U.S. patent application number 14/114049 was filed with the patent office on 2014-02-20 for novel bisaminoquinoline compounds, pharmaceutical compositions prepared therefrom and their use.
The applicant listed for this patent is Ravi K. Amaravadi, Jeffrey Winkler. Invention is credited to Ravi K. Amaravadi, Jeffrey Winkler.
Application Number | 20140050696 14/114049 |
Document ID | / |
Family ID | 47073068 |
Filed Date | 2014-02-20 |
United States Patent
Application |
20140050696 |
Kind Code |
A1 |
Amaravadi; Ravi K. ; et
al. |
February 20, 2014 |
NOVEL BISAMINOQUINOLINE COMPOUNDS, PHARMACEUTICAL COMPOSITIONS
PREPARED THEREFROM AND THEIR USE
Abstract
The present invention relates to novel bisaminoquinoline
compounds, pharmaceutical compositions comprising these novel
compounds and methods for inhibiting autophagy in biological
systems. Methods of treating cancer in patients in need using
compounds and/or compositions according to the present invention
alone or in combination with at least one additional anticancer
agent represent additional aspects of the invention. Methods of
treating disease states and/or conditions in which inhibition of
autophagy plays a favorable treatment role including rheumatoid
arthritis, malaria, antiphospholipid antibody syndrome, lupus,
chronic urticaria and Sjogren's disease, with compounds according
to the present invention represent additional aspects of the
invention.
Inventors: |
Amaravadi; Ravi K.; (Media,
PA) ; Winkler; Jeffrey; (Wynnewood, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Amaravadi; Ravi K.
Winkler; Jeffrey |
Media
Wynnewood |
PA
PA |
US
US |
|
|
Family ID: |
47073068 |
Appl. No.: |
14/114049 |
Filed: |
April 26, 2012 |
PCT Filed: |
April 26, 2012 |
PCT NO: |
PCT/US12/35251 |
371 Date: |
October 25, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61480641 |
Apr 29, 2011 |
|
|
|
Current U.S.
Class: |
424/85.2 ;
514/313; 546/163 |
Current CPC
Class: |
A61K 31/4709 20130101;
C07D 401/14 20130101; A61K 45/06 20130101; C07D 215/46 20130101;
Y02A 50/30 20180101; Y02A 50/411 20180101; A61P 33/06 20180101;
C07D 401/12 20130101; A61P 35/00 20180101; A61P 29/00 20180101;
A61K 31/4725 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/85.2 ;
546/163; 514/313 |
International
Class: |
A61K 31/4709 20060101
A61K031/4709; C07D 401/14 20060101 C07D401/14; A61K 45/06 20060101
A61K045/06; C07D 401/12 20060101 C07D401/12 |
Goverment Interests
[0002] This invention was made with government support under the
Abramson Cancer Center Pilot grant awarded by the National Cancer
Institute. The government has certain rights in the invention.
Claims
1. A compound according to the chemical structure I ##STR00019##
Wherein R.sup.1 and R.sup.1' are each independently H, halo (F, Cl,
Br or I), CN, NO.sub.2, optionally substituted C.sub.1-C.sub.6
alkyl (when substituted, preferably substituted with 1 or 2
hydroxyl groups or 3-5 fluoro groups), optionally substituted
O--C.sub.1-C.sub.6 alkyl (preferably, OCH.sub.3), optionally
substituted C.sub.2-C.sub.7 acyl (preferably acetyl) or optionally
substituted C.sub.2-C.sub.7 ester (oxycarbonyl ester or
carboxyester, preferably carboxyester); R and R' are each
independently H, a C.sub.1-C.sub.6 optionally substituted alkyl
group, a C.sub.1-C.sub.7 (preferably C.sub.2-C.sub.7) optionally
substituted acyl group, a C.sub.2-C.sub.7 optionally substituted
carboxy ester group (which forms a urethane group with the nitrogen
atom to which R or R' is bonded); L is a ##STR00020## group or a
##STR00021## group wherein either A or A' may be bonded to either
of the two amine groups in compound I and wherein at least one of
the CH.sub.2 groups in L is optionally substituted with a
C.sub.1-C.sub.3 alkyl group which itself is optionally substituted
with one or two hydroxyl groups; X is absent, (CH.sub.2).sub.jO, S
or N--R''; Y is absent, CH.sub.2, O, CH.sub.2O or N--R'' and Y' is
absent CH.sub.2, O, OCH.sub.2 or N--R'', with the proviso that when
one or more of X, Y and Y' is present, each of X and Y, X and Y' or
Y and Y', when present, forms a stable bond; R'' is H or an
optionally substituted C.sub.1-C.sub.6 (preferably C.sub.1-C.sub.3)
alkyl group; j is 1, 2 or 3 (preferably 1 or 2); n is 0, 1, 2, 3 or
4, with the proviso that when n is 0, X is (CH.sub.2).sub.j where j
is at least 1 and at least one CH.sub.2 group is optionally
substituted with a C.sub.1-C.sub.3 alkyl group which itself is
optionally substituted with one or two hydroxyl groups; A is absent
or (CH.sub.2).sub.j and A' is (CH.sub.2).sub.j wherein at least one
CH.sub.2 group in A or A' is optionally substituted with a
C.sub.1-C.sub.3 alkyl group which is itself optionally substituted
with one or two hydroxyl groups; Z is O or N--R.sup.Z; R.sup.Z is H
or an optionally substituted C.sub.1-C.sub.3 alkyl group, or a
pharmaceutically acceptable salt, enantiomer, diastereomer, solvate
or polymorph thereof.
2. The compound according to claim 1, wherein R.sup.1 and R.sup.1'
are each independently H, a halo group, a nitro group or a
trifluoromethyl group.
3. The compound according to claim 1 wherein R and R' are each
independently H, a C.sub.1-C.sub.3 alkyl group substituted with at
least one hydroxyl group, alkoxy group, an amine, monoalkyl amine
or dialkyl amine group, wherein said amine group or said monoalkyl
amine group is optionally substituted on the amine position with a
7-substituted-4-quinolinyl group wherein the amine binds to the
4-position of the quinolinyl group and the 7 position of said
quinolinyl group is substituted by R.sup.1 or R1' of claim 1, or
one or both alkyl groups of said monoalkyl amine or dialkyl amine
is itself further optionally substituted with at least one hydroxyl
group, alkoxy group, an amine, a monoalkyl amine, a dialkyl amine
wherein the amine or monoalkyl amine is optionally substituted on
the amine position with one or two 7-substituted-quinolinyl
group(s) wherein the amine binds to the 4-position of the
quinolinyl group and the 7 position of said quinolinyl group is
substituted by R.sup.1 or R1' of claim 1, and each of said alkoxy
groups may be further substituted with an alkoxy group, preferably
a methoxy group (thus forming a diether substituent).
4. The compound according to claim 1 wherein L is a ##STR00022##
group, where X is N--R''; n is 1, 2, or 3; Y and Y' are each
independently absent or CH.sub.2; and R'' is H or a C.sub.1-C.sub.3
alkyl group which is optionally substituted with at least one
hydroxyl group, an alkoxy group, an amine, monoalkyl amine, dialkyl
amine group, wherein said amine group or said monoalkyl amine group
is optionally substituted on the amine position with a
7-substituted-4-quinolinyl group wherein the amine binds to the
4-position of the quinolinyl group and the 7 position of said
quinolinyl group is substituted by R.sup.1 or R1' of claim 1, or
one or both alkyl groups of said monoalkyl amine or dialkyl amine
is itself further optionally substituted with at least one hydroxyl
group, alkoxy group, an amine, a monoalkyl amine or a dialkyl amine
wherein the amine or monoalkyl amine is optionally substituted on
the amine position with one or two 7-substituted-quinolinyl
group(s) wherein the amine binds to the 4-position of the
quinolinyl group and the 7 position of said quinolinyl group is
substituted by R.sup.1 or R1' of claim 1, and each of said alkoxy
group may be further substituted with a second alkoxy group, thus
forming a diether substituent.
5. A compound according to claim 3 wherein said C.sub.1-C.sub.3
alkyl group is substituted with an amine group which itself is
substituted with one 7-substituted-4-quinolinyl group.
6. The compound according to claim 5 wherein said alkyl group is a
C.sub.2 alkyl group.
7. The compound according to claim 3 wherein said C.sub.1-C.sub.3
alkyl group is substituted with an alkoxy group which itself is
substituted with a second alkoxy group thus forming a diether
group.
8. The compound according to claim 7 wherein said second alkoxy
group is a methoxy group or an ethoxy group.
9. A compound as depicted in Scheme 1 or Scheme 3-10 hereof.
10. A compound of claim 9 which is any one of compounds 28-40 of
scheme 3 hereof.
11. A compound of claim 9 which is any one of compounds 41-46 of
scheme 4 hereof.
12. A compound of claim 9 which is either of compounds 47-48 of
scheme 5 hereof.
13. A compound of claim 9 which is either of compounds 49-50 of
scheme 6 hereof.
14. A compound of claim 9 which is any one of compounds 51-58 of
scheme 7 hereof.
15. A compound of claim 9 which is any one of compounds 63-70 of
scheme 8 hereof.
16. A compound of claim 9 which is any one of compounds 71-78 of
scheme 9 hereof.
17. A compound of claim 9 which is any one of compounds 79-82 of
scheme 10 hereof.
18. A compound according to claim 9 which is compound 3 of scheme 1
hereof.
19. A compound as depicted in FIG. 14, 15 or 15A hereof.
20. The compound:
N.sup.1-(7-chloroquinolin-4-yl)-N.sup.2-(2-((7-chloroquinolin-4-yl)amino)-
ethyl)-N.sup.2-methylethane-1,2-diamine;
N.sup.1-(7-chloroquinolin-4-yl)-N.sup.2-(2-((7-chloroquinolin-4-yl)amino)-
ethyl)ethane-1,2-diamine;
N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))bis(7-chloroquinolin--
4-amine);
N'-(7-methoxyquinolin-4-yl)-N.sup.2-(2-((7-methoxyquinolin-4-yl)-
amino)ethyl)-N.sup.2-methylethane-1,2-diamine;
N,N-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))bis(7-chloroquinolin-4-
-amine);
N.sup.1-(7-chloroquinolin-4-yl)-N.sup.2-(2-((7-chloroquinolin-4-y-
l)amino)ethyl)-N.sup.2-methylethane-1,2-diamine trihydrochloride;
N.sup.1-(7-chloroquinolin-4-yl)-N.sup.2-(2-((7-chloroquinolin-4-yl)(methy-
l)amino)ethyl)-N.sup.1,N.sup.2-dimethylethane-1,2-diamine;
N,N-((methylazanediyl)bis(ethane-2,1-diyl))bis(N-(7-chloroquinolin-4-yl)a-
cetamide);
(S)--N.sup.2-(7-chloroquinolin-4-yl)-N--((S)-2-((7-chloroquinol-
in-4-yl)amino)propyl)-N-methylpropane-1,2-diamine;
2-(bis(2-((7-chloroquinolin-4-yl)amino)ethyl)amino)ethanol;
N.sup.1-(7-chloroquinolin-4-yl)-N.sup.2,N.sup.2-bis(2-((7-chloroquinolin--
4-yl)amino)ethyl)ethane-1,2-diamine, or a pharmaceutically
acceptable salt thereof.
21. The compound according to claim 1 which is
N.sup.1-(7-chloroquinolin-4-yl)-N.sup.2-(2-((7-chloroquinolin-4-yl)amino)-
ethyl)-N.sup.2-methylethane-1,2-diamine or a pharmaceutically
acceptable salt thereof.
22. The compound according to claim 1 which is
N'-(7-chloroquinolin-4-yl)-N.sup.2-(2-((7-chloroquinolin-4-yl)amino)ethyl-
)-N.sup.2-methylethane-1,2-diamine trihydrochloride.
23. A pharmaceutical composition comprising an effective amount of
at least one compound according to claim 1 in combination with a
pharmaceutically acceptable carrier, additive or excipient and
optionally in combination with at least one additional anticancer
agent.
24. A method of inhibiting autophagy in a biological system in
which inhibition of autophagy is desired, said method comprising
exposing said biological system to an effective amount of at least
one compound according to claim 1 hereof.
25. A method of inhibiting or treating cancer in a patient in need
comprising administering to said patient an effective amount of at
least one compound according to claim 1, optionally in combination
with at least one additional anticancer agent.
26. The method according to claim 25 wherein said cancer is
metastatic.
27. The method according to claim 26 wherein said cancer is a
drug-resistant cancer.
28. A method of reducing the likelihood that cancer will occur in a
patient or that a cancer will metastasize in a patient comprising
administering at least one compound according to claim 1,
optionally in combination with at least one additional anticancer
agent.
29. The method according to claim 25 wherein said cancer is a
carcinoma, cancer of the esophagus, head, kidney, liver, lung,
nasopharyngeal, neck, ovary, pancreas, prostate, and stomach; a
leukemia, a malignant lymphoma, a malignant melanoma;
myeloproliferative diseases; a sarcoma, a tumor of the central
nervous system, a germ-line tumor, lung cancer, ovarian cancer,
testicular cancer, thyroid cancer, astrocytoma, esophageal cancer,
pancreatic cancer, stomach cancer, liver cancer, colon cancer,
melanoma, a mixed type of neoplasia,
30. (canceled)
31. (canceled)
32. (canceled)
33. (canceled)
34. (canceled)
35. (canceled)
36. (canceled)
37. (canceled)
38. (canceled)
39. The method according to claim 25 wherein said additional
anticancer agent is selected from the group consisting of
everolimus, trabectedin, abraxane, TLK 286, AV-299, DN-101,
pazopanib, GSK690693, RTA 744, ON 0910.Na, AZD 6244 (ARRY-142886),
AMN-107, TKI-258, GSK461364, AZD 1152, enzastaurin, vandetanib,
ARQ-197, MK-0457, MLN8054, PIA-739358, R-763, AT-9263, pemetrexed,
erlotinib, dasatanib, nilotinib, decatanib, panitumumab,
amnrubicin, oregovomab, Lep-etu, nolatrexed, azd2171, batabulin,
ofatumumab, zanolimumab, edotecarin, tetrandrine, rubitecan,
tesmilifene, oblimersen, ticilimumab, ipilimumab, gossypol, Bio
111, 131-I-TM-601, ALT-110, BIO 140, CC 8490, cilengitide,
gimatecan, IL 13-PE38QQR, INO 1001, IPdR.sub.1KRX-0402, lucanthone,
LY 317615, neuradiab, vitespan, Rta 744, Sdx 102, talampanel,
atrasentan, Xr 311, romidepsin, ADS-100380, sunitinib,
5-fluorouracil, vorinostat, etoposide, gemcitabine, doxorubicin,
irinotecan, liposomal doxorubicin, 5'-deoxy-5-fluorouridine,
vincristine, temozolomide, ZK-304709, seliciclib; PD0325901,
AZD-6244, capecitabine, L-Glutamic acid,
N-[4-[2-(2-amino-4,7-dihydro-4-oxo-1H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-
benzoyl]-, disodium salt, heptahydrate, camptothecin, PEG-labeled
irinotecan, tamoxifen, toremifene citrate, anastrazole, exemestane,
letrozole, DES(diethylstilbestrol), estradiol, estrogen, conjugated
estrogen, bevacizumab, IMC-1C11, CHIR-258,); 3-[5-(methyl
sulfonylpiperadinemethyl)-indolyl]-quinolone, vatalanib, AG-013736,
AVE-0005, the acetate salt of [D-Ser(Bu t) 6,Azgly 10]
(pyro-Glu-His-Trp-Ser-Tyr-D-Ser(Bu t)-Leu-Arg-Pro-Azgly-NH.sub.2
acetate
[C.sub.59H.sub.84N.sub.18Oi.sub.4-(C.sub.2H.sub.4O.sub.2).sub.x
where x=1 to 2.4], goserelin acetate, leuprolide acetate,
triptorelin pamoate, medroxyprogesterone acetate,
hydroxyprogesterone caproate, megestrol acetate, raloxifene,
bicalutamide, flutamide, nilutamide, megestrol acetate, CP-724714;
TAK-165, HKI-272, erlotinib, lapatanib, canertinib, ABX-EGF
antibody, erbitux, EKB-569, PKI-166, GW-572016, Ionafamib,
BMS-214662, tipifarnib; amifostine, NVP-LAQ824, suberoyl analide
hydroxamic acid, valproic acid, trichostatin A, FK-228, SUI 1248,
sorafenib, KRN951, aminoglutethimide, arnsacrine, anagrelide,
L-asparaginase, Bacillus Calmette-Guerin (BCG) vaccine, bleomycin,
buserelin, busulfan, carboplatin, carmustine, chlorambucil,
cisplatin, cladribine, clodronate, cyproterone, cytarabine,
dacarbazine, dactinomycin, daunorubicin, diethylstilbestrol,
epirubicin, fludarabine, fludrocortisone, fluoxylnesterone,
flutamide, gemcitabine, gleevac, hydroxyurea, idarubicin,
ifosfamide, imatinib, leuprolide, levamisole, lomustine,
mechlorethamine, melphalan, 6-mercaptopurine, mesna, methotrexate,
mitomycin, mitotane, mitoxantrone, nilutamide, octreotide,
oxaliplatin, pamidronate, pentostatin, plicamycin, porfimer,
procarbazine, raltitrexed, rituximab, streptozocin, teniposide,
testosterone, thalidomide, thioguanine, thiotepa, tretinoin,
vindesine, 13-cis-retinoic acid, phenylalanine mustard, uracil
mustard, estramustine, altretamine, floxuridine, 5-deooxyuridine,
cytosine arabinoside, 6-mercaptopurine, deoxycoformycin,
calcitriol, valrubicin, mithramycin, vinblastine, vinorelbine,
topotecan, razoxin, marimastat, COL-3, neovastat, BMS-275291,
squalamine, endostatin, SU5416, SU6668, EMD 121974, interleukin-12,
IM862, angiostatin, vitaxin, droloxifene, idoxyfene,
spironolactone, finasteride, cimitidine, trastuzumab, denileukin
diftitox, gefitinib, bortezimib, paclitaxel, irinotecan, topotecan,
doxorubicin, docetaxel, vinorelbine, bevacizumab (monoclonal
antibody) and erbitux, cremophor-free paclitaxel, epithilone B,
BMS-247550, BMS-310705, droloxifene, 4-hydroxytamoxifen,
pipendoxifene, ERA-923, arzoxifene, fulvestrant, acolbifene,
lasofoxifene, idoxifene, TSE-424, HMR-3339, ZK186619, PTK787/ZK
222584, VX-745, PD 184352, rapamycin,
40-O-(2-hydroxyethyl)-rapamycin, temsirolimus, AP-23573, RAD001,
ABT-578, BC-210, LY294002, LY292223, LY292696, LY293684, LY293646,
wortmannin, ZM336372, L-779,450, PEG-filgrastim, darbepoetin,
erythropoietin, granulocyte colony-stimulating factor,
zolendronate, prednisone, cetuximab, granulocyte macrophage
colony-stimulating factor, histrelin, pegylated interferon alfa-2a,
interferon alfa-2a, pegylated interferon alfa-2b, interferon
alfa-2b, azacitidine, PEG-L-asparaginase, lenalidomide, gemtuzumab,
hydrocortisone, interleukin-11, dexrazoxane, alemtuzumab,
all-transretinoic acid, ketoconazole, interleukin-2, megestrol,
immune globulin, nitrogen mustard, methylprednisolone, ibritgumomab
tiuxetan, androgens, decitabine, hexamethylmelamine, bexarotene,
tositumomab, arsenic trioxide, cortisone, editronate, mitotane,
cyclosporine, liposomal daunorubicin, Edwina-asparaginase,
strontium 89, casopitant, netupitant, an NK-1 receptor antagonists,
palonosetron, aprepitant, diphenhydramine, hydroxyzine,
metoclopramide, lorazepam, alprazolam, haloperidol, droperidol,
dronabinol, dexamethasone, methylprednisolone, prochlorperazine,
granisetron, ondansetron, dolasetron, tropisetron, sspegfilgrastim,
erythropoietin, epoetin alfa and darbepoetin alfa, ipilumumab,
vemurafenib and mixtures thereof.
40. The method according to claim 25 wherein said additional
anticancer agent is a FLT-3 inhibitor, a VEGFR inhibitor, an EGFR
TK inhibitor, an aurora kinase inhibitor, a PIK-1 modulator, a
Bcl-2 inhibitor, an HDAC inhibitor, a c-MET inhibitor, a PARP
inhibitor, a Cdk inhibitor, an EGFR TK inhibitor, an IGFR-TK
inhibitor, an anti-HGF antibody, a PI3 kinase inhibitors, an AKT
inhibitor, a JAK/STAT inhibitor, a checkpoint-1 or 2 inhibitor, a
focal adhesion kinase inhibitor, a Map kinase kinase (mek)
inhibitor, a VEGF trap antibody or a mixture thereof.
41. A method of treating a disease state or condition in a patient
in need wherein said disease state or condition responds favorably
to inhibition of autophagy comprising administering to said patient
an effective amount of a compound according to claim 1 to said
patient.
42. The method according to claim 41 wherein said disease state or
condition is rheumatoid arthritis, malaria, antiphospholipid
antibody syndrome, lupus, chronic urticaria or Sjogren's
disease.
43. The method according to claim 42 wherein said disease state is
malaria.
44.-63. (canceled)
Description
RELATED APPLICATIONS AND GRANT SUPPORT
[0001] The present application claims the benefit of priority of
U.S. provisional application No. 61/480,641, filed Apr. 29, 2011,
of identical title to the present application, the entire contents
of which are incorporated by reference herein.
FIELD OF THE INVENTION
[0003] The present invention relates to novel bisaminoquinoline
compounds, pharmaceutical compositions comprising these novel
compounds and methods for inhibiting autophagy in biological
systems. Methods of treating cancer in patients in need using
compounds and/or compositions according to the present invention
alone or in combination with at least one additional anticancer
agent represent additional aspects of the invention. The use of
radiation therapy in combination with the present compounds, alone
or in combination with an additional anticancer agent as otherwise
disclosed herein, represents an additional aspect of the invention.
Methods of treating disease states and/or conditions in which
inhibition of autophagy plays a favorable treatment role including
rheumatoid arthritis, malaria, antiphospholipid antibody syndrome,
lupus, chronic urticaria and Sjogren's disease, with compounds
according to the present invention represent additional aspects of
the invention.
BACKGROUND OF THE INVENTION
[0004] Autophagy consists of the sequestration of organelles and
proteins in autophagic vesicles (AV) and degradation of this cargo
through lysosomal fusion (1). Autophagy allows tumor cells to
survive metabolic and therapeutic stresses (2-5). Multiple
publications indicate therapy-induced autophagy is a key resistance
mechanism to many anti-cancer agents. Chloroquine (CQ) (Compound 1,
FIG. 1) derivatives block autophagy by inhibiting the lysosome (3,
6, 7). A randomized phase III trial of CQ versus placebo with
carmustine and radiation in patients with glioma reported a trend
towards a doubling in duration of survival in the patients treated
with CQ (8). Based on these findings, clinical trials combining
cancer therapies with hydroxychloroquine (HCQ; FIG. 1 Compound 2),
(which is safer than CQ to dose escalate) have been launched.
Preliminary results indicate these combinations have activity (9),
but it is still unclear if this activity is consistently due to the
addition of HCQ. High micromolar concentrations of HCQ are required
to inhibit autophagy. While there is some pharmacodynamic evidence
of autophagy inhibition with HCQ in cancer patients, it is
inconsistent because adequate concentrations are not achieved in
all patients (10). There is an unmet need to develop more potent
inhibitors of autophagy. The design and synthesis of dimeric
analogs of CQ, that exploit the thermodynamic advantages imparted
by polyvalency (11, 12), has been a subject of intensive study for
over 10 years (13-15). An early report by Vennerstrom(14) described
the synthesis of heteroalkane-bridged bisquinolines as potential
antimalarials, but none of the compounds had sufficient
antimalarial activity to warrant further investigation.
Subsequently, Sergheraert (13) reported that tetraquinolines, i.e.,
dimers of bisquinolines, afforded potent antimalarials, confirming
the possibility that the application of the polyvalency strategy
could afford increased potency, at least with respect to
antimalarial activity.
[0005] More recently, Lee(16) has described the potentiation of AKT
inhibitors by fluorinated quinoline analogs. Solomon(17) has
reported the preparation of "repositioned" chloroquine dimers,
based on the use of a piperazine connector. These results suggest
that these chloroquine analogs could serve as bases for the
development of a new group of effective cancer
chemotherapeutics.
OBJECTS OF THE INVENTION
[0006] It is an object of the invention to provide novel compounds
for inhibiting autophagy in biological systems, especially
including patients or subjects in need.
[0007] It is another object of the invention to treat disease
states and/or conditions in which inhibition of autophagy is
beneficial to the disease states and/or conditions a patient or
subject.
[0008] It is yet a further object of the invention to provide
pharmaceutical compositions which may be used to inhibit autophagy,
especially autophagy associated with disease states and/or
conditions including cancer and its metastasis.
[0009] It is still a further object of the invention to inhibit,
treat or prevent cancer, including the metastasis of cancer in
patients or subjects in need utilizing compounds, compositions
and/or methods which are presented herein.
[0010] It is still another object of the invention to inhibit,
treat or prevent diseases in which the inhibition of autophagy
provides a favorable effect, including rheumatoid arthritis,
malaria, antiphospholipid antibody syndrome, lupus, chronic
urticaria and Sjogren's disease, among others.
[0011] Any one or more of these and/or other objects of the
invention may be readily gleaned from a description of the
invention which follows.
BRIEF DESCRIPTION OF THE INVENTION
[0012] The present invention relates to compounds according to the
chemical structure I
##STR00001##
Wherein R.sup.1 and R.sup.1' are each independently H, halo (F, Cl,
Br or I), CN, NO.sub.2, optionally substituted C.sub.1-C.sub.6
alkyl (when substituted, preferably substituted with 1 or 2
hydroxyl groups or 3-5 fluoro groups), optionally substituted
O--C.sub.1-C.sub.6 alkyl (preferably, OCH.sub.3), optionally
substituted C.sub.2-C.sub.7 acyl (preferably acetyl) or optionally
substituted C.sub.2-C.sub.7 ester (oxycarbonyl ester or
carboxyester, preferably carboxyester); R and R' are each
independently H, a C.sub.1-C.sub.6 optionally substituted alkyl
group, a C.sub.1-C.sub.7 (preferably C.sub.2-C.sub.7) optionally
substituted acyl group, a C.sub.2-C.sub.7 optionally substituted
carboxy ester group (which forms a urethane group with the nitrogen
atom to which R or R' is bonded);
L is a
##STR00002##
[0013] group or a
##STR00003##
group (either A or A' may be bonded to either of the two amine
groups in compound I) wherein at least one of the CH.sub.2 groups
in L is optionally substituted with a C.sub.1-C.sub.3 alkyl group
which itself is optionally substituted with one or two hydroxyl
groups; X is absent, (CH.sub.2).sub.j O, S or N--R''; Y is absent,
CH.sub.2, O, CH.sub.2O or N--R'' and Y' is absent CH.sub.2, O,
OCH.sub.2 or N--R'', with the proviso that when one or more of X, Y
and Y' is present, each of X and Y, X and Y' or Y and Y', when
present, forms a stable bond; R'' is H or an optionally substituted
C.sub.1-C.sub.6 (preferably C.sub.1-C.sub.3) alkyl group; j is 1, 2
or 3 (preferably 1 or 2); n is 0, 1, 2, 3 or 4, with the proviso
that when n is 0, X is (CH.sub.2).sub.j where j is at least 1 and
at least one CH.sub.2 group is optionally substituted with a
C.sub.1-C.sub.3 alkyl group which itself is optionally substituted
with one or two hydroxyl groups; A is absent or (CH.sub.2).sub.j
and A' is (CH.sub.2).sub.j wherein at least one CH.sub.2 group in A
or A' is optionally substituted with a C.sub.1-C.sub.3 alkyl group
which is itself optionally substituted with one or two hydroxyl
groups;
Z is O or N--R.sup.Z;
[0014] R.sup.Z is H or an optionally substituted C.sub.1-C.sub.3
alkyl group, or a pharmaceutically acceptable salt, enantiomer,
diastereomer, solvent or polymorph thereof.
[0015] In preferred aspects of the invention, R.sup.1 and R.sup.1'
are each independently H, a halo group, a nitro group or a
trifluoromethyl group, preferably a chloro group. R and R' are
preferably each independently H, a C.sub.1-C.sub.3 optionally
substituted alkyl group itself preferably substituted with at least
one hydroxyl group, an alkoxy group, an amine, monoalkyl amine or
dialkyl amine group, wherein said amine group or said monoalkyl
amine group is optionally substituted on the amine position with
one or two 7-substituted-4-quinolinyl group(s) wherein the amine
binds to the 4-position of the quinolinyl group and the 7-position
of each quinolinyl group is optionally substituted, preferably with
a R.sup.1 and R.sup.1' group as broadly described for generic
structure I above, or one or both alkyl groups of said monoalkyl
amine or dialkyl amine is itself further optionally substituted
with at least one hydroxyl group, an alkoxy group, an amine, a
monoalkyl amine or a dialkyl amine wherein the amine or monoalkyl
amine is optionally substituted on the amine position with one or
two 7-substituted-quinolinyl group(s) wherein the amine binds to
the 4-position of the quinolinyl group and the 7-position of each
quinolinyl group is optionally substituted, preferably with R.sup.1
and/or R.sup.1' as broadly described for generic structure I above,
and each of said alkoxy groups (e.g. methoxy or ethoxy) is
optionally further substituted with an alkoxy group, preferably a
methoxy group, thus forming a diether substituent.
[0016] In certain preferred aspects of the invention L is a
##STR00004##
group, where X is N--R'', Y and Y' are each independently absent or
CH.sub.2, and R'' is H or a C.sub.1-C.sub.3 alkyl group which is
optionally substituted with at least one hydroxyl group, an alkoxy
group, an amine, monoalkyl amine or dialkyl amine group, wherein
said amine group or said monoalkyl amine group is optionally
substituted on the amine position with one or two
7-substituted-4-quinolinyl group wherein the amine binds to the
4-position of the quinolinyl group and the 7-position of each
quinolinyl group is optionally substituted, preferably with R.sup.1
and/or R.sup.1' as broadly described for generic structure I above,
or one or both alkyl groups of said monoalkyl amine or dialkyl
amine is itself further optionally substituted with at least one
hydroxyl group, an alkoxy group, an amine, a monoalkyl amine or a
dialkyl amine wherein the amine or monoalkyl amine is optionally
substituted on the amine position with one or two
7-substituted-quinolinyl group(s) wherein the amine binds to the
4-position of the quinolinyl group and the 7-position of each
quinolinyl group is optionally substituted, preferably with R.sup.1
and/or R.sup.1' as broadly described for generic structure I above,
and each of said alkoxy groups (e.g. methoxy or ethoxy) is
optionally further substituted with an alkoxy group, preferably a
methoxy group, thus forming a diether substituent.
[0017] Further preferred compounds according to the present
invention include those which are presented in the various schemes
which are presented in Scheme 1 and Schemes 3-10 and FIGS. 14, 15
and 15 A as presented herein.
[0018] In another aspect of the invention, a pharmaceutical
composition comprises a compound according to formula I above or as
otherwise described herein in combination with a pharmaceutically
acceptable carrier, additive or excipient, optionally in
combination with at least one additional anticancer agent.
[0019] Methods of inhibiting autophagy in a biological system, in
particular a patient or subject is a further aspects of this
invention. In this aspect of the invention, a bisaminoquinoline
compound as otherwise described herein is presented to the
biological system, including administration to a patient or subject
in need, in order to inhibit autophagy. The resulting inhibition
may be monitored or applied in the biological system to effect a
favorable result, including the inhibition, treatment and/or
prevention of cancer, including metastasis of cancer, or the
inhibition, treatment and/or prevention of one or more disease
states or conditions in which the inhibition of autophagy provides
a favorable result including rheumatoid arthritis, malaria,
antiphospholipid antibody syndrome, lupus, chronic urticaria and
Sjogren's disease, among others.
[0020] Methods of inhibiting, treating and/or reducing the
likelihood of cancer, including metastasis of cancer and drug
resistant cancer, comprises administering to a patient in need at
least one compound according to the present invention, optionally
in combination with at least one additional anticancer agent as
otherwise described herein.
[0021] The present invention also relates to treating, inhibiting
and/or preventing diseases, diseases states and/or conditions in a
patient in need in which the inhibition of autophagy provides a
favorable outcome, including rheumatoid arthritis, malaria,
antiphospholipid antibody syndrome, lupus, chronic urticaria and
Sjogren's disease, the method comprising administering to said
patient at least one compound according to the present
invention.
BRIEF DESCRIPTION OF THE FIGURES
[0022] FIG. 1. Chemical structure of mono and
bisaminoquinolines.
[0023] FIG. 2. Synthesis scheme for bisaminoquinolines
[0024] FIG. 3. Effects of Lys01-Lys04 on LC3 immunoblotting
Immunoblotting and quantification of LC3II/LC3I ratio in lysates
from LN229 cells treated for 4 hours. The graphs show (mean+/-SD)
LC3II/LC3I ratios of each treatment normalized to the LC3II/LC3I
ratio of control treated cells for each experiment.
[0025] FIG. 4. Autophagy inhibition and cytotoxicity of Lys01
compared to HCQ. (A) Representative images of LN229 GFP-LC3 cells
treated as indicated for 4 hours. White arrows: small puncta; red
arrows: dense puncta. Graphs show mean+/-SEM puncta/cell. (B)
Representative electron micrographs of LN229-GFP-LC3 cells treated
(4 hours) with DMSO, HCQ 10 .mu.M, or Lys01 10 .mu.M Arrows:
Autophagic vesicles. (C) LC3 immunoblotting of LN229 cells treated
for 24 hours as indicated; calculated ratio of LC3II/LC3I ratios
for bafilomycin versus control co-treatment. Above the dashed line
indicates an autophagy inducer or control, below the dashed line
indicates an autophagy inhibitor. (D) MTT assay (72 hours) for 4
cell lines. Red: Lys01, Blue: Lys02, Purple: Lys03 Green: Lys04
Orange: HCQ Values presented are means+/-SEM with 5 replicates per
treatment.
[0026] FIG. 5. Autophagy inhibition and cytotoxicity of Lys05, the
water soluble salt of Ly01. (A) Immunoblotting against LC3 and p62
in c8161 cells treated as indicated (B) MTT assay in c8161 cells at
72 hours. HCQ: Hydroxychloroquine. Values presented are mean+/-SD
with 5 replicates per treatment condition. *No remaining cells for
analysis.
[0027] FIG. 6. In vivo autophagy inhibition and antitumor activity
of Lys05. (A) Representative electron micrographs (12,000.times.)
of c8161 xenograft tumors harvested after 2 days of daily i.p.
treatment with PBS, HCQ 60 mg/kg, or Lys05 76 mg/kg. Arrows:
autophagic vesicles; scale bar 2 .mu.m (B) Quantification of
mean.+-.SEM number of autophagic vesicles/cell from two
representative tumors from each treatment group. (C-D) 1205Lu
xenografts were treated with PBS (blue), HCQ 60 mg/kg ip (green),
or Lys05 76 mg/kg (red) i.p. every 3/5 days (C) Tumor volumes over
14 days (D) Daily tumor growth rate. (E-G) HT29 xenografts were
generated in the flanks of nude mice and treated with PBS, Lys05 10
mg/kg ip daily, Lys05 40 mg/kg ip daily, or Lys05 80 mg/kg ip every
3/5 days. (E) Average daily tumor growth rate (F) Tumor volumes
over 14 days (G) Weight of excised tumors, *p<0.05.
[0028] FIG. 7. Autophagy inhibition and tumor necrosis in melanoma
and colon cancer xenografts treated with Lys05 or HCQ. (A)
Immunoblotting against LC3 in lysates from individual c8161 tumors
treated as indicated with daily i.p. injections for 48 hours.
Quantification of LC3II/Lc3Iratio (mean+/-SEM) (B) Tumor necrosis
(arrows) in H&E stained sections of 1205Lu tumor xenografts
harvested after 14 days of treatment; Electron micrographs
(7000-12000.times.) of melanoma tumor cells. Arrows: Autophagic
vesicle (white); apoptotic cell (orange) (C) Immunoblotting against
LC3 in HT29 xenografts treated with daily dosing (10, 40 mg/kg) or
3/5 days (80 mg/kg) for 14 days.
[0029] FIG. 8. Toxicity associated with Lys05 76 mgkg ip 3/5 days.
(A) Mice were lethargic with arched backs after 3 days of dosing.
(B) 3/10 mice developed bowel obstruction. (C) Dysmorphic paneth
cells (arrows) in the terminal ileum of one mouse.
[0030] FIG. 9. Lys05 treatment at the highest dose reproduces the
intestinal phenotype of a genetic autophagy deficiency. (A-F)
Weight and intestines were analyzed for mice bearing HT29
xenografts treated with PBS, Lys05 10-80 mg/kg. (A) Daily weight
(B) Representative excised gastrointestinal tracts after 14 days of
treatment (C) Representative images (40.times.) of hemotoxylin and
eosin stained ileal crypts from mice bearing HT29 xenografts (14
days), (40.times.) arrows: paneth cells. (D) Paneth cell number per
crypt (E) Paneth cell dysfunction score, *p<0.05 (F) Scoring of
lysozyme positive cells, *p=0.001. Representative images of
lysosozyme immunofluorescence (green) of ileum in mice treated with
PBS and Lys05 80 mg/kg ip 3/5 days.
[0031] FIG. 10. Paneth cell dysfunction scale. Under 40.times.
power the size and number of eosinophilic granules per Paneth cell
was scored for 10 Paneth cells per sample: A0=normal size and
number A1: Decreased size, normal number; A2: Normal size,
decreased number A3: decreased size and number.
[0032] FIG. 11. Lys05 inhibits autophagy by accumulating in and
deacidifying the lysosome. (A) 1205Lu cells (treated with PBS, HCQ
10 .mu.M, or Lys05 10 .mu.M for 24 hours) and harvested 1205 Lu
xenograft tumors (treated with PBS, HCQ 60 mg/kg i.p. 3/5 days, or
Lys05 76 mg/kg i.p. 3/5 days for 14 days) were homogenized and
fractionated into whole cell (WC) and Lysosomal (L) fractions.
LAMP2 immunoblotting confirmed isolation of concentrated lysosomes
for analysis. (B) Concentrations of HCQ or Lys05 in cells and tumor
whole cell and lysosomal homogenates. (C) Fluorescence imaging of
1205 Lu cells treated as indicated for 30 minutes and stained with
Lysotracker Red. Lysotracker puncta (red) per cell was scored for
three high powered fields. Blue: nuclear DAPI staining. Data
presented is mean.+-.SEM. (D) Fluorescence imaging of c8161 cells
treated as indicated for 24 hours and stained with acridine orange
(AO): orange: aggregated AO, green: diffuse AO.
[0033] FIG. 12. High performance liquid chromatography tandem mass
spectrometry assay for HCQ and Lys05. 1205Lu cells (24 hours) and
1205Lu tumors (14 days). WC: Whole cell homogenate L: Lysosomal
subfraction HCQ: hydroxychloroquine
[0034] FIG. 13. Impairment of lysosomal enzymes and extralysosomal
leakage associated with Lys05 treatment. (A) Acid phosphatase
activity and (B) Cathepsin D immunoblotting in whole cell (white,
WC) and lysosomal (Black; L) fractions of 1205 Lu cells treated
with PBS, HCQ 10 .mu.M, Lys05 10 .mu.M for 24 hours. Graphs show
the mean+/-SEM for three independent experiments. (C) Acid
phosphatase activity and (D) Cathepsin D immunoblotting in whole
cell (white, WC) and lysosomal (Black; L) fractions of 1205 Lu
xenografts treated with PBS, HCQ 60 mg/kg, Lys05 76 mg/kg i.p. 3/5
days (tumors). Whole cell homogenates (white) and lysosomal
homogenates (black) were prepared from three separate tumors were
pooled together. *p<0.05.
[0035] FIG. 14. Chemical structures of synthesized compounds
Lys06-Lys 12; The chemical structures of Lys06-Lys 112 are
shown.
[0036] FIG. 15. Chemical structures of synthesized compounds Lys
13-Lys18; The chemical structures of Lys 13-Lys18 are shown.
[0037] FIG. 15A shows a number of additional bisaminoquinoline
autophagy inhibitors under investigation.
[0038] FIG. 16, Table 1 provides MTT IC.sub.50 values in LN229 for
select compounds of the present invention as presented.
[0039] FIG. 17, Table 12 provides IC.sub.50 (M) values of a number
of compounds according to the present invention in P.
falciparum.
DETAILED DESCRIPTION OF THE INVENTION
[0040] The following terms shall be used throughout the
specification to describe the present invention. Where a term is
not specifically defined herein, that term shall be understood to
be used in a manner consistent with its use by those of ordinary
skill in the art.
[0041] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limit of that range and any other stated or intervening
value in that stated range is encompassed within the invention. The
upper and lower limits of these smaller ranges may independently be
included in the smaller ranges is also encompassed within the
invention, subject to any specifically excluded limit in the stated
range. Where the stated range includes one or both of the limits,
ranges excluding either both of those included limits are also
included in the invention. In instances where a substituent is a
possibility in one or more Markush groups, it is understood that
only those substituents which form stable bonds are to be used.
[0042] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can also be used in the practice or testing of the present
invention, the preferred methods and materials are now
described.
[0043] It must be noted that as used herein and in the appended
claims, the singular forms "a," "and" and "the" include plural
references unless the context clearly dictates otherwise.
[0044] Furthermore, the following terms shall have the definitions
set out below.
[0045] The term "patient" or "subject" is used throughout the
specification within context to describe an animal, generally a
mammal, especially including a domesticated animal and preferably a
human, to whom treatment, including prophylactic treatment
(prophylaxis), with the compounds or 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. In most instances, the patient or subject of the
present invention is a human patient of either or both genders.
[0046] The term "effective" is used herein, unless otherwise
indicated, to describe an amount of a compound or component which,
when used within the context of its use, produces or effects an
intended result, whether that result relates to the prophylaxis
and/or therapy of an infection and/or disease state or as otherwise
described herein. The term effective subsumes all other effective
amount or effective concentration terms (including the term
"therapeutically effective") which are otherwise described or used
in the present application.
[0047] The term "compound" is used herein to describe any specific
compound or bioactive agent disclosed herein, including any and all
stereoisomers (including diasteromers), individual optical isomers
(enantiomers) or racemic mixtures, pharmaceutically acceptable
salts and prodrug forms. The term compound herein refers to stable
compounds. Within its use in context, the term compound may refer
to a single compound or a mixture of compounds as otherwise
described herein. It is understood that the choice of substituents
or bonds within a Markush or other group of substituents or bonds
is provided to form a stable compound from those choices within
that Markush or other group.
[0048] The term "bioactive agent" refers to any biologically active
compound or drug which may be formulated for use in the present
invention. Exemplary bioactive agents include the compounds
according to the present invention which are used to inhibit
autophagy and to treat cancer as well as other compounds or agents
which are otherwise described herein.
[0049] The terms "treat", "treating", and "treatment", are used
synonymously to refer to any action providing a benefit to a
patient at risk for or afflicted with a disease, including
improvement in the condition through lessening or suppression of at
least one symptom, delay in progression of the disease, prevention
or delay in the onset of the disease, etc.
[0050] Treatment, as used herein, encompasses both prophylactic and
therapeutic treatment, principally of cancer. Compounds according
to the present invention can, for example, be administered
prophylactically to a mammal in advance of the occurrence of
disease to reduce the likelihood of that disease. Prophylactic
administration is effective to reduce or decrease the likelihood of
the subsequent occurrence of disease in the mammal, or decrease the
severity of disease that subsequently occurs, especially including
metastasis of cancer. Alternatively, compounds according to the
present invention can, for example, be administered therapeutically
to a mammal that is already afflicted by disease. In one embodiment
of therapeutic administration, administration of the present
compounds is effective to eliminate the disease and produce a
remission or substantially eliminate the likelihood of metastasis
of a cancer. Administration of the compounds according to the
present invention is effective to decrease the severity of the
disease or lengthen the lifespan of the mammal so afflicted, in the
case of cancer.
[0051] The term "pharmaceutically acceptable" as used herein means
that the compound or composition is suitable for administration to
a subject to achieve the treatments described herein, without
unduly deleterious side effects in light of the severity of the
disease and necessity of the treatment.
[0052] The term "inhibit" as used herein refers to the partial or
complete elimination of a potential effect, while inhibitors are
compounds that have the ability to inhibit.
[0053] The term "prevention" when used in context shall mean
"reducing the likelihood" or preventing a disease, condition or
disease state from occurring as a consequence of administration or
concurrent administration of one or more compounds or compositions
according to the present invention, alone or in combination with
another agent. It is noted that prophylaxis will rarely be 100%
effective; consequently the terms prevention and reducing the
likelihood are used to denote the fact that within a given
population of patients or subjects, administration with compounds
according to the present invention will reduce the likelihood or
inhibit a particular condition or disease state (in particular, the
worsening of a disease state such as the growth or metastasis of
cancer) or other accepted indicators of disease progression from
occurring.
[0054] The term "autophagy" or "autophagocytosis" is used to
describe a catabolic process in cells which involves the
degradation of a cell's own components through lysosomes.
[0055] Autophagy is a highly regulated process of biological
systems that plays a normal part in cell growth development and
homeostasis helping to maintain a balance between the synthesis,
degradation, and subsequent recycling of cellular products. It is a
major mechanism by which a cell allocates nutrients from
unnecessary processes to more-essential processes.
[0056] A number of autophagic processes occur in nature, all of
which have the degradation of intracellular components via the
lysosome as a common feature. A well-known mechanism of autophagy
involves the formation of a membrane around a targeted region of a
cell, separating the contents from the rest of the cytoplasm. The
resultant vesicle then fuses with a lysosome which subsequently
degrades the contents.
[0057] Autophagy consists of the sequestration of organelles and
proteins in autophagic vesicles (AV) and degradation of this cargo
through lysosomal fusion (1). Autophagy allows tumor cells to
survive metabolic and therapeutic stresses (2-5). Multiple
publications indicate therapy-induced autophagy is a key resistance
mechanism to many anti-cancer agents.
[0058] Diseases, disease states and/or conditions which benefit
from the inhibition of autophagy include cancer (including the
metastasis of cancer), rheumatoid arthritis, malaria,
antiphospholipid antibody syndrome, lupus, chronic urticaria and
Sjogren's disease.
[0059] The term "cancer" shall refer to a proliferation of tumor
cells having the unique trait of loss of normal controls, resulting
in unregulated growth, lack of differentiation, local tissue
invasion, and/or metastasis. As used herein, neoplasms include,
without limitation, morphological irregularities in cells in tissue
of a subject or host, as well as pathologic proliferation of cells
in tissue of a subject, as compared with normal proliferation in
the same type of tissue. Additionally, neoplasms include benign
tumors and malignant tumors (e.g., colon tumors) that are either
invasive or noninvasive. Malignant neoplasms are distinguished from
benign neoplasms in that the former show a greater degree of
dysplasia, or loss of differentiation and orientation of cells, and
have the properties of invasion and metastasis.
[0060] The term cancer also within context, includes drug resistant
cancers, including multiple drug resistant cancers. Examples of
neoplasms or neoplasias from which the target cell of the present
invention may be derived include, without limitation, carcinomas
(e.g., squamous-cell carcinomas, adenocarcinomas, hepatocellular
carcinomas, and renal cell carcinomas), particularly those of the
bladder, bone, bowel, breast, cervix, colon (colorectal),
esophagus, head, kidney, liver, lung, nasopharyngeal, neck, ovary,
pancreas, prostate, and stomach; leukemias, such as acute
myelogenous leukemia, acute lymphocytic leukemia, acute
promyelocytic leukemia (APL), acute T-cell lymphoblastic leukemia,
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; benign and
malignant lymphomas, particularly Burkitt's lymphoma, Non-Hodgkin's
lymphoma and B-cell lymphoma; benign and malignant melanomas;
myeloproliferative diseases; sarcomas, particularly Ewing's
sarcoma, hemangiosarcoma, Kaposi's sarcoma, liposarcoma,
myosarcomas, peripheral neuroepithelioma, and synovial sarcoma;
tumors of the central nervous system (e.g., gliomas, astrocytomas,
oligodendrogliomas, ependymomas, gliobastomas, neuroblastomas,
ganglioneuromas, gangliogliomas, medulloblastomas, pineal cell
tumors, meningiomas, meningeal sarcomas, neurofibromas, and
Schwannomas); germ-line tumors (e.g., bowel cancer, breast cancer,
prostate cancer, cervical cancer, uterine cancer, lung cancer
(e.g., small cell lung cancer, mixed small cell and non-small cell
cancer, pleural mesothelioma, including metastatic pleural
mesothelioma small cell lung cancer and non-small cell lung
cancer), ovarian cancer, testicular cancer, thyroid cancer,
astrocytoma, esophageal cancer, pancreatic cancer, stomach cancer,
liver cancer, colon cancer, and melanoma; mixed types of
neoplasias, particularly carcinosarcoma and Hodgkin's disease; and
tumors of mixed origin, such as Wilms' tumor and teratocarcinomas,
among others. It is noted that certain epithelial tumors including
ovarian, breast, colon, head and neck, medulloblastoma and B-cell
lymphoma, among others are shown to exhibit increased autophagy and
are principal target cancers for compounds and therapies according
to the present invention.
[0061] The term "additional anti-cancer agent" is used to describe
an additional compound which may be coadministered with one or more
compounds of the present invention in the treatment of cancer. Such
agents include, for example, everolimus, trabectedin, abraxane, TLK
286, AV-299, DN-101, pazopanib, GSK690693, RTA 744, ON 0910.Na, AZD
6244 (ARRY-142886), AMN-107, TKI-258, GSK461364, AZD 1152,
enzastaurin, vandetanib, ARQ-197, MK-0457, MLN8054, PHA-739358,
R-763, AT-9263, a FLT-3 inhibitor, a VEGFR inhibitor, an EGFR TK
inhibitor, an aurora kinase inhibitor, a PIK-1 modulator, a Bcl-2
inhibitor, an HDAC inhibitor, a c-MET inhibitor, a PARP inhibitor,
a Cdk inhibitor, an EGFR TK inhibitor, an IGFR-TK inhibitor, an
anti-HGF antibody, a PI3 kinase inhibitors, an AKT inhibitor, a
JAK/STAT inhibitor, a checkpoint-1 or 2 inhibitor, a focal adhesion
kinase inhibitor, a Map kinase kinase (mek) inhibitor, a VEGF trap
antibody, pemetrexed, erlotinib, dasatanib, nilotinib, decatanib,
panitumumab, amrubicin, oregovomab, Lep-etu, nolatrexed, azd2171,
batabulin, ofatumumab, zanolimumab, edotecarin, tetrandrine,
rubitecan, tesmilifene, oblimersen, ticilimumab, ipilimumab,
gossypol, Bio 111, 131-I-TM-601, ALT-110, BIO 140, CC 8490,
cilengitide, gimatecan, IL13-PE38QQR, INO 1001, IPdR.sub.1
KRX-0402, lucanthone. LY 317615, neuradiab, vitespan, Rta 744, Sdx
102, talampanel, atrasentan, Xr 311, romidepsin, ADS-100380,
sunitinib, 5-fluorouracil, vorinostat, etoposide, gemcitabine,
doxorubicin, irinotecan, liposomal doxorubicin,
5'-deoxy-5-fluorouridine, vincristine, temozolomide, ZK-304709,
seliciclib; PD0325901, AZD-6244, capecitabine, L-Glutamic acid,
N-[4-[2-(2-amino-4,7-dihydro-4-oxo-1H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-
benzoyl]-, disodium salt, heptahydrate, camptothecin, PEG-labeled
irinotecan, tamoxifen, toremifene citrate, anastrazole, exemestane,
letrozole, DES(diethylstilbestrol), estradiol, estrogen, conjugated
estrogen, bevacizumab, IMC-1C11, CHIR-258,);
3-[5-(methylsulfonylpiperadinemethyl)-indolyl]-quinolone,
vatalanib, AG-013736, AVE-0005, the acetate salt of [D-Ser(Bu t)
6,Azgly 10](pyro-Glu-His-Trp-Ser-Tyr-D-Ser(Bu
t)-Leu-Arg-Pro-Azgly-NH.sub.2 acetate
[C.sub.59H.sub.84N.sub.18Oi.sub.4-(C.sub.2H.sup.4O.sub.2).sub.x
where x=1 to 2.4], goserelin acetate, leuprolide acetate,
triplorelin pamoate, medroxyprogesterone acetate,
hydroxyprogesterone caproate, megestrol acetate, raloxifene,
bicalutamide, flutamide, nilutamide, megestrol acetate, CP-724714;
TAK-165, HKI-272, erlotinib, lapatanib, canertinib, ABX-EGF
antibody, erbitux, EKB-569, PKI-166, GW-572016, Ionafarnib,
BMS-214662, tipifarnib; amnifostine. NVP-LAQ824, suberoyl analide
hydroxamic acid, valproic acid, trichostatin A, FK-228, SU11248,
sorafenib, KRN951, aminoglutethimide, arnsacrine, anagrelide,
L-asparaginase, Bacillus Calmette-Guerin (BCG) vaccine, bleomycin,
buserelin, busulfan, carboplatin, carmustine, chlorambucil,
cisplatin, cladribine, clodronate, cyproterone, cytarabine,
dacarbazine, dactinomycin, daunorubicin, diethylstilbestrol,
epirubicin, fludarabine, fludrocortisone, fluoxymesterone,
flutamide, gemcitabine, gleevac, hydroxyurea, idarubicin,
ifosfamide, imatinib, leuprolide, levamisole, lomustine,
mechlorethamine, melphalan, 6-mercaptopurine, mesna, methotrexate,
mitomycin, mitotane, mitoxantrone, nilutamide, octreotide,
oxaliplatin, pamidronate, pentostatin, plicamycin, porfimer,
procarbazine, raltitrexed, rituximab, streptozocin, teniposide,
testosterone, thalidomide, thioguanine, thiotepa, tretinoin,
vindesine, 13-cis-retinoic acid, phenylalanine mustard, uracil
mustard, estramustine, altretamine, floxuridine, 5-deooxyuridine,
cytosine arabinoside, 6-mercaptopurine, deoxycoformycin,
calcitriol, valrubicin, mithramycin, vinblastine, vinorelbine,
topotecan, razoxin, marimastat, COL-3, neovastat, BMS-275291,
squalamine, endostatin, SU5416, SU6668, EMD121974, interleukin-12,
IM862, angiostatin, vitaxin, droloxifene, idoxyfene,
spironolactone, finasteride, cimitidine, trastuzumab, denileukin
diftitox, gefitinib, bortezimib, paclitaxel, irinotecan, topotecan,
doxorubicin, docetaxel, vinorelbine, bevacizumab (monoclonal
antibody) and erbitux, cremophor-free paclitaxel, epithilone B,
BMS-247550, BMS-310705, droloxifene, 4-hydroxytamoxifen,
pipendoxifene, ERA-923, arzoxifene, fulvestrant, acolbifene,
lasofoxifene, idoxifene, TSE-424, HMR-3339, ZK186619, PTK787/ZK
222584, VX-745, PD 184352, rapamycin,
40-O-(2-hydroxyethyl)-rapamycin, temsirolimus, AP-23573, RAD001,
ABT-578, BC-210, LY294002, LY292223, LY292696, LY293684, LY293646,
wortmannin, ZM336372, L-779,450, PEG-filgrastim, darbepoetin,
erythropoietin, granulocyte colony-stimulating factor,
zolendronate, prednisone, cetuximab, granulocyte macrophage
colony-stimulating factor, histrelin, pegylated interferon alfa-2a,
interferon alfa-2a, pegylated interferon alfa-2b, interferon
alfa-2b, azacitidine, PEG-L-asparaginase, lenalidomide, gemtuzumab,
hydrocortisone, interleukin-11, dexrazoxane, alemtuzumab,
all-transretinoic acid, ketoconazole, interleukin-2, megestrol,
immune globulin, nitrogen mustard, methylprednisolone, ibritgumomab
tiuxetan, androgens, decitabine, hexamethylmelamine, bexarotene,
tositumomab, arsenic trioxide, cortisone, editronate, mitotane,
cyclosporine, liposomal daunorubicin, Edwina-asparaginase,
strontium 89, casopitant, netupitant, an NK-1 receptor antagonists,
palonosetron, aprepitant, diphenhydramine, hydroxyzine,
metoclopramide, lorazepam, alprazolam, haloperidol, droperidol,
dronabinol, dexamethasone, methylprednisolone, prochlorperazine,
granisetron, ondansetron, dolasetron, tropisetron, sspegfilgrastim,
erythropoietin, epoetin alfa and darbepoetin alfa, ipilumumab,
vemurafenib among others.
[0062] The term "alkyl" is used herein to refer to a fully
saturated monovalent radical containing carbon and hydrogen (up to
10 carbon atoms or as otherwise indicated), and which may be a
straight chain, branched or cyclic. Examples of alkyl groups are
methyl, ethyl, n-butyl, n-heptyl, isopropyl, 2-methyl propyl,
tert-butyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, etc.
[0063] The term "substituted" as that term relates to alkyl groups
which are described above include one or more functional groups
such as lower alkyl groups containing 1-6 carbon atoms which are
optionally substituted with 1 or 2 hydroxyl groups or between 1 and
5 (preferably 3-5) fluoro groups, acyl (C.sub.1-C.sub.6), halogen
(F, Cl, Br, I, e.g., alkyl halos, e.g., CF.sub.3), amido, hydroxyl,
carboxy/carboxylic acid, thioamido, cyano, nitro, alkenyl
(C.sub.2-C.sub.6) alkynyl (C.sub.2-C.sub.6), azido, alkoxy
(C.sub.1-C.sub.6), (including alkoxy groups which are further
substituted with a C.sub.1-C.sub.6 alkoxy group thus producing a
diether group), amino, C.sub.1-C.sub.6 alkylamino and
dialkyl-amino, where the alkyl groups may be optionally substituted
with 1 or 2 hydroxyl groups or an amine, aminoalkyl or dialkyl
group which itself is substituted one or two alkyl groups or a
7-substituted-4-quinolinyl group, C.sub.2-C.sub.6 acylamino,
C.sub.2-C.sub.6 oxyacylester or carboxyester, aryloxy,
aryloxy(C.sub.1-C.sub.6)alkyl, carboxamido, thio, C.sub.2-C.sub.6
ether or thioether, a 7-substituted-4-aminoquinolinyl group (or a
substitution on an amine group which forms a
7-substituted-4-aminoqunolinyl group) and the like. Preferred
substituents on alkyl groups (within context, especially on the
amino group of the 7-substituted-4-aminoquinoline) or a linker
which contains at least one amine group, include, for example, at
least one hydroxyl group, an amine, monoalkyl amine or dialkyl
amine (where one or both alkyl groups is itself further optionally
substituted with a dialkyl amine or an amine substituted with one
or two (preferably one) 7-substituted-4-quinolinyl group(s) where
the amine group is bonded to the 4-position of the quinolinyl
group) or an alkoxy group (e.g. methoxy or ethoxy) which may be
further substituted with an alkoxy group, preferably a methoxy
group, thus forming a diether substituent.
[0064] The term "aryl" refers to a substituted or unsubstituted
monovalent aromatic radical having a single ring (e.g., phenyl) or
multiple condensed rings (e.g., naphthyl). Other examples include
heterocyclic aromatic (heteroaromatic or heteroaryl) ring groups
having one or more nitrogen, oxygen, or sulfur atoms in the ring,
in particular, quinoline groups, in particular, 7-substituted-amino
quinoline groups, as well as other groups.
[0065] The term "substituted" as used in the term "substituted
aryl, substituted aromatic, substituted heteroaryl, or substituted
heteroaromatic" herein signifies that a substitution on the
7-position of 4-aminoquinoline may be present, said substituents
being selected from atoms and groups, which when present enhance
the activity of the compound as an inhibitor of autophagy. Examples
of substituents that may be present in a substituted aromatic or
heteroaromatic group include, but are not limited to, groups such
as H, halo (F, Cl, Br or I), CN, NO.sub.2, optionally substituted
C.sub.1-C.sub.6 alkyl (when substituted, preferably substituted
with 1 or 2 hydroxyl groups or 3-5 fluoro groups), optionally
substituted O--C.sub.1-C.sub.6 alkyl (preferably, OCH.sub.3),
optionally substituted C.sub.2-C.sub.7 acyl (preferably acetyl) or
optionally substituted C.sub.2-C.sub.7 ester (oxycarbonyl ester or
carboxyester, preferably carboxyester);. It is noted that each of
the substituents disclosed herein may themselves be
substituted.
[0066] The term "co-administration" or "adjunct therapy" shall mean
that at least two compounds or compositions are administered to the
patient at the same time, such that effective amounts or
concentrations of each of the two or more compounds may be found in
the patient at a given point in time. Although compounds according
to the present invention may be co-administered to a patient at the
same time, the term embraces both administration of two or more
agents at the same time or at different times, including sequential
administration. Preferably, effective concentrations of all
co-administered compounds or compositions are found in the subject
at a given time. The term co-administration or adjunct therapy also
contemplates other bioactive agents being coadministered with
pharmaceutical compositions according to the present invention,
especially where a cancer has metastasized or is at risk for
metastasis.
[0067] The term "radiotherapy" or "radiation therapy" is used to
describe therapy for cancer which may be used in conjunction with
the present compounds. Radiation therapy uses high doses of
radiation, such as X-rays, or other energy sources such as
radioisotopes (gamma, beta or alpha emitters), to destroy cancer
cells. The radiation damages the genetic material of the cells so
that they can't grow. Although radiation damages normal cells as
well as cancer cells, the normal cells can repair themselves and
function, while the cancer cells cannot.
[0068] Radiation therapy may be used in combination with the
presently claimed compounds, alone or in combination with
additional anticancer compounds as otherwise disclosed herein,
depending on the cancer to be treated. Radiotherapy therapy is most
effective in treating cancers that have not spread outside the area
of the original cancer, but it also may be used if the cancer has
spread to nearby tissue. Radiotherapy is sometimes used after
surgery to destroy any remaining cancer cells and to relieve pain
from metastatic cancer.
Pharmaceutical Compositions
[0069] Compounds according to the present invention may be readily
formulated into pharmaceutical compositions, useful in the
inhibition of autophagy in a biological system and/or the
inhibition, treatment or prevention of diseases states and/or
conditions which benefit from the inhibition of autophagy including
cancer (and its metastasis), rheumatoid arthritis, malaria,
antiphospholipid antibody syndrome, lupus (systemic lupus
erythematosus), chronic urticaria and Sjogren's disease.
Pharmaceutical compositions comprise an effective amount of one or
more compounds according to the present invention in combination
with a pharmaceutically acceptable carrier, additive or excipient,
optionally in combination with at least one additional agent, in
the case of cancer, preferably an anticancer agent as otherwise
described herein.
[0070] As noted above, the compounds and method of the invention
may be used to inhibit autophagy as otherwise described herein, and
are useful for the inhibition (including prophylaxis) and/or
treatment of cancer and its metastasis, rheumatoid arthritis,
malaria, antiphospholipid antibody syndrome, lupus (systemic lupus
erythematosus), chronic urticaria and Sjogren's disease. The
treatment of cancer or malaria are important aspects of the present
invention.
[0071] In methods according to the present invention, subjects or
patients in need are treated with the present compounds,
pharmaceutical compositions in order to inhibit, reduce the
likelihood or treat a disease state, condition and/or infection as
otherwise described herein. The disease states, conditions and
infections treated by the present compounds and compositions are
readily recognized and diagnosed by those of ordinary skill in the
art and treated by administering to the patient an effective amount
of one or more compounds according to the present invention.
[0072] Generally; dosages and routes of administration of the
compound are determined according to the size and condition of the
subject, according to standard pharmaceutical practices. Dose
levels employed can vary widely, and can readily be determined by
those of skill in the art. Typically, amounts in the milligram up
to gram quantities are employed. The composition may be
administered to a subject by various routes, e.g. orally,
transdermally, perineurally or parenterally, that is, by
intravenous, subcutaneous, intraperitoneal, or intramuscular
injection, among others, including buccal, rectal and transdermal
administration. Subjects contemplated for treatment according to
the method of the invention include humans, companion animals,
laboratory animals, and the like.
[0073] Formulations containing the compounds according to the
present invention may take the form of solid, semi-solid,
lyophilized powder, or liquid dosage forms, such as, for example,
tablets, capsules, powders, sustained-release formulations,
solutions, suspensions, emulsions, suppositories, creams,
ointments, lotions, aerosols, patches or the like, preferably in
unit dosage forms suitable for simple administration of precise
dosages.
[0074] Pharmaceutical compositions according to the present
invention typically include a conventional pharmaceutical carrier
or excipient and may additionally include other medicinal agents,
carriers, adjuvants, additives and the like. Preferably, the
composition is about 0.1% to about 85%, about 0.5% to about 75% by
weight of a compound or compounds of the invention, with the
remainder consisting essentially of suitable pharmaceutical
excipients. For oral administration, such excipients include
pharmaceutical grades of mannitol, lactose, starch, magnesium
stearate, sodium saccharine, talcum, cellulose, glucose, gelatin,
sucrose, magnesium carbonate, and the like. If desired, the
composition may also contain minor amounts of non-toxic auxiliary
substances such as wetting agents, emulsifying agents, or
buffers.
[0075] Liquid compositions can be prepared by dissolving or
dispersing the compounds (about 0.5% to about 20% by weight or
more), and optional pharmaceutical adjuvants, in a carrier, such
as, for example, aqueous saline, aqueous dextrose, glycerol, or
ethanol, to form a solution or suspension. For use in oral liquid
preparation, the composition may be prepared as a solution,
suspension, emulsion, or syrup, being supplied either in liquid
form or a dried form suitable for hydration in water or normal
saline.
[0076] When the composition is employed in the form of solid
preparations for oral administration, the preparations may be
tablets, granules, powders, capsules or the like. In a tablet
formulation, the composition is typically formulated with
additives, e.g. an excipient such as a saccharide or cellulose
preparation, a binder such as starch paste or methyl cellulose, a
filler, a disintegrator, and other additives typically used in the
manufacture of medical preparations.
[0077] An injectable composition for parenteral administration will
typically contain the compound in a suitable i.v. solution, such as
sterile physiological salt solution. The composition may also be
formulated as a suspension in a lipid or phospholipid, in a
liposomal suspension, or in an aqueous emulsion.
[0078] Methods for preparing such dosage forms are known or is
apparent to those skilled in the art; for example, see Remington's
Pharmaceutical Sciences (17th Ed., Mack Pub. Co., 1985). The
composition to be administered will contain a quantity of the
selected compound in a pharmaceutically effective amount for
inhibiting autophagy in a biological system, including a patient or
subject according to the present invention.
Synthesis of Compounds According to the Present Invention
Strategy for Synthesis of Bivalent Aminoquinoline Autophagy
Inhibitors
[0079] The inventors have examined the application of the strategy
of polyvalency (11, 12) to the synthesis of novel autophagy
inhibitors by preparing a dimeric chloroquine (FIG. 1, compound 3:
Lys01), from commercially available materials. Based on literature
precedent(14), we envisioned the preparation of compound 3 from one
equivalent of compound 5 and two equivalents of compound 6 (14), as
outlined retrosynthetically in FIG. 2.
[0080] While compound 4 (R.dbd.Cl) is a known compound (14), the
bisquinoline compound 3 (R.sub.2=Me) has not been described in the
literature. Due its putative lysosomotropism, we refer to compound
3 as Lys01. Reaction of compound 5 with two equivalents of compound
4 led to the formation of a mixture of the desired product compound
3 along with some of the monoquinoline compound 7 (FIG. 1, Lys02,
FIG. 2), the synthesis of which was previously reported by Higuchi
(18). To examine the role of the C-7 chlorine substituents in
compound 3, we prepared compound 9 (FIG. 1, compound 9: Lys 03),
the dimethoxy analog of compound 3.
[0081] To determine the importance of the polyamine connector of
compound 3, we prepared the polyether analog compound 11 (FIG. 1,
compound 11: Lys04) of compound 3 from the commercially available
2,2'-(ethylenedioxy)bis(ethylamine) 10 (see FIG. 2).
[0082] In an effort to obtain SAR data on the lead compound 3
(Lys01), the inventors examined the systematic modification of the
structure of compound 3 (i.e., 12 R.dbd.Cl), as outlined in Scheme
A of FIG. 15A. Initial efforts focused on changes in three
different areas of the structure of compound 12 as examined: 1)
modification of the C-7 chlorine substituent present in compound 3
(X in compound 12 of FIG. 15A); 2) modification of the C-4 nitrogen
substituent, i.e., N-alkylation or acylation, as well as the
adjacent carbon atom (that contains a stereocenter in CQ and HCQ
(FIG. 1); and 3) modification of the N-methyl group in compound 12
(FIG. 15A).
[0083] Each of the requisite starting compounds outlined in FIG.
15A (compounds 13-16) are known or commercially available,
facilitating the synthesis of a family of analogs that differ from
compound 3 (12 R.dbd.Cl) by incorporating different
electron-withdrawing groups, based on the work of Lee (16).
[0084] The inventors also examine the biological activity of
N-alkyated and N-acylated analogs of compound 12, compound 17 and
compound 18 (FIG. 15A). The preparation of these novel compounds
would proceed directly from 12 (R.dbd.Cl), either by direct
alkylation, reductive alkylation, or acylation.
[0085] The inventors also examine the introduction into compound 12
of the chirality that is present in CQ and HCQ (FIG. 1) as outlined
in compound 19 (Scheme C of FIG. 15A). The requisite linker
compound 20 could be obtained using the method of Kokotos (J. Chem.
Res, Synopses 1992, 12, 391).
[0086] Finally, the modification of the structure of compound 12
via replacement of the N-methyl group (Scheme A of FIG. 15A) with
other functionalities is examined. Two intriguing possibilities are
shown in Scheme D of FIG. 15A, i.e., compound 21 and compound 22,
in which the N-methyl group is replaced with the hydroxyethyl group
present in HCQ, and in which the N-methyl group is replaced by
another quinoline moiety to produce tris-quinoline compound 22. The
synthetic routes for the formation of 21 and 22 are based closely
on the work of Lee (19) and Solomon(17), respectively.
##STR00005##
[0087] While characterization of the second generation compounds
described continue, there are additional compounds which are
believed to represent more potent autophagy inhibitors. Taking the
lead compound as compound 12 (scheme 1; Lys01), the inventors
describe more extensive systematic modification of three parts of
the structure of 12, as outlined in the generic structure 23 (see
above) as the next logical step in SAR analysis. Each of the three
parts of the structure (R1, R2 and R3) is modified, as outlined
below.
The Role of R1
[0088] The effect of substituting the chlorine moiety in
chloroquine 1 (scheme 2) has been examined
##STR00006##
by Egan and coworkers (20), who established that
electron-withdrawing groups are important for the antiplasmodial
activity of these 7-substituted quinolines. The inventors therefore
examined these same substitutions in the case of the generic
structure 23, substituting both R.sup.1 and R.sup.1' in 23, and
also examining the effect of monosubstitution, in which the R1'
substituent is then either Cl as in 12 or hydrogen (X.dbd.H). The
Egan study indicates that all of the requisite
4-chloro-7-substituted-quinolines are known, thereby facilitating
the preparation of each of the compounds shown in Scheme 3. None of
the compounds outlined in scheme 3 are known, although the
synthesis of linked bis-quinolines (R1=H) has been described
(21).
##STR00007##
The Role of R.sup.2
[0089] The role of R.sup.2.dbd.H in lead structure 3 is examined by
either acetylation (R2=MeCO--; mono- or di-) or methylation
(R.sup.2=Me; mono- or di-) to give the structures 41-44 shown in
Scheme 4. While the homologous analogs 45 and 46 (containing one or
two propylene chains between the nitrogen atoms) are known (FR
1345573; CAN 60:68181), each of the analogs 41-44 represents a
novel structure.
##STR00008##
[0090] Well-tolerated substitution at R.sup.2 further directs
substitution of R.sup.2 with a hydroxyethyl moiety, as found in
hydroxychloroquine 2 (scheme 5). The reaction of anilines with
ethylene oxide to give the corresponding hydroxyethyl compounds is
well-precedented (22), so that conversion of the lead structure 3
to either mono- or di-hydroxyethylated analogs 47 and 48 is readily
achieved.
##STR00009##
[0091] The inventors also examine the preparation of tri- and
tetra-quinoline containing structures by oxidation of the primary
alcohols in 47 and 48 to give the corresponding aldehydes, which on
reductive alkylation with 7-chloro-4-aminoquinoline gives the tri-
and tetraquinolines 49 and 50, based on the work of Bailey and
coworkers (23), as shown in scheme 6.
##STR00010##
[0092] The inventors also examine the effect of incorporation of
both lipophilic groups, i.e., long chain alkyl, as well as more
polar substituents in the place of R.sup.2 in the lead structure in
Scheme 7, via alkylation of the secondary amines (R.sup.2.dbd.H)
with the commercially available alkylating agents 59-62, leading to
the preparation of 51-58 via mono- and dialkylation of the two
secondary amine functionalities, based on the work of Drefahl and
Konig (Chem. Ber. 1954, 87, 1632-4).
##STR00011##
[0093] The inventors examine the effect of changing R.sup.3 in the
lead structure 23 (see scheme 1, above). Certain substitutions for
R.sup.3 are already known, such as shown in 63-65 in Scheme 8,
below. The inventors prepare a series of new analogs, based on the
alkylation of the known secondary amine 63 (13) with the alkylating
agents shown in scheme 7 (59-62) to generate the novel structures
(66-69). The substrate 70 in which R.sup.3.dbd.CH.sub.2CH.sub.2OH,
i.e., the analog of 63 that corresponds most closely to
hydroxychloroquine is examined. This compound is available by the
same sequence used to prepare the analogs shown in Scheme 5, or by
demethylation of 66.
##STR00012##
[0094] Another important difference between the lead structure 3
and chloroquine 1 (scheme 9) is the presence of the stereocenter
next to the nitrogen atom in 1. The inventors also prepare hybrid
structures, in which either one of both of the sides of 23 more
closely resemble 1, such as 71/72 and 73/74. The requisite diamines
in each case are prepared starting from (L) glutamic acid,
following the procedure employed by Craig in the stereoselective
synthesis of chloroquine (J. Org. Chem. 1988, 53, 1167-1170).
##STR00013##
[0095] The inventors prepare the analog of the lead structure 3
containing the stereocenter present in chloroquine 1, i.e., 75/76
and 77/78, which are available from alanine and serine,
respectively, using the method of Charlton and coworkers (24) via
reductive alkylation.
[0096] Finally, the inventors examine a series of compounds that
contain four nitrogen atoms in the tether connecting the quinoline
rings, instead of the three nitrogen atoms that are present in the
connector chain in 3 (scheme 9), as illustrated in scheme 10,
below. Denny and coworkers (24) have described the synthesis of the
requisite tetramines 81 and 82. Attachment of the additional
chloroquine moieties present in 80 takes place via the same
methodology employed in Scheme 8 for the synthesis of 65.
##STR00014##
Method of Treatment
[0097] According to one aspect of the invention, a method is
provided for treating a mammalian patient or subject to inhibit
autophagy in that patient or subject. Compounds according to the
present invention described herein may be used to inhibit autophagy
in a manner consistent with inhibiting, treating and/or preventing
disease states and/or conditions including cancer (including
metastasis of cancer), rheumatoid arthritis, malaria,
antiphospholipid antibody syndrome, lupus, chronic urticaria and
Sjogren's disease.
[0098] According to the present invention, in patients or subjects
in need thereof, are treated by administering to the patient or
subject an effective amount of one or more compounds according to
the present invention, optionally in combination with at least one
additional bioactive agent useful for treating the same disease
state or condition. Compounds according to the present invention
may be used to inhibit, reduce the likelihood or treat cancer,
including the metastasis of cancer in a patient or subject in need
of such treatment. The treatment is useful for any cancer for which
inhibition of autophagy represents a favorable result or for which
metastasis is a risk element. Therapy with at least one additional
anticancer agent as otherwise described herein is also contemplated
in the present methods. The numerous cancers which may be treated
pursuant to the present method are described hereinabove.
[0099] In another aspect the present invention is directed to a
method for treating a disease state and/or condition which benefits
from the inhibition of autophagy, including rheumatoid arthritis,
malaria, antiphospholipid antibody syndrome, lupus, chronic
urticaria and Sjorgen's disease. In this method, a patient or
subject in need of treatment is administered an effective amount of
a compound as otherwise described herein optionally in combination
with a pharmaceutically acceptable carrier, additive or excipient
in order to inhibit, treat and/or prevent the above disease states
of conditions.
[0100] In the present invention, the method of treatment comprises
administering to the subject in need of treatment, in a
pharmaceutically acceptable carrier, an effective amount of a
compound according to I below:
##STR00015##
Wherein R.sup.1 and R.sup.1' are each independently H, halo (F, Cl,
Br or I), CN, NO.sub.2, optionally substituted C.sub.1-C.sub.6
alkyl (when substituted, preferably substituted with 1 or 2
hydroxyl groups or 3-5 fluoro groups), optionally substituted
O--C.sub.1-C.sub.6 alkyl (preferably, OCH.sub.3), optionally
substituted C.sub.2-C.sub.7 acyl (preferably acetyl) or optionally
substituted C.sub.2-C.sub.7 ester (oxycarbonyl ester or
carboxyester, preferably carboxyester); R and R' are each
independently H, a C.sub.1-C.sub.6 optionally substituted alkyl
group, a C.sub.1-C.sub.7 (preferably C.sub.2-C.sub.7) optionally
substituted acyl group, a C.sub.2-C.sub.7 optionally substituted
carboxy ester group (which forms a urethane group with the nitrogen
atom to which R or R' is bonded);
[0101] L is a
##STR00016##
group or a
##STR00017##
group (either A or A' may be bonded to either of the two amine
groups in compound I) wherein at least one of the CH.sub.2 groups
in L is optionally substituted with a C.sub.1-C.sub.3 alkyl group
which itself is optionally substituted with one or two hydroxyl
groups; X is absent, (CH.sub.2).sub.j O, S or N--R''; Y is absent,
CH.sub.2, O, CH.sub.2O or N--R'' and Y' is absent CH.sub.2, O,
OCH.sub.2 or N--R'', with the proviso that when one or more of X, Y
and Y' is present, each of X and Y, X and Y' or Y and Y', when
present, forms a stable bond; R'' is H or an optionally substituted
C.sub.1-C.sub.6 (preferably C.sub.1-C.sub.3) alkyl group; j is 1, 2
or 3 (preferably 1 or 2); n is 0, 1, 2, 3 or 4, with the proviso
that when n is 0, X is (CH.sub.2).sub.j where j is at least 1 and
at least one CH.sub.2 group is optionally substituted with a
C.sub.1-C.sub.3 alkyl group which itself is optionally substituted
with one or two hydroxyl groups; A is absent or (CH.sub.2).sub.j
and A' is (CH.sub.2).sub.j wherein at least one CH.sub.2 group in A
or A' is optionally substituted with a C.sub.1-C.sub.3 alkyl group
which is itself optionally substituted with one or two hydroxyl
groups;
Z is O or N--R.sup.Z;
[0102] R.sup.Z is H or an optionally substituted C.sub.1-C.sub.3
alkyl group, or a pharmaceutically acceptable salt, enantiomer,
diastereomer, solvent or polymorph thereof.
[0103] In certain preferred methods of the invention, R.sup.1 and
R.sup.1' are each independently H, a halo group, a nitro group or a
trifluoromethyl group, preferably a chloro group. R and R' are
preferably each independently H, a C.sub.1-C.sub.3 optionally
substituted alkyl group itself preferably substituted with at least
one hydroxyl group, an amine, monoalkyl amine or dialkyl amine
group, wherein said amine group or said monoalkyl amine group is
optionally substituted on the amine position with a
7-substituted-4-quinolinyl group wherein the amine binds to the
4-position of the quinolinyl group, or one or both alkyl groups of
said monoalkyl amine or dialkyl amine is itself further optionally
substituted with at least one hydroxyl group, an amine, a monoalkyl
amine or a dialkyl amine wherein the amine or monoalkyl amine is
optionally substituted on the amine position with one or two
7-substituted-quinolinyl group(s) (the 7-position of each
quinolinyl group may be substituted with R.sup.1 and/or R.sup.1' as
broadly described for generic structure I above), or an alkoxy
group (e.g. methoxy or ethoxy) which alkoxy group may be further
substituted with an alkoxy group, preferably a methoxy group (thus
forming a diether substituent).
[0104] In other preferred methods of the invention L is a
##STR00018##
group, where X is N--R'', Y and Y' are each independently absent or
CH.sub.2, and R'' is H or a C.sub.1-C.sub.3 alkyl group which is
optionally substituted with at least one hydroxyl group, an amine,
monoalkyl amine or dialkyl amine group, wherein said amine group or
said monoalkyl amine group is optionally substituted on the amine
position with a 7-substituted-4-quinolinyl group wherein the amine
binds to the 4-position of the quinolinyl group, or one or both
alkyl groups of said monoalkyl amine or dialkyl amine is itself
further optionally substituted with at least one hydroxyl group, an
amine, a monoalkyl amine or a dialkyl amine wherein the amine or
monoalkyl amine is optionally substituted on the amine position
with one or two 7-substituted-quinolinyl group(s) (the 7-position
of each quinolinyl group may be substituted with R.sup.1 and/or
R.sup.1' as broadly described for generic structure I above) for
the), or an alkoxy group (e.g. methoxy or ethoxy) which alkoxy
group may be further substituted with an alkoxy group, preferably a
methoxy group (thus forming a diether substituent).
[0105] Further preferred methods relate to the use/administration
of the compounds according to the present invention which are
presented in the various schemes which are presented in Scheme 1
and Schemes 3-10 and FIGS. 14, 15 and 15A as presented herein.
[0106] In the methods treating or inhibiting cancer or the
metastasis of cancer, the compounds described above may be
coadministered with at least one additional anticancer agent
including, for example, everolimus, trabectedin, abraxane, TLK 286,
AV-299, DN-101, pazopanib, GSK690693, RTA 744, ON 0910.Na, AZD 6244
(ARRY-142886), AMN-107, TKI-258, GSK461364, AZD 1152, enzastaurin,
vandetanib, ARQ-197, MK-0457, MLN8054, PHA-739358, R-763, AT-9263,
a FLT-3 inhibitor, a VEGFR inhibitor, an EGFR TK inhibitor, an
aurora kinase inhibitor, a PIK-1 modulator, a Bcl-2 inhibitor, an
HDAC inhibitor, a c-MET inhibitor, a PARP inhibitor, a Cdk
inhibitor, an EGFR TK inhibitor, an IGFR-TK inhibitor, an anti-HGF
antibody, a PI3 kinase inhibitors, an AKT inhibitor, a JAK/STAT
inhibitor, a checkpoint-1 or 2 inhibitor, a focal adhesion kinase
inhibitor, a Map kinase kinase (mek) inhibitor, a VEGF trap
antibody, pemetrexed, erlotinib, dasatanib, nilotinib, decatanib,
panitumumab, amrubicin, oregovomab, Lep-etu, nolatrexed, azd2171,
batabulin, SUBSTITUTE SHEET (RULE 26) ofatumumab, zanolimumab,
edotecarin, tetrandrine, rubitecan, tesmilifene, oblimersen,
ticilimumab, ipilimumab, gossypol, Bio 111, 131-I-TM-601, ALT-110,
BIO 140, CC 8490, cilengitide, gimatecan, IL13-PE38QQR, INO 1001,
IPdR.sub.1KRX-0402, lucanthone, LY 317615, neuradiab, vitespan, Rta
744, Sdx 102, talampanel, atrasentan, Xr 311, romidepsin,
ADS-100380, sunitinib, 5-fluorouracil, vorinostat, etoposide,
gemcitabine, doxorubicin, irinotecan, liposomal doxorubicin,
5'-deoxy-5-fluorouridine, vincristine, temozolomide, ZK-304709,
seliciclib; PD0325901, AZD-6244, capecitabine, L-Glutamic acid,
N-[4-[2-(2-amino-4,7-dihydro-4-oxo-1H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-
benzoyl]-, disodium salt, heptahydrate, camptothecin, PEG-labeled
irinotecan, tamoxifen, toremifene citrate, anastrazole, exemestane,
letrozole, DES(diethylstilbestrol), estradiol, estrogen, conjugated
estrogen, bevacizumab, IMC-1C11, ClHIR-258,);
3-[5-(methylsulfonylpiperadinemethyl)-indolyl]-quinolone,
vatalanib, AG-013736, AVE-0005, the acetate salt of [D-Ser(Bu t)
6,Azgly 10](pyro-Glu-His-Trp-Ser-Tyr-D-Ser(Bu
t)-Leu-Arg-Pro-Azgly-NH.sub.2 acetate
[C.sub.59H.sub.84N.sub.18Oi.sub.4-(C.sub.2H.sub.4O.sub.2).sub.x
where x=1 to 2.4], goserelin acetate, leuprolide acetate,
triptorelin pamoate, medroxyprogesterone acetate,
hydroxyprogesterone caproate, megestrol acetate, raloxifene,
bicalutamide, flutamide, nilutamide, megestrol acetate, CP-724714;
TAK-165, HKI-272, erlotinib, lapatanib, canertinib, ABX-EGF
antibody, erbitux, EKB-569, PKI-166, GW-572016, lonafarnib,
BMS-214662, tipifarnib; amifostine, NVP-LAQ824, suberoyl analide
hydroxamic acid, valproic acid, trichostatin A, FK-228, SU111248,
sorafenib, KRN951, aminoglutethimide, arnsacrine, anagrelide,
L-asparaginase, Bacillus Calmette-Guerin (BCG) vaccine, bleomycin,
buserelin, busulfan, carboplatin, cannustine, chlorambucil,
cisplatin, cladribine, clodronate, cyproterone, cytarabine,
dacarbazine, dactinomycin, daunorubicin, diethylstilbestrol,
epirubicin, fludarabine, fludrocortisone, fluoxymesterone,
flutamide, gemcitabine, gleevac, hydroxyurea, idarubicin,
ifoslamide, imatinib, leuprolide, levarnisole, lomustine,
mechlorethamine, melphalan, 6-mercaptopurine, mesna, methotrexate,
mitomycin, mitotane, mitoxantrone, nilutamide, octreotide,
oxaliplatin, pamidronate, pentostatin, plicamycin, porfimer,
procarbazine, raltitrexed, rituximab, streptozocin, teniposide,
testosterone, thalidomide, thioguanine, thiotepa, tretinoin,
vindesine, 13-cis-retinoic acid, phenylalanine mustard, uracil
mustard, estramustine, altretamine, floxuridine, 5-deooxyuridine,
cytosine arabinoside, 6-mercaptopurine, deoxycoformycin,
calcitriol, valrubicin, mithramycin, vinblastine, vinorelbine,
topotecan, razoxin, marimastat, COL-3, neovastat, BMS-275291,
squalamine, endostatin, SU5416, SU6668, EMD121974, interleukin-12,
IM862, angiostatin, vitaxin, droloxifene, idoxyfene,
spironolactone, linasteride, cimitidine, trastuzumab, denileukin
diftitox, gefitinib, bortezimib, paclitaxel, irinotecan, topotecan,
doxorubicin, docetaxel, vinorelbine, bevacizumab (monoclonal
antibody) and erbitux, cremophor-free paclitaxel, epithilone B,
BMS-247550, BMS-310705, droloxifene, 4-hydroxytamoxifen,
pipendoxifene, ERA-923, arzoxifene, fulvestrant, acolbifene,
lasofoxifene, idoxifene, TSE-424, HMR-3339, ZK186619, PTK787/ZK
222584, VX-745, PD 184352, rapamycin,
40-O-(2-hydroxyethyl)-rapamycin, temsirolimus, AP-23573, RAD001,
ABT-578, BC-210, LY294002, LY292223, LY292696, LY293684, LY293646,
wortmannin, ZM336372, L-779,450, PEG-filgrastim, darbepoetin,
erythropoietin, granulocyte colony-stimulating factor,
zolendronate, prednisone, cetuximab, granulocyte macrophage
colony-stimulating factor, histrelin, pegylated interferon alfa-2a,
interferon alfa-2a, pegylated interferon alfa-2b, interferon
alfa-2b, azacitidine, PEG-L-asparaginase, lenalidomide, gemtuzumab,
hydrocortisone, interleukin-11, dexrazoxane, alemtuzumab,
all-transretinoic acid, ketoconazole, interleukin-2, megestrol,
immune globulin, nitrogen mustard, methylprednisolone, ibritgumomab
tiuxetan, androgens, decitabine, hexamethylmelamine, bexarotene,
tositumomab, arsenic trioxide, cortisone, editronate, mitotane,
cyclosporine, liposomal daunorubicin, Edwina-asparaginase,
strontium 89, casopitant, netupitant, an NK-1 receptor antagonists,
palonosetron, aprepitant, diphenhydramine, hydroxyzine,
metoclopramide, lorazepam, alprazolam, haloperidol, droperidol,
dronabinol, dexamethasone, methylprednisolone, prochlorperazine,
granisetron, ondansetron, dolasetron, tropisetron, pegfilgrastim,
erythropoietin, epoetin alfa and darbepoetin alfa, among others,
and mixtures thereof.
[0107] In methods involving infections, disease states and/or
conditions caused by rheumatoid arthritis, malaria,
antiphospholipid antibody syndrome, lupus, chronic urticaria and
Sjogren's disease, the compounds according to the present invention
may be coadministered with additional agents which are
traditionally used in therapy for these disease states and/or
conditions.
EXAMPLES
[0108] The following examples illustrate and describe the present
invention but are not intended to limit the invention in any
way.
[0109] Synthesis of Compound 3 (Lys01).
[0110] A round-bottom flask was charged with the
4-bromo-7-chloroquinoline (compound 5) (734 mg, 3.0 mmol),
Pd(OAc).sub.2 (23 mg, 0.1 mmol), BINAP (125 mg, 0.2 mmol),
K.sub.3PO.sub.4 (1.06 g, 5.0 mmol), and triamine (compound 6) (117
mg, 1.0 mmol). Dioxane (10 mL) was introduced through the septum.
The resulting suspension was stirred under argon at 90.degree. C.
for 18 h and cooled. The mixture was adsorbed onto silica gel and
purified by flash chromatography (CH.sub.2Cl.sub.2/MeOH: 90/9/1) to
afford compound 3 (387 mg, 88%) as a yellow solid. mp
199-200.degree. C.; R.sub.f=0.28 (silica gel,
CH.sub.2Cl.sub.2/MeOH/NH.sub.4OH: 90/9/1);): .sup.1H NMR (500 MHz,
CDCl.sub.3: .delta. 8.53 (d, J=5.5 Hz, 2H), 7.94 (d, J=2.0 Hz, 2H),
7.41 (d, J=9.0 Hz, 2H), 6.98 (dd, J=9.0, 2.0 Hz, 2H), 6.39 (d,
J=5.0 Hz, 2H), 5.44 (s, 2H). 3.42 (q, J=5.0 Hz, 4H), 2.90 (t, J=6.0
Hz, 2H), 2.46 (s, 9H). .sup.13C NMR (125 MHz, CDCl.sub.3): .delta.
152.1, 149.5, 149.1, 135.1, 128.9, 125.5, 120.6, 117.1, 99.3, 55.5,
42.4, 40.3 FTIR (thin film): 3215, 2917, 1609, 1579, 1449. HRMS-ESI
(m/z): calcd for C.sub.23H.sub.24N.sub.5Cl.sub.2 [M+H].sup.+:
440.1409. found: 440.1406.
[0111] Synthesis of Compound 11 (Lys 05).
[0112] To generate a water soluble salt of compound 3, a suspension
of compound 3 (896 mg, 2.04 mmol) in MeOH (40 mL) was bubbled with
HCl gas for 10 min at 0.degree. C. The mixture was stirred for
another 12 h at room temperature. The solvent was removed by rotary
evaporation and the residue was dried under vacuum at 50.degree. C.
overnight to afford the salt 3 (1.13 g, 100%) as a yellow solid. mp
270.degree. C. (decomp.); .sup.1H NMR (500 MHz, D.sub.2O): .delta.
8.12 (d, J=7.0 Hz, 2H), 7.73 (d, J=9.0 Hz, 2H), 7.58 (d, J=2.0 Hz,
2H), 7.26 (dd, J=9.0, 2.0 Hz, 2H), 6.62 (d, J=2.0 Hz, 2H), 3.89
(br, 4H), 3.68 (br, 4H), 3.12 (s, 3H). .sup.13C NMR (125 MHz,
D.sub.2O): .delta. 155.8, 142.8, 140.2, 137.2, 128.1, 123.8, 119.1,
114.8, 98.7, 52.9, 42.7, 38.2. FTIR (thin film): 3376, 3019, 2914,
1631, 1612, 1215 cm.sup.-1. HRMS-ESI (m/z): calcd for
C.sub.23H.sub.24N.sub.5Cl.sub.2 [M-3HCl+H].sup.+: 440.1409. found:
440.1408.
Biological Testing
[0113] Lys01 is a More Potent Autophagy Inhibitor than HCQ or
CQ.
[0114] LN229 (human glioblastoma) were treated with Lys01 and
derivatives Lys02, Lys03, Lys04, HCQ and CQ. Near complete cell
death of cultured cells was observed in cells treated with Lys01 at
concentrations of 10 .mu.M or higher between 4-24 hours. LC3 is a
ubiquitin-like protein which exists as an unconjugated form (LC3I)
or conjugated to AV membranes (LC3II)(25). The ratio of LC3II/LC3I
reflects the accumulation of AV in cells, and therefore effective
autophagy inhibition. LC3 immunoblotting (FIG. 3) demonstrated that
Lys01 is a >10-fold more potent autophagy inhibitor than HCQ or
CQ at a concentration of 10 .mu.M. Lys02 and Lys03 produced
dose-response relationships for LC3 immunoblotting similar to HCQ
or CQ, whereas Lys04, which retains the two chloroquinoline rings
present in Lys01, demonstrated intermediate potency in the LC3
autophagy assay
[0115] To further characterize the effects of Lys01 on autophagy,
LN229 GFP-LC3 cells were treated with Lys01 or HCQ (FIG. 4A).
Within 4 hours of treatment, in cells treated with HCQ 1 .mu.M,
punctate fluorescence, indicating an accumulation of ineffective
autophagic vesicles, was observed in a minority of cells. HCQ 10
.mu.M produced numerous small puncta, and HCQ 100 .mu.M resulted in
larger dense puncta that represent fusion of accumulated autophagic
vesicles. Lys01 1 .mu.M produced numerous small puncta, whereas in
cells treated with Lys01 10 .mu.M dense puncta similar in
appearance to those observed in cells treated with HCQ 100 .mu.M
were apparent. All cells treated with Lys01 100 .mu.M were dead by
4 hours. Quantification of the GFP-LC3 puncta per cell demonstrated
a significant 5-fold increase in GFP-LC3 puncta between 10 .mu.M
Lys01 compared to 10 .mu.M HCQ treatments. The average number of
vesicles per cell in cells treated with Lys01 10 .mu.M was higher
than in cells treated with 100 .mu.M HCQ (FIG. 4A). Electron
micrographs of LN229 GFP-LC3 cells treated with DMSO, HCQ, or Lys01
further characterized the significant morphological difference in
the size and number of vesicles produced by blockade of autophagy
with these agents (FIG. 4B). Thus, Lys01 produces morphological
changes more pronounced than HCQ, a known lysosomal inhibitor, at
10-fold lower concentrations. To determine if Lys01 treatment was
inducing production of new autophagic vesicles (an autophagy
inducer) or blocking the clearance of autophagy vesicles (an
autophagy inhibitor), a bafilomycin clamp experiment was performed
(FIG. 4C). LN229 GFP-LC3 cells were treated with DMSO, rapamycin,
HCQ 10 .mu.M, and Lys01 10 .mu.M, in the absence or presence of
bafilomycin. At 24 hours, rapamycin treatment resulted in a further
increase in the LC3II/LC3 ratio in bafilomycin treated cells
compared to control cells whereas HCQ- or Lys05-treated cells did
not demonstrate an increase in LC3II/LC3I ratio in bafilomycin
treated cells compared to control, providing further evidence that
Lys01 is an autophagy inhibitor (FIG. 4C).
[0116] To determine the implications of more potent autophagy
inhibition on cytotoxicity, LN229 (glioma), 1205Lu (melanoma),
HT-29 (colon) and c8161 (melanoma) cells were treated with Lys01,
Lys02, Lys03 Lys04, and HCQ at concentrations between 0.01-100
.mu.M (FIG. 4D). The MTT assay was used to assess viable cells at
72 hours. In the 4 cell lines tested, the IC50 of Lys01 was 4-8
.mu.M (Supplemental Table 2). Near complete cell death after 24
hours was observed in 1205Lu and HCC827 (cell lines which are
highly resistant to HCQ) cells treated with 10 .mu.M Lys01. In
contrast the IC50 for Lys02, which is a monofunctional CQ
derivative (35-91 .mu.M), Lys03, the bisaminoquinoline with methoxy
groups replacing chlorine (24-53 .mu.M), or HCQ (15-42 .mu.M) were
collectively 9-30-fold less potent than Lys01. Lys04, which retains
the bivalent aminoquinoline rings but has an altered linker, had
intermediate activity, with IC50 of 10-17 .mu.M. These studies
demonstrate that Lys01 is consistently more cytotoxic than other
aminoquinolines tested or HCQ. Together with the LC3 western blot
data, these results indicate that the most potent cytotoxic
autophagy inhibitors contain two aminoquinoline rings, the triamine
linker present in Lys01 and a chlorine substituent at the C-7
position of the aminoquinoline ring.
In Vivo Autophagy Inhibition and Antitumor Efficacy Lys05.
[0117] Lys05, the trihydrochloride salt of Lys01 was synthesized to
enhance aqueous solubility and to enable in vivo studies. Lys01 and
Lys05 produced equivalent dose-dependent increases in the
LC3II/LC3I ratio, and accumulation of the autophagy cargo protein
p62 (26), and identical IC50 values in the MTT assay, (FIG. 5A, B).
To investigate the safety of Lys05 and its in vivo effects on
autophagy, c8161 xenografts matched for tumor size were treated
with intraperitoneal (i.p.) daily PBS, or equimolar doses of HCQ or
Lys05 (HCQ 60 mg/kg (138 nmoles/g), Lys05 76 mg/kg (138 nmoles/g))
for 48 hours. With this high dose, short term treatment no mice
died, but after 2 days of dosing, mice treated with Lys05 76 mg/kg
i.p. were observed to have arched backs and lethargy. After 48
hours of treatment mice were euthanized, and tumors were processed
for electron microscopy (EM). Morphologically, EM demonstrated that
cells with intact nuclear and cytoplasmic membranes contained large
AV in Lys05-treated tumors (FIG. 6A). Quantification of the mean
number of AV/cell in two representative tumor from each treatment
group found a significant >2-fold increase in the mean number of
AV/cell in Lys05 treated tumors compared to control- or HCQ-treated
tumors (FIG. 6B). Significantly higher LC3II/LC3I levels were
observed in Lys05-treated tumors compared to control- or
HCQ-treated tumors providing further evidence of in vivo autophagy
inhibition (FIG. 7A). After 48 hours of treatment, cleaved caspase
3 levels indicative of apoptosis were elevated in Lys05 treated
tumors compared to HCQ- or PBS-treated tumors.
[0118] Except for certain models of pancreatic cancer (27), in many
animal tumor models, where high levels of autophagy are likely
present in untreated tumors(4, 28), treatment with single agent HCQ
does not impair tumor growth(29, 30). To determine if a more potent
autophagy inhibitor such as Lys05 could significantly impair tumor
growth as a single agent 1205Lu xenografts were generated in the
flanks of nude mice. For chronic treatment experiments the 1205Lu
melanoma model was chosen over the c8161 xenograft model because
c8161 xenografts tend to spontaneously ulcerate confounding tumor
measurements and safety analysis. Ten mice bearing 1205Lu
xenografts were matched for tumor volume per cohort were assigned
to either PBS, HCQ 60 mg/kg i.p. or Lys05 76 mg/kg i.p. (equimolar
dosing) dosed for 3 days of daily treatment with 2 days off
treatment (3/5 days) for all 3 treatment groups, to allow for
symptom recovery and to avoid excess toxicity. This schedule was
tolerated well for a 14 day period. Tumor growth curves for each of
the 3 groups indicated tumor growth was significantly impaired in
Lys05 treated tumors compared to controls (FIG. 6C). Lys05
treatment resulted in a 53% reduction in the average daily tumor
growth rate compared to vehicle treated controls (31.2 v. 14.6
mm.sup.3/day; p=0.002; FIG. 6D). A significant accumulation of AV
was observed at the end of 14 days of treatment in both HCQ- and
Lys05-treated tumors, but Lys05 treated tumors had a 6-fold
increase in AV/cell whereas HCQ-treated tumors had a 3-fold
increase in AV/cell compared to control treated tumors (FIG. 7B).
Extensive tumor necrosis was observed in the center of Lys05
treated tumors (FIG. 7B).
[0119] To determine if lower doses of Lys05 could produce antitumor
activity, mice bearing HT-29 colon cancer xenografts were treated
with PBS, or Lys05 at 10 mg/kg i.p. daily, 40 mg/kg i.p. daily, or
80 mg/kg i.p. 3/5 days off. Clinical toxicity was observed only in
the 80 mg/kg cohort, with 2/8 mice euthanized early for bowel
obstruction. Daily dosing for the 10 mg/kg and 40 mg/kg cohorts was
well tolerated. The average daily tumor growth rate was
significantly impaired in a dose-dependent fashion with Lys05
treatment (FIG. 6E). Tumor growth curves demonstrated that all 3
doses of Lys05 produced significant tumor growth impairment
compared to control (FIG. 6F). At the end of the experiment excised
tumor weights demonstrated that significant antitumor activity was
observed with 10 mg/kg daily dosing (FIG. 6G). Immunoblotting
against LC3 in tumor lysates harvested after 14 days of treatment
revealed a significant increase in LC3II/LC3I ratio in all Lys05
treated tumors including the 10 mg/kg dosed tumors compared to
control (FIG. 7C).
Intestinal Toxicity at the Maximal Administered Dose of Lys05
Resembles a Genetic Autophagy Deficiency.
[0120] In the 1205lu xenograft experiment, individual animals
treated with Lys05 76 mg/kg i.p. 3/5 days appeared lethargic with
arched backs (FIG. 8A). Three out often mice treated with Lys05
developed signs of bowel obstruction (FIG. 8B). Inspection of the
bowel found dilated proximal small intestine with a pseudostricture
of the terminal ileum. Histological examination of the ileum
revealed no evidence of excess inflammation, fibrosis, or
mechanical obstruction, indicating that the obstructive signs
observed in the mice were due to pseudo-obstruction or functional
ileus. While intestinal villi and crypt architecture were intact,
dysmorphic Paneth cells (FIG. 8C) were observed. Paneth cell
dysfunction, including reduced size and number of eosinophilic
lysozyme-containing granules, has previously been described as the
pathognomonic sign of autophagy deficiency in mice and a subset of
Crohn's disease patients that have a genetic deficiency in the
essential autophagy gene ATG16L1 (31).
[0121] In the HT29 dose-finding xenograft experiment there was no
significant weight loss observed in any dose cohort (FIG. 9A).
Resection of the entire gastrointestinal tract from mice bearing
HT-29 tumors after 14 days of treatment demonstrated bowel
thickening and obstruction was limited to 80 mg/kg dose cohort
(FIG. 9B). Histological examination of the terminal ileum resected
from mice bearing HT-29 xenografts treated with PBS ip daily, or
Lys05 10 mg/kg ip daily, 40 mg/kg ip daily, and 80 mg/kg ip daily
every 3/5 days for 14 days demonstrated dose dependent effects on
Paneth cell morphology (FIG. 9C). While the number of Paneth
cells/crypt did not change with treatment (FIG. 9D), the size and
number of granules decreased in a dose dependent manner. Scoring on
a Paneth cell dysfunction scale (FIG. 10) indicated that Paneth
cell dysfunction was observed at all doses tested of Lys05, despite
signs and symptoms of toxicity being restricted to the 80 mg/kg
dose (FIG. 9E). In mice treated with Lys05 40 mg/kg or 80 mg/kg,
but not 10 mg/kg, lysozyme was significantly reduced or absent in
Paneth cells (FIG. 9F). Taken together these findings indicate that
Lys05-associated Paneth cell dysfunction mimics ATG16L1 deficiency,
and lower doses of Lys05 produce significant antitumor activity
without dose-limiting toxicity.
Lys05 Inhibits Autophagy by Deacidifying the Lysosome.
[0122] To compare the relative lysosomal accumulation of Lys05
compared to HCQ, lysosomes were subfractionated from 1205Lu cells
treated with PBS, HCQ 10 .mu.M, or Lys05 10 PM, and 1205Lu tumors
harvested after 14 days of treatment with PBS, HCQ 60 mg/kg i.p.,
or Lys05 76 mg./kg i.p. every 3/5 days. Immunoblotting against the
lysosomal marker LAMP2 confirmed adequate separation of the
lysosomal and whole cell population in both cells and tumor samples
(FIG. 12A). HPLC tandem mass spectrometry (MS/MS) measurements
(FIG. 11) determined that the concentrations of Lys05 and HCQ in
the whole cell homogenate treated with Lys05 10 .mu.M or HCQ 10
.mu.M for 24 hours were 57 .mu.M and 8 .mu.M respectively,
indicating an 6-fold higher concentration of Lys05 within the cell
compared to HCQ. The concentration of Lys05 and HCQ in the
lysosomal fraction of cells treated with Lys05 10 .mu.M or HCQ 10
.mu.M were 105 .mu.M and 13 .mu.M respectively, indicating an
8-fold higher concentration of Lys05 in the lysosome compared to
HCQ. This difference in cellular and lysosomal accumulation of
Lys05 and HCQ was more marked in tumor tissue. There was an 11-fold
higher concentration and a 34-fold higher concentration of Lys05
compared to HCQ in whole cell homogenates and lysosomes,
respectively, within tumors (FIG. 12B).
[0123] Having established that Lys05 more effectively accumulates
in the lysosome than HCQ, the functional effects of this
accumulation were investigated. 1205Lu cells were treated with
vehicle, Lys05 and HCQ, and stained the Lysotracker Red (FIG.
12C).
[0124] Within 30 minutes of treatment, fewer Lysotracker-positive
puncta were observed in Lys05 treated cells at both 10 .mu.M and
100 .mu.M concentrations. In contrast, a significant decrease in
Lysotracker positive puncta was observed in cells treated with HCQ
100 .mu.M, but not observed in cells treated with HCQ 10 .mu.M. To
understand the implications of this more potent and complete
lysosomal inhibition, 1205Lu cells were treated with vehicle,
Lys05, or HCQ and stained with acridine orange (AO; a dye which
aggregates in all endovesicular acidic compartments) at 24 hours
(FIG. 12D). HCQ produced a dose-dependent accumulation of acidic
vesicles. In contrast, Lys05 caused an accumulation of acidic
vesicles at lower doses (10 .mu.M), but at higher doses (50 .mu.M),
no acidic vesicles were observed, indicating the complete
deacidification of the endovesicular system.
[0125] Finally, the functional consequences of lysosomal
deacidification were investigated by measuring enzymatic activity
of acid phosphatase. In 1205Lu cells treated with PBS, HCQ 10 .mu.M
or Lys05 10 .mu.M, within 24 hours there is a 43% reduction in acid
phosphatase activity in the lysosomal fraction of Lys05 treated
versus PBS treated cells (FIG. 13A). Leakage of certain lysosomal
enzymes such as activated cathepsins, could lead to an
autophagy-independent cell death Within 24 hours of treatment of
1205Lu cells with PBS, HCQ or Lys05, there is decreased
acid-dependent processing of immature cathepsin D to the mature
activated form within the lysosome in Lys05-treated compared to
HCQ- or PBS-treated cells (FIG. 13B), In 1205Lu xenograft tumors,
after 14 days of treatment there was a 1.75-fold increase in
extralysosomal acid phosphatase activity in the Lys05-treated
tumors, suggesting that chronic treatment can lead to
extralysosomal leakage of enzymes (FIG. 13C). but increased
acid-dependent processing of cathepsin D within the whole cell
homogenate was not observed in Lys05 treated tumors (FIG. 13D).
These results indicate that high doses of Lys05 cause lysosomal
dysfunction by deacidifying the lysosome, leading to impairment of
lysosomal enzymes, and effective autophagy inhibition, whereas high
doses of HCQ incompletely deacidify the lysosome, leading to
incomplete autophagy inhibition associated with less cell
death.
Additional Compounds Lys06-Lys18
[0126] Additional Lys01 derivatives have been synthesized and
tested (FIG. 14, FIG. 15). In 72 hour MTT assays the IC50's of
compounds Lys01-Lys 13 demonstrate increased or decreased activity
compared to HCQ and CQ (Table 1, FIG. 16). In most cases the Lys01
derivatives are more active than CQ or HCQ. These findings further
refine the starting point for further drug development of Lys01
derivatives.
Activity of Lys01 Derivatives in Malaria.
[0127] Table 2, FIG. 17 shows the IC50 values for Lys01
derivative-induced cell death in the human cancer cell LN229 and a
number of strains of P. Falciparum grown in in vitro in human RBC.
There is a similar activity profile for anticancer activity of
Lys01 derivatives and malaria cytotoxicity. Lys01 was more active
than artesunate in some CQ-resistant cell lines.
CONCLUSIONS
[0128] Potential Commercial Uses and Applications: Lys01 and Lys05
are lead compounds with great potential to be optimized further for
potency as a novel autophagy inhibitor. Autophagy inhibition is a
new therapeutic strategy in cancer that is applicable to every
cancer. There are currently over 30 HCQ trials in cancer patients
involving nearly every tumor type. Due to its low potency and poor
pharmacology, in humans HCQ will likely not yield the promising
augmentation of anticancer therapy observed in laboratory models.
An optimized derivative of Lys01 could be developed as a second
generation autophagy inhibitor. The GI toxicity associated with
Paneth cell dysfunction observed at LD30 doses of Lys05, support
the mechanism of action of the drug, and also suggests that colon
cancers, which often share features with Paneth cells, may be a
tumor type that may be especially sensitive to Lys05 and its
optimized derivatives. Additional cancers worth investigating
include melanoma, and non small cell lung cancer, since melanoma
cell lines demonstrated the highest difference in sensitivity to
Lys01 compared to HCQ, and an EGFR mutated lung cancer cell line
demonstrated sensitivity to both HCQ and Lys05. The synthesis of
Lys01 was designed such that there is no overlap with other
patented and/or published aminoquinoline compounds. Further
mechanistic studies are planned that are to identify
pharmacodynamics assays that guide drug development.
Pharmacokinetic studies planned in mice establish initial in vivo
profile.
[0129] Other similar technologies and competing products: Novel
chloroquine derivatives for use as anticancer agents is an active
area of investigation (16). Autophagy has been identified as one of
the top ten areas of research in which the NIH will invest in the
next few years. No studies to date have leveraged the potential of
bivalency as the inventors provide here. In addition, most studies
lack the in vivo studies and the mechanistic studies herein
reported that can guide further development of optimize lead
compounds for drug development.
[0130] Advantages over other similar technologies and products:
Thus, the present application has shows that the disclosed series
of bisaminoquinolines are potent autophagy inhibitors that have
single agent antitumor activity in an in vivo tumor model.
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