U.S. patent application number 14/587925 was filed with the patent office on 2015-07-16 for selective efflux inhibitors and related pharmaceutical compositions and methods of treatment.
The applicant listed for this patent is STC.UNM, UNIVERSITY OF KANSAS. Invention is credited to Jeffrey Aube, Bruce S. Edwards, Jennifer Elizabeth Golden, Irena Ivnitski-Steele, Hydya M. Khawaja, Richard Smith Larson, Jerec Warren Ricci, Chad E. Schroeder, Larry A. Sklar, Juan Jacob Strouse, Warren S. Weiner, Tuanli Yao.
Application Number | 20150197526 14/587925 |
Document ID | / |
Family ID | 53279769 |
Filed Date | 2015-07-16 |
United States Patent
Application |
20150197526 |
Kind Code |
A1 |
Larson; Richard Smith ; et
al. |
July 16, 2015 |
SELECTIVE EFFLUX INHIBITORS AND RELATED PHARMACEUTICAL COMPOSITIONS
AND METHODS OF TREATMENT
Abstract
The present invention provides novel compounds which inhibit
cancer-associated transporter proteins, methods of treating or
preventing the onset of a cancer-associated transporter
protein-mediated disease by administering such compounds, and
pharmaceutical compositions comprising such compounds. In one
embodiment, the invention provides novel pyrazolo[1,5-a]pyrimidine
efflux inhibitors that are selective toward ABCG2 over ABCB1.
Inventors: |
Larson; Richard Smith;
(Albuquerque, NM) ; Sklar; Larry A.; (Albuquerque,
NM) ; Edwards; Bruce S.; (Albuquerque, NM) ;
Strouse; Juan Jacob; (Albuquerque, NM) ;
Ivnitski-Steele; Irena; (Coral Springs, FL) ;
Khawaja; Hydya M.; (Albuquerque, NM) ; Ricci; Jerec
Warren; (Albuquerque, NM) ; Aube; Jeffrey;
(Lawrence, KS) ; Golden; Jennifer Elizabeth;
(Olathe, KS) ; Yao; Tuanli; (Lawrence, KS)
; Weiner; Warren S.; (Lawrence, KS) ; Schroeder;
Chad E.; (Lawrence, KS) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
STC.UNM
UNIVERSITY OF KANSAS |
Albuquerque
Lawrence |
NM
KS |
US
US |
|
|
Family ID: |
53279769 |
Appl. No.: |
14/587925 |
Filed: |
December 31, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13622651 |
Sep 19, 2012 |
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14587925 |
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61537199 |
Sep 21, 2011 |
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61680899 |
Aug 8, 2012 |
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Current U.S.
Class: |
514/252.16 ;
514/259.3; 544/281 |
Current CPC
Class: |
C07D 487/04 20130101;
A61K 31/519 20130101; A61K 31/4745 20130101; A61K 45/06 20130101;
A61K 31/437 20130101 |
International
Class: |
C07D 487/04 20060101
C07D487/04; A61K 31/4745 20060101 A61K031/4745; A61K 45/06 20060101
A61K045/06; A61K 31/519 20060101 A61K031/519 |
Goverment Interests
RELATED APPLICATIONS AND FEDERALLY SPONSORED RESEARCH
[0002] The present invention was made with government support under
Grant Nos. 5R01CA114589-01, 1RO3MH081228-01A1, U54MH084690 and
U54HG005031 awarded by the National Institutes of Health (NIH).
Consequently, the Government has certain rights in this invention.
Claims
1. A compound of formula (I), or an enantiomer, diastereomer,
tautomer, or pharmaceutically-acceptable salt or hydrate thereof:
##STR00023## wherein R.sub.1 is selected from the group consisting
of substituted or unsubstituted alkyl, substituted or unsubstituted
alkylene, substituted or unsubstituted alkynyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl,
substituted or unsubstituted heteroaryl, substituted or
unsubstituted arylalkyl, and substituted or unsubstituted
heteroarylalkyl; R.sub.2 and R.sub.4 are independently selected
from the group consisting of hydrogen, halogen, hydroxy, carboxyl,
acyl, amino, amide, substituted or unsubstituted alkyl, substituted
or unsubstituted alkylene, substituted or unsubstituted alkynyl,
substituted or unsubstituted heterocycloalkyl, substituted or
unsubstituted aryl, substituted or unsubstituted heteroaryl,
substituted or unsubstituted arylalkyl, and substituted or
unsubstituted heteroarylalkyl; R.sub.3 is selected from the group
consisting of hydrogen, halogen, hydroxy, carboxyl, acyl, amino,
amide, substituted or unsubstituted alkyl, substituted or
unsubstituted alkylene, or substituted or unsubstituted alkynyl;
and wherein R.sub.a, R.sub.b, R.sub.c, and R.sub.d are
independently selected from the group consisting of hydrogen,
halogen, hydroxy, carboxyl, oxo, acyl, amino, amide, substituted or
unsubstituted alkyl, substituted or unsubstituted alkylene, or
substituted or unsubstituted alkynyl, or one or more of R.sub.a,
R.sub.b, R.sub.c, and R.sub.d, together with the carbon ring atom
to which it is bound, forms carbonyl.
2. (canceled)
3. (canceled)
4. (canceled)
5. Cancelled
6. (canceled)
7. (canceled)
8. (canceled)
9. (canceled)
10. (canceled)
11. (canceled)
12. (canceled)
13. (canceled)
14. (canceled)
15. (canceled)
16. (canceled)
17. A method of treating a patient who suffers from, or who is at
risk of developing, a cancer-associated transporter protein
mediated disease, the method comprising administering to the
patient a pharmaceutically effective amount of a compound of claim
1, or an enantiomer, diastereomer, tautomer, or
pharmaceutically-acceptable salt or hydrate thereof.
18. The method of treatment of claim 17, wherein one or more
additional anti-cancer agents are co-administered to the
patient.
19. The method of claim 18 wherein said additional anti-cancer
agent is an antimetabolite, or a topoisomerase I and/or
topoisomerase II inhibitor.
20. The method according to claim 18 wherein said additional
anti-cancer agent is Ara C, etoposide, doxorubicin, taxol,
hydroxyurea, vincristine, cytoxan (cyclophosphamide), mitomycin C,
adriamycin, topotecan, campothecin, irinotecan, gemcitabine,
campothecin, cisplatin and mixtures thereof.
21. The method of claim 18 wherein said additional anti-cancer
agent is adriamycin, anastrozole, arsenic trioxide, asparaginase,
azacytidine, BCG Live, bevacizumab, bexarotene capsules, bexarotene
gel, bleomycin, bortezombi, busulfan intravenous, busulfan oral,
calusterone, campothecin, capecitabine, carboplatin, carmustine,
carmustine with polifeprosan 20 implant, celecoxib, cetuximab,
chlorambucil, cisplatin, cladribine, clofarabine, cyclophosphamide,
cytarabine, cytoxan, cytarabine liposomal, dacarbazine,
dactinomycin, actinomycin D, dalteparin sodium, darbepoetin alfa,
dasatinib, daunorubicin liposomal, daunorubicin, daunomycin,
decitabine, denileukin, denileukin diftitox, dexrazoxane,
dexrazoxane, docetaxel, doxorubicin, doxorubicin liposomal,
dromostanolone propionate, eculizumab, Elliott's B Solution,
epirubicin, epirubicin hcl, epoetin alfa, erlotinib, estramustine,
etoposide phosphate, etoposide VP-16, exemestane, fentanyl citrate,
filgrastim, floxuridine (intraarterial), fludarabine, fluorouracil
5-FU, fulvestrant, gefitinib, gemcitabine, gemcitabine hcl,
gemicitabine, gemtuzumab ozogamicin, goserelin acetate, goserelin
acetate, histrelin acetate, hydroxyurea, ibritumomab tiuxetan,
idarubicin, ifosfamide, imatinib mesylate, interferon alfa 2a,
interferon alfa-2b, irinotecan, lapatinib ditosylate, lenalidomide,
letrozole, leucovorin, leuprolide acetate, levamisole, lomustine
CCNU, meclorethamine, nitrogen mustard, megestrol acetate,
melphalan L-PAM, mercaptopurine 6-MP, mesna, methotrexate,
methoxsalen, mitomycin C, mitotane, mitoxantrone, nandrolone
phenpropionate, nelarabine, nofetumomab, oprelvekin, oxaliplatin,
paclitaxel, paclitaxel protein-bound particles, palifermin,
pamidronate, panitumumab, pegademase, pegaspargase, pegfilgrastim,
peginterferon alfa-2b, pemetrexed disodium, pentostatin,
pipobroman, plicamycin, mithramycin, porfimer sodium, procarbazine,
quinacrine, rasburicase, rituximab, sargramostim, sorafenib,
streptozocin, sunitinib, sunitinib maleate, talc, tamoxifen,
temozolomide, teniposide VM-26, testolactone, thalidomide,
thioguanine 6-TG, thiotepa, topotecan, topotecan hcl, toremifene,
tositumomab, tositumomab/I-131 tositumomab, trastuzumab, tretinoin
ATRA, uracil mustard, valrubicin, vinblastine, vincristine,
vinorelbine, vorinostat, zoledronate, zoledronic acid and mixtures
thereof.
22. The method of treatment of claim 18, wherein topotecan is
co-administered to the patient.
23. (canceled)
24. (canceled)
25. A method of treating a patient who suffers from a tumor which
contains ABCG2 resistant tumor cells, the method comprising
administering to the patient a pharmaceutically effective amount of
a compound of claim 1, or an enantiomer, diastereomer, tautomer, or
a pharmaceutically-acceptable salt or hydrate thereof.
26. The method of treatment of claim 25, wherein a
pharmaceutically-effective amount of an additional anti-cancer
agent is co-administered to the patient.
27. The method of claim 26, wherein said additional anti-cancer
agent is an antimetabolite, or a topoisomerase I and/or
topoisomerase II inhibitor.
28. The method of claim 26 wherein said additional anti-cancer
agent is Ara C, etoposide, doxorubicin, taxol, hydroxyurea,
vincristine, cytoxan (cyclophosphamide), mitomycin C, adriamycin,
to+potecan, campothecin, irinotecan, gemcitabine, campothecin,
cisplatin and mixtures thereof.
29. The method of claim 26 wherein said additional anti-cancer
agent is adriamycin, anastrozole, arsenic trioxide, asparaginase,
azacytidine, BCG Live, bevacizumab, bexarotene capsules, bexarotene
gel, bleomycin, bortezombi, busulfan intravenous, busulfan oral,
calusterone, campothecin, capecitabine, carboplatin, carmustine,
carmustine with polifeprosan 20 implant, celecoxib, cetuximab,
chlorambucil, cisplatin, cladribine, clofarabine, cyclophosphamide,
cytarabine, cytoxan, cytarabine liposomal, dacarbazine,
dactinomycin, actinomycin D, dalteparin sodium, darbepoetin alfa,
dasatinib, daunorubicin liposomal, daunorubicin, daunomycin,
decitabine, denileukin, denileukin diftitox, dexrazoxane,
dexrazoxane, docetaxel, doxorubicin, doxorubicin liposomal,
dromostanolone propionate, eculizumab, Elliott's B Solution,
epirubicin, epirubicin hcl, epoetin alfa, erlotinib, estramustine,
etoposide phosphate, etoposide VP-16, exemestane, fentanyl citrate,
filgrastim, floxuridine (intraarterial), fludarabine, fluorouracil
5-FU, fulvestrant, gefitinib, gemcitabine, gemcitabine hcl,
gemicitabine, gemtuzumab ozogamicin, goserelin acetate, goserelin
acetate, histrelin acetate, hydroxyurea, ibritumomab tiuxetan,
idarubicin, Ifosfamide, imatinib mesylate, interferon alfa 2a,
interferon alfa-2b, irinotecan, lapatinib ditosylate, lenalidomide,
letrozole, leucovorin, leuprolide acetate, levamisole, lomustine
CCNU, meclorethamine, nitrogen mustard, megestrol acetate,
melphalan L-PAM, mercaptopurine 6-MP, mesna, methotrexate,
methoxsalen, mitomycin C, mitotane, mitoxantrone, nandrolone
phenpropionate, nelarabine, nofetumomab, oprelvekin, oxaliplatin,
paclitaxel, paclitaxel protein-bound particles, palifermin,
pamidronate, panitumumab, pegademase, pegaspargase, pegfilgrastim,
peginterferon alfa-2b, pemetrexed disodium, pentostatin,
pipobroman, plicamycin, mithramycin, porfimer sodium, procarbazine,
quinacrine, rasburicase, rituximab, sargramostim, sorafenib,
streptozocin, sunitinib, sunitinib maleate, talc, tamoxifen,
temozolomide, teniposide VM-26, testolactone, thalidomide,
thioguanine 6-TG, thiotepa, topotecan, topotecan hcl, toremifene,
tositumomab, tositumomab/I-131 tositumomab, trastuzumab, tretinoin
ATRA, uracil mustard, valrubicin, vinblastine, vincristine,
vinorelbine, vorinostat, zoledronate, zoledronic acid and mixtures
thereof.
30. (canceled)
31. (canceled)
32. (canceled)
33. The method of treatment of any of claims 26, wherein the
patient's cancer has previously proven to be non-responsive to
chemotherapy.
34. A pharmaceutical composition comprising a compound of claim 1,
or an enantiomer, diastereomer, tautomer, or
pharmaceutically-acceptable salt or hydrate thereof, and optionally
one or more pharmaceutically-acceptable excipients.
35. The pharmaceutical composition of claim 34, wherein the
composition further comprises an additional anti-cancer agent.
36. (canceled)
37. The pharmaceutical composition of claim 35 wherein said
additional anti-cancer agent is Ara C, etoposide, doxorubicin,
taxol, hydroxyurea, vincristine, cytoxan (cyclophosphamide),
mitomycin C, adriamycin, topotecan, campothecin, irinotecan,
gemcitabine, campothecin, cisplatin and mixtures thereof.
38. The pharmaceutical composition of claim 35 wherein said
additional anti-cancer agent is adriamycin, anastrozole, arsenic
trioxide, asparaginase, azacytidine, BCG Live, bevacizumab,
bexarotene capsules, bexarotene gel, bleomycin, bortezombi,
busulfan intravenous, busulfan oral, calusterone, campothecin,
capecitabine, carboplatin, carmustine, carmustine with polifeprosan
20 implant, celecoxib, cetuximab, chlorambucil, cisplatin,
cladribine, clofarabine, cyclophosphamide, cytarabine, cytoxan,
cytarabine liposomal, dacarbazine, dactinomycin, actinomycin D,
dalteparin sodium, darbepoetin alfa, dasatinib, daunorubicin
liposomal, daunorubicin, daunomycin, decitabine, denileukin,
denileukin diftitox, dexrazoxane, dexrazoxane, docetaxel,
doxorubicin, doxorubicin liposomal, dromostanolone propionate,
eculizumab, Elliott's B Solution, epirubicin, epirubicin hcl,
epoetin alfa, erlotinib, estramustine, etoposide phosphate,
etoposide VP-16, exemestane, fentanyl citrate, filgrastim,
floxuridine (intraarterial), fludarabine, fluorouracil 5-FU,
fulvestrant, gefitinib, gemcitabine, gemcitabine hcl, gemicitabine,
gemtuzumab ozogamicin, goserelin acetate, goserelin acetate,
histrelin acetate, hydroxyurea, ibritumomab tiuxetan, idarubicin,
ifosfamide, imatinib mesylate, interferon alfa 2a, interferon
alfa-2b, irinotecan, lapatinib ditosylate, lenalidomide, letrozole,
leucovorin, leuprolide acetate, levamisole, lomustine CCNU,
meclorethamine, nitrogen mustard, megestrol acetate, melphalan
L-PAM, mercaptopurine 6-MP, mesna, methotrexate, methoxsalen,
mitomycin C, mitotane, mitoxantrone, nandrolone phenpropionate,
nelarabine, nofetumomab, oprelvekin, oxaliplatin, paclitaxel,
paclitaxel protein-bound particles, palifermin, pamidronate,
panitumumab, pegademase, pegaspargase, pegfilgrastim, peginterferon
alfa-2b, pemetrexed disodium, pentostatin, pipobroman, plicamycin,
mithramycin, porfimer sodium, procarbazine, quinacrine,
rasburicase, rituximab, sargramostim, sorafenib, streptozocin,
sunitinib, sunitinib maleate, talc, tamoxifen, temozolomide,
teniposide VM-26, testolactone, thalidomide, thioguanine 6-TG,
thiotepa, topotecan, topotecan hcl, toremifene, tositumomab,
tositumomab/I-131 tositumomab, trastuzumab, tretinoin ATRA, uracil
mustard, valrubicin, vinblastine, vincristine, vinorelbine,
vorinostat, zoledronate, zoledronic acid and mixtures thereof.
39. The pharmaceutical composition of claim 35, wherein the
additional anti-cancer agent is topotecan.
Description
[0001] This application claims priority from U.S. Provisional
Application Ser. No. 61/537,199, entitled "Selective Efflux
Inhibitors and Related Pharmaceutical Compositions and Methods of
Treatment, filed Sep. 21, 2011, and U.S. Provisional Application
Ser. No. 61/680,899, entitled "Selective ATP-binding Cassette
Sub-Family G Member 2 Efflux Inhibitor Revealed Via High Throughput
Flow Cytometry", filed Aug. 8, 2012, the entire contents of which
are hereby incorporated by reference in their entirety.
FIELD OF THE INVENTION
[0003] The present invention provides novel compounds which inhibit
cancer-associated transporter proteins, methods of treating or
preventing the onset of a cancer-associated transporter
protein-mediated disease by administering such compounds, and
pharmaceutical compositions comprising such compounds. In one
embodiment, the invention provides novel pyrazolo[1,5-a]pyrimidine
efflux inhibitors that are selective toward ABCG2 over ABCB1.
BACKGROUND OF THE INVENTION
[0004] More than 48 members of the ABC transporter superfamily have
been identified and three major subfamilies (ABCB, ABCC, and ABCG)
are related to human multidrug resistance (MDR) and influence oral
absorption and disposition of a wide variety of drugs, and as a
result their expression levels have important consequences for
susceptibility to drug-induced side effects, interactions, and
treatment efficacy. The specific subclass members ABCB1(MDR1/Pgp),
ABCC1 (MRP1), and ABCG2 (BCRP) are known to significantly influence
the efficacy of drugs and have unambiguously been shown to
contribute to cancer multidrug resistance..sup.1-2 Although a large
number of compounds have been identified possessing ABC transporter
inhibitory properties, only a few of these agents are appropriate
candidates for clinical use as MDR reversing agents..sup.3-4 Dual
treatment with ABC transporter inhibitors in conjunction with
chemotherapeutics is a common treatment strategy to circumvent MDR
in cancers..sup.5-6 However, the failure of current classes provide
ample justification for identifying new classes of modulators and
exploring the biology around them. These efflux pumps are expressed
in many human tumors where they likely contribute to resistance to
chemotherapy treatment. ABCB1, ABCC1, and ABCG2 are highly
expressed in the gut, liver, and kidneys and they may restrict the
oral bioavailability of administered drugs. ABCB1 and ABCG2 are
also expressed in the epithelia of the brain and placenta and also
in stem cells, where they perform a barrier function..sup.7 More
specifically, ABCG2 relevance as a clinical target has been well
documented..sup.8 This includes a mouse model using a human ovarian
xenograft with Igrove1/T8 tumors,.sup.9 a system utilizing
flavopiridol-resistant human breast cancer cells,.sup.10 an FTC/Ko
143 inhibition in vitro and mouse intestine model," and a phase
I/II trial with lapatinib in glioblastoma multiforme..sup.12
[0005] Early clinical failures with ABCB1 inhibitors initially
resulted in diminished enthusiasm. However, progress over the last
decade has renewed activity in the field and a variety of
modulators have been identified. ABC efflux transporter inhibition
is now in its third generation with the majority of focus still on
ABCB1. It has been observed that a large number of structurally and
functionally diverse compounds act as substrates or modulators of
these pumps with numerous publications dedicated to the
subject..sup.13-16 A subset of these compounds will be discussed
here. The first-generation of chemosensitizers were discovered from
already approved drugs and included the calcium channel blocker
verapamil (as well asnicardipine), cyclosporin A, and progesterone
but dose-related toxicity and other adverse effects (i.e.
solubility limitations) prevented progress into the
clinic..sup.17-24Second and third generation inhibitors were drawn
predominantly from the derivatization of first-generation molecules
as well as from combinatorial chemistry targeted primarily at
ABCB1. Some of the higher profile examples include: the cyclosporin
A derivative valspodar (PSC-833).sup.25; Vertex Pharmaceuticals'
biricodar (VX-710).sup.26-28; the anthranilamide based modulators
XR9051.sup.29, tariquidar (XR9576)30-32, XR9577.sup.33-34, and
WK-X-34.sup.34 35; the acridonecarboxamidederivativeelacridar
(GF120918).sup.36; the heteroaryloxypropanolamineszosuquidar
(LY335979).sup.37-39 and dofequidar (MS-209).sup.40-41 (and the
structurally related laniquidar (R101933).sup.42-43), and
diarylimidazoleontogen (OC144-093, ONT-093).sup.44-47. The late
generation inhibitors tended to be more potent and less toxic than
the first-generation compounds, however, multiple issues
remain.
[0006] Although much of the work to date is targeted at ABCB1, the
selectivity profile of these inhibitors is significantly varied.
Valspodar, tariquidar, elacridar, zosuquidar and ontogeny have been
reported to be selective (though not necessarily specific) for
ABCB1..sup.25-28,30-31,40 Those specific for ABCC1 include the
quinoline based MK571 and the uricosuric drug
probenecide..sup.48-49 Although there is significant progress with
ABCB1 inhibitors, similar progress has not been made with ABCG2
inhibitors. The Aspergillusfumigatusmycotoxinfumitremorgin C (FTC)
and its analogs Ko 132, Ko 134, and Ko 143 have been demonstrated
to be selective inhibitors for ABCG2..sup.11,50-52 Other
imidazoline and .beta.-carboline amino acid benzyl ester conjugates
analogous to FTC were labeled `dual-acting` due to a cytotoxicity
that was coupled to their resistance reversing
activity..sup.53Examples of cross pump inhibitors include
verapamil, cyclosporin A, dofequidar, and reversanfor ABCB1/ABCC1
and biricodar and nicardipinefor
ABCB1/ABCC1/ABCG2..sup.28,54-56
[0007] Structural information for all mammalian ABC transporter
family members is relatively sparse, with ABCB1 being the most
extensively studied. The presence of multiple, potentially
overlapping, binding sites and possible interactions between them
may account for diverse specificity of structurally and
functionally unrelated modulators and substrates. This
polyspecificity also raises questions as to which substrate should
be used to demonstrate inhibitory potential of a new chemical
entity. In order to understand the mechanism and to design more
effective modulators, great effort has been made to study the
interaction of substrates and modulators with these
transporters..sup.57 It was shown that most ABCB1 inhibitors are
additionally substrates of the efflux pump..sup.58 It is important
to not only evaluate inhibitor potency in a given transporter, but
also to profile its activity with other transporters as well as its
interrelationship with substrate drugs. For instance, strong
inhibition of ABCB1 by drugs like cyclosporine or verapamil in in
vitro models proved to be limited in in vivo studies due to toxic
pharmacological effects of the inhibitors..sup.2 Our recent work
further demonstrated differential cross-reactivity of inhibitors
across ABCB1, ABCC1, and ABCG2 transporters and we demonstrated
cross-reactivity of both these inhibitors across all three
transporters, which could help explain such severe toxicity
effects..sup.56 Such interactions can be quite complex, since the
array of substrate/non-substrate and inhibitor/non-inhibitor is
further clouded by the possibility of multiple interaction sites
and unwarranted cytotoxicity.
[0008] Several of the aforementioned small molecule inhibitors were
selected to help profile the compounds of interest in vitro (FIG.
1). Compounds were chosen specifically for their reported
selectivity profiles. The sub-micromolar modulator of ABCB1 XR9051
(a precursor to tariquidar) has been shown to reverse resistance to
cytotoxic drugs such as doxorubicin and vincristine..sup.3,29 The
previously mentioned MK 571 is documented as a specific inhibitor
of ABCC1..sup.59 For direct comparison of selective inhibition of
ABCG2 both FTC and Ko 143 were chosen..sup.11,50-52 Also, the
pyrazolopyrimidine reversan, with a similar core to our inhibitor
class, was identified as an active inhibitor of ABCB1 and
ABCC1..sup.54
SUMMARY OF THE INVENTION
[0009] The present invention relates to compounds which inhibit
cancer-associated transporter proteins and methods of treatment
that use such compounds to mediate a variety of disease states, in
particular cancer, and especially drug resistant (DR) and multiple
drug resistant (MDR) cancer. In one embodiment, the invention
provides novel pyrazolo[1,5-a]pyrimidine efflux inhibitors that are
selective toward ABCG2 over ABCB1. These compounds have low in
vitro cellular toxicity, are soluble, and are stable.
[0010] Accordingly, the invention provides a compound of formula
(I), or an enantiomer, diastereomer, tautomer, or
pharmaceutically-acceptable salt or hydrate thereof:
##STR00001##
wherein [0011] R.sub.1 is selected from the group consisting of
substituted or unsubstituted alkyl, substituted or unsubstituted
alkylene, substituted or unsubstituted alkynyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl,
substituted or unsubstituted heteroaryl, substituted or
unsubstituted arylalkyl, and substituted or unsubstituted
heteroarylalkyl; [0012] R.sub.2 and R.sub.4 are independently
selected from the group consisting of hydrogen, halogen, hydroxy,
carboxyl, acyl, amino, amide, substituted or unsubstituted alkyl,
substituted or unsubstituted alkylene, substituted or unsubstituted
alkynyl, substituted or unsubstituted heterocycloalkyl, substituted
or unsubstituted aryl, substituted or unsubstituted heteroaryl,
substituted or unsubstituted arylalkyl, and substituted or
unsubstituted heteroarylalkyl; [0013] R.sub.3 is selected from the
group consisting of hydrogen, halogen, hydroxy, carboxyl, acyl,
amino, amide, substituted or unsubstituted alkyl, substituted or
unsubstituted alkylene, or substituted or unsubstituted alkynyl;
and wherein [0014] R.sub.a, R.sub.b, R.sub.c, and R.sub.d are
independently selected from the group consisting of hydrogen,
halogen, hydroxy, carboxyl, oxo, acyl, amino, amide, substituted or
unsubstituted alkyl, substituted or unsubstituted alkylene, or
substituted or unsubstituted alkynyl, or one or more of R.sub.a,
R.sub.b, R.sub.c, and R.sub.d, together with the carbon ring atom
to which it is bound, forms carbonyl.
[0015] In a preferred embodiment of the compounds of formula (I),
R.sub.1 is selected from the group consisting of substituted or
unsubstituted aryl, R.sub.2 is selected from the group consisting
of substituted or unsubstituted alkyl, substituted or unsubstituted
aryl, and substituted or unsubstituted heteroaryl, R.sub.3 is
selected from the group consisting of hydrogen and substituted or
unsubstituted alkyl, R.sub.4 is either (1) an acyl group which
includes either a substituted or unsubstituted aryl or a
substituted or unsubstituted heteroaryl moiety, or (2) is a
substituted or unsubstituted arylalkyl or a substituted or
unsubstituted heteroarylalkyl, and R.sub.a, R.sub.b, R.sub.c, and
R.sub.d are hydrogen.
[0016] In a more preferred embodiment of the compounds of formula
(I), R.sub.1 is substituted or unsubstituted phenyl, R.sub.2 is a
substituted or unsubstituted C.sub.1-C.sub.3 alkyl or is a
substituted or unsubstituted heteroaryl, R.sub.3 is hydrogen,
R.sub.4 is R.sub.xR.sub.y, where R.sub.x is bound to the nitrogen
ring atom and is either carbonyl or CH.sub.2 and R.sub.y is a 5 or
6-membered, substituted or unsubstituted heteroaryl, and R.sub.a,
R.sub.b, R.sub.c, and R.sub.d are hydrogen.
[0017] In a still more preferred embodiment of the compounds of
formula (I), R.sub.1 is substituted or unsubstituted phenyl,
R.sub.2 is a substituted or unsubstituted 5 or 6-membered
heteroaryl, R.sub.3 is hydrogen, R.sub.4 is R.sub.xR.sub.y, where
R.sub.x is bound to the nitrogen ring atom and is either carbonyl
or CH.sub.2 and R.sub.y is a 5 or 6-membered, substituted or
unsubstituted heteroaryl, and R.sub.a, R.sub.b, R.sub.c, and
R.sub.d are hydrogen.
[0018] In an even more preferred embodiment of the compounds of
formula (I), R.sub.1 is substituted or unsubstituted phenyl,
R.sub.2 is a substituted or unsubstituted furan, R.sub.3 is
hydrogen, R.sub.4 is R.sub.xR.sub.y, where R.sub.x is bound to the
nitrogen ring atom and is either carbonyl or CH.sub.2 and R.sub.y
is substituted or unsubstituted furan, and R.sub.a, R.sub.b,
R.sub.c, and R.sub.d are hydrogen.
[0019] In another preferred embodiment, the invention provides a
compound of formula (I), or an enantiomer, diastereomer, tautomer,
or pharmaceutically-acceptable salt or hydrate thereof, wherein the
compound is:
##STR00002##
[0020] In another preferred embodiment, the invention provides a
compound of formula (I), or an enantiomer, diastereomer, tautomer,
or pharmaceutically-acceptable salt or hydrate thereof, wherein the
compound is:
##STR00003##
[0021] In another preferred embodiment, the invention provides a
compound of formula (I), or an enantiomer, diastereomer, tautomer,
or pharmaceutically-acceptable salt or hydrate thereof, wherein the
compound is:
##STR00004##
[0022] In another preferred embodiment, the invention provides a
compound of formula (I), or an enantiomer, diastereomer, tautomer,
or pharmaceutically-acceptable salt or hydrate thereof, wherein
R.sub.1 and R.sub.2 are substituted or unsubstituted phenyl,
R.sub.3 is hydrogen, R.sub.4 is CO-3-pyridyl, and R.sub.a, R.sub.b,
R.sub.c, and R.sub.d are hydrogen.
[0023] In another preferred embodiment, the invention provides a
compound of formula (I), or an enantiomer, diastereomer, tautomer,
or pharmaceutically-acceptable salt or hydrate thereof, wherein
R.sub.1 is 3-Cl-phenyl, R.sub.2 is phenyl, R.sub.3 is hydrogen,
R.sub.4 is CO-3-pyridyl, and R.sub.a, R.sub.b, R.sub.c, and R.sub.d
are hydrogen.
[0024] In another preferred embodiment, the invention provides a
compound of formula (I), or an enantiomer, diastereomer, tautomer,
or pharmaceutically-acceptable salt or hydrate thereof, wherein
R.sub.1 is phenyl, R.sub.2 is 2-F-phenyl, R.sub.3 is hydrogen,
R.sub.4 is CO-3-furyl, and R.sub.a, R.sub.b, R.sub.c, and R.sub.d
are hydrogen.
[0025] In another preferred embodiment, the invention provides a
compound of formula (I), or an enantiomer, diastereomer, tautomer,
or pharmaceutically-acceptable salt or hydrate thereof, wherein
R.sub.1 is 3-Cl-phenyl, R.sub.2 is 3-pyridyl, R.sub.3 is hydrogen,
R.sub.4 is CO-3-furyl, and R.sub.a, R.sub.b, R.sub.c, and R.sub.d
are hydrogen.
[0026] In another preferred embodiment, the invention provides a
compound of formula (I), or an enantiomer, diastereomer, tautomer,
or pharmaceutically-acceptable salt or hydrate thereof, wherein
R.sub.1 is phenyl, R.sub.2 is 3-MeO-phenyl, R.sub.3 is hydrogen,
R.sub.4 is CO-3-furyl, and R.sub.a, R.sub.b, R.sub.c, and R.sub.d
are hydrogen.
[0027] In another preferred embodiment, the invention provides a
compound of formula (IA), or an enantiomer, diastereomer, tautomer,
or pharmaceutically-acceptable salt or hydrate thereof:
##STR00005##
wherein R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are as defined for
formula (I).
[0028] In another preferred embodiment, the invention provides a
compound of formula (IA), or an enantiomer, diastereomer, tautomer,
or pharmaceutically-acceptable salt or hydrate thereof, wherein
R.sub.1 is substituted or unsubstituted phenyl; R.sub.2 is alkyl
(preferably methyl), substituted or unsubstituted phenyl or a
substituted or unsubstituted five-membered heteroaryl containing
one or two ring heteroatoms selected from the group consisting of O
and N; R.sub.3 is hydrogen or alkyl (preferably methyl); and
R.sub.4 is alkyl-phenyl or an acyl group containing either a
substituted or unsubstituted phenyl or a five-membered, substituted
or unsubstituted heteroaryl containing one or two ring heteroatoms
selected from the group consisting of O and N.
[0029] In another preferred embodiment, the invention provides a
compound of formula (IA), or an enantiomer, diastereomer, tautomer,
or pharmaceutically-acceptable salt or hydrate thereof, wherein
R.sub.1 is phenyl substituted by either halogen (most preferably Cl
or F) or lower alkoxy (most preferably methoxy), or R.sub.1 is
unsubstituted phenyl; R.sub.2 is alkyl (preferably methyl), phenyl
substituted by either halogen (most preferably F) or lower alkoxy
(most preferably methoxy), or R.sub.2 is furyl; R.sub.3 is hydrogen
or alkyl (most preferably methyl); and R.sub.4 is alkyl-phenyl,
benzoyl substituted by halogen (most preferably Cl) or lower alkoxy
(most preferably methoxy), or R.sub.4 is furoyl.
[0030] In another preferred embodiment, the invention provides a
compound of formula (IA), or an enantiomer, diastereomer, tautomer,
or pharmaceutically-acceptable salt or hydrate thereof, wherein
R.sub.1 is unsubstituted phenyl; R.sub.2 is phenyl substituted by
either halogen (most preferably F) or lower alkoxy (most preferably
methoxy), or R.sub.2 is furyl; R.sub.3 is hydrogen; and R.sub.4 is
benzoyl substituted by either halogen (most preferably CO or lower
alkoxy (most preferably methoxy), or R.sub.4 is furoyl.
[0031] In one aspect of the invention, a compound of formulae (I)
or (IA), or an enantiomer, diastereomer, tautomer, or
pharmaceutically-acceptable salt or hydrate thereof, is
administered to a patient in need thereof to treat or prevent the
onset of a cancer-associated transporter protein mediated
disease.
[0032] In another aspect of the invention, a compound of formula
(I) or (IA), or an enantiomer, diastereomer, tautomer, or
pharmaceutically-acceptable salt or hydrate thereof, and an
adjuvant anti-cancer therapy, are co-administered to a patient in
need thereof to treat or prevent the onset of a cancer-associated
transporter protein mediated disease.
[0033] In still another aspect, the invention provides a method of
treating a patient who suffers from a tumor containing ABCG2
resistant tumor cells by administering to the patient a
pharmaceutically effective amount of a compound of formulae (I) or
(IA), or an enantiomer, diastereomer, tautomer, or a
pharmaceutically-acceptable salt or hydrate thereof. In certain
aspects, the patient's tumor has proven non-responsive to
chemotherapy prior to treatment.
[0034] Pharmaceutical compositions which comprise an effective
amount of a compound of formula (I) or (IA) or as otherwise
disclosed in the present application, or an enantiomer,
diastereomer, tautomer, or pharmaceutically-acceptable salt or
hydrate thereof, are also provided. In some embodiments, these
pharmaceutical compositions include an effective amount at least
one additional anti-cancer agent, especially as otherwise described
herein, optionally in combination with a pharmaceutically
acceptable carrier, additive or excipient.
[0035] These and other aspects of the invention are described
further hereinafter in the detailed description of the
invention.
BRIEF DESCRIPTION OF THE FIGURES
[0036] FIG. 1 depicts structures of small molecules chosen for
direct experimental comparison in connection with the making of the
claimed, invention.
[0037] FIG. 2 illustrates a general synthetic route useful in
making compounds of the invention.
[0038] FIG. 3 depicts the aqueous stability of compound SID
88095709 in PBS and no acetonitrile (closed circles), and the
aqueous stability of compound SID 88095709 in PBS with the addition
of acetonitrile (50% v/v final, closed triangles). Reagents: (a)
methyl 3-(furan-2-yl)-3-oxopropanoate, AcOH, 100.degree. C., 2 h;
(b) POCl3, BnEt3NCl, PhNMe2, CH3CN, 80.degree. C., 16 h; (c)
furan-3-yl(piperazin-1-yl)methanone, DIPEA, CH3CN, 100.degree. C.,
16 h; (d) diethylmalonate, 21% NaOEt, EtOH, 80.degree. C., 3 h,
75%; (e) POCl3, N,N-dimethylaniline, 115.degree. C., 16 h, 42%; (f)
potassium aryltrifluoroborate salt, Pd(OAc)2, RuPhos, Na2CO3, EtOH,
MWI, 90.degree. C., 6 h.
[0039] FIG. 4 shows that SID 85240370 had attractive efflux potency
towards ABCG2 and marginal selectivity over ABCB1.
[0040] FIG. 5 illustrates how, in SAR studies, the
pyrazolo[1,5-a]pyrimidine core was preserved, and the exchange of
the peripheral substituents were surveyed, as depicted by the
highlighted regions.
[0041] FIG. 6 illustrates SAR-based modification of parent hit SID
85240370 to a new lead, SID 88095709.
[0042] FIG. 7 illustrates the effect of R.sub.3 alkyl substitution
on compounds of the invention.
[0043] FIG. 8 depicts refinement of structure based on efflux and
associated potentiation and toxicity data.
[0044] FIG. 9 illustrates efflux inhibition and chemotherapeutic
potentiation of SID 88095709. [0045] A) A representative curve
showing efflux inhibition of ABCB1 in Jurkat-DNR cells (closed
circles). The average IC.sub.50 (n=3) is 4.7.+-.0.5 B) A
representative curve showing efflux inhibition of ABCG2 in Ig-MXP3
cells (open circles). The average IC.sub.50 (n=2) is 0.13.+-.0.30
.mu.M) Potentiation of DNR mediated killing in Jurkat-DNR cells
with SID 88095709 (n=2 per data point). The CR.sub.50 (closed
triangles) is 0.55 .mu.M while the TD.sub.50 (closed squares) is
5.5 .mu.M. Minimum allowable toxicity is set at 15 .mu.M, thus the
toxicity here is below the cut-off. D) Potentiation of MTX mediated
killing in Ig-MXP3 cells (n=2 per data point). The CR.sub.50 (open
triangles) is 0.31 .mu.M while the TD.sub.50 (open squares) is 18.3
.mu.M. The minimum toxicity and the CR.sub.50/TD.sub.50 ratio
(equal to 59) meet the cut-off criteria for a desirable compound in
the chemoreversal secondary ABCG2 assay.
[0046] FIG. 10 depicts the response of ABCG2 resistant tumors in
mice to combination therapy of TPT and ABCG2 inhibitors. The tumor
size at 0, 24, 48, and 72 hours is indicated.
[0047] FIG. 11 summarizes a comparison of known compounds to SID
88095709, 85752814, and 97301789.
[0048] FIG. 12 is a map of the computed solubility of selected
compounds in the PLS principal component space. Only prior art
compounds and compounds tested in the secondary assays have been
included. Most of the newly synthesized compounds show higher
computed solubility (green) than prior art compounds which show a
low one (red). SID 88095709 is marked with a red circle.
[0049] FIG. 13 (data on page 1 of disclosure) presents SAR data for
SID 88095709, SID 85752814, and SID 97301789.
[0050] FIG. 14 shows Table 1, which summarizes SAR expansion on
initial hit SID 85240370.
[0051] FIG. 15 shows Table 2, which summarizes a continuation of
SAR expansion on initial hit SID 85240370.
[0052] FIG. 16 shows Table 3, which summarizes a series of
modifications of formula (I) variable R.sub.1.
[0053] FIG. 17 shows Table 4, which summarizes a series of
modifications of formula (I) variable R.sub.2.
[0054] FIG. 18 shows Table 5, which summarizes a series of
modifications of formula (I) variable R.sub.4.
[0055] FIG. 19 shows Table 6, which summarizes piperazine and
combined R4 modifications (X).
[0056] FIG. 20 shows Table 7, which summarizes analogues with
R.sub.1-R.sub.4 substitution patterns in combination.
[0057] FIG. 21 shows Table 8, which summarizes the percent of
activity remaining for various kinases when inhibited by SID
88095709.
[0058] Figure A1 illustrates LCMS purity data at 215 nm for SID
88095709; LCMS retention time: 3.20 min; purity at 215 nm=100%.
[0059] Figure A2 shows HRMS data for SID 88095709; HRMS m/z
calculated for C.sub.25H.sub.22N.sub.5O.sub.3 [M.sup.++H]:
440.1717, found 440.1715.
[0060] FIG. 1A (Example 2). Structures of small molecules chosen
for direct experimental comparison. Probe compound CID44640177 (1),
ABCB1 inhibitor XR9051 (2), ABCG2 inhibitors FTC (3) and Ko143 (4),
ABCC1 inhibitor MK571 (5), and the pyrazolopyrimidine reversan
(6).
[0061] FIG. 2A (Example 2). Representative synthetic route for
compound 1. (A) methyl 3-(furan-2-yl)-3-oxopropanoate, AcOH,
100.degree. C., 2 hr (65% yield). (B) POCl.sub.3, BnEt.sub.3NCl,
PhNMe.sub.2, CH.sub.3CN, 80.degree. C., 16 hr (84% yield). (C)
furan-3-yl(piperazin-1-yl)methanone, DIPEA, CH.sub.3CN, 100.degree.
C., 16 hr (99% yield). (D) diethylmalonate (21% yield) NaOEt, EtOH,
80.degree. C., 3 hr (75% yield). (E) POCl.sub.3,
N,N-dimethylaniline, 115.degree. C., 16 hr (42% yield). (F)
potassium aryltrifluoroborate salt, Pd(OAc).sub.2, RuPhos,
Na.sub.2CO.sub.3, EtOH, MWI, 90.degree. C., 6 hr.
[0062] FIG. 3A (Example 2). General scheme for the duplex HTS flow
cytometric screening campaign. (A) In 384 well format, 1 .mu.M JC-1
in PBS is added to the assay wells. (B) A volume of 100 nL of test
compound is added to each well via pintool transfer (final
concentration of 6.6 .mu.M). (C) A 3.times.10.sup.6 cells mL.sup.-1
mixture of both cell lines is added to each well. The ABCB1 cell
line was previously color-coded with CellTrace.TM. Far Red DDAO-SE
prior to mixture with the unlabeled ABCG2 line. (D) Flow cytometric
data of light scatter and fluorescence emission at 530+/-20 nm (488
nm excitation, FL1) and 665+/-10 nm (633 nm excitation, FL8) are
then collected via HyperCyt.RTM.. Each population is gated in FL8
allowing for analysis of FL1 in individual time bins for each cell
line. The JC-1 retention can then be quantified as an indication of
efflux inhibition by test compound(s). The FL1 versus time excerpt
shown represents 24 binned wells of a 384 well plate.
[0063] FIG. 4A (Example 2). Scaffold modification summary from
primary hit to probe compound. (A) Screening hit compound 7
(CID1434724) and regions of targeted SAR optimization (shaded
areas). (B) Compound 8 (CID1441553) obtained from first-generation
SAR optimization. (C) SAR refinement of ABCG2 selectivity leading
to compound 1 (CID44640177).
[0064] FIG. 5A (Example 2). Efflux inhibition and chemotherapeutic
potentiation of 1 (CID 44640177). (A) A representative curve
showing efflux inhibition of ABCB1 in Jurkat-DNR cells (closed
circles). The average IC.sub.50 (n=3) is 4.65.+-.0.74 .mu.M. (B) A
representative curve showing efflux inhibition of ABCG2 in Ig-MXP3
cells (open circles). The average IC.sub.50 (n=2) is 0.13.+-.0.03
.mu.M (C) Potentiation of daunorubicin (DNR) mediated killing in
Jurkat-DNR cells with 1 (n=2 per data point). The CR.sub.50 (closed
triangles) is 0.55 .mu.M while the TD.sub.50 (closed squares) is
5.52 .mu.M. Minimum allowable toxicity is set at 15 .mu.M, thus the
toxicity here is below the cut-off (D) Potentiation of mitoxantrone
(MTX) mediated killing in Ig-MXP3 cells (n=2 per data point). The
CR.sub.50 (open triangles) is 0.31 .mu.M while the TD.sub.50 (open
squares) is 18.30 .mu.M. The minimum toxicity and the
CR.sub.50/TD.sub.50 ratio (equal to 59) meet the cut-off criteria
for a desirable compound in the chemoreversal secondary ABCG2
assay.
[0065] FIG. 6A (Example 2). Response of ABCG2 resistant Igrov1/T8
derived tumors in mice to combination therapy of 150 nM topotecan
(TPT). The tumor size at 0, 24, 48, 72, and 96 hr is indicated
(n=3) along with the standard error of the mean (SEM). The
significant difference between the mean values from 0 to 96 hr is
indicated by an asterisk (p<0.001). Inhibitor concentration was
selected based on potentiation efficacy balanced with apparent
cellular toxicity. Significant tumor reduction was noted in both
cases. (A) Compound 7 (original MLSMR hit) at 500 nM in conjunction
with TPT. (B) Probe compound 1 at 100 nM with TPT.
[0066] FIG. 1B (Example 3). Time course of injection of 100 nM
Topotecan in conjunction with 100 nM compound SAI 88095709 into
mice (n=5). Chemotherapeutic (topotecan) resistant Igrov1/TB cells
over-expressing ATP binding Cassette G2 (ABCG2) were xenografted
subcutaneously into the hind limbs of CB-17/SCID mice. The mice
were injected intra-tumorally with 100 nM topotecan in conjunction
with 100 nM compound 709 (SID 88095709) every 24 hours. The affect
of this combination therapy is shown over a period of 6 days (144
hours). Tumor size was reduced by 80% (p<0.001). No reduction in
size was observed in tumors treated with either 100 nM topotecan or
100 nM compound 709 alone.
[0067] FIG. 2B (Example 3). Time course of injection of 100 nM
Topotecan in conjunction with 500 nM compound SAI 85752814 into
mice (n=5). Chemotherapeutic (topotecan) resistant Igrov1/T8 cells
over-expressing ATP Binding Cassette G2 (ABCG2) were xenografted
subcutaneously into the hind limbs of CB-17/SCID mice. The mice
were injected intra-tumorally with 100 nM topotecan in conjunction
with 500 nM compound #37 (SID 85752814) every 24 hours. The affect
of this combination therapy is shown above a period of 5 days (120
hours). Tumor size was reduced by 81% (p<0.001). No reduction in
size was observed in tumors treated with either 100 nM topotecan or
500 nM compound #37 alone.
[0068] FIG. 3B (Example 3). Time course of injection of 100 nM
Topotecan in conjunction with 100 nM compound SAI 97301789 into
mice (n=5). Chemotherapeutic (topotecan) resistant Igrov1/T8 cells
over-expressing ATP Binding Cassette G2 (ABCG2) were xenografted
subcutaneously into the hind limbs of CB-17/SCID mice. The mice
were injected intra-tumorally with 100 nM topotecan in conjunction
with 100 nM compound 789 (SID 97301789) every 24 hours. The affect
of this combination therapy is shown above a period of 5 days (120
hours). Tumor size was reduced by 55% (p<0.001). No reduction in
size was observed in tumors treated with either 100 nM topotecan or
500 nM compound 789 alone.
DEFINITIONS
[0069] The following terms are used throughout the specification to
describe the present invention. Where a term is not given a
specific definition herein, that term is to be given the same
meaning as understood by those of ordinary skill in the art. The
definitions given to the disease states or conditions which may be
treated using one or more of the compounds according to the present
invention are those which are generally known in the art.
[0070] "Alkyl" refers to a fully saturated monovalent radical
containing carbon (C.sub.1-C.sub.12) and hydrogen, and which may be
cyclic, branched or a straight chain. Examples of alkyl groups are
methyl, ethyl, n-butyl, n-hexyl, n-heptyl, n-octyl, n-nonyl,
n-decyl, isopropyl, 2-methylpropyl, cyclopropyl, cyclopropylmethyl,
cyclobutyl, cyclopentyl, cyclopentylethyl, cyclohexylethyl and
cyclohexyl. Preferred alkyl groups are C.sub.1-C.sub.6 alkyl
groups. "Alkylene" refers to a fully saturated hydrocarbon which is
divalent (may be linear, branched or cyclic) and which is
optionally substituted. "Alkenyl" refers to an unsaturated
aliphatic hydrocarbon containing at least one double bond.
"Alkynyl" means an unsaturated aliphatic hydrocarbon containing at
least one triple bond. Preferred alkylene groups include
C.sub.1-C.sub.6 alkylene groups. Other terms used to indicate
substituent groups in compounds according to the present invention
are as conventionally used in the art.
[0071] "Aryl" or "aromatic", in context, refers to a substituted or
unsubstituted monovalent aromatic radical having a single ring
(e.g., benzene) or multiple condensed rings (e.g., naphthyl,
anthracenyl, phenanthryl) and can be can be bound to the compound
according to the present invention at any position on the ring(s)
or as otherwise indicated in the chemical structure presented.
Other examples of aryl groups, in context, may include heterocyclic
aromatic ring systems, "heteroaryl" groups having one or more
nitrogen, oxygen, or sulfur atoms in the ring (moncyclic) such as
imidazole, furyl, pyrrole, pyridyl, furanyl, thiene, thiazole,
pyridine, pyrimidine, pyrazine, triazole, oxazole, indole or fused
ring systems (bicyclic, tricyclic), among others, which may be
substituted or unsubstituted as otherwise described herein.
[0072] The term "cyclic" shall refer to an optionally substituted
carbocyclic or heterocyclic group, preferably a 5- or 6-membered
ring or fused rings (two or three rings) preferably containing from
8 to 14 atoms. A heterocyclic ring or group shall contain at least
one monocyclic ring containing between 3 and 7 atoms of which up to
four of those atoms are other than carbon and are selected from
nitrogen, sulfur and oxygen. Carbocyclic and heterocyclic rings
according to the present invention may be unsaturated or saturated.
Preferred carbocyclic groups are unsaturated, and include phenyl
groups, among other groups. Preferred heterocyclic groups are
heteroaryl or heteroaromatic.
[0073] The term "heterocyclic group" as used throughout the present
specification refers to an aromatic or non-aromatic cyclic group
having 3 to 14 atoms, preferably 5 to 14 atoms forming the cyclic
ring(s) and including at least one hetero atom such as nitrogen,
sulfur or oxygen among the atoms forming the cyclic ring, which is
an aromatic heterocyclic group (also, "heteroaryl" or
"heteroaromatic") in the former case and a "non-aromatic
heterocyclic group" in the latter case. Specific examples of the
heterocyclic group therefore include specific examples of the
aromatic heterocyclic group and specific examples of the
non-aromatic heterocyclic group, both of which groups fall under
the rubric "heterocyclic group" as otherwise described herein.
Among the heterocyclic groups which may be mentioned for use in the
present invention within context include nitrogen-containing
aromatic heterocycles such as pyrrole, pyridine, pyridone,
pyridazine, pyrimidine, pyrazine, pyrazole, imidazole, triazole,
tetrazole, indole, isoindole, indolizine, purine, indazole,
quinoline, isoquinoline, quinolizine, phthalazine, naphthyridine,
quinoxaline, quinazoline, cinnoline, pteridine, imidazopyridine,
imidazotriazine, pyrazinopyridazine, acridine, phenanthridine,
carbazole, carbazoline, perimidine, phenanthroline, phenacene,
oxadiazole, benzimidazole, pyrrolopyridine, pyrrolopyrimidine and
pyridopyrimidine; sulfur-containing aromatic heterocycles such as
thiophene and benzothiophene; oxygen-containing aromatic
heterocycles such as furan, pyran, cyclopentapyran, benzofuran and
isobenzofuran; and aromatic heterocycles comprising 2 or more
hetero atoms selected from among nitrogen, sulfur and oxygen, such
as thiazole, thiadizole, isothiazole, benzoxazole, benzothiazole,
benzothiadiazole, phenothiazine, isoxazole, furazan, phenoxazine,
pyrazoloxazole, imidazothiazole, thienofuran, furopyrrole,
pyridoxazine, furopyridine, furopyrimidine, thienopyrimidine and
oxazole. As examples of the "5- to 14-membered aromatic
heterocyclic group" there may be mentioned preferably, pyridine,
triazine, pyridone, pyrimidine, imidazole, indole, quinoline,
isoquinoline, quinolizine, phthalazine, naphthyridine, quinazoline,
cinnoline, acridine, phenacene, thiophene, benzothiophene, furan,
pyran, benzofuran, thiazole, benzthiazole, phenothiazine,
pyrrolopyrimidine, furopyridine and thienopyrimidine, more
preferably pyridine, thiophene, benzothiophene, thiazole,
benzothiazole, quinoline, quinazoline, cinnoline,
pyrrolopyrimidine, pyrimidine, furopyridine and thienopyrimidine.
The term "heterocyclic group" shall generally refer to 3 to
14-membered heterocyclic groups and all subsets of heterocyclic
groups (including non-heteroaromatic or heteroaromatic) subsumed
under the definition of heterocyclic group.
[0074] Among the heterocyclic groups for use in the present
invention may preferably include pyrrolidinyl, piperidinyl,
morpholinyl, pyrrole, pyridine, pyridone, pyrimidine, imidazole,
indole, quinoline, isoquinoline, quinolizine, phthalazine,
naphthyridine, quinazoline, cinnoline, acridine, phenacene,
thiophene, benzothiophene, furan, pyran, benzofuran, thiazole,
benzothiazole, phenothiazine and carbostyryl, more preferably
pyrrolidinyl, piperidinyl, morpholinyl, pyrrole, pyridine,
pyridine-N-oxide, thiophene, benzothiophene, thiazole,
benzothiazole, quinoline, quinazoline, cinnoline and carbostyryl,
and even more preferably thiazole, quinoline, quinazoline,
cinnoline and carbostyryl, among others.
[0075] Among the bicyclic or tricyclic heterocyclic groups which
may be used in the present invention include indole, isoindole,
indolizine, purine, indazole, quinoline, isoquinoline, quinolizine,
phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline,
pteridine, imidazopyridine, imidazotriazine, pyrazinopyridazine,
acridine, phenanthridine, carbazole, carbazoline, perimidine,
phenanthroline, phenacene, benzimidazole, pyrrolopyridine,
pyrrolopyrimidine and pyridopyrimidine; sulfur-containing aromatic
heterocycles such as thiophene and benzothiophene;
oxygen-containing aromatic heterocycles such as cyclopentapyran,
benzofuran and isobenzofuran; and aromatic heterocycles comprising
2 or more hetero atoms selected from among nitrogen, sulfur and
oxygen, such as benzoxazole, benzothiazole, benzothiadiazole,
phenothiazine, benzofurazan, phenoxazine, pyrazoloxazole,
imidazothiazole, thienofuran, furopyrrole, pyridoxazine,
furopyridine, furopyrimidine and thienopyrimidine, among
others.
[0076] The term "substituted" shall mean substituted at a carbon
(or nitrogen) position within context, hydroxyl, carboxyl, cyano
(CN), nitro (NO.sub.2), halogen (preferably, 1, 2 or 3 halogens,
especially on an alkyl, especially a methyl group such as a
trifluoromethyl), thiol, an optionally substituted alkyl, alkene or
alkyne group (preferably, C.sub.1-C.sub.6, C.sub.2-C.sub.6, more
preferably C.sub.1-C.sub.3, C.sub.2-C.sub.3), optionally
substituted aryl (especially optionally substituted phenyl or
benzyl), optionally substituted heterocyclic (especially optionally
substituted heteroaryl for example, pyridyl (2-, 3-, 4-),
pyrimidinyl, thienyl (2- or 3-), furanyl (2- or 3-), alkoxy
(preferably, C.sub.1-C.sub.6 alkyl or aryl), optionally substituted
ether (preferably, C.sub.1-C.sub.10 alkyl ether, alkenylether,
alkynyl ether or aryl ether, including phenyl or benzyl ether),
acyl (preferably C.sub.2-C.sub.8 acyl which may include an aryl
substituted acyl), optionally substituted ester (preferably,
C.sub.1-C.sub.6 alkyl or aryl) including alkylene, alkenyl or
alkynyl ester (alkylene attachment to compound), ketoester
(carbonyl attachment to compound) or hydroxyester (oxygen
attachment to compound), thioether (preferably, C.sub.1-C.sub.6
alkyl or aryl), thioester (preferably C .sub.1-C.sub.6 alkyl or
aryl), amine (including a five- or six-membered cyclic alkylene
amine, including an optionally substituted C.sub.1-C.sub.6 alkyl
amine (e.g., monoalkanolamine) or an optionally substituted
C.sub.1-C.sub.6 dialkyl amine (e.g. dialkanolamine), alkanol
(preferably, C.sub.1-C.sub.6 alkyl or aryl), or alkanoic acid
(preferably, C.sub.1-C.sub.6 alkyl or aryl), optionally substituted
carboxyamide (carbonyl attached to the carbon atom with one or two
substituents on the amine group--preferably H or an optionally
substituted C.sub.1-C.sub.6 alkyl group), amido group (amine group
with H or C.sub.1-C.sub.3 alkyl group attached to the carbon atom
with a single group, preferably H or an optionally substituted
C.sub.1-C.sub.6 alkyl group on the keto group) or an optionally
substituted urethane group (with either the amine or the O-carboxy
group attached to a carbon atom to which the urethane is a
substituent--the amine group being substituted with one or two H or
one or two C.sub.1-C.sub.6 alkyl groups). Preferably, the term
"substituted" shall mean within the context of its use alkyl,
alkoxy, halogen, hydroxyl, carboxylic acid, cyano, ether, ester,
acyl, nitro, amine (including mono- or di-alkyl substituted amines)
and amide, as otherwise described above. Any substitutable position
in a compound according to the present invention may be substituted
in the present invention. Preferably no more than 5, more
preferably no more than 3 substituents are present on a single ring
or ring system. Preferably, the term "unsubstituted" shall mean
substituted with one or more H atoms. It is noted that in
describing a substituent, all stable permutations of the
substituent are intended.
[0077] Preferred substituents for use in the present invention
include, for example, F, Cl, CN, NO.sub.2, CH.sub.3, CH.sub.2OH,
CH.sub.2CH.sub.3, CH.sub.2OCH.sub.3, CF.sub.3, CO.sub.2CH.sub.3,
optionally substituted thienyl, optionally substituted furanyl
(especially CH.sub.2OCH.sub.2-furanyl), optionally substituted
pyridyl (especially CH.sub.2OCH.sub.2-pyridyl), optionally
substituted pyrimidyl and optionally substituted phenyl, including
benzyl (CH.sub.2OCH.sub.2-phenyl).
[0078] The term "patient" or "subject" is used throughout the
specification to describe an animal, preferably a human, to whom
treatment, including prophylactic treatment, with the compositions
according to the present invention is provided. For treatment of
those infections, conditions or disease states which are specific
for a specific animal such as a human patient, the term patient
refers to that specific animal.
[0079] The term "compound" is used herein to refer to any specific
chemical compound disclosed herein. Within its use in context, the
term generally refers to a single small molecule as disclosed
herein, but in certain instances may also refer to stereoisomers
and/or optical isomers (including racemic mixtures) of disclosed
compounds. The term compound or "agent" includes active metabolites
of compounds and/or pharmaceutically active salts thereof.
[0080] The term "inhibitor" is used herein to refer to any compound
which inhibits cancer-associated transporter proteins (e.g.
especially inhibition of ABCG2, but also, in certain instances,
ABCC1 or ABCB1, or inhibition which is selective toward ABCG2 over
ABCB1 or which produces an inhibition of ABCB1 transporter protein
by any mechanism, direct or indirect, whether it be by inhibition
of the interaction of ABCG2 and/or ABCB1 transporter proteins with
their intended receptor or other target or whether it be by
inhibition of the expression of ABCG2 and/or ABCB1 transporter
proteins).
[0081] The term "effective amount" is used throughout the
specification to describe concentrations or amounts of compounds or
other components which are used in amounts, within the context of
their use, to produce an intended effect according to the present
invention. The compound or component may be used to produce a
favorable change in a disease or condition treated, whether that
change is a remission, a favorable physiological result, a reversal
or attenuation of a disease state or condition treated, the
prevention or the reduction in the likelihood of a condition or
disease-state occurring, depending upon the disease or condition
treated. Where compounds are used in combination, each of the
compounds is used in an effective amount, wherein an effective
amount may include a synergistic amount. In many instances, the
term effective amount refers to that amount which inhibits
expression of ABCG2 and/or ABCB1 transporter proteins and
consequently, results in a diminution of resistance to a
therapeutic approach, to symptoms or results in an actual cure of a
disease state such as cancer, which cancer may include drug
resistant cancer, especially a multiple drug resistant (MDR)
cancer, a cancer such as a leukemia or a cancerous tumor,
especially including T-lineage Acute lymphoblastic Leukemia
(T-ALL), T-lineage lymphoblastic Lymphoma (T-LL), Peripheral T-cell
lymphoma, Adult T-cell Leukemia, Pre-B ALL, Pre-B Lymphomas, Large
B-cell Lymphoma, Burkitts Lymphoma, B-cell ALL, Philadelphia
chromosome positive ALL and Philadelphia chromosome positive CML,
among others.
[0082] In the present invention all compounds are used in effective
amounts to provide activity relevant to the use of the compound. In
combination therapy, the cancer-associated transporter
protein-inhibiting compounds (e.g. ABCG2 and/or ABCB1 transporter
protein inhibitors) and the anticancer agent are both used in
effective amounts. The amount of compound used in the present
invention may vary according to the nature of the compound, the age
and weight of the patient and numerous other factors which may
influence the bioavailability and pharmacokinetics of the compound,
the amount of compound which is administered to a patient generally
ranges from about 0.001 mg/kg to about 50 mg/kg or more, about 0.5
mg/kg to about 25 mg/kg, about 0.1 to about 15 mg/kg, about 1 mg to
about 10 mg/kg per day and otherwise described herein. The person
of ordinary skill may easily recognize variations in dosage
schedules or amounts to be made during the course of therapy.
[0083] The term "cancer-associated transporter protein mediated
disease" is used throughout the specification to describe a disease
which is mediated through the action or overexpression of any
cancer-related transporter protein, e.g. any or all of the ABCG2,
ABCC1 and ABCB1 transporter proteins, or where the overexpression
of such transporter proteins occurs in conjunction with the disease
state. Diseases which may be treated according to the present
invention include a cancerous disease state, in particular, a drug
resistant cancer, a multiple drug resistant cancer, a leukemia or
related hematopoietic cancer, including T-ALL and related
leukemias, especially drug resistant (multiple) leukemias, such as
T-ALL, and numerous cancerous tumors as otherwise described herein.
These diseases may include any one or more of hematopoietic
neoplasms and metastasis of such neoplasms, including Hodgkin's
disease, non-Hodgkin's lymphoma, leukemias, including non-acute and
acute leukemias, such as acute myelogenous leukemia, acute
lymphocytic leukemia, acute promyelocytic leukemia (APL), acute
T-cell lymphoblastic leukemia, T-lineage acute lymphoblastic
leukemia (T-ALL), adult T-cell leukemia, basophilic leukemia,
eosinophilic leukemia, granulocytic leukemia, hairy cell leukemia,
leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia,
lymphocytic leukemia, megakaryocytic leukemia, micromyeloblastic
leukemia, monocytic leukemia, neutrophilic leukemia and stem cell
leukemia. Other cancers, including cancerous tumors, which may be
treated using the present invention include for example, stomach
(especially including gastric stromal cells), colon, rectal, liver,
pancreatic, lung, breast, cervix uteri, corpus uteri, ovary,
prostate, testis, bladder, renal, brain/CNS, head and neck, throat,
Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma,
leukemia, skin cancer, including melanoma and non-melanoma, acute
lymphocytic leukemia, acute myelogenous leukemia, Ewing's sarcoma,
small cell lung cancer, choriocarcinoma, rhabdomyosarcoma, Wilms'
tumor, neuroblastoma, hairy cell leukemia, mouth/pharynx,
oesophagus, larynx, kidney cancer and lymphoma, among others.
Additional cancers which may be particularly responsive to
therapeutic methods according to the present invention include for
example, T-lineage Acute lymphoblastic Leukemia (T-ALL), T-lineage
lymphoblastic Lymphoma (T-LL), Peripheral T-cell lymphoma, Adult
T-cell Leukemia, Pre-B ALL, Pre-B Lymphomas, Large B-cell Lymphoma,
Burkitts Lymphoma, B-cell ALL, Philadelphia chromosome positive ALL
and Philadelphia chromosome positive CML, breast cancer, Ewing's
sarcoma, osteosarcoma and undifferentiated high-grade sarcomas,
among others.
[0084] The term "neoplasia" or "neoplasm" is used throughout the
specification to refer to the pathological process that results in
the formation and growth of a cancerous or malignant neoplasm,
i.e., abnormal tissue that grows by cellular proliferation, often
more rapidly than normal and continues to grow after the stimuli
that initiated the new growth cease. Malignant neoplasms show
partial or complete lack of structural organization and functional
coordination with the normal tissue and may invade surrounding
tissues. As used herein, the term neoplasia/neoplasm is used to
describe all cancerous disease states and embraces or encompasses
the pathological process associated with cancer, including
hematopoietic cancers, numerous cancerous tumors and their
metastasis.
[0085] A "hematopoietic neoplasm" or "hematopoietic cancer" is a
neoplasm or cancer of hematopoeitic cells of the blood or lymph
system and includes disease states such as Hodgkin's disease,
non-Hodgkin's lymphoma, leukemias, including non-acute and acute
leukemias, such as acute myelogenous leukemia, acute lymphocytic
leukemia, acute promyelocytic leukemia (APL), adult T-cell
leukemia, T-lineage acute lymphoblastic leukemia (T-ALL),
basophilic leukemia, eosinophilic leukemia, granulocytic leukemia,
hairy cell leukemia, leukopenic leukemia, lymphatic leukemia,
lymphoblastic leukemia, lymphocytic leukemia, megakaryocytic
leukemia, micromyeloblastic leukemia, monocytic leukemia,
neutrophilic leukemia and stem cell leukemia, among others.
[0086] The present method may be used to treat all cancers
(including drug resistant and multiple drug resistant cancers),
especially the above hematopoietic and tumorigenic cancers which
exhibit an overexpression of ABCG2 and/or ABCB1 transporter
proteins. While T-ALL and especially multiple drug resistant T-ALL
are particularly relevant disease targets for the methods of the
present invention, virtually any cancer implicating any
cancer-related transporter protein, e.g. any or all of the ABCG2,
ABCC1 and ABCB1 transporter proteins, or where any of the ABCG2,
ABCC1 and ABCB1 transporter proteins are implicated in instilling
drug resistance or multiple drug resistance to the cancer and/or
tumor is an appropriate target of the present therapeutic methods
and compositions according to the present invention. Cancers which
are particularly response to the present invention include
T-lineage Acute lymphoblastic Leukemia (T-ALL), T-lineage
lymphoblastic Lymphoma (T-LL), Peripheral T-cell lymphoma, Adult
T-cell Leukemia, Pre-B ALL, Pre-B Lymphomas, Large B-cell Lymphoma,
Burkitts Lymphoma, B-cell ALL, Philadelphia chromosome positive ALL
and Philadelphia chromosome positive CML, breast cancer, Ewing's
sarcoma, osteosarcoma and undifferentiated high-grade sarcomas,
among others. Other cancers which may be treated according to the
present invention include for example, stomach (especially
including gastric stromal cells), colon, rectal, liver, pancreatic,
lung, breast, cervix uteri, corpus uteri, ovary, prostate, testis,
bladder, renal, brain/CNS, head and neck, throat, Hodgkin's
disease, non-Hodgkin's lymphoma, multiple myeloma, leukemia, skin
cancer, including melanoma and non-melanoma, acute lymphocytic
leukemia, acute myelogenous leukemia, Ewing's sarcoma, small cell
lung cancer, choriocarcinoma, rhabdomyosarcoma, Wilms' tumor,
neuroblastoma, hairy cell leukemia, mouth/pharynx, oesophagus,
larynx, kidney cancer and lymphoma, among others, including drug
resistant (DR) and multiple drug resistant (MDR) forms of each of
these cancers.
[0087] The term "prophylactic" is used to describe the use of a
compound described herein which reduces the likelihood of an
occurrence of a condition or disease state in a patient or subject.
The term "reducing the likelihood" refers to the fact that in a
given population of patients, the present invention may be used to
reduce the likelihood of an occurrence, recurrence or metastasis of
disease in one or more patients within that population of all
patients, rather than prevent, in all patients, the occurrence,
recurrence or metastasis of a disease state.
[0088] The term "pharmaceutically acceptable" refers to a salt form
or other derivative (such as an active metabolite or prodrug form)
of the present compounds or a carrier, additive or excipient which
is not unacceptably toxic to the subject to which it is
administered.
[0089] The term "cancer" is used throughout the specification to
refer to the pathological process that results in the formation and
growth of a cancerous or malignant neoplasm, i.e., abnormal tissue
that grows by cellular proliferation, often more rapidly than
normal and continues to grow after the stimuli that initiated the
new growth cease. Malignant neoplasms show partial or complete lack
of structural organization and functional coordination with the
normal tissue and most invade surrounding tissues, metastasize to
several sites, and are likely to recur after attempted removal and
to cause the death of the patient unless adequately treated. As
used herein, the term neoplasia is used to describe all cancerous
disease states and embraces or encompasses the pathological process
associated with malignant hematogenous, ascetic and solid
tumors.
[0090] It is noted that, as used in this specification and the
appended claims, the singular forms "a," "an," and "the," include
plural referents unless expressly and unequivocally limited to one
referent. Thus, for example, reference to "a compound" includes two
or more different compound. As used herein, the term "include" and
its grammatical variants are intended to be non-limiting, such that
recitation of items in a list is not to the exclusion of other like
items that can be substituted or other items that can be added to
the listed items.
DETAILED DESCRIPTION OF THE INVENTION
[0091] The present disclosure provides novel small molecules that
inhibit cancer-related transporter proteins, e.g. ABCG2, ABCC1 and
ABCB1 transporter proteins, in cancer disease states, especially
hematopoietic cancers and cancerous tumors as otherwise described
herewith especially including those which are drug resistant,
especially those disease states which are drug resistant as a
consequence of overexpression of ABCG2, ABCC1 or ABCB1 transporter
protein. Various cancers as otherwise described herein may be
treated using the methods of the present invention, especially
including cancers exhibiting multiple drug resistance. Particularly
responsive cancers to the present methods include, for example,
T-lineage Acute lymphoblastic Leukemia (T-ALL), T-lineage
lymphoblastic Lymphoma (T-LL), Peripheral T-cell lymphoma, Adult
T-cell Leukemia, Pre-B ALL, Pre-B Lymphomas, Large B-cell Lymphoma,
Burkitts Lymphoma, B-cell ALL, Philadelphia chromosome positive ALL
and Philadelphia chromosome positive CML, breast cancer, Ewing's
sarcoma, osteosarcoma and undifferentiated high-grade sarcomas,
among others.
[0092] The present invention also relates to an identification of
the unexpected activity of compounds which are well known in the
art, but have heretofore not been known to be inhibitors of ABCG2,
ABCC1 and ABCB1. Methods of making these compounds and
incorporating these compounds into pharmaceutical compositions are
well known in the art. Pharmaceutically acceptable salts prepared
from the active compounds are readily prepared. The present
invention is not limited in any way by the method of synthesis of
compounds, but encompasses all small molecules otherwise identified
that may be produced by any suitable method of synthesis. Compounds
may be synthesized step-wise by first synthesizing various synthons
and then condensing the synthons together to produce compounds
according to the invention. The synthesis of compounds according to
the present invention is well within the routine skill of the
person of ordinary skill. If desired, intermediates and products
may be purified by chromatography and/or recrystallization.
Starting materials, intermediates and reagents are either
commercially available or may be prepared by one skilled in the art
using methods described in the relevant chemical literature. Most
of the compounds which are used therapeutically in the present
invention are known in the art, as are the methods of their
synthesis.
[0093] The present invention includes, where applicable, the
compositions comprising the pharmaceutically acceptable salts, in
particular, acid or base addition salts of compounds of the present
invention. The acids which are used to prepare the pharmaceutically
acceptable acid addition salts of the aforementioned base compounds
useful in this invention are those which form non-toxic acid
addition salts, i.e., salts containing pharmacologically acceptable
anions, such as the hydrochloride, hydrobromide, hydroiodide,
nitrate, sulfate, bisulfate, phosphate, acid phosphate, acetate,
lactate, citrate, acid citrate, tartrate, bitartrate, succinate,
maleate, fumarate, gluconate, saccharate, benzoate,
methanesulfonate, ethanesulfonate, benzenesulfonate,
p-toluenesulfonate and pamoate [i.e.,
1,1'-methylene-bis-(2-hydroxy-3 naphthoate)]salts, among numerous
others.
[0094] Pharmaceutically acceptable base addition salts may also be
used to produce pharmaceutically acceptable salt forms of the
compounds or derivatives according to the present invention. The
chemical bases that may be used as reagents to prepare
pharmaceutically acceptable base salts of the present compounds
that are acidic in nature are those that form non-toxic base salts
with such compounds. Such non-toxic base salts include, but are not
limited to those derived from such pharmacologically acceptable
cations such as alkali metal cations (eg., potassium and sodium)
and alkaline earth metal cations (eg, calcium, zinc and magnesium),
ammonium or water-soluble amine addition salts such as
N-methylglucamine-(meglumine), and the lower alkanolammonium and
other base salts of pharmaceutically acceptable organic amines,
among others.
[0095] The compounds of the present invention may, in accordance
with the invention, be administered in single or divided doses by
the oral, parenteral or topical routes. Administration of the
active compound may range from continuous (intravenous drip) to
several oral administrations per day (for example, Q.I.D.) and may
include oral, topical, parenteral, intramuscular, intravenous,
sub-cutaneous, transdermal (which may include a penetration
enhancement agent), buccal, sublingual and suppository
administration, among other routes of administration. Enteric
coated oral tablets may also be used to enhance bioavailability of
the compounds from an oral route of administration. The most
effective dosage form will depend upon the pharmacokinetics of the
particular agent chosen as well as the severity of disease in the
patient. Administration of compounds according to the present
invention as sprays, mists, or aerosols for intra-nasal,
intra-tracheal or pulmonary administration may also be used. The
present invention therefore also is directed to pharmaceutical
compositions comprising an effective amount of compound according
to the present invention, optionally in combination with a
pharmaceutically acceptable carrier, additive or excipient.
Compounds according to the present invention may be administered in
immediate release, intermediate release or sustained or controlled
release forms. Sustained or controlled release forms are preferably
administered orally, but also in suppository and transdermal or
other topical forms. Intramuscular injections in liposomal form may
also be used to control or sustain the release of compound at an
injection site.
[0096] The amount compound which is used in the present invention,
whether that compound is an ABCG2, ABCC1 or ABCB1 transporter
protein inhibitor or an anticancer compound is that amount
effective within the context of the administration of the
compound(s). A suitable oral dosage for a compound of the present
invention would be in the range of about 0.01 mg to 10 g or more
per day, preferably about 0.1 mg to about 1 g per day. In
parenteral formulations, a suitable dosage unit may contain from
about 0.1 to about 250-500 mg of said compounds, which may be
administered continuously or from one to four times per day,
whereas for topical administration, formulations containing 0.01 to
1% or more by weight active ingredient are preferred. It should be
understood, however, that the dosage administered from patient to
patient will vary and the dosage for any particular patient will
depend upon the clinician's judgment, who will use as criteria for
fixing a proper dosage the size and condition of the patient as
well as the patient's response to the drug.
[0097] When the compounds of the present invention are to be
administered by oral route, they may be administered as medicaments
in the form of pharmaceutical preparations which contain them in
association with a compatible pharmaceutical carrier material. Such
carrier material can be an inert organic or inorganic carrier
material suitable for oral administration. Examples of such carrier
materials are water, gelatin, talc, starch, magnesium stearate, gum
arabic, vegetable oils, polyalkylene-glycols, petroleum jelly and
the like. Immediate release, intermediate release and sustained
and/or controlled release formulations are contemplated by the
present invention.
[0098] The pharmaceutical formulations/preparations according to
the present invention can be prepared in a conventional manner and
finished dosage forms can be solid dosage forms, for example,
tablets, dragees, capsules, and other like oral dosage forms, or
liquid dosage forms, for example solutions, suspensions, emulsions
and the like.
[0099] The pharmaceutical formulations/preparations may be
subjected to conventional pharmaceutical operations such as
sterilization. Further, the pharmaceutical preparations may contain
conventional additives and excipients such as preservatives,
stabilizers, emulsifiers, flavor-improvers, wetting agents,
buffers, salts for varying the osmotic pressure and the like. Solid
carrier material which can be used include, for example, starch,
lactose, mannitol, methyl cellulose, microcrystalline cellulose,
talc, silica, dibasic calcium phosphate, and high molecular weight
polymers (such as polyethylene glycol).
[0100] For parenteral use, a compound according to the present
invention can be administered in an aqueous (saline) or non-aqueous
solution, suspension or emulsion in a pharmaceutically acceptable
oil or a mixture of liquids, which may contain bacteriostatic
agents, antioxidants, preservatives, buffers or other solutes to
render the solution isotonic with the blood, thickening agents,
suspending agents or other pharmaceutically acceptable additives.
Additives of this type include, for example, tartrate, citrate and
acetate buffers, ethanol, propylene glycol, polyethylene glycol,
complex formers (such as EDTA), antioxidants (such as sodium
bisulfite, sodium metabisulfite, and ascorbic acid), high molecular
weight polymers (such as liquid polyethylene oxides) for viscosity
regulation and polyethylene derivatives of sorbitol anhydrides.
Preservatives may also be added if necessary, such as benzoic acid,
methyl or propyl paraben, benzalkonium chloride and other
quaternary ammonium compounds.
[0101] The compounds of this invention may also be administered as
solutions for nasal application and may contain in addition to the
compounds of this invention suitable buffers, tonicity adjusters,
microbial preservatives, antioxidants and viscosity-increasing
agents in an aqueous vehicle. Examples of agents used to increase
viscosity are polyvinyl alcohol, cellulose derivatives,
polyvinylpyrrolidone, polysorbates or glycerin. Preservatives added
may include benzalkonium chloride, chlorobutanol or phenylethyl
alcohol, among numerous others.
[0102] In certain aspects according to the present invention, where
various cancers are to be treated, the compounds may be
co-administered with at least one other anti-cancer agent such as
antimetabolites, Ara C, etoposide, doxorubicin, taxol, hydroxyurea,
vincristine, cytoxan (cyclophosphamide) or mitomycin C, among
numerous others, including topoisomerase I and topoisomerase II
inhibitors, such as adriamycin, topotecan, campothecin and
irinotecan, other agent such as gemcitabine and agents based upon
campothecin and cisplatin. By "co-administer" it is meant that the
present compounds are administered to a patient such that the
present compounds as well as the co-administered compound may be
found in the patient's bloodstream at the same time, regardless
when the compounds are actually administered, including
simultaneously. In many instances, the co-administration of the
present compounds with traditional anticancer agents produces a
synergistic (i.e., more than additive) result which is
unexpected.
[0103] Additional compounds which may be used in combination with
the compounds uncovered in the present invention include for
example: adriamycin, anastrozole, arsenic trioxide, asparaginase,
azacytidine, BCG Live, bevacizumab, bexarotene capsules, bexarotene
gel, bleomycin, bortezombi, busulfan intravenous, busulfan oral,
calusterone, campothecin, capecitabine, carboplatin, carmustine,
carmustine with polifeprosan 20 implant, celecoxib, cetuximab,
chlorambucil, cisplatin, cladribine, clofarabine, cyclophosphamide,
cytarabine, cytoxan, cytarabine liposomal, dacarbazine,
dactinomycin, actinomycin D, dalteparin sodium, darbepoetin alfa,
dasatinib, daunorubicin liposomal, daunorubicin, daunomycin,
decitabine, denileukin, denileukin diftitox, dexrazoxane,
dexrazoxane, docetaxel, doxorubicin, doxorubicin liposomal,
dromostanolone propionate, eculizumab, Elliott's B Solution,
epirubicin, epirubicin hcl, epoetin alfa, erlotinib, estramustine,
etoposide phosphate, etoposide VP-16, exemestane, fentanyl citrate,
filgrastim, floxuridine (intraarterial), fludarabine, fluorouracil
5-FU, fulvestrant, gefitinib, gemcitabine, gemcitabine hcl,
gemicitabine, gemtuzumab ozogamicin, goserelin acetate, goserelin
acetate, histrelin acetate, hydroxyurea, ibritumomab tiuxetan,
idarubicin, ifosfamide, imatinib mesylate, interferon alfa 2a,
interferon alfa-2b, irinotecan, lapatinib ditosylate, lenalidomide,
letrozole, leucovorin, leuprolide acetate, levamisole, lomustine
CCNU, meclorethamine, nitrogen mustard, megestrol acetate,
melphalan L-PAM, mercaptopurine 6-MP, mesna, methotrexate,
methoxsalen, mitomycin C, mitotane, mitoxantrone, nandrolone
phenpropionate, nelarabine, nofetumomab, oprelvekin, oxaliplatin,
paclitaxel, paclitaxel protein-bound particles, palifermin,
pamidronate, panitumumab, pegademase, pegaspargase, pegfilgrastim,
peginterferon alfa-2b, pemetrexed disodium, pentostatin,
pipobroman, plicamycin, mithramycin, porfimer sodium, procarbazine,
quinacrine, rasburicase, rituximab, sargramostim, sorafenib,
streptozocin, sunitinib, sunitinib maleate, talc, tamoxifen,
temozolomide, teniposide VM-26, testolactone, thalidomide,
thioguanine 6-TG, thiotepa, topotecan, topotecan hcl, toremifene,
tositumomab, tositumomab/I-131 tositumomab, trastuzumab, tretinoin
ATRA, uracil mustard, valrubicin, vinblastine, vincristine,
vinorelbine, vorinostat, zoledronate, zoledronic acid and mixtures
thereof.
[0104] In a pharmaceutical composition aspect of the present
invention at least one compound selected from the group consisting
of bepridil, lidoflazine, nicardipine, propafenone, rescinnamine,
GBR 12909, ellipticine, hexestrol, loxapine, pimozide, acacetin,
mometasone furoate or its active 6-.beta.-hydroxy metabolite,
ketoconazole and cyclosporin A, and mixtures thereof or their
pharmaceutically acceptable salts (preferably, bepridil,
nicardipine, propafenone, rescinnamine, ketoconazole, cyclosporine
A, loxapine, pimozide, acacetin, mometasone furoate, its active
6.beta.-hydroxy metabolite and mixtures thereof) is combined with
at least one compound according to the present invention and an
additional anticancer compound (agent) in an effective amount in
combination with a pharmaceutically acceptable carrier, additive or
excipient to treat cancer, or to reduce the likelihood of an
occurrence, a recurrence or metastasis of any one or more of the
cancers specifically identified in the present application.
[0105] The above identified compound(s) may be combined with at
least one agent selected from the group consisting of
antimetabolites, Ara C, etoposide, doxorubicin, taxol, hydroxyurea,
vincristine, cytoxan (cyclophosphamide) or mitomycin C, among
numerous others, including topoisomerase I and topoisomerase II
inhibitors, such as adriamycin, topotecan, campothecin and
irinotecan, other agent such as gemcitabine and agents based upon
campothecin and cisplatin for the treatment of cancer, as otherwise
described herein. Additional agents which may be combined in
pharmaceutical compositions according to the present invention
include, for example, adriamycin, anastrozole, arsenic trioxide,
asparaginase, azacytidine, BCG Live, bevacizumab, bexarotene
capsules, bexarotene gel, bleomycin, bortezombi, busulfan
intravenous, busulfan oral, calusterone, campothecin, capecitabine,
carboplatin, carmustine, carmustine with polifeprosan 20 implant,
celecoxib, cetuximab, chlorambucil, cisplatin, cladribine,
clofarabine, cyclophosphamide, cytarabine, cytoxan, cytarabine
liposomal, dacarbazine, dactinomycin, actinomycin D, dalteparin
sodium, darbepoetin alfa, dasatinib, daunorubicin liposomal,
daunorubicin, daunomycin, decitabine, denileukin, denileukin
diftitox, dexrazoxane, dexrazoxane, docetaxel, doxorubicin,
doxorubicin liposomal, dromostanolone propionate, eculizumab,
Elliott's B Solution, epirubicin, epirubicin hcl, epoetin alfa,
erlotinib, estramustine, etoposide phosphate, etoposide VP-16,
exemestane, fentanyl citrate, filgrastim, floxuridine
(intraarterial), fludarabine, fluorouracil 5-FU, fulvestrant,
gefitinib, gemcitabine, gemcitabine hcl, gemicitabine, gemtuzumab
ozogamicin, goserelin acetate, goserelin acetate, histrelin
acetate, hydroxyurea, ibritumomab tiuxetan, idarubicin, ifosfamide,
imatinib mesylate, interferon alfa 2a, interferon alfa-2b,
irinotecan, lapatinib ditosylate, lenalidomide, letrozole,
leucovorin, leuprolide acetate, levamisole, lomustine CCNU,
meclorethamine, nitrogen mustard, megestrol acetate, melphalan
L-PAM, mercaptopurine 6-MP, mesna, methotrexate, methoxsalen,
mitomycin C, mitotane, mitoxantrone, nandrolone phenpropionate,
nelarabine, nofetumomab, oprelvekin, oxaliplatin, paclitaxel,
paclitaxel protein-bound particles, palifermin, pamidronate,
panitumumab, pegademase, pegaspargase, pegfilgrastim, peginterferon
alfa-2b, pemetrexed disodium, pentostatin, pipobroman, plicamycin,
mithramycin, porfimer sodium, procarbazine, quinacrine,
rasburicase, rituximab, sargramostim, sorafenib, streptozocin,
sunitinib, sunitinib maleate, talc, tamoxifen, temozolomide,
teniposide VM-26, testolactone, thalidomide, thioguanine 6-TG,
thiotepa, topotecan, topotecan hcl, toremifene, tositumomab,
tositumomab/I-131 tositumomab, trastuzumab, tretinoin ATRA, uracil
mustard, valrubicin, vinblastine, vincristine, vinorelbine,
vorinostat, zoledronate, zoledronic acid and mixtures thereof.
[0106] Alternative pharmaceutical compositions according to the
present invention may comprise an effective amount of a compound
according to the present invention and at least one compound
selected from the group consisting of bepridil, nicardipine,
propafenone, rescinnamine, ketoconazole, cyclosporine A, loxapine,
pimozide, acacetin, mometasone furoate, its active 6.beta.-hydroxy
metabolite and mixtures thereof, or their pharmaceutically
acceptable salts and mixtures thereof (preferably, including at
least mometasone furoate, its active 6.beta.-hydroxy metabolite or
a pharmaceutically acceptable salt thereof), in combination with
one or more anticancer agents as otherwise disclosed herein and in
particular, at least one compound selected from the group
consisting of anthracyclines (daunorubin, doxorubicin, epirubicin,
idarubicin, and valrubicin), the vinca alkaloids (vincristine,
vinblastine, vindesine and vinorelbine), taxanes (paclitaxel or
taxol, and docetoxel or taxotere), epidopodophyllotoxins (etoposide
or VP-16 and tenoposide), nelarabine and imatinib, or a
pharmaceutically acceptable salt thereof, among others.
[0107] At the first onset of cancer or at the first indication that
a patient is at risk for the occurrence or recurrence of cancer,
for example because of the isolation and analysis of precancerous
cells or other conditions which evidence that a precancerous
condition may worsen into a cancer disease state or alternatively,
metastasize to other tissue, an effective amount of at least one
ABCB1 inhibitor as otherwise described herein is coadministered
with at least one anticancer agent as described herein to treat the
patient for a time and in a manner which is appropriate for
avoiding the cancer or metastasis of the cancer and/or causing the
cancer to go into remission or at least to extend the life of the
patient. Although the present method may be used quite effectively
to treat cancers which are drug resistant and especially those
exhibiting multiple drug resistance, the present method is used to
treat any cancer in order to reduce the likelihood that a cancer
will develop drug resistance during treatment, reduce the
likelihood that the cancer will recur and reduce the likelihood
that should such cancer recur, that the recurring cancer is drug
resistant or will exhibit multiple drug resistance.
[0108] Thus, the present compounds and compositions may be used
quite effectively to treat cancers, especially those which are drug
resistant or exhibit multiple drug resistance and which provide an
exceptionally effective treatment modality to reduce the risk of
occurrence, recurrence and/or metastasis of a cancer, especially a
drug resistant cancer or a cancer which exhibits multiple drug
resistance.
[0109] The invention also provides methods of treatment useful for
treating diseases in which transporter proteins mediate the disease
state in particular cancer, especially drug resistant (DR) and
multiple drug resistant (MDR) cancer. The treatment of cancer,
including the treating of various leukemias, especially T-lineage
acute lymphoblastic leukemia, especially forms which are multiple
drug resistant, are important features of the present invention.
Pharmaceutical compositions which comprise novel
pyrazolo[1,5-a]pyrimidine efflux inhibitors are also provided; in
some embodiments these compositions include at least one additional
anticancer agent, optionally in combination with a pharmaceutically
acceptable carrier, additive or excipient.
[0110] The invention is described further in the following
examples, which are illustrative and in no way limiting.
EXAMPLES
Example 1
[0111] Efflux Inhibitors with Varying Selectivity Toward ABCG2 Over
ABCB1
Experimental Overview
[0112] Although many mechanisms exist, resistance of tumors to
cancer therapy drugs is the principal reason for treatment failure
and the majority of clinical and experimental data indicates that
multidrug transporters such ABCB1 (Pgp, MDR1) and ABCG2 (BCRP,
MRP1)) play a leading role by preventing cytotoxic intracellular
drug concentrations. Inhibition of the function of these drug
efflux pumps presents a promising approach to treat cancer using
existing drugs. To date, clinical trials with such adjuvant
therapies have been relatively unsuccessful. One likely
contributing factor to these failed clinical applications is
limited understanding of specific substrate/inhibitor/pump
interactions. We have identified selective efflux inhibitors by
profiling multiple ABC transporter efflux pumps against a library
of small molecules could result in molecular probes that could
further explore such interactions. Using JC-1 as a dual-pump
fluorescent reporter substrate in our primary screening protocol we
observed a piperazine substituted pyrazolo[1,5-a]pyrimidine
substructure with promise for selective efflux inhibition. As a
result of a focused structure activity relationship driven
chemistry effort we describe below efflux inhibitors with varying
selective toward ABCG2 over ABCB1. These compounds have low in
vitro cellular toxicity, as well as adequate solubility and
stability under appropriate experimental conditions. To our
knowledge, low nanomolar chemoreversal activity coupled with direct
evidence of efflux inhibition for ABCG2 inhibitors is
unprecedented. The compounds also appear to have an IP landscape
with space to operate. In vitro chemotherapeutic potentiation
further illustrates the utility of the compounds and other related
members. The scaffold and analogs show promise for extension into
in vitro animal models. In fact, preliminary studies in our ABCG2
over-expressing tumor indicate that the at least two of the
compounds significantly reduces tumor size in combination with the
chemotherapeutic topotecan. Tumors that were grown for 4 weeks
effectively disappeared by the fourth day of treatment.
Materials and Methods
General Information:
[0113] The ABCB1 over-expressing drug-resistant cell line, CCRF-Adr
5000, and its parental CCRF-CEM cells were kindly provided by Dr.
T. Efferth (Pharmaceutical Biology, German Cancer Research Center,
Heidelberg, Germany). We have previously described the generation
of the Jurkat-DNR ABCB1 over-expressing cell line..sup.60 We have
also developed and previously characterized a SupT1-vincristine
(Vin) drug-resistant cell line that selectively over-expresses
ABCC1..sup.61 Ovarian Ig-MXP3(ABCG2) and its parental
Igrov1-sensitive cells were kindly provided by Dr. D. Ross
(Department of Medicine, University of Maryland Greenebaum Cancer
Center, Baltimore, Md.). Cells are grown in RPMI-1640 medium
supplemented with 10% fetal bovine serum (FBS, Hyclone, Logan,
Utah), 2 mM L-glutamine, 10 mM HEPES, 10 U/mL penicillin, 10
.mu.g/mL streptomycin, and 4 .mu.g/mL ciprofloxacin. Selective
pressure for the ABCB1 over-expressing CCRF-ADR 5000 and Jurkat-DNR
cells is maintained by growth in 20 nM daunorubicin (DNR).
Selective pressure for the ABCG2 over-expressing Ig-MXP3 cells is
maintained by treatment with 340 nM mitoxantrone (MTX) for 1 hr.
prior to harvest. Selective pressure for the ABCC1 over-expressing
SupT1-Vin cells is maintained by growth in 150 nM vincristine
(Vin).
[0114] The fluorescent reporter dye JC-1 and cell type
differentiation dye CellTrace.TM. Far Red DDAO-SE were obtained
from Invitrogen.TM. (Eugene, Oreg.). Nicardipine hydrochloride,
daunorubicin hydrochloride, mitoxantronedihydrochloride,
vincristine sulfate, and Fumitremorgin C were purchased from
Sigma-Aldrich (St. Louis, Mo.). XR9051, reversan, MK 571, and Ko
143 were purchased from Tocris Bioscience (St. Louis, Mo.).
Compounds ordered for SAR by commerce were purchased from ChemDiv
(San Diego, Calif.) and Ryan Scientific (Mt. Pleasant, S.C.).
Unless otherwise indicated, all compound solutions were maintained
and diluted in DMSO prior to addition to assay wells. Final DMSO
concentrations were no more than 1% v/v. A Biomek.RTM. NX
Multichannel (Beckman-Coulter, Fullerton, Calif.) was used for all
cell and compound solution transfers for volumes greater than 1
.mu.L. Low volume transfers (100 nL) were done via pintool (V&P
Scientific, Inc., San Diego, Calif.). Compound dose response plates
were generated with the Biomek.RTM. NX Span-8 (Beckman-Coulter,
Fullerton, Calif.).
[0115] The HyperCyt.RTM. high throughput flow cytometry platform
(IntelliCyt.TM., Albuquerque, N. Mex.) was used to sequentially
sample cells from 384-well microplates (2 .mu.L/sample) for flow
cytometer presentation at a rate of 40 samples per
minute..sup.62-63 Flow cytometric analysis was performed on a
CyAn.TM. flow cytometer (Beckman-Coulter, Fullerton, Calif.). The
resulting time-gated data files were analyzed with HyperView.RTM.
software to determine compound activity in each well. Inhibition
response curves were fitted by Prism.RTM. software (GraphPad
Software, Inc., San Diego, Calif.) using nonlinear least-squares
regression in a sigmoidal dose response model with variable slope,
also known as the four-parameter logistic equation. This type of
time-gated flow cytometric data analysis was described in detail
for a previous ABC transporter screen from our group..sup.56
[0116] .sup.1H and .sup.13C NMR spectra were recorded on a Bruker
AM 400 spectrometer (operating at 400 and 101 MHz respectively) or
Bruker AM 500 spectrometer (operating at 500 and 125 MHz
respectively) in CDCl.sub.3 with 0.03% TMS as an internal standard
or DMSO-d.sub.6. The chemical shifts (.delta.) reported are given
in parts per million (ppm) and the coupling constants (J) are in
Hertz (Hz). The spin multiplicities are reported as s=singlet, br.
s=broad singlet, d=doublet, t=triplet, q=quartet, dd=doublet of
doublet and m=multiplet. The LCMS analysis was performed on an
Agilent 1200 RRL chromatograph with photodiode array UV detection
and an Agilent 6224 TOF mass spectrometer. The chromatographic
method utilized the following parameters: a Waters Acquity BEH C-18
2.1.times.50 mm, 1.7 um column; UV detection wavelength=214 nm;
flow rate=0.4 mL/min; gradient=5-100% acetonitrile over 3 minutes
with a hold of 0.8 minutes at 100% acetonitrile; the aqueous mobile
phase contained 0.15% ammonium hydroxide (v/v). The mass
spectrometer utilized the following parameters: an Agilent
multimode source which simultaneously acquires ESI+/APCI+; a
reference mass solution consisting of purine and hexakis (1H, 1H,
3H-tetrafluoropropoxy)phosphazine; and a make-up solvent of
90:10:0.1 MeOH:Water:Formic Acid which was introduced to the LC
flow prior to the source to assist ionization. The melting point
was determined on a Stanford Research Systems OptiMelt
apparatus.
Primary Assay:
[0117] Single point, duplex: The assay is conducted in 384-well
format microplates in a total volume of 15.1 .mu.L dispensed
sequentially as follows: 1) JC-1 substrate (10 .mu.L/well); 2) test
compound (100 nL/well); 3) drug-resistant cells (5 .mu.L/well).
CCRF-Adr cells (ABCB1) are color-coded with 0.5 ng/mL CellTrace.TM.
Far Red DDAO-SE for 15 minutes at room temperature, washed twice by
centrifugation, and then combined with unlabeled Ig-MXP3 cells
(ABCG2) in the assay buffer. Final in-well concentration of test
compound is 6.6 .mu.M, JC-1 concentration is .about.1 .mu.M, and
the cell concentration is 3.times.10.sup.6 cells/mL (1:1 ratio of
the two cell types). Nicardipine is used as an on plate control for
both pumps at 50 .mu.M. The plate contents are mixed, rotated
end-over-end at 4 RPM and 25.degree. C. for 10 minutes, and then
cell samples are immediately analyzed. Approximately 2 .mu.L
volumes from each well are collected at a rate of approximately 40
samples per minute. This results in analysis of approximately 1,000
cells of each cell type from each well. Flow cytometric data of
light scatter and fluorescence emission at 530+/-20 nm (488 nm
excitation, FL1) and 665+/-10 nm (633 nm excitation, FL8) are
collected.
[0118] Dose response, single-plex: The assay provider's ABCB1
over-expressing Jurkat-DNR cell line is used for confirmatory
follow up instead of the CCRF-Adr cells. Each cell line (Jurkat-DNR
and Ig-MXP3) is run separately against all compounds (no
differential cell staining) in dose response. The assay is
conducted in 384-well format in a total volume of 15.1 .mu.L. Cells
and reagents are added sequentially as follows: 1) PBS buffer (5
.mu.L/well); 2) test compound (100 nL/well); 3) drug-resistant
cells (10 .mu.L/well) pre-stained with the JC-1 substrate at 1
.mu.M. Final in-well concentrations of test compound range from 50
.mu.M to 69 nM over an 18 point dose response and the cell
concentration is 1.times.10.sup.6 cells/mL. Nicardipine (50 .mu.M)
is added to each plate as a pan-inhibition positive control. The
plate is rotated end-over-end at 4 RPM and 25.degree. C. for 30
minutes and then cell samples are analyzed and flow cytometric data
of light scatter and fluorescence emission at 530+/-20 nm (488 nm
excitation, FL1) are collected.
Chemoreversal Assay:
[0119] Cells (ABCB1, Jurkat-DNR or ABCG2 Ig-MXP3) are incubated
with the test compound (3 order of magnitude concentration range)
over a 3-day and 7-day period in the presence of the inhibitor and
chemotherapeutic (ABCB1, DNR or ABCG2 MTX), such that a cell
concentration of at least 1.times.10.sup.5 cells/mL is maintained.
Cell viability is determined by trypan blue staining and
enumeration under light microscopy. At day 3, wells with greater
than 2.times.10.sup.5 cells are refreshed, to include readjustment
of chemotherapeutic and inhibitor concentration. A chemoreversal
index (Chemoreversal 50, CR.sub.50) is determined from the
viability assessment. Using a similar approach, a direct
cytotoxicity index (Toxic Dose 50, TD.sub.50) is determined by
assessment of cell death of cells grown in media alone. Results are
compared with the survival of parental cells in the presence of the
selective agent (chemotherapeutic; 100% cell death), as well as
survival of drug-resistant cells in the presence of the
chemotherapeutic drug (control yields 100% viability). The
difference between the CR.sub.50 and the TD.sub.50 give an
approximation of the in vitro therapeutic index for the test
compound.
In Vivo Animal Study Methods:
[0120] Model design: Specific-pathogen-free adult CB-17 female SCID
mice (5-6 weeks of age) weighing 20-25 g, are purchased. Briefly,
ABCG2 over-expressing Igrov1/T8 cells are injected
(5.times.10.sup.6 cells/mouse). Prior to injection, Igrov1/T8 cells
are maintained in topotecan in order to assure continuous
expression of ABCG2. Tumors are allowed to grow for 3-4 weeks or
until tumor volumes are greater than .about.100 cubic millimeters,
at which time they are stratified into treatment groups. Treatment
is administered and we observe the size of the tumor and weight of
the mouse over a 7 day period. At the end of 7 days, we: 1)
sacrifice the animal; 2) measure and weigh the tumor (if present);
3) analyze the histology of the tumor as well as other organs for
evidence of toxicity; and 4) measure the expression of ABCG2
receptors on the tumor to determine if in vivo growth has altered
ABCG2 expression. In our initial studies, we have generally
observed high survival at 4 weeks (>90%) which escalates rapidly
with near 100% mortality by 6 weeks. This approach is well
described elsewhere..sup.9
[0121] In the proposed experimentation, three groups of mice will
be studied: 1) mice with Igrov11T8 tumors and injected with ABCG2
inhibitor; 2) mice with Igrov11T8 tumors that are sham injected;
and 3) mice with parental cells that are not resistant to
topotecan. In the first set of studies, we will determine the
optimal dose of the ABCG2 inhibitor using primarily Group 1 mice.
Using the in vitro cell killing data as a guide, we will test 6
doses of the ABCG2 inhibitor (SID 103911215) over 2 logs of final
blood concentration (10 nM to 1 .mu.M). In order to assure
statistical significance, we will study 5 mice in each group.
Results will be compared against one group of mice from Groups 2
and 3. The reduction of the size of the tumor will be used as the
principal factor in determining the optimal dose, although toxicity
will also be considered.
[0122] In the second set of studies, we will employ all three
groups of mice using the optimal dose of ABCG2 inhibitor for Group
1. We will examine mice for overall survival at 6 weeks (including
tumor size and body weight), acute toxicity due to the
administration of the ABCG2 inhibitor, and tumors to make sure that
the level of ABCG2 expression is not altered by in vivo growth.
[0123] Acute toxicity: Since prior information regarding the acute
toxicity of the ABCG2 inhibitor is not available, body weight and
histology of liver, kidneys, spleen, lungs, and heart will be
obtained and analyzed. In addition to standard histologic stains,
immunohistochemical stains to detect apoptosis will be obtained in
order to compare organs and toxicity from sham and ABCG2 injected
animals.
[0124] ABCG2 expression by RT-PCR: We will also measure the RNA
expression of ABCG2 by RT-PCR in 10 tumors from treated and
sham-injected mice in order to measure the effects of in vivo
growth and treatment on the expression of ABCG2 in these tumors.
Finally, we will perform limited toxicity studies and any acute
toxicity leading to cell necrosis or other histologic change will
be observed. Pharmacokinetic sampling and analysis will be
performed in subsequent studies outside of this proposal.
[0125] Regulatory: Mice will be studied and maintained in
accordance with guidelines of our Institutional Animal Research
Committee at UNM HSC. We have already obtained IACUC approval.
Representative Synthesis and Chemical Characterization:
[0126] SID 88095709 and many analogues are synthesized by the
method shown (sequence a-c, FIG. 2). Commercial or readily-obtained
substituted aminopyrazoles 1 are treated with the appropriate
dialkylmalonate or .beta.-ketoester to give intermediate 2,
followed by chlorination to afford the pyrazolo[1,5-a]pyrimidine
core intermediate 3. Installation of the piperazine moiety afforded
SID 88095709 compound directly. In some cases, a Suzuki or Molander
type coupling was preferred to install aryl functionality at a late
stage in the synthesis (see sequence d-f). With minor modification
(including utilizing different starting materials and/or
intermediates), the compounds according to the present invention
are readily afforded.
[0127] 5-(furan-2-yl)-2-phenylpyrazolo[1,5-a]pyrimidin-7(4H)-one
(2): A mixture of 3-phenyl-1H-pyrazol-5-amine (1: 0.318 g, 2.0
mmol, 1.0 eq) and methyl 3-(furan-2-yl)-3-oxopropanoate (0.370 g,
2.2 mmol, 1.10 eq) was heated in acetic acid (2.0 mL) at
100.degree. C. for 4 hr. After cooling down to rt, the precipitate
was collected by filtration. The precipitate was rinsed with EtOH
(15 mL) and dried under air to afford
5-(furan-2-yl)-2-phenylpyrazolo[1,5-a]pyrimidin-7(4H)-one (0.358 g,
65%) as a white solid. .sup.1H-NMR (400 MHz, DMSO-d.sub.6) .delta.
12.70 (s, 1H), 8.06 (m, 1H), 8.00 (m, 1H), 7.98 (m, 1H), 7.51-7.47
(m, 3H), 7.44-7.42 (m, 1H), 6.81 (dd, J=3.7, 1.8 Hz, 1H), 6.64 (s,
1H), 6.15 (s, 1H).
[0128] 7-chloro-5-(furan-2-yl)-2-phenylpyrazolo[1,5-a]pyrimidine
(3): A mixture of
5-(furan-2-yl)-2-phenylpyrazolo[1,5-a]pyrimidin-7(4H)-one (0.277 g,
1.0 mmol, 1.0 eq), POCl.sub.3 (0.613 g, 4.0 mmol, 4.0 eq),
N-benzyl-N,N,N-triethylethanaminium chloride (0.456 g, 2.0 mmol,
2.0 eq) and N,N-dimethylaniline (0.121 g, 1.0 mmol, 1.0 eq) in
acetonitrile (5.0 mL) was heated at 80.degree. C. for 4 hr. The
completed reaction was diluted with CHCl.sub.3 (20 mL), washed with
H.sub.2O (10 mL), and the separated organic layers were dried
(MgSO.sub.4) and concentrated. The residue was purified by
chromatography (Biotage 25 g, EtOAc/Hexane) to afford
7-chloro-5-(furan-2-yl)-2-phenylpyrazolo[1,5-a]pyrimidine (0.247 g,
84%) as a yellow solid. .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta.
7.97-7.94 (m, 2H), 7.56 (m, 1H), 7.43-7.39 (m, 2H), 7.36-7.34 (m,
1H), 7.19 (s, 1H), 7.17 (dd, J=3.5, 0.5 Hz, 1H), 6.97 (s, 1H), 6.54
(dd, J=3.5, 1.7 Hz, 1H).
[0129]
(4-(5-(furan-2-yl)-2-phenylpyrazolo[1,5-a]pyrimidin-7-yl)piperazin--
1-yl)(furan-3-yl)methanone (SID 88095709): A mixture of
7-chloro-5-(furan-2-yl)-2-phenylpyrazolo[1,5-a]pyrimidine (0.148 g,
0.5 mmol, 1.0 eq), furan-3-yl(piperazin-1-yl)methanone (0.180 g,
1.0 mmol, 2.0 eq,) and N-ethyl-N-isopropylpropan-2-amine (0.129 g,
1.0 mmol, 2.0 eq) in acetonitrile (5.0 mL) was heated at
100.degree. C. for 3 hr. The completed reaction was purified by
chromatography (Biotage 25 g, EtOAc/Hexane) to afford
(4-(5-(furan-2-yl)-2-phenylpyrazolo[1,5-a]pyrimidin-7-yl)piperazin-1-yl)(-
furan-3-yl)methanone (0.218 g, 99%) as a white solid..sup.1H-NMR
(400 MHz, CDCl.sub.3) .delta. 8.03-8.00 (m, 2H), 7.83 (m, 1H), 7.62
(m, 1H), 7.52-7.47 (m, 3H), 7.45-7.41 (m, 1H), 7.23 (dd, J=3.5, 0.7
Hz, 1H), 6.93 (s, 1H), 6.65 (m, 1H), 6.62 (dd, J=3.5, 1.8 Hz, 1H),
6.60 (s, 1H), 4.08 (b, 4H), 3.92 (b, 4H). .sup.13C NMR (125 MHz,
CDCl.sub.3) .delta. 164.1, 155.4, 152.4, 151.8, 150.1, 148.5,
144.3, 143.8, 143.2, 132.9, 129.0, 128.7, 126.4, 120.6, 112.6,
110.9, 110.1, 92.9, 88.9, 48.3. LCMS retention time: 3.20 min;
purity at 215 nm=100%. HRMS m/z calculated for
C.sub.24H.sub.27N.sub.5O.sub.2 ([M+H].sup.+): 440.1717, found
440.1715.
[0130] Solubility: Aqueous solubility was measured in phosphate
buffered saline (PBS) at room temperature (23.degree. C.). PBS by
definition is 137 mM NaCl, 2.7 mM KCl, 10 mM sodium phosphate
dibasic, 2 mM potassium phosphate monobasic and a pH of 7.4. The
solubility of SID 88095709 was determined to be 0.51
.mu.g/mL..sup.64
[0131] Applying a flow cytometric kinetic solubility
protocol,.sup.65 we were also able to compare solubility across
primary, secondary, and profiling buffer conditions in house. This
method evaluates precipitation as a function of side scatter
changes (above the buffer baseline); we observed 2 .mu.g/mL
solubility in PBS alone. This is approximately three fold higher
than reported above; however, our protocol uses 1% DMSO as compared
to 0.1%. Such changes in the concentration of DMSO can
significantly increase overall solubility of a compound. The
presence of fetal bovine serum also affects compound availability
and in the primary assay screening buffer (6.7% FBS in PBS, 1%
DSMO) the precipitation was inhibited slightly with no particulate
noted at 3 .mu.g/mL. The growth media indicated in the methods
section (RPMI with 10% FBS, 1% DMSO) showed no precipitate forming
at 6.mu.g/mL. It should also be noted that compounds with poor
solubility at high concentrations (i.e. nicardipine above 50 .mu.M)
tend to show a response drop-off in the primary dose response
conditions and this phenomenon was not significantly observed with
the SID 88095709 or related compounds. In general, we are confident
that compound availability is sufficient for the concentration
ranges discussed for this scaffold set in our described biological
systems.
[0132] Stability: Aqueous stability was measured at room
temperature (23.degree. C.) in PBS (no antioxidants or other
protectants and DMSO concentration below 0.1%). The stabilityofSlD
88095709, determined as the percent of compound remaining after 48
hours, was 10%..sup.64Stability data are depicted as a graph
showing the loss of compound with time over a 48 hour period with a
minimum of 6 time points and provide the percent remaining compound
at end of the 48 hours (FIG. 3, closed circles).
[0133] It has been reported by several MLPCN partners and our
collaborators at the Sanford-Burnham Institute who perform this
assay, that the conditions for the stability assay as described
above is fundamentally unreliable for compounds that exhibit
moderate to poor aqueous solubility. Given the low solubility of
SID 88095709 (0.51 .mu.g/mL, reported in Section C above), the
stability experiment was repeated with the addition of acetonitrile
(50% v/v final) which has been reported to routinely resolve any
contributions due to insolubility. The stability data under these
conditions overlain in the FIG. 3 and is also depicted as a graph
showing the loss of compound with time over a 48 hour period with a
minimum of 6 time points and provide the percent remaining compound
at end of the 48 hours (FIG. 3, closed triangles). The addition of
acetonitrile appears to have solubilized the compound and provided
a more accurate reading of the stability, which is now 100%
remaining after 48 hours.
Results
Summary of Screening Results:
[0134] A total of 194,394 compounds were tested in the primary
single point assay conditions with 200 and 130 actives noted in
ABCB1 and ABCG2 respectively. Compounds were deemed active if the
percent inhibition was greater than 80%. Activity was determined on
the basis of the median fluorescence intensity (MFI) of JC-1.
Percent inhibition was calculated as 100.times.[1-(MFI_PC-MFI
Test)/(MFI_PC-MFI_NC)] in which MFI Test, MFI_PC and MFI_NC
represent the MFI of cells in wells containing test compound, the
average MFI of cells in positive control wells (maximum
fluorescence intensity) and the average MFI of cells in negative
control wells (minimum fluorescence intensity), respectively. The
ACTIVITY_SCORE is equal to the calculated percent inhibition,
except when percent inhibition is greater than 100 and less than
0.
[0135] A subsequent Small Molecule Repository cherry pick resulted
in single point confirmatory testing of 273 compounds resulting in
18 and 16 actives in ABCB1 and ABCG2, respectively. Innate
fluorescence of test compound was subtracted out based on MFI of
unstained cells before calculation of percent inhibition of efflux
pump activity. The following equations were used to calculate
percent inhibition: % Inhibition=100.times.(FluorDelta
Sample-FluorDelta_NC)/(FluorDelta_PC-FluorDelta_NC) in which
FluorDelta_Sample (FluorDelta=MFI_JC-1-MFI non-JC-1 in which
MFI_JC-1 are the MFI of cells in wells in the presence of JC-1 and
MFI_non-JC-1 are the MFI of cells in wells without JC-1) are the
differences of JC-1 minus non-JC-1 from wells with test compound,
FluorDelta_NC are differences from negative control wells (cells
with DMSO, minimum fluorescence intensity) and FluorDelta_PC are
differences from nicardipine positive control wells. These dose
response of % Inhibition data were fitted via GraphPad Prism to a
sigmoidal dose response curve with variable hillslope: %
Inhibition=Bottom+(Top-Bottom)/(1+10 ((Log EC50-Log
Cmpd)*HillSlope)) where Log Cmpd is the log of compound
concentration in micromolar and Top-Bottom is the FIT_PERCENT_SPAN.
Prism reports estimated values and fitted statistics for the four
parameters (Bottom, Top, Hillslope and EC.sub.50). Dose response
fits assessed as decent were those with Residual square<0.5,
Hillslope<5, Standard deviation of estimated Log EC.sub.50<3,
and standard deviation of top/estimate for top<0.5. Only the
fits that passed this filter were reported. The activity score was
calculated based on two weighted criteria; EC.sub.50<10
micromolar and FIT_PERCENT_SPAN>20% by the following equation:
Activity
Score=75*(EC.sub.50Cutoff-EC.sub.50)/EC.sub.50Cutoff+25*(Span-SpanCutoff)-
/SpanCutoff. Active compounds have activity scores greater than 52,
inactive compounds have scores less than 52. Values above 100 or
below 0 were adjusted to 100 or 0 respectively.
Structure Activity Relationship Information:
[0136] SAR by commerce: Limited SAR was revealed through the first
round of cherry pick analysis, and about a third of the compounds
were observed to be fluorescent artifacts. However, preliminary
secondary screening efforts confirmed activity of several compounds
including MLS000527783 (SID 17388272) with which micromolar
potentiation and low toxicity was observed, but little pump
specificity (data not shown). Resupply of this compound (new SID
85752814) confirmed the efflux inhibition, and a series of
compounds similar in structure was ordered around this SMR hit.
These 31 compounds were tested in dose response in two
over-expressing cell lines: Jurkat-DNR (ABCB1) and Ig-MXP3 (ABCG2).
Expansion of this piperazine substituted pyrazolo[1,5-a]pyrimidine
substructure resulted in a selectivity profile significantly biased
toward ABCG2. Of the hits that were identified through this
commercial SAR endeavor, SID 85240370 had attractive efflux potency
towards ABCG2 and marginal selectivity over ABCB1 (FIG. 4). The KU
SCC launched an SAR campaign aimed at further understanding the
origin of potency and selectivity and set out to optimize the
compound profile to meet the appropriate criteria for potency
(<1 .mu.M) and selectivity (>10 fold) in the efflux assay.
Compounds meeting these criteria were then screened in a subsequent
chemoreversal assay, a cell killing secondary assay that shows
potentiation of specific chemotherapeutics for each cell line as
compared to the compound's inherent toxicity. The chemoreversal
assays quantitatively show the potentiation of known killing agents
for each cell line with efflux inhibitory compounds.
[0137] Chemoreversal potency criteria were set such that micromolar
potentiation is desired and the TD.sub.50/CR.sub.50 ratio must be
>10 with overall toxicity >15 .mu.M. One compound was found
to meet these secondary assay criteria for ABCB1, while 5 compounds
matching this profile were identified for ABCG2. Structure activity
relationships remained unclear at this stage. As compared to the
efflux inhibition activity for SID 85240370, selectivity in the
potentiation assay maintained a slight selectivity for ABCG2 with
CR.sub.50=0.14 .mu.M vs. 0.70 .mu.M in ABCB1. However, there was
significant toxicity noted for both cell lines (TD.sub.50=6.0 and
3.2 .mu.M for ABCG2 and ABCB1 respectively). With these preliminary
results in hand, an extensive SAR initiative was undertaken.
[0138] SAR by synthesis: A total of 165 compounds were assessed in
the primary efflux assay and of these, 126 were synthesized by the
KU SCC. A subset of compounds relating to the parent scaffold were
purchased and assessed as part of the HTS effort at UNMCMD, prior
to the KU SCC involvement. Of the .about.30 commercial compounds
assessed by the UNMCMD, a few showed modest ABCG2 selectivity (FIG.
4, SID 85240370), but gaps in the collection did not resolve the
structural functionality responsible for any significant efflux
potency or selectivity towards ABCG2 or ABCB1. Due to the diversity
of structural changes present in the commercial set, additional
compounds were needed to construct meaningful SAR, and this was
done using the primary efflux data as it was more readily
obtainable. The commercial set contained several members with
conserved functionality that provided the basis for establishing a
methodical SAR assessment. The pyrazolo[1,5-a]pyrimidine core was
preserved, and exchange of the peripheral substituents were
surveyed as depicted by the highlighted regions in FIG. 5. Several
compounds of the purchased collection contained a 3-chlorophenyl
substituent at R1, which also reflected the R1 moiety present in
hit SID 85240370. As such, initially a series of compounds were
prepared with these features maintained while adjusting R2-R4
(Table 1).
[0139] One compound cluster was constructed with R1-R3 groups
identical to that of the parent hit SID 85240370 (entries 3-18)
while modulating R4. Notably, the substitution of the acyl-2 furan
for acyl-3-furan (entry 4) produced an enhancement in selectivity
for ABCG2 (8.6-fold), predominately due to erosion of ABCB1
potency, while only modestly attenuating ABCG2 potency as compared
to the parent hit. Improved ABCG2 potency was achieved with
installation of an acyl-3-pyridine; however, the selectivity
deteriorated essentially to pan inhibition (entry 13). Following
incorporation of the acyl-3-furan as the more optimal R4
substituent, a survey was then done on the R2 group while holding
constant R1 and R3 (entries 19-25). Substantial potency for ABCG2
was gained when R4 was 3-pyridine (entry 23); however, once again,
selectivity was negatively impacted.
[0140] Alterations in the 3-chlorophenyl R1 substituent were then
made while assessing several R4 head groups, specifically toggling
between acyl-2-furan, acyl-3-furan, or benzoyl functionalities
(entries 1-11, Table 2). No substantial improvements were noted
with these changes; however, when R1 was changed from
3-chlorophenyl to phenyl, and R2 was varied (entries 12-18), it was
discovered that a 2-furan at R2 in concert with the optimized
acyl-3-furan afforded a significant boost in both ABCG2 potency as
well as overall ABCG2 selectivity (entry 12, SID 88095709, ABCB1
EC.sub.50=4.65 .mu.M; ABCG2 EC.sub.50=0.13 .mu.M, selectivity=36
fold).
[0141] With this information in hand, the team followed up with an
SAR effort aimed at demonstrating supportive SAR for compounds
bearing an R2=2-furyl group while also attempting to improve upon
the profile of the new lead, SID 88095709 (FIG. 6).
[0142] The new lead scaffold, represented by SID 88095709, was
further studied by adjusting physiochemical and spatial elements in
R1 (Table 3).
[0143] Switching out the phenyl ring of the lead with a t-butyl
group erased much of the gains towards ABCG2 selectivity (entry 2,
Table 3). Traditional phenyl replacements such as thiophene or
furan were tolerated, but only led to modest selectivity and
potencies. The installation of a 4-chlorophenyl substituent led to
a reduced impact on ABCB1, resulting in selectivity in the efflux
assay of 22-fold (entry 6); however, the change also marginalized
the potency on ABCG2. Renovating the phenyl substituent with
electron donating groups did not appear to be beneficial.
[0144] An examination of 2-furan replacements at R2 was also
undertaken (Table 4). Simple alkyl units such as methyl or t-butyl
degraded potency and fold-selectivity for both transporters.
Notably, use of t-butyl actually reversed selectivity for ABCB1,
albeit at the expense of potency (entry 5).
[0145] Some R2 revisions resulted in impressive ABCG2 selectivities
and potencies. The choice of 2-F-phenyl (entry 10) slightly
degraded potency for ABCB1 as compared to the parent (entry 1),
leading to a 10-fold selectivity in favor of ABCG2. For the
fluorinated series (entries 10-12), the potency for both
transporters decreased as the fluorine atom was migrated from the
2- to 3- to 4-position of the aromatic R2 ring. Interestingly, the
use of the use of a 3-MeO-phenyl group impeded potency for ABCB1
activity while retaining submicromolar ABCG2 potency on par with
the parent, leading to an improved 83-fold selectivity between the
transporters (entry 8). In this series (entries 7-9), however, a
trend was not observed as the substituent was shifted from each
position. Additional compounds prepared with the 3-MeO-phenyl group
at R2 did not show a consistent SAR (data not shown).
[0146] The commercial set of compounds contained a few scaffolds
bearing a methyl group at R3. SAR generated in the early phases
demonstrated some benefit to the presence of small alkyl groups at
R3; however, this was highly dependent on the identity of groups at
R1, R2 and R4. For the purposes of expanding the SAR around SID
88095709, one analogue was prepared to quickly evaluate the effect
of this substitution pattern in concert with our chosen
functionalities at R1, R2 and R4 (FIG. 7). ABCB1 potency was
encouragingly impaired, but not without also effecting G2,
resulting in a marginal selectivity profile.
[0147] Attention was then turned to investigating the effect of
different R4 functionality appended to the piperazine (Table 5). In
earlier SAR sets, activity was found to be sensitive to the
identity of R4 and the pairing of groups at R2 and R3. In the
context of our new lead, SID 88095709, we wanted to better
understand the effect of R4 with the chosen substituents. It was
confirmed that an acyl-3-furan was preferred to an acyl-2-furan
(entry 2), and simple alkyl substitution of the 3-furan (entries
5-7) or a larger benzofuran (entry 8), while tolerated, did not
reveal any benefits. However, the most influential effects on ABCB1
were observed when the acyl furan was exchanged for a benzyl ester
(entry 11). While ABCG2 potency was compromised compared to the
lead, ABCB1 potency was completely lost, yielding a selectivity of
19 fold. In a more aggressive effort, the entire "top piece" of the
scaffold, consisting of the piperazine and the R4 group, was
modified (Table 6). Ring-opened piperazine equivalents, truncated
amino groups, piperidine amides, ring-expanded amines (not shown)
and various structural variations on a theme did not produce a
profile superior to that which had already been observed.
[0148] In the process of evaluating these structural modifications,
several compounds were prepared singly to target possible
oversights in SAR, as every possible R1-R4 combination cannot be
prepared and assessed in a timely way. Others were targeted as a
means of inserting the best combinations as gleaned from the
preceding generations of SAR. These compounds were more recently
pursued for specific structural combinations summarized in Table 7.
Data obtained early on had indicated that the acetyl group at R4
was more advantageous than other changes that had been surveyed
(including the benzyl ester modification), though later refinement
of these data does not now stand out as particular SAR of interest.
Based on the information in hand at the time, substituted phenyl
derivatives possessing a disparate electronic nature at R1 were
incorporated with the acetyl R4 group in place (entries 2 and 3).
No advantages were found.
[0149] We were interested in also surveying the effect of reducing
the carbonyl of the R4 head group to give an amine in place of the
amide when some of the preferred R1-R3 substituents were
incorporated (entry 4) and, as an experimental one-off compound, in
other pockets of SAR that seemed to be unrelated but were still
interesting (entry 6). While the effect did not enhance the profile
in the case of the most closely related analogue to the lead SID
88095709 (entry 4, SID 99376134), a profound effect was observed
for the alternatively substituted compound with SID 97301789 (entry
6). As previously mentioned, the combination of R1-R4 has been
found to radically influence the profile of the compounds towards
ABCB1 or ABCG2. For example, methylation at R2 and R3 in concert
with previously surveyed R1 and R4 moieties delivered an analogue
which amounts to pan inhibition (entry 5). The combination effect
cannot be underestimated, as shown with entry 6, in which
methylation at R2 and R3, in concert with the newly discovered R4
head group of CH.sub.2-3-furan, led to a 233-fold selectivity for
G2 with comparable potency to the lead compound and abolished
activity for ABCB1 in the efflux assay! The improved selectivity
observed with the CH.sub.2-3-furan in the R4 position could be due
to a number of effects. Removal of the carbonyl of the parental
acyl-3-furan R4 group confers basicity to the head group that was
previously missing. Removal of the carbonyl also changes the
conformation of the R4 group in relation of the piperidine ring
(sp.sup.3 hybridization) versus when the amide of the parent is
intact (sp.sup.2 hybridization). Although these aspects are likely
not the only contributors to the observed benefits in selectivity,
as a previously screened benzyl R4 group did not result in
analogous improvements. Still, these effects are advantageous only
when put in play with a select group of R1-R3 substitutions.
[0150] In parallel to the above efforts, compounds were also
assessed in potentiation secondary assays and associated data are
presented in the preceding tables; however, the chemoreversal assay
is a very low throughput assay and compound data from this assay
could not be used to drive the SAR program. Key data have been
collected for some of the most promising compounds (FIG. 8).
Evaluation of SID 88095709 shows submicromolar ABCG2 efflux
inhibition activity with .about.36-fold selectivity toward ABCG2
over ABCB1. Potent submicromolar activity has also been
demonstrated in the potentiated killing of both over-expressing
cell lines with preference for ABCG2 from a toxicity perspective,
though the degree of selectivity observed in the cell killing assay
is removed (1.8 fold in chemoreversal vs. 36-fold in efflux assay).
The most recently advanced analogue, SID 97301789, presents as an
exceedingly potent compound in the potentiation assays with a
4.5-fold window between the observed potencies for the two
transporters, but in favor of ABCB1. Interestingly, the analogue
appears to be devoid of toxicity (>100 .mu.M) and represents a
promising tool for further refinement.
In Vivo Data:
[0151] To specifically demonstrate the effects of the drugs with
our approach, we administered by IP injection a dose of TPT (150 nM
in 75 .mu.L) that results in the killing of parental cells, but not
the resistant Igrov1/T8 cells (EC.sub.50 for parental cells is 7 nM
vs. 311 nM for resistant cells). To demonstrate efficacy of the
inhibitors, we grew ABCG2 resistant cells in mice for 28 days.
Tumor-bearing mice were injected with either 150 nM TPT, 100 nM SID
88095709 (probe resupply), or 500 nM SID 85752814 (original SMR
hit). Tumor size remained the same or increased in each case. In
contrast, injecting a combination of 150 nM TPT and 100 nM of SID
88095709 or 500 nM of SID 85752814 dramatically reduced tumor size
and eliminated evidence of tumor within 2 to 3 days (FIG. 10),
indicating that tumor sensitivity to TPT returned when one of the
ABCG2-blocking compounds was present.
Scaffold/Moiety Chemical Liabilities:
[0152] The pyrazolo[1,5-a]pyrimidine scaffold and its derivatives
have been easily handled in terms of stability to reaction
conditions, exposure to acid or base, heating, and general
manipulation. Most are isolated as stable solid materials. We have
not observed decomposition nor have we experienced any chemical
liability with these compounds. The structure does not contain
moieties that are known generally to be reactive. Stability
assessment was performed in 1.times. PBS buffer at pH 7.4 and room
temperature. After 48 hours, it was determined that only 10 percent
of the parent compound remained when the experiment was performed
in PBS buffer alone, thus possibly indicating some structural
liability that at this point in time is unknown. However, as
previously mentioned, the stability assay used with PBS alone is
likely not suitable for compounds with lower solubility. The
experiment was repeated with acetonitrile to help solubilize the
compound, leading to a favorable profile in which 100% of the
sample was remaining after 48 hours and indicating no loss of
integrity. Comparative assessment of the solubility in multiple
assay conditions indicates moderate solubility; however this does
not appear to be an issue for either primary or secondary screening
protocols based both on direct observation of activity as well as
the flow based experimentation briefly described above.
Kinase Profiling of SID 88095709:
[0153] Since ABC transporters are ATP dependent efflux pumps and
several kinase targets have been implicated in the patent
literature with structurally similar scaffolds, SID 88095709 was
profiled against 50 kinases at a single concentration of 10 .mu.M
to assess promiscuity of the chemotype..sup.66 SID 88095709 was
dissolved in DMSO and tested at a final concentration of 10 .mu.M.
Prior to initiating a profiling campaign, the compound was
evaluated for false positive against split-luciferase. Profiling
was done in duplicate for SID 88095709 against each kinase. The
Percent Inhibition and Percent Activity Remaining are calculated
using the following equation:
% Inhibition=ALUControl-ALUSample.times.100
ALUControl
% Activity Remaining=100-% Inhibition
The team has also submitted SID 88095709 and the analog SID
97301789 to the NIH National Cancer Institute to elucidate the
effect of SID 88095709 on the cancer cell line panel.
Discussion
[0154] ABCB1, ABCC1, and ABCG2 transporters are known to
significantly influence the ADME-Tox properties of drugs,.sup.4 and
although a large number of compounds have been identified
possessing ABC transporter inhibitory properties, only a few of
these agents are appropriate candidates for clinical use as MDR
reversing agents..sup.67 Clinical trials with late generation
modulators (e.g. biricodar, zosuquidar, dofequidar, and laniquidar)
specifically developed for MDR reversal are ongoing..sup.38,41,68
Efflux pumps are by design highly adaptive and potentially able to
adjust to a wide array of chemotypes owing to a relatively large
cavity (6000 .ANG..sup.3) and at least three non-overlapping
binding site configurations..sup.13 For example, rhodamine 123 has
been used in combination with Hoechst 33342 to describe two
functional transport sites in ABCB1 with complex allosteric
interactions..sup.69Concurrently, rhodamine 123 may bind to a
different overlapping region, or potentially within the same large
flexible binding site, as LDS 751..sup.70 It has also been shown
that ABCB1 possesses two allosterically coupled drug acceptor sites
where one binds vinblastine, doxorubucin, etoposide and cyclosporin
A, and the other binds dexniguldipine and other
1,4-dihydropyridines..sup.71 It is thus possible that within a
single chemical sub-structure class there could be multiple binding
patterns leading to both the difficulty of direct structure
activity relationship comparisons but also to the possibility of a
tuneable synthetic system allowing selectivity and/or
cross-transporter inhibition. It should be noted, however, that
acquired mutations in transporter genes introduce even more
complexity, altering the pattern of resistance and improving the
ability of the mutants to efflux new drugs..sup.72 It was reported
that various drug-selected human tumor cell lines expressed
different ABCG2 variants, which were suggested to be
gain-of-function mutations acquired during the course of drug
exposure..sup.73 Single amino-acid changes cause an altered drug
resistance profile and substrate specificity compared to the wild
type ABCG2 transporter..sup.74 The lesson to be learned is that
high efficacy and good selectivity need to be carefully compared in
analogous systems and cell lines. This is readily apparent in the
discrepant activity seen with FTC and Ko 143 (discussed below).
[0155] FIG. 11 summarizes the prior art comparison to SID 88095709,
85752814, and 97301789. XR9051 and MK571 were chosen to verify
ABCB1 activity and counterscreen ABCC1 activity, respectively in
our system. XR9051 does inhibit the efflux of JC-1 in both ABCB1
and ABCG2 over-expressing cell lines (0.6 and 2.3 .mu.M
respectively). Potentiation data indicates submicromolar
chemoreversal in both Jurkat-DNR and Ig-MXP3 cells with a bias
toward ABCB1 at 10 nM as compared to 700 nM for ABCG2. Not
surprisingly, we didn't observe any inhibition in ABCB1 or ABCG2
with MK571 but we remain interested in the MK571 response in ABCC1
over-expressing SupT1-Vin cells. It is likely that the
"potentiation" seen with MK571 is simply due to general toxicity
since in both cell lines the CR.sub.50 is equivalent to the
TD.sub.50 (curves not shown).
[0156] Direct comparison of reversan in our efflux inhibition
system shows low micromolar inhibition of both ABCB1 and ABCG2 (4.4
and 0.8 .mu.M respectively) with moderate selectivity for ABCG2. In
our chemoreversal potentiation assay, reversan showed micromolar
activity and no apparent selectivity (2.2 and 3.6 .mu.M in ABCB1
and ABCG2, respectively). This was coupled with significant
toxicity in the Jurkat-DNR cell line. FTC showed no activity in
either cell line in the efflux inhibition assay (data not shown)
and was not tested in the potentiation assay. Ko 143 has been shown
to potentiate mitoxantrone (MTX) at nanomolar levels in ABCG2
over-expressing cells..sup.11 In our potentiation assay there
actually seemed to be a selectivity for ABCB1 over ABCG2
(CR.sub.50=1.0 and 5.9 M respectively) with considerable toxicity
in both cell lines. The efflux inhibition activity did not mirror
this, showing no activity in ABCB1 and only 13.6 .mu.M inhibition
in ABCG2, potentially indicating a binding site difference versus
JC-1.
[0157] The entire SAR series was also structurally compared via PLS
analysis to the inhibitors described in FIG. 11 as well as the
clinically relevant potentiators; Cyclosporin A, Biricodar,
Tariquidar, Zosuquidar, Elacridar, Laniquidar, Dofequidar, and
ONT093. The eight biological parameters listed in Table 1 have been
used as dependent variables in the Y block of the PLS analysis
which was performed. They included the normalized percent response
and IC.sub.50 values in the primary assay, and the CR.sub.50 values
in the secondary assay for both ABCB1 and ABCG2 transporters, and
also the associated toxicity TD.sub.50 data for these two targets.
The goal of this analysis was not only to cluster the compounds in
the physicochemical molecular descriptors space, but also to map
these biological parameters and selected physicochemical parameters
(computed solubility, log D7.4, MW, hydrogen bond donors and
acceptors, etc) in the principal components space.
[0158] FIG. 12 illustrates the clustering. This resulted in
effectively three groupings based on the similarity comparisons.
SID 88095709 and analogs with aryl and heteroaryl R1 and R2 groups
(excluding those with modifications to the piperazine) obviously
clustered together and those like SID 97301789 and SID85752814 (and
to a lesser extent reversan) with alkyl R2 substituents were
grouped together. The third set is included in the remainder of
compared structures that did not show significant similarity to one
another or the related class scaffolds. In conjunction with the
unique chemical space covered, the calculated solubility for the
SAR structures appear to be potentially better than those
previously described in clinical trials. Interestingly, the
calculated solubility of compounds with greater selectivity toward
ABCG2 efflux inhibition of JC-1 seem to generally be poorer (more
lipophilic) than those for ABCB1 inhibition where greater
solubility/hydrophilicity appears to be required. Although the
experimental solubility of SID 88095709 is less than ultimately
desirable, we are confident that the availability is sufficient for
all biological conditions outlined in this report. SID 88095709
also shows good solution stability and appears to not be a kinase
inhibitor based on the profile outlined Section 3.6.
[0159] The primary screening conditions outlined here are a model
system where JC-1 is an efflux inhibition surrogate for
chemotherapeutics and it must be noted that there is not
necessarily a one to one comparison of substrate recognition by
either efflux pump. Previous experimentation from our group has
validated the utility of such a model..sup.56,75 However, this does
not exclude the possibility that JC-1 efflux inhibition will not
match more phenotypic cell killing assay conditions such as the
potentiation assays outlined in this report. Our group has
extensively looked at profiling dozens of fluorescent substrates
against a panel of known efflux inhibitors in several ABC
transporters and observed distinctly different activities dependent
on the substrate inhibitor pairing in each over-expression system
(unpublished results, manuscript in preparation). This, however,
does not diminish the utility of small molecules with specific
efflux inhibition profiles. Such inhibitors can be useful tools to
look more closely at the nature of such pump poly-specific
substrate recognition, ultimately allowing for generation of better
model systems.
[0160] SID 88095709 demonstrates a 36-fold better efflux inhibition
of JC-1 efflux in ABCG2 over ABCB1, thus establishing its
usefulness in exploration of the system from a biochemical
perspective. This result, coupled with the noted cellular activity
in the potentiation assay justifies the overall utility of SID
88095709 as a chemical probe for ABCG2. SID 88095709 showed greater
potency and ABCG2 selectivity than any of the aforementioned
literature precedent compounds in the efflux inhibition screening
conditions. Only XR9051 seems to have better activity in the
potentiation assay although with reversed selectivity toward ABCB1.
SID 85752814, which was the original primary screening hit, does
not possess the high level ABCG2 efflux inhibition selectivity of
SID 88095709 but submicromolar potentiation and preliminary tumor
reduction data indicate potential clinical opportunities.
Interestingly, the structurally-related analogue that has an
improved selectivity toward ABCG2 in the efflux assessment,
compound SID 97301789, was shown to have significantly more potent
cell killing activity in the chemoreversal assay (with no observed
toxicity), however, the efflux inhibition selectivity appears to be
lost. The emergence of this compound occurred after extensive
supporting SAR had been established for SID 88095709, thus limiting
the expansion of another arm of SAR in support of SID 97301789;
however, this development provides an exciting opportunity for
refinement, particularly in the context of the observed ABCG2 tumor
reduction preliminary results for SID 88095709 and SID
85752814.
References for Background of the Invention and Example 1
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Example 2
A Selective ATP-Binding Cassette Sub-Family G Member 2 Efflux
Inhibitor Revealed Via High-Throughput Flow Cytometry.
[0236] As a result of a focused SAR-driven chemistry effort we
describe compound 1 (CID44640177), an efflux inhibitor with
selectivity toward ABCG2 over ABCB1. Compound 1 is also shown to
potentiate the activity of mitoxantrone in vitro as well as
preliminarily in vivo in an ABCG2 over-expressing tumor model. At
least two analogs significantly reduce tumor size in combination
with the chemotherapeutic topotecan. To our knowledge, low
nanomolar chemoreversal activity coupled with direct evidence of
efflux inhibition for ABCG2 is unprecedented.
[0237] We set out to develop new small molecule scaffolds with
distinct efflux inhibition selectivity profiles based on multiplex
transporter target assays. Early in the post-screen follow-up it
was evident that ABCG2 was the desirable focus for a probe campaign
based on promising preliminary selectivity. Although there has been
significant progress with ABCB1 inhibitors, similar progress has
not been achieved with ABCG2 inhibitors. An example was noted with
the Aspergillus fumigates mycotoxin fumitremorgin C (FTC, 3) and
its analogs Ko132, Ko134, and Ko143 (4) which have been
demonstrated to be selective inhibitors for ABCG2..sup.17-18 Other
reported ABCG2 inhibitors engage non-selectively to include
biricodar and nicardipine which are cross-pump inhibitors for
ABCB1, ABCC1, and ABCG2..sup.7,19 Further, specific relevance for
ABCG2 as a clinical target has been well documented..sup.20 This
includes a mouse model using a human ovarian xenograft with
Igrove1/T8 tumors,.sup.21 a system utilizing flavopiridol-resistant
human breast cancer cells,.sup.22 FTC (3) and Ko143 (4) inhibition
in vitro and mouse intestine model,.sup.17 and a phase I/II trial
with lapatinib in glioblastoma multiforme..sup.23
[0238] Given the absence of clinically relevant ABCB1 or ABCG2
specific inhibitors and as there remain gaps in understanding how
inhibition of these efflux pumps can be best exploited for
therapeutic gain, our team focused on vetting and optimizing novel
hit scaffolds with promising preliminary ABCG2 or ABCB1 selectivity
and potency. As part of that effort, several bench mark compounds
were chosen for comparison during development of the
pyrazolopyrimidinylpiperazine scaffold, 1. Bench mark compounds
were chosen for differential selectivities on ABCB1, ABCC1 and
ABCG2, so as to represent a broad panel against which analogs of 1
could be evaluated (FIG. 1A). For direct comparison of selective
ABCG2 inhibition, both 3 and 4 were chosen..sup.17-18 The
submicromolar ABCB1 modulator 2 was chosen as it is known to
reverse resistance to cytotoxic drugs such as doxorubicin and
vincristine..sup.8,24 Quinoline MK571 (5), a specific inhibitor of
ABCC1, was necessary to gauge any ABCC1 activity..sup.25 Also,
reversan (6), identified as an active inhibitor of ABCB1 and ABCC1,
was included as it contained a similar, pyrazolopyrimidine
core..sup.26
Materials and Methods
General Information
[0239] The ABCB1 over-expressing drug-resistant cell line, CCRF-Adr
5000, and its parental CCRF-CEM cells were kindly provided by Dr.
T. Efferth (Pharmaceutical Biology, German Cancer Research Center,
Heidelberg, Germany). We have previously described the generation
of the Jurkat-DNR ABCB1 over-expressing cell line..sup.27 Ovarian
ABCG2 over-expressing Ig-MXP3 and Igrov1/T8 cells as well as the
parental Igrov1-sensitive cells were kindly provided by Dr. D. Ross
(Department of Medicine, University of Maryland Greenebaum Cancer
Center, Baltimore, Md.). Cells were grown in RPMI-1640 medium
supplemented with 10% fetal bovine serum (FBS, Hyclone, Logan,
Utah), 2 mM L-glutamine, 10 mM HEPES, 10 U mL.sup.-1 penicillin, 10
.mu.g mL.sup.-1 streptomycin, and 4 .mu.g mL.sup.-1 ciprofloxacin.
Selective pressure for the ABCB1 over-expressing CCRF-ADR 5000 and
Jurkat-DNR cells was maintained by growth in 20 nM daunorubicin
hydrochloride (DNR). Selective pressure for the ABCG2
over-expressing Ig-MXP3 cells is maintained by treatment with 340
nM mitoxantrone dihydrochloride (MTX) for 1 hr. prior to
harvest.
[0240] The fluorescent reporter dye JC-1 and cell type
differentiation dye CellTrace.TM. Far Red DDAO-SE were obtained
from Invitrogen.TM. (Carlsbad, Calif.). Nicardipine hydrochloride,
DNR, MTX, topotecan hydrochloride hydrate (TPT) and FTC (3) were
purchased from Sigma-Aldrich (St. Louis, Mo.). XR9051 (2), reversan
(6), MK571 (5), and Ko143 (4) were purchased from Tocris Bioscience
(Minneapolis, Minn.). Compounds ordered for SAR by commerce were
purchased from ChemDiv (San Diego, Calif.) and Ryan Scientific (Mt.
Pleasant, S.C.). Unless otherwise indicated, all compound solutions
were maintained and diluted in DMSO prior to addition to assay
wells. Final DMSO concentrations were no more than 1% (v/v). A
Biomek.RTM. NX Multichannel (Beckman-Coulter, Brea, Calif.) was
used for all cell and compound solution transfers for volumes
greater than 1 .mu.L. Low volume transfers (100 nL) were done via
pintool (V&P Scientific, San Diego, Calif.). Compound
dose-response plates were generated with the Biomek.RTM. NX Span-8
(Beckman-Coulter, Brea Calif.).
[0241] The HyperCyt.RTM. high throughput flow cytometry platform
(IntelliCyt.TM., Albuquerque, N. Mex.) was used to sequentially
sample cells from 384-well microplates (2 .mu.L per sample) for
flow cytometer presentation at a rate of .about.40 samples per
minute..sup.28-29 Flow cytometric analysis was performed on a
CyAn.TM. flow cytometer (Beckman-Coulter, Brea, Calif.). The
resulting time-gated data files were analyzed with HyperView.RTM.
software to determine compound activity in each well. Inhibition
response curves were fitted by Prism.RTM. software (GraphPad
Software, Inc., La Jolla, Calif.) using nonlinear least-squares
regression in a sigmoidal dose-response model with variable slope,
also known as the four-parameter logistic equation. This type of
time-gated flow cytometric data analysis was described in detail
for a previous ABC transporter screen from our group..sup.30
Primary Assay Conditions
[0242] To facilitate a shortened screening timeline the single
point assay was performed as a duplex allowing for data from both
cell lines to be collected in one screening campaign. The assay was
conducted in 384-well format microplates in a total volume of 15.1
.mu.L dispensed sequentially as follows: 1) JC-1 substrate (10
.mu.L per well); 2) test compound (100 nL per well); 3)
drug-resistant cells (5 .mu.L per well). CCRF-Adr cells (ABCB1)
were color-coded with 0.5 ng mL.sup.-1 CellTrace.TM. Far Red
DDAO-SE for 15 minutes at room temperature, washed twice by
centrifugation, and then combined with unlabeled Ig-MXP3 cells
(ABCG2) in the assay buffer. Final in-well concentration of test
compound was 6.6 .mu.M, JC-1 concentration was .about.1 .mu.M. JC-1
previously proved to be an ideal fluorescent reporter substrate for
both ABCB1 and ABCG2..sup.30 The cell concentration was
3.times.10.sup.6 cells mL.sup.-1 (1:1 ratio of the two cell types).
Nicardipine was used as an on-plate control for both pumps at 50
.mu.M. The plate contents were mixed, rotated end-over-end at 4 RPM
at 25.degree. C. for 10 minutes, and then cell samples were
immediately analyzed. This resulted in analysis of approximately
1,000 cells of each cell type from each well. Flow cytometric data
of light scatter and fluorescence emission at 530+/-20 nm (488 nm
excitation, FL1) and 665+/-10 nm (633 nm excitation, FL8) were
collected.
[0243] The CCRF-Adr cell line proved optimal for the duplex-Far Red
DDAO-SE staining protocol but in the single-plex follow-up we
preferentially used the assay provider's ABCB1 over-expressing
Jurkat-DNR cell line for confirmatory dose-response. Each cell line
(Jurkat-DNR and Ig-MXP3) was run separately against all compounds
(no differential cell staining) in dose-response. The protocol
differed from the single point screen as describe here. Cells and
reagents were added sequentially as follows: 1) PBS buffer (5 .mu.L
per well); 2) test compound (100 nL per well); 3) drug-resistant
cells (10 .mu.L per well) pre-exposed to the JC-1 substrate at 1
.mu.M just prior to the well addition. Final in-well concentrations
of test compound ranged from 50 .mu.M to 69 nM over an 18 point
dose-response and the cell concentration was 1.times.10.sup.6 cells
mL.sup.-1. Dose response IC.sub.50 values were average for multiple
runs (average n of 2 to 4).
Chemoreversal Secondary Assay
[0244] Cells (ABCB1, Jurkat-DNR or ABCG2 Ig-MXP3) were incubated
with the test compound in a 3 order of magnitude concentration
range over three and seven day periods in the presence of the
inhibitor and chemotherapeutic (ABCB1, 100 nM DNR or ABCG2 30 nM
MTX), such that a cell concentration of at least 1.times.10.sup.5
cells mL.sup.-1 was maintained. Cell viability was determined by
trypan blue staining and enumeration under light microscopy. At day
3, wells with greater than 2.times.10.sup.5 cells were refreshed
with medium, to include readjustment of chemotherapeutic and
inhibitor concentration. A chemoreversal index (Chemoreversal 50,
CR.sub.50) was determined from the viability assessment. Using a
similar approach, a direct cytotoxicity index (Toxic Dose 50,
TD.sub.50) was determined by assessment of cell death of cells
grown in media alone. Results were compared with the survival of
parental cells in the presence of the selective agent
(chemotherapeutic; 100% cell death), as well as survival of
drug-resistant cells in the presence of the chemotherapeutic drug
(control yields 100% viability). As previously described by our
group for an ABCB1-reversal agent the difference between the
CR.sub.50 and the TD.sub.50 affords an approximation of the in
vitro therapeutic index for the test compound..sup.31 The threshold
for a "good" therapeutic window when comparing CR.sub.50 and
TD.sub.50 somewhat depends on the endpoint use. For cancer
treatment a low threshold, in the 10 fold (or greater) range, can
still be considered acceptable due to the severity and life
threatening nature of the disease.
Preliminary in Vivo Study
[0245] Igrov1/T8 cells were injected into the hind limbs of CB-17
SCID mice at a concentration of 1.times.10.sup.7 cells in 200
.mu.L, n of 3 per condition. The tumor was grown until the volume
was in the range of 75 mm.sup.3 to 250 mm.sup.3. The volume of the
tumor was verified with a Scienceware.RTM. Digi-Max.TM. slide
caliper obtained from Sigma-Aldrich, and the tumor volumes were
calculated by the equation: (W.sup.2/2)*L..sup.21 Tumor-bearing
mice were injected intratumorally with 150 nM topotecan alone, as
well as with either 100 nM of 1 or 500 nM of 7. Injections were
repeated daily and the size of the tumor was determined as a
fraction of the starting size. After four days the mice were
sacrificed and any recurring tumor was verified by light microscopy
examination of histological sections. Mice were studied and
maintained in accordance with guidelines of our Institutional
Animal Research Committee at UNM HSC.
Representative Synthesis
[0246] Probe compound 1 and many analogues were synthesized by the
method shown (FIG. 2, sequence A-C). Commercial substituted
aminopyrazoles 90 (R.sub.1=phenyl, substituted phenyl, other
substituents found on a different chemical positions) were treated
with the appropriate dialkylmalonate or .beta.-ketoester to give
intermediate(s) 91, followed by chlorination to afford the
pyrazolo[1,5-a]pyrimidine core intermediate(s) 92. Intermediate(s)
92 is an active compound which allows nucleophilic displacement on
the chloro position of a piperazine moiety to provide Installation
of the piperazine moiety afforded 1 directly. In some cases, a
Suzuki or Molander type coupling was preferred to install aryl
functionality at a late stage in the synthesis (FIG. 2A, sequence
D-F). More detailed synthetic methods and spectral data can be
found in the supplementary material.
Results and Discussion
Primary Screening
[0247] A high throughput, no wash, duplex assay was constructed in
which both the ABCB1 and ABCG2 transporters were evaluated in
parallel using fluorescent JC-1 as the efflux reporting substrate.
ABCB1 over-expressing CCRF-Adr cells were color-coded to allow
their distinction from Ig-MXP3 ABCG2 over-expressing cells as
previously described..sup.30 FIG. 3A briefly summarizes the
primary, duplex screening protocol. The primary screening results
were uploaded as PubChem BioAssay Database Identifiers (AID) 1325
and 1326 for ABCG2 and ABCB1 respectively (Summary AID
1818)..sup.32 A total of 194,393 Molecular Libraries Small Molecule
Repository (MLSMR, http://mlsmr.glpg.com) compounds were tested
with Z' values of 0.74.+-.0.10 and 0.64.+-.0.13 for ABCB1 and ABCG2
respectively. A total of 200 and 130 actives were noted in ABCG2
and ABCB1 respectively. Compounds were deemed active if the percent
inhibition was greater than 80%. A subsequent cherry pick resulted
in single point confirmatory testing of 273 compounds (AIDs 1453
and 1451) resulting in 16 and 18 actives in ABCG2 and ABCB1,
respectively. As a fluorescence counter-screen, a set of related
488/530 nm fluorescence compound profiling data was also associated
with the SMR cherry pick set in which compound fluorescence was
assessed in the absence of JC-1 to rule out false positives versus
actual efflux inhibitors. These data were uploaded as two AIDs
(1480 and 1483) where the 273 compounds were tested with 89 and 83
compounds noted as active (i.e. fluorescent) in ABCG2 and ABCB1,
respectively. Based on this single point screening data
confirmatory dose-response analysis was subsequently performed on
40 compounds (AIDs 1690 and 1689) resulting in 16 actives for ABCB1
and 9 actives for ABCG2.
Efflux Inhibition Driven SAR
[0248] No discernible structure activity relationship (SAR) was
revealed through the first round of cherry pick analysis or the
powder resupply, and many of the compounds were observed to be
fluorescent artifacts. However, preliminary chemoreversal secondary
screening efforts confirmed activity of several compounds,
including 7 (CID 1434724), where micromolar potentiation and low
toxicity were observed but with little pump specificity. Secondary
potentiation data for compound 7 and fourteen other confirmed
compounds were reported in AIDs 2830 and 2833. Resynthesis,
purification and retesting of 7 confirmed the efflux inhibition,
and a series of compounds similar in structure was ordered around
this original hit. These 31 compounds were tested in dose-response
in the two efflux pump over-expressing cell lines: Jurkat-DNR
(ABCB1) and Ig-MXP3 (ABCG2). A few compounds showed modest ABCG2
selectivity, but gaps in the collection did not resolve the
structural functionality responsible for any significant efflux
potency or selectivity towards ABCG2 or ABCB1. Due to the diversity
of structural changes present in the commercial set, additional
compounds were needed to construct meaningful SAR. The commercial
powder set contained several members with conserved functionality
that provided the basis for establishing a methodical SAR
assessment.
[0249] The pyrazolo[1,5-a]pyrimidine core was preserved, and
exchange of the peripheral substituents were surveyed, depicted as
shaded regions (FIG. 4A, panel A). Exploratory commercial SAR
expansion resulted in compounds with selectivity profiles
significantly biased toward ABCG2. The initial set of hit-related
compounds screened from the MLSMR and purchased from vendors
predominately possessed structural differences in R.sub.1-R.sub.3
and the furan ring of R.sub.4. Based on these structural variations
around the core, we chose functional groups that would fill in the
SAR gaps and further reveal pharmacological preferences based on
steric interactions, lipophilicity, hydrogen bond donating or
accepting character, and modulating the electronic nature of aryl
substituents. Of the hits that were identified through this
endeavor, 8 (CID1441553) had attractive efflux potency towards
ABCG2 and marginal selectivity over ABCB1 (FIG. 5A, panel B). The
KU SCC launched an SAR campaign aimed at further understanding the
origin of potency and selectivity and set out to optimize the
compound profile to meet the MLPCN probe criteria for potency and
selectivity in the efflux assay. A suitable probe was defined as a
compound that effected micromolar potentiation with a
toxic-dose.sub.50/chemoreversal.sub.50 (TD.sub.50/CR.sub.50) ratio
of greater than 10 and with overall toxicity greater than 15
.mu.M.
[0250] Of the approximately 160 compounds assessed in the primary
efflux dose-response assay (AIDs 489002, 489003, 504566, and
504569), 126 were synthesized by the KU SCC. Several compounds in
the purchased collection contained a 3-chlorophenyl substituent at
R.sub.1, analogous to the R.sub.1 moiety present in hit 8. As such,
an initial series of compounds was prepared with this feature
maintained while adjusting R.sub.2-R.sub.4 (Table 1). One compound
cluster was constructed with R.sub.1-R.sub.3 groups (11-25)
identical to that of the parent hit 8 while modulating R.sub.4.
Notably, the substitution of the acyl-2 furan for acyl-3-furan (11)
produced a 7-fold enhancement in selectivity for ABCG2,
predominately due to erosion of ABCB1 potency, while only modestly
attenuating ABCG2 potency as compared to the parent hit. Improved
ABCG2 potency was achieved with installation of an acyl-3-pyridine;
however, the selectivity deteriorated essentially to pan inhibition
(20). Following incorporation of the acyl-3-furan as the more
optimal R.sub.4 substituent, a survey was then done on the R.sub.2
group while holding constant R.sub.1 and R.sub.3 (26-32).
Substantial potency for ABCG2 was gained when R.sub.4 was
3-pyridine (30); however, once again, selectivity was negatively
impacted.
[0251] Alterations in the 3-chlorophenyl R.sub.1 substituent were
then made while assessing three R.sub.4 head groups, specifically
alternating between acyl-2-furan, acyl-3-furan, or benzoyl
functionalities (33-43). No substantial improvements were noted
with these changes; however, when R.sub.1 was changed from
3-chlorophenyl to phenyl, and R.sub.2 was varied (1, 44-49), it was
discovered that a 2-furan at R.sub.2 in concert with the optimized
acyl-3-furan afforded a significant boost in both ABCG2 potency as
well as overall ABCG2 selectivity (1, ABCB1 EC.sub.50=4.65 .mu.M;
ABCG2 EC.sub.50=0.13 .mu.M, selectivity=36 fold). Representative
dose-response curves for 1 are compared (FIG. 5A).
[0252] With this information in hand, the team followed up with an
SAR effort aimed at demonstrating supportive SAR for compounds
bearing an R.sub.2=2-furyl group while also attempting to improve
upon the profile of the most promising analog, 1 (FIG. 4A, panel
C). The modified scaffold, represented by 1, was further studied by
adjusting physiochemical and spatial elements in R.sub.1
(Supplemental Table S1, compounds 1, 50-60). Replacing the phenyl
ring of the lead with a t-butyl group erased much of the gains
towards ABCG2 selectivity (50). Traditional phenyl replacements
such as thiophene or furan were tolerated, but only led to modest
selectivity and potencies. The installation of a 4-chlorophenyl
substituent led to a reduced inhibition of ABCB1, resulting in
selectivity in the efflux assay of 22-fold (54); however, the
change also marginalized the potency on ABCG2. Renovating the
phenyl substituent with electron donating groups was not
beneficial.
[0253] An examination of 2-furan replacements at R.sub.2 was also
undertaken (Supplemental Table S1, compounds 1, 44-49, 61-66).
Simple alkyl units such as methyl or t-butyl degraded potency and
fold-selectivity for both transporters. Notably, use of t-butyl
actually reversed selectivity for ABCB1, albeit at the expense of
potency (49). Some R.sub.2 revisions resulted in impressive ABCG2
selectivities and potencies. The choice of 2-F-phenyl (46) slightly
degraded potency for ABCB1 as compared to the parent (1), leading
to a 10-fold selectivity in favor of ABCG2. For the fluorinated
series (46-48), the potency for both transporters decreased as the
fluorine atom was migrated from the 2- to 3- to 4-position of the
aromatic R.sub.2 ring. Interestingly, the use of a 3-MeO-phenyl
group impeded potency for ABCB1 activity while retaining
submicromolar ABCG2 potency on par with the parent, leading to an
improved 83-fold selectivity between the transporters (65). In this
series (44, 45, 65), however, a trend was not observed as the
substituent was shifted from each position. Additional compounds
prepared with the 3-MeO-phenyl group at R.sub.2 did not show a
consistent SAR (data not shown).
[0254] The commercial set of compounds contained a few scaffolds
bearing a methyl group at R.sub.3. SAR data generated in the early
experimental phases demonstrated some benefit to the presence of
small alkyl groups at R.sub.3; however, this was highly dependent
on the identity of groups at R.sub.1, R.sub.2 and R.sub.4. To
understand the functionality changes around 1, one analogue was
prepared to quickly evaluate the effect of this substitution
pattern in concert with our chosen functionalities at R.sub.1,
R.sub.2 and R.sub.4). ABCB1 potency was encouragingly impaired, but
not without also effecting G2, resulting in a marginal selectivity
profile (data not shown).
[0255] Attention was then turned to investigating the effect of
different R.sub.4 functionality appended to the piperazine
(Supplemental Table S1, compounds 1, 67-76). In earlier SAR sets,
activity was found to be sensitive to the identity of R.sub.4 and
the pairing of groups at R.sub.2 and R.sub.3. In the context of our
new lead, 1, we wanted to better understand the effect of R.sub.4
with the chosen substituents. It was confirmed that an acyl-3-furan
was preferred to an acyl-2-furan (67), and simple alkyl
substitution of the 3-furan (70-72) or a larger benzofuran (73),
while tolerated, did not reveal any benefits. However, the most
influential effects on ABCB1 were observed when the acyl furan was
exchanged for a benzyl ester (76). While ABCG2 potency was
compromised compared to the lead, ABCB1 potency was completely
lost, yielding a selectivity of .about.19 fold.
[0256] In a more aggressive effort, the entire "top piece" of the
scaffold, consisting of the piperazine and the R.sub.4 group, was
modified (Supplemental Table S2, compounds 77-86). Ring-opened
piperazine equivalents, truncated amino groups, piperidine amides,
ring-expanded amines (not shown) and various structural variations
on a theme did not produce a profile superior to that which had
already been observed.
[0257] In the process of evaluating these structural modifications,
several compounds were prepared singly to target possible
oversights in SAR, as every possible R.sub.1-R.sub.4 combination
cannot be prepared and assessed in a timely way. Others were
targeted as a means of inserting the best combinations as gleaned
from the preceding generations of SAR. These compounds were more
recently pursued to probe specific structural combinations and are
summarized. (Supplemental Table S1, compounds 87-89). Data obtained
early in the project had indicated that the acetyl group at R.sub.4
was more advantageous than other changes that had been surveyed
(including the benzyl ester modification), though later refinement
of these data does not now stand out as particular SAR of interest.
Based on the information available at the time, substituted phenyl
derivatives with varying electronic features at R.sub.1 were
incorporated with the acetyl R.sub.4 group in place (87, 88). No
advantages were found.
Prior Art Comparison and Potentiation
[0258] In parallel to the above efforts, select compounds were also
assessed in secondary assays; however, this chemoreversal assay
based on potentiation of a given chemotherapeutic is a very low
throughput assay and compound data from this assay could not be
used to drive the SAR program. Key data have been collected (AIDs
504476 and 504477) for some of the most promising compounds (Table
2). Evaluation of probe compound 7 showed submicromolar effective
potentiated killing of both over-expressing cell lines with
preference for ABCG2 from a toxicity perspective, though the degree
of selectivity observed in the cell killing assay is removed (1.8
fold in chemoreversal vs. 36-fold in efflux assay). Though clear
SAR trends could not be gleaned from the chemoreversal and toxicity
data, these experiments provided the basis for evaluating how
select compounds would perform in a cellular context. Most of the
compounds evaluated in the chemoreversal assay for ABCB1 registered
in the 100 to 600 nM range, with only a few outliers. For ABCG2,
the CR.sub.50 was more broad, ranging from .about.20 to 1400 nM.
These data, coupled with several compounds that showed TD.sub.50
values>100, provided support for the selection of compounds for
preliminary in vivo studies.
[0259] Prior art for 1 includes 2 (XR9051) and MK571 which were
chosen to verify both ABCB1 activity and counterscreen ABCC1
activity, respectively in our system (Table 2). Compound 2 inhibits
the efflux of JC-1 in both ABCB1 and ABCG2 over-expressing cell
lines (0.61.+-.0.43 and 2.27.+-.2.05 .mu.M respectively).
Potentiation data indicate submicromolar chemoreversal in both
Jurkat-DNR and Ig-MXP3 cells with a bias toward ABCB1 at 10 nM as
compared to 650 nM for ABCG2. Not surprisingly, we didn't observe
any inhibition of ABCB1 or ABCG2 with MK571 (5) up to 50 .mu.M and
activity noted in the secondary assays mirrored toxicity indicating
no potentiation. Direct comparison of reversan (6) in our efflux
inhibition system shows low micromolar inhibition of both ABCB1 and
ABCG2 (4.41.+-.2.90 and 0.84.+-.0.03 .mu.M respectively) with
moderate selectivity for ABCG2. In our chemoreversal potentiation
assay, 6 showed micromolar activity and no ABCG2 selectivity (0.22
and 3.61 .mu.M in ABCB1 and ABCG2, respectively). This was coupled
with significant toxicity in the Jurkat-DNR cell line. FTC (3)
showed no activity in either cell line in the efflux inhibition
assay and was not tested in the potentiation assay. Ko 143 (4) has
been shown to potentiate mitoxantrone (MTX) at nanomolar levels in
ABCG2 over-expressing cells.sup.17. In our potentiation assay there
appeared to be a selectivity for ABCB1 over ABCG2 (CR.sub.50=0.99
and 5.94 .mu.M respectively) with considerable toxicity in both
cell lines. The efflux inhibition activity did not emulate this,
showing no activity for ABCB1 and only 13.55 .mu.M inhibition for
ABCG2, potentially indicating a binding site difference versus
JC-1. Probe compound 1 demonstrates a 36-fold better inhibition of
JC-1 efflux for ABCG2 over ABCB1, thus establishing its usefulness
for biochemical exploration. This result, coupled with the noted
cellular activity in the potentiation assay justifies the overall
utility of 1 as a probe for ABCG2. Compound 1 showed greater
potency and ABCG2 selectivity than any of the aforementioned
literature precedent compounds in the efflux inhibition screening
conditions. Only 2 appears to have better activity in the
potentiation assay although with reversed selectivity toward
ABCB1.
Preliminary in Vivo Mouse Data
[0260] To specifically demonstrate the direct effect of a
chemotherapeutic agent relevant to these studies, we administered
an intratumoral dose of topotecan (TPT, 150 nM). This concentration
of TPT was slow to kill non-resistant (ABCG2 non-expressing)
parental tumor cells, but not the drug resistant (ABCG2 expressing)
tumor cells (EC.sub.50 for parental is 7 nM vs. 311 nM for
resistant cells). To demonstrate efficacy of the inhibitors, we
grew ABCG2 resistant tumor cells to a volume between 75 and 250
mm.sup.3 in CB-17 SCID mice. Tumor size was determined immediately
prior to the first injection. Tumor-bearing mice were injected with
150 nM TPT and either 100 nM of 1 or 500 nM of 7. Such treatment
dramatically reduced tumor size the over a 96 hour observatory
period (FIG. 6A), indicating that tumor sensitivity of TPT returned
when either of the ABCG2-blocking compounds were present
(p<0.001). A concentration of 150 nM TPT alone did not affect
the growth of tumors (data not shown). Compounds 1 and 7 were
chosen as representative molecules from this scaffold, with 7 being
the initial hit from the screening campaign and 1 being the efflux
inhibition optimized molecule. As previously stated, the low
throughput nature of the in vitro chemoreversal secondary assay did
not allow for exhaustive testing of all members in the scaffold and
thus specific correlation between efflux inhibition and potentiated
activity of known chemotherapeutics was not readily accessible.
However, similar in vivo activity of 1 and 7 (note there is a
five-fold concentration difference for the two, see FIG. 6A)
despite dissimilar efflux inhibition profiles indicates that the
efflux reporting via JC-1 may not represent activity for both MTX
and DNR in Ig-MXP3 and Jurkat-DNR cell lines respectively. This in
conjunction with the reported in vitro chemoreversal data in
Supplemental Table S1 indicates a need for further analysis of the
in vitro to in vivo correlation. A subsequent manuscript detailing
activity of molecules from this scaffold in this animal model is
forthcoming.
Summary
[0261] As a result of a duplex, high-throughput flow cytometric
screening campaign and subsequent medicinal chemistry optimization
we report herein the discovery of an ABCG2 efflux inhibitor 1 which
demonstrates a 36-fold selectivity over ABCB1 toward blocking
efflux of the fluorescent substrate JC-1. Furthermore, in the same
JC-1 efflux reporter system, 1 maintains approximately 100-fold
higher potency in ABCG2 than the prior art Ko143. Subsequent in
vitro assays using the known chemotherapeutic mitoxantrone
demonstrate the ability of 1 to potentiate cell death of the ABCG2
expressing Ig-MXP3 cells at submicromolar concentration levels. We
believe that selective transport inhibitors may allow for more
targeted and effective therapy. Preliminary in vivo mouse model
data indicate dramatic tumor reduction with co-treatment of TPT
with 1. Future studies will focus on several related scaffold
members as well as on selectivity profiles in both in vitro and in
vivo potentiation.
TABLE-US-00001 TABLE 1 (Example 2). SAR expansion on initial hit
compound 8 (CID1441553). ##STR00006## IC.sub.50 (.mu.M).sup.a ~Fold
G2 Cpd CID R.sub.1 R.sub.2 R.sub.3 R.sub.4 ABCB1 ABCG2
Selective.sup.b 8 CID1441553 3-Cl--Ph Ph H CO-2-furyl 6.18 .+-.
4.92 0.96 .+-. 0.37 6.4 9 CID644556 3-Cl--Ph Me Me CO-2-furyl 8.40
.+-. 0.70 2.65 .+-. 1.07 3.2 10 CID652994 3-Cl--Ph Me H CO-2-furyl
5.88 .+-. 1.17 2.78 .+-. 0.82 2.1 11 CID44640182 3-Cl--Ph Ph H
CO-3-furyl 8.58 .+-. 0.63 1.19 .+-. 0.26 7.2 12 CID44607976
3-Cl--Ph Ph H CO-3-thiophene 3.97 .+-. 0.80 1.40 .+-. 0.32 2.8 13
CID44602407 3-Cl--Ph Ph H CO--Ph 3.42 .+-. 1.64 1.45 .+-. 0.59 2.4
14 CID45105078 3-Cl--Ph Ph H CO-2-MeO--Ph 4.73 .+-. 1.78 4.92 .+-.
4.28 1.0 15 CID45105074 3-Cl--Ph Ph H CO-3-MeO--Ph 2.64 .+-. 0.64
4.45 .+-. 4.68 0.6 16 CID45105073 3-Cl--Ph Ph H CO-4-MeO--Ph 2.89
.+-. 0.55 2.82 .+-. 1.91 1.0 17 CID45105075 3-Cl--Ph Ph H
CO-2-Cl--Ph 1.85 .+-. 0.75 1.31 .+-. 0.23 1.4 18 CID45105080
3-Cl--Ph Ph H CO-3-Cl--Ph 4.19 .+-. 1.88 2.95 .+-. 0.57 1.4 19
CID45105081 3-Cl--Ph Ph H CO-4-Cl--Ph 2.36 .+-. 0.70 2.67 .+-. 0.85
0.9 20 CID44623842 3-Cl--Ph Ph H CO-3-pyridyl 0.57 .+-. 0.08 0.23
.+-. 0.15 2.5 21 CID44630540 3-Cl--Ph Ph H CO-4-pyridyl 1.16 .+-.
0.30 1.03 .+-. 0.23 1.1 22 CID45281172 3-Cl--Ph Ph H CO-cyclohexyl
3.31 .+-. 1.86 2.67 .+-. 1.26 1.2 23 CID44602405 3-Cl--Ph Ph H
COCH.sub.3 5.67 .+-. 2.21 6.07 .+-. 0.73 0.9 24 CID45105082
3-Cl--Ph Ph H CO-Bn 4.68 .+-. 2.69 5.76 .+-. 3.40 0.8 25
CID44607585 3-Cl--Ph Ph H SO.sub.2--Ph >50 >50 NA 26
CID44631077 3-Cl--Ph 2-F--Ph H CO-3-furyl 4.69 .+-. 1.50 1.93 .+-.
0.48 2.4 27 CID44629741 3-Cl--Ph 4-F--Ph H CO-3-furyl 5.65 .+-.
0.11 2.41 .+-. 0.52 2.3 28 CID44630538 3-Cl--Ph 3-MeO--Ph H
CO-3-furyl 2.17 .+-. 1.02 1.14 .+-. 0.54 1.9 29 CID44629740
3-Cl--Ph 4-MeO--Ph H CO-3-furyl 4.12 .+-. 1.05 1.71 .+-. 0.58 2.4
30 CID44629742 3-Cl--Ph 3-pyridyl H CO-3-furyl 0.77 .+-. 0.28 0.27
.+-. 0.12 2.9 31 CID44630541 3-Cl--Ph 4-pyridyl H CO-3-furyl 3.68
.+-. 0.35 1.46 .+-. 0.35 2.5 32 CID44631078 3-Cl--Ph ethynyl H
CO-3-furyl 10.16 .+-. 3.99 2.36 .+-. 0.53 4.3 33 CID44623844
4-Br--Ph Ph H CO-3-furyl 1.31 .+-. 0.35 0.76 .+-. 0.43 1.7 34
CID44640183 2-MeO--Ph Ph H CO-3-furyl 2.17 .+-. 0.76 0.45 .+-. 0.17
4.8 35 CID44623840 3-MeO--Ph Ph H CO-3-furyl 2.26 .+-. 0.83 1.14
.+-. 0.59 2.0 36 CID44607592 4-MeO--Ph Ph H CO-3-furyl 1.44 .+-.
0.70 1.16 .+-. 0.66 1.2 37 CID44640176 2-MeO--Ph Ph H CO-2-furyl
3.00 .+-. 0.57 1.03 .+-. 0.17 2.9 38 CID44968166 3-MeO--Ph Ph H
CO-2-furyl 4.77 .+-. 0.63 1.40 .+-. 0.32 3.4 39 CID492424 4-Cl--Ph
Ph H CO-2-furyl 3.64 .+-. 1.47 1.37 .+-. 0.53 2.7 40 CID45105079
3-Me--Ph Ph H CO-2-furyl 3.88 .+-. 1.67 2.46 .+-. 1.09 1.6 41
CID44640179 2-MeO--Ph Ph H CO--Ph 2.31 .+-. 0.79 0.76 .+-. 0.06 3.0
42 CID44968164 3-F--Ph Ph H CO--Ph 4.34 .+-. 2.09 2.27 .+-. 0.06
1.9 43 CID44968158 4-F--Ph Ph H CO--Ph 3.99 .+-. 2.31 2.03 .+-.
0.11 2.0 1 CID44640177 Ph 2-furyl H CO-3-furyl 4.65 .+-. 0.74 0.13
.+-. 0.03 35.8 44 CID46905002 Ph 2-MeO--Ph H CO-3-furyl 4.52 3.66
.+-. 2.54 1.2 45 CID46905009 Ph 4-MeO--Ph H CO-3-furyl 4.55 2.37
.+-. 1.80 1.9 46 CID46904993 Ph 2-F--Ph H CO-3-furyl 5.61 0.56 10.0
47 CID46905008 Ph 3-F--Ph H CO-3-furyl 7.07 1.68 .+-. 0.61 4.2 48
CID46904994 Ph 4-F--Ph H CO-3-furyl 11.82 6.45 1.8 49 CID46905006
Ph .sup.tBu H CO-3-furyl 5.62 11.50 0.5 .sup.aEfflux inhibition
activity (IC.sub.50) of the JC-1 substrate is reported using
Jurkat-DNR cells over-expressing ABCB1 and Ig-MXP3 cells
over-expressing ABCG2. Replicates of two to four are reported as
averages. .sup.bSelectivity is indicated by the quotient of the
average ABCB1 IC.sub.50 and the average of ABCG2 IC.sub.50.
TABLE-US-00002 TABLE 2 (Example 2). Potentiation data and prior art
comparison. ABCB1 ABCG2 IC.sub.50.sup.a CR.sub.50.sup.b
TD.sub.50.sup.c IC.sub.50.sup.a CR.sub.50.sup.b TD.sub.50.sup.c Cpd
CID (.mu.M) (.mu.M) (.mu.M) (.mu.M) (.mu.M) (.mu.M) 1 CID44640177
4.65 0.55 5.52 0.13 0.31 18.30 8 CID1441553 6.18 0.25 6.77 0.96
0.14 6.00 9 CID644556 8.40 1.58 >100 2.65 0.17 47.40 15
CID45105074 2.64 0.35 >100 4.45 0.21 >100 20 CID44623842 0.57
0.17 4.72 0.23 0.13 10.30 21 CID44630540 1.16 0.25 10.90 1.03 0.15
28.30 24 CID45105082 4.68 0.51 17.80 5.76 0.60 >100 30
CID44629742 0.77 0.20 5.33 0.27 0.02 5.77 31 CID44630541 3.68 0.60
13.50 1.46 0.89 57.80 34 CID44640183 2.17 0.45 4.37 0.45 0.49 6.50
36 CID44607592 1.44 0.23 5.54 1.16 0.38 >100 44 CID46905002 4.52
0.17 3.73 3.66 0.68 7.62 52 CID46905000 1.70 0.19 1.93 2.35 1.21
>100 53 CID45105077 6.42 0.53 8.53 2.55 1.38 10.80 56
CID45281176 7.40 0.47 5.52 2.26 1.00 >100 57 CID45489721 3.45
5.77 >100 0.89 0.76 >100 61 CID46905005 1.60 0.42 7.33 1.40
0.76 18.30 65 CID46904996 16.42 0.18 5.77 0.20 0.68 67.80 74
CID46173053 3.64 0.19 10.10 0.52 0.93 >100 87 CID46912089 0.80
0.08 17.60 0.56 0.58 >100 88 CID46912090 1.64 0.32 49.30 1.90
1.18 >100 2 XR9051 0.61 0.01 1.97 2.27 0.65 21.40 4 Ko143 >50
0.99 2.84 13.55 5.94 20.00 5 MK571 >50 20.40 20.40 >50 57.40
57.40 6 reversan 4.41 0.22 7.04 0.84 3.61 >100 .sup.aEfflux
inhibition activity (IC.sub.50) of the JC-1 substrate is reported
using Jurkat-DNR cells over-expressing ABCB1 and Ig-MXP3 cells
over-expressing ABCG2. Replicates of two to four are reported as
averages (standard deviation has been removed in this table for
clarity). .sup.bChemoreversal.sub.50 values are calculated as
compared to day zero viability across varied dose of inhibitor and
constant dose of chemotherapeutic (100 nM DNR for ABCB1 and 30 nM
MTX for ABCG2). Each data point was the average of two replicates.
.sup.cToxic-dose.sub.50 values are calculated as compared to day
zero viability across varied dose of inhibitor without
chemotherapeutic.
[0262] Chemicals, reagents, and general methods for synthesis.
.sup.1H and .sup.13C NMR spectra were recorded on a Bruker AM 400
spectrometer (operating at 400 and 101 MHz respectively) or Bruker
AM 500 spectrometer (operating at 500 and 125 MHz respectively) in
CDCl.sub.3 with 0.03% TMS as an internal standard or DMSO-d.sub.6.
The chemical shifts (.delta.) reported are given in parts per
million (ppm) and the coupling constants (J) are in Hertz (Hz). The
spin multiplicities are reported as s=singlet, br. s=broad singlet,
d=doublet, t=triplet, q=quartet, dd=doublet of doublet and
m=multiplet. The LCMS analysis was performed on an Agilent 1200 RRL
chromatograph with photodiode array UV detection and an Agilent
6224 TOF mass spectrometer. The chromatographic method utilized the
following parameters: a Waters Acquity BEH C-18 2.1.times.50 mm,
1.7 .mu.m column; UV detection wavelength=214 nm; flow rate=0.4 mL
min.sup.-1; gradient=5-100% acetonitrile over 3 min with a hold of
0.8 min at 100% acetonitrile; the aqueous mobile phase contained
0.15% ammonium hydroxide (v/v). The mass spectrometer utilized the
following parameters: an Agilent multimode source which
simultaneously acquires ESI+/APCI+; a reference mass solution
consisting of purine and hexakis (1H, 1H, 3H-tetrafluoropropoxy)
phosphazine; and a make-up solvent of 90:10:0.1 MeOH:Water:Formic
Acid which was introduced to the LC flow prior to the source to
assist ionization. The melting point was determined on a Stanford
Research Systems OptiMelt apparatus.
5-(furan-2-yl)-2-phenylpyrazolo[1,5-a]pyrimidin-7(4H)-one (91)
##STR00007##
[0264] A mixture of 3-phenyl-1H-pyrazol-5-amine (90: 0.318 g, 2.0
mmol, 1.0 eq) and methyl 3-(furan-2-yl)-3-oxopropanoate (0.370 g,
2.2 mmol, 1.10 eq) was heated in acetic acid (2.0 mL) at
100.degree. C. for 4 hr. After cooling down to rt, the precipitate
was collected by filtration. The precipitate was rinsed with EtOH
(15 mL) and dried under air to afford
5-(furan-2-yl)-2-phenylpyrazolo[1,5-a]pyrimidin-7(4H)-one (0.358 g,
65%) as a white solid. .sup.1H-NMR (400 MHz, DMSO-d.sub.6) .delta.
12.70 (s, 1H), 8.06 (m, 1H), 8.00 (m, 1H), 7.98 (m, 1H), 7.51-7.47
(m, 3H), 7.44-7.42 (m, 1H), 6.81 (dd, J=3.7, 1.8 Hz, 1H), 6.64 (s,
1H), 6.15 (s, 1H).
7-chloro-5-(furan-2-yl)-2-phenylpyrazolo[1,5-a]pyrimidine (92)
##STR00008##
[0266] A mixture of
5-(furan-2-yl)-2-phenylpyrazolo[1,5-a]pyrimidin-7(4H)-one (0.277 g,
1.0 mmol, 1.0 eq), POCl.sub.3 (0.613 g, 4.0 mmol, 4.0 eq),
N-benzyl-N,N,N-triethylethanaminium chloride (0.456 g, 2.0 mmol,
2.0 eq) and N,N-dimethylaniline (0.121 g, 1.0 mmol, 1.0 eq) in
acetonitrile (5.0 mL) was heated at 80.degree. C. for 4 hr. The
completed reaction was diluted with CHCl.sub.3 (20 mL), washed with
H.sub.2O (10 mL), and the separated organic layers were dried
(MgSO.sub.4) and concentrated. The residue was purified by
chromatography (Biotage 25 g, EtOAc/Hexane) to afford
7-chloro-5-(furan-2-yl)-2-phenylpyrazolo[1,5-a]pyrimidine (0.247 g,
84%) as a yellow solid. .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta.
7.97-7.94 (m, 2H), 7.56 (m, 1H), 7.43-7.39 (m, 2H), 7.36-7.34 (m,
1H), 7.19 (s, 1H), 7.17 (dd, J=3.5, 0.5 Hz, 1H), 6.97 (s, 1H),
6.54. (dd, J=3.5, 1.7 Hz, 1H).
(4-(5-(furan-2-yl)-2-phenylpyrazolo[1,5-a]pyrimidin-7-yl)piperazin-1-yl)(f-
uran-3-yl)methanone (1)
##STR00009##
[0268] A mixture of
7-chloro-5-(furan-2-yl)-2-phenylpyrazolo[1,5-a]pyrimidine (0.148 g,
0.5 mmol, 1.0 eq), furan-3-yl(piperazin-1-yl)methanone (0.180 g,
1.0 mmol, 2.0 eq,) and N-ethyl-N-isopropylpropan-2-amine (0.129 g,
1.0 mmol, 2.0 eq) in acetonitrile (5.0 mL) was heated at
100.degree. C. for 3 hr. The completed reaction was purified by
chromatography (Biotage 25 g, EtOAc/Hexane) to afford
(4-(5-(furan-2-yl)-2-phenylpyrazolo[1,5-a]pyrimidin-7-yl)piperazin-1-yl)(-
furan-3-yl)methanone (0.218 g, 99%) as a white solid..sup.1H-NMR
(400 MHz, CDCl.sub.3) .delta. 8.03-8.00 (m, 2H), 7.83 (m, 1H), 7.62
(m, 1H), 7.52-7.47 (m, 3H), 7.45-7.41 (m, 1H), 7.23 (dd, J=3.5, 0.7
Hz, 1H), 6.93 (s, 1H), 6.65 (m, 1H), 6.62 (dd, J=3.5, 1.8 Hz, 1H),
6.60 (s, 1H), 4.08 (b, 4H), 3.92 (b, 4H). .sup.13C NMR (125 MHz,
CDCl.sub.3) .delta. 164.1, 155.4, 152.4, 151.8, 150.1, 148.5,
144.3, 143.8, 143.2, 132.9, 129.0, 128.7, 126.4, 120.6, 112.6,
110.9, 110.1, 92.9, 88.9, 48.3. LCMS retention time: 3.20 min;
purity at 215 nm=100%. HRMS m/z calculated for
C.sub.24H.sub.27N.sub.5O.sub.2 ([M+H].sup.+): 440.1717, found
440.1715.
TABLE-US-00003 TABLE S1 Continuation of SAR expansion, R1-R4
modification. ##STR00010## IC.sub.50 (.mu.M).sup.a CR.sub.50
(.mu.M).sup.b TD.sub.50 (.mu.M).sup.c Cpd CID R.sub.1 R.sub.2
R.sub.3 R.sub.4 ABCB1 ABCG2 B1/G2.sup.d ABCB1 ABCG2 ABCB1 ABCG2 1
CID44640177 Ph 2-furyl H CO-3-furyl 4.65 .+-. 0.74 0.13 .+-. 0.03
35.8 0.55 0.31 6.52 18.30 8 CID1441553 3-Cl--Ph Ph H CO-2-furyl
6.18 .+-. 4.92 0.96 .+-. 0.37 6.4 0.25 0.14 6.77 6.00 9 CID644556
3-Cl--Ph Me Me CO-2-furyl 8.40 .+-. 0.70 2.65 .+-. 1.07 3.2 1.58
0.17 >100 47.40 10 CID652994 3-Cl--Ph Me H CO-2-furyl 5.88 .+-.
1.17 2.78 .+-. 0.82 2.1 NT NT NT NT 11 CID44640182 3-Cl--Ph Ph H
CO-3-furyl 8.58 .+-. 0.63 1.19 .+-. 0.26 7.2 NT NT NT NT 12
CID44607976 3-Cl--Ph Ph H CO-3-thiopheneyl 3.97 .+-. 0.80 1.40 .+-.
0.32 2.8 NT NT NT NT 13 CID44602407 3-Cl--Ph Ph H CO--Ph 3.42 .+-.
1.64 1.45 .+-. 0.59 2.4 NT NT NT NT 14 CID45105078 3-Cl--Ph Ph H
CO-2-MeO--Ph 4.73 .+-. 1.78 4.92 .+-. 4.28 1.0 NT NT NT NT 15
CID45105074 3-Cl--Ph Ph H CO-3-MeO--Ph 2.64 .+-. 0.64 4.45 .+-.
4.68 0.6 0.35 0.21 >100 >100 16 CID45105073 3-Cl--Ph Ph H
CO-4-MeO--Ph 2.89 .+-. 0.55 2.82 .+-. 1.91 1.0 NT NT NT NT 17
CID45105075 3-Cl--Ph Ph H CO-2-Cl--Ph 1.85 .+-. 0.75 1.31 .+-. 0.23
1.4 NT NT NT NT 18 CID45105080 3-Cl--Ph Ph H CO-3-Cl--Ph 4.19 .+-.
1.88 2.95 .+-. 0.57 1.4 NT NT NT NT 19 CID45105081 3-Cl--Ph Ph H
CO-4-Cl--Ph 2.36 .+-. 0.70 2.67 .+-. 0.85 0.9 NT NT NT NT 20
CID44623842 3-Cl--Ph Ph H CO-3-pyridyl 0.57 .+-. 0.08 0.23 .+-.
0.15 2.5 0.17 0.13 4.72 10.30 21 CID44630540 3-Cl--Ph Ph H
CO-4-pyridyl 1.16 .+-. 0.30 1.03 .+-. 0.23 1.1 0.25 0.15 10.90
28.30 22 CID45281172 3-Cl--Ph Ph H CO-cyclohexyl 3.31 .+-. 1.86
2.67 .+-. 1.26 1.2 NT NT NT NT 23 CID44602405 3-Cl--Ph Ph H
COCH.sub.3 5.67 .+-. 2.21 6.07 .+-. 0.73 0.9 NT NT NT NT 24
CID45105082 3-Cl--Ph Ph H CO--Bn 4.68 .+-. 2.69 5.76 .+-. 3.40 0.8
0.51 0.60 17.80 >100 25 CID44607585 3-Cl--Ph Ph H SO.sub.2--Ph
>50 >50 NA NT NT NT NT 26 CID44631077 3-Cl--Ph 2-F--Ph H
CO-3-furyl 4.69 .+-. 1.50 1.93 .+-. 0.48 2.4 NT NT NT NT 27
CID44629741 3-Cl--Ph 4-F--Ph H CO-3-furyl 5.65 .+-. 0.11 2.41 .+-.
0.52 2.3 NT NT NT NT 28 CID44630538 3-Cl--Ph 3-MeO--Ph H CO-3-furyl
2.17 .+-. 1.02 1.14 .+-. 0.54 1.9 NT NT NT NT 29 CID44629740
3-Cl--Ph 4-MeO--Ph H CO-3-furyl 4.12 .+-. 1.05 1.71 .+-. 0.58 2.4
NT NT NT NT 30 CID44629742 3-Cl--Ph 3-pyridyl H CO-3-furyl 0.77
.+-. 0.28 0.27 .+-. 0.12 2.9 0.20 0.02 5.33 5.77 31 CID44630541
3-Cl--Ph 4-pyridyl H CO-3-furyl 3.68 .+-. 0.35 1.46 .+-. 0.35 2.5
0.60 0.89 13.50 57.80 32 CID44631078 3-Cl--Ph ethynyl H CO-3-furyl
10.16 .+-. 3.99 2.36 .+-. 0.53 4.3 NT NT NT NT 33 CID44623844
4-Br--Ph Ph H CO-3-furyl 1.31 .+-. 0.35 0.76 .+-. 0.43 1.7 NT NT NT
NT 34 CID44640183 2-MeO--Ph Ph H CO-3-furyl 2.17 .+-. 0.76 0.45
.+-. 0.17 4.8 0.45 0.49 4.37 6.50 35 CID44623840 3-MeO--Ph Ph H
CO-3-furyl 2.26 .+-. 0.83 1.14 .+-. 0.59 2.0 NT NT NT NT 36
CID44607592 4-MeO--Ph Ph H CO-3-furyl 1.44 .+-. 0.70 1.16 .+-. 0.66
1.2 0.23 0.38 5.54 >100 37 CID44640176 2-MeO--Ph Ph H CO-3-furyl
3.00 .+-. 0.57 1.03 .+-. 0.17 2.9 NT NT NT NT 38 CID44968166
3-MeO--Ph Ph H CO-2-furyl 4.77 .+-. 0.63 1.40 .+-. 0.32 3.4 NT NT
NT NT 39 CID1441456 4-Cl--Ph Ph H CO-2-furyl 3.64 .+-. 1.47 1.37
.+-. 0.53 2.7 NT NT NT NT 40 CID45105079 3-Me--Ph Ph H CO-2-furyl
3.88 .+-. 1.67 2.46 .+-. 1.09 1.6 NT NT NT NT 41 CID44640179
2-MeO--Ph Ph H CO--Ph 2.31 .+-. 0.79 0.76 .+-. 0.06 3.0 NT NT NT NT
42 CID44968164 3-F--Ph Ph H CO--Ph 4.34 .+-. 2.09 2.27 .+-. 0.06
1.9 NT NT NT NT 43 CID44968158 4-F--Ph Ph H CO--Ph 3.99 .+-. 2.31
2.03 .+-. 0.11 2.0 NT NT NT NT 44 CID46905002 Ph 2-MeO--Ph H
CO-3-furyl 4.52 3.66 .+-. 2.64 1.2 0.17 0.68 3.73 7.62 45
CID46905009 Ph 4-MeO--Ph H CO-3-furyl 4.55 2.37 .+-. 1.80 1.9 NT NT
NT NT 46 CID46904993 Ph 2-F--Ph H CO-3-furyl 5.61 0.56 10.0 NT NT
NT NT 47 CID46905008 Ph 3-F--Ph H CO-3-furyl 7.07 1.68 .+-. 0.61
4.2 NT NT NT NT 48 CID46904994 Ph 4-F--Ph H CO-3-furyl 11.82 6.46
1.8 NT NT NT NT 49 CID46905006 Ph .sup.tBu H CO-3-furyl 5.62 11.50
0.5 NT NT NT NT 50 CID46912088 .sup.tBu 2-furyl H CO-3-furyl 3.31
1.97 1.7 NT NT NT NT 51 CID46905003 2-thiopheneyl 2-furyl H
CO-3-furyl 8.42 1.56 .+-. 1.30 5.4 NT NT NT NT 52 CID46905000
2-furyl 2-furyl H CO-3-furyl 1.70 2.35 .+-. 2.33 0.7 0.19 1.21 1.93
>100 53 CID45105077 3-Cl--Ph 2-furyl H CO-3-furyl 6.42 .+-. 2.87
2.55 .+-. 2.22 2.5 0.53 1.38 8.53 10.80 54 CID48904995 4-Cl--Ph
2-furyl H CO-3-furyl 41.54 1.89 .+-. 1.18 22.0 NT NT NT NT 55
CID46905004 2-MeO--Ph 2-furyl H CO-3-furyl 5.57 2.76 .+-. 2.14 2.0
NT NT NT NT 56 CID45281176 3-MeO--Ph 2-furyl H CO-3-furyl 7.40 .+-.
3.34 2.26 .+-. 1.00 3.3 0.47 1.00 5.52 >100 57 CID45489721
4-MeO--Ph 2-furyl H CO-3-furyl 3.45 .+-. 1.11 0.89 .+-. 0.61 3.9
5.77 0.76 >100 >100 58 CID46905007 3-Me--Ph 2-furyl H
CO-3-furyl 3.45 0.58 5.9 NT NT NT NT 59 CID46904998 4-Me--Ph
2-furyl H CO-3-furyl 1.127 5.19 .+-. 4.22 2.2 NT NT NT NT 60
CID44640180 3-(2-furyl)-Ph 2-furyl H CO-3-furyl 3.98 .+-. 1.30 0.93
.+-. 0.27 4.3 NT NT NT NT 61 CID46905005 Ph 3-furyl H CO-3-furyl
1.60 1.40 .+-. 0.27 1.1 0.42 0.76 7.33 18.30 62 CID44602406 Ph Ph H
CO-3-furyl 4.79 .+-. 4.23 1.58 .+-. 0.63 3.0 NT NT NT NT 63
CID44607586 Ph Me H CO-3-furyl 15.81 .+-. 3.33 7.27 .+-. 3.01 2.2
NT NT NT NT 64 CID46905001 Ph 2-thiopheneyl H CO-3-furyl 3.46 1.64
.+-. 0.03 2.1 NT NT NT NT 65 CID46904996 Ph 3-MeO--Ph H CO-3-furyl
16.42 0.20 82.1 0.18 0.68 5.77 67.80 66 CID46904999 Ph
2-(5-Me-furyl) H CO-3-furyl 0.68 0.84 .+-. 0.87 0.8 NT NT NT NT 67
CID46173049 Ph 2-furyl H CO-2-furyl 6.57 .+-. 2.13 2.77 .+-. 4.01
2.4 NT NT NT NT 68 CID46173055 Ph 2-furyl H CO-3-thiopheneyl 4.93
.+-. 1.76 1.38 .+-. 1.04 3.6 NT NT NT NT 69 CID46173043 Ph 2-furyl
H CO--Ph 5.56 .+-. 1.25 1.62 .+-. 1.27 3.4 NT NT NT NT 70
CID46173050 Ph 2-furyl H CO-3-(2-Me-furyl) 11.90 .+-. 7.40 2.33
.+-. 2.23 5.1 NT NT NT NT 71 CID46173047 Ph 2-furyl H
CO-3-(2,4-diMe-furyl) 7.32 .+-. 2.52 3.68 .+-. 2.21 2.0 NT NT NT NT
72 CID46173046 Ph 2-furyl H CO-3-(2,5-diMe-furyl) 12.00 .+-. 6.11
3.37 .+-. 1.19 3.6 NT NT NT NT 73 CID46173052 Ph 2-furyl H
CO-3-benzofuryl 8.83 .+-. 6.63 2.84 .+-. 0.96 3.1 NT NT NT NT 74
CID46173053 Ph 2-furyl H CO--Me 3.64 .+-. 2.55 0.52 .+-. 0.16 7.0
0.19 0.93 10.0 >100 75 CID45489722 Ph 2-furyl H CH.sub.2--Ph
8.99 .+-. 3.67 3.07 .+-. 1.88 2.9 NT NT NT NT 76 CID45489719 Ph
2-furyl H CO.sub.2-Bn >50 2.71 .+-. 1.95 18.5 NT NT NT NT 87
CID46912089 3-Cl--Ph 2-furyl H CO--Me 0.80 0.56 .+-. 0.09 1.4 0.06
0.58 17.60 >100 88 CID46912090 3-MeO--Ph 2-furyl H CO--Me 1.64
1.90 .+-. 1.42 0.9 0.32 1.18 49.30 >100 89 CID44629743 Ph Me Me
CO-3-furyl 1.63 .+-. 0.59 2.18 .+-. 1.93 0.7 NT NT NT NT
.sup.aEfflux inhibition activity (IC.sub.50) of the JC-1 substrate
is reported using Jurkat-DNR cells over-expressing ABCB1 and
Ig-MXP3 cells over-expressing ABCG2. Replicates of two to four are
reported as averages (standard deviation has been removed in this
table for clarity). .sup.bChemoreversal.sub.50 values are
calculated as compared to day zero viability across varied dose of
inhibitor and constant dose of chemotherapeutic (100 nM DNR for
ABCB1 and 30 nM MTX for ABCG2). .sup.cToxic-dose.sub.50 values are
calculated as compared to day zero viabilty across varied dose of
inhibitor without chemotherapeutic. .sup.dSelectivity is indicated
by the quotient of the average ABCB1 IC.sub.50 and the average of
ABCG2 IC.sub.50.
TABLE-US-00004 TABLE S2 SAR expansion, modifications to piperazine
moiety. ##STR00011## IC.sub.50 (.mu.M).sup.a ~Fold G2 Cpd CID X
ABCB1 ABCG2 Selective.sup.b 1 CID44640177 ##STR00012## 4.65 .+-.
0.74 0.13 .+-. 0.03 35.8 77 CID45489714 ##STR00013## 23.56 .+-.
12.07 5.71 .+-. 1.80 4.1 78 CID45489720 ##STR00014## >50 8.88
.+-. 2.82 5.6 79 CID45489718 ##STR00015## 11.92 .+-. 8.94 25.47
.+-. 13.39 0.5 80 CID45489715 ##STR00016## 17.54 .+-. 5.88 13.05
.+-. 7.88 1.3 81 CID46173045 ##STR00017## 13.06 .+-. 6.27 5.43 .+-.
4.11 2.4 82 CID45489712 ##STR00018## 27.67 .+-. 14.47 7.68 .+-.
1.69 3.6 83 CID45489716 ##STR00019## 4.06 .+-. 1.78 1.64 .+-. 0.52
2.5 84 CID46173044 ##STR00020## >50 8.03 .+-. 2.18 6.2 85
CID46173054 ##STR00021## 19.14 .+-. 9.52 2.25 .+-. 1.90 8.5 86
CID46245506 ##STR00022## 3.27 .+-. 1.43 4.65 .+-. 3.96 0.7
.sup.aEfflux inhibition activity (IC.sub.50) of the JC-1 substrate
is reported using Jurkat-DNR cells over-expressing ABCB1 and
Ig-MXP3 cells over-expressing ABCG2. Replicates of two to four are
reported as averages (standard deviation has been removed in this
table for clarity). .sup.bSelectivity is indicated by the quotient
of the average ABCB1 IC.sub.50 and the average of ABCG2
IC.sub.50.
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Example 3
Combination Therapies
[0301] FIG. 1B depicts the time course of injection of 100 nM
Topotecan in conjunction with 100 nM compound 709 (SID 88095709)
into mice (n=5). Experimental conditions were as described in the
figure legend. The effect of this combination therapy is shown in
FIG. 1B over a period of six days (144 hours). Tumor size was
reduced by 89% (p less than 0.001). No reduction in size was
observed in tumors treated with either 100 nM Topotecan or 100 nM
compound 709 alone.
[0302] FIG. 2B depicts the time course of injection of 100 nM
Topotecan in conjunction with 500 nM compound 37 (SID 85752814)
into mice (n=5). Experimental conditions were as described in the
figure legend. The effect of this combination therapy is shown in
FIG. 2B over a period of five days (120 hours). Tumor size was
reduced by 81% (p less than 0.001). No reduction in size was
observed in tumors treated with either 100 nM Topotecan or 100 nM
compound 37 alone.
[0303] FIG. 3B depicts the time course of injection of 100 nM
Topotecan in conjunction with 100 nM compound 789 (SID 97301789)
into mice (n=5). Experimental conditions were as described in the
figure legend. The effect of this combination therapy is shown in
FIG. 3B over a period of five days (120 hours). Tumor size was
reduced by 55% (p less than 0.001). No reduction in size was
observed in tumors treated with either 100 nM Topotecan or 100 nM
compound 789 alone.
* * * * *
References