasma elimination of rifabutin is biphasic with
an initial half-life of approximately 4 hours, followed by a mean
terminal half-life of 45 (.+-.17) hours (range 16 to 69 hours). The
rifabutin derivative RTI-79 is expected to present similar results.
Accordingly, appropriate dosages for variations of this example
using intravenously injected rifabutin or RTI-79 rather than orally
administered forms may be calculated.
[0266] In an alternative embodiment, rifamycin or a rifamycin
derivative, such as rifabutin or RTI-79, may be administered in a
method that matches the pharmokinetics of the rifamycin or
rifamycin derivative to that of the chemotherapeutic also
administered to the patient. For example, maximal doxorubicin
tissue absorption occurs 48 hours after administration. Maximal
RTI-79 plasma concentration is reached within 3 hours of
administration. Accordingly, administering RTI-79 orally 24 and 48
hours after intravenous doxorubicin administration may maximize
efficacy.
[0267] In another alternative embodiment, rifamycin or a rifamycin
derivative, such as rifabutin or RTI-79, may be administered in
amounts similar to those described herein after the cessation of
chemotherapy to reduce or prevent metastasis.
[0268] Although only exemplary embodiments of the invention are
specifically described above, it will be appreciated that
modifications and variations of these examples are possible without
departing from the spirit and intended scope of the invention. For
example, various specific formulations including components not
listed herein and specific methods of administering such
formulations may be developed using the ordinary skill in the art.
Numeric amounts expressed herein will be understood by one of
ordinary skill in the art to include amounts that are approximately
or about those expressed. Furthermore, the term "or" as used herein
is not intended to express exclusive options (either/or) unless the
context specifically indicates that exclusivity is required; rather
"or" is intended to be inclusive (and/or).
* * * * *