U.S. patent application number 13/173480 was filed with the patent office on 2012-02-09 for chimeric small molecules for the recruitment of antibodies to cancer cells.
Invention is credited to Ryan Murelli, David Spiegel, Andrew Zhang.
Application Number | 20120034295 13/173480 |
Document ID | / |
Family ID | 45556331 |
Filed Date | 2012-02-09 |
United States Patent
Application |
20120034295 |
Kind Code |
A1 |
Spiegel; David ; et
al. |
February 9, 2012 |
CHIMERIC SMALL MOLECULES FOR THE RECRUITMENT OF ANTIBODIES TO
CANCER CELLS
Abstract
The present invention relates to chimeric chemical compounds
which are used to recruit antibodies to cancer cells, in
particular, prostate cancer cells or metastasized prostate cancer
cells. The compounds according to the present invention comprise an
antibody binding terminus (ABT) moiety covalently bonded to a cell
binding terminus (CBT) through a linker and optionally, a connector
molecule.
Inventors: |
Spiegel; David; (New Haven,
CT) ; Murelli; Ryan; (Torrington, CT) ; Zhang;
Andrew; (New Haven, CT) |
Family ID: |
45556331 |
Appl. No.: |
13/173480 |
Filed: |
June 30, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12991926 |
Apr 12, 2011 |
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PCT/US2009/002957 |
May 13, 2009 |
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13173480 |
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61127539 |
May 13, 2008 |
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61360732 |
Jul 1, 2010 |
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Current U.S.
Class: |
424/450 |
Current CPC
Class: |
A61K 47/549 20170801;
C07D 249/04 20130101; A61K 45/06 20130101; A61K 47/6803 20170801;
A61K 47/646 20170801; A61K 47/6891 20170801; C07K 16/44 20130101;
A61K 47/6869 20170801; C07K 16/3069 20130101; A61K 31/4192
20130101; A61K 9/0019 20130101; C07K 2317/31 20130101; A61K 47/55
20170801; A61K 47/6873 20170801; A61K 47/54 20170801; A61K 47/68
20170801 |
Class at
Publication: |
424/450 |
International
Class: |
A61K 9/127 20060101
A61K009/127 |
Goverment Interests
GRANT SUPPORT
[0002] This invention was supported by a grant from the National
Institutes of Health, grant no. 1DP2OD002913-01. Consequently, the
government retains certain rights in the invention.
Claims
1. A compound according to the chemical structure: ##STR00092##
Wherein A is an antibody binding moiety comprising a hapten which
is capable of binding to an antibody in a patient; B is a cell
binding moiety capable of binding to prostate specific membrane
antigen on the cell surface of cells in said patient; L is a linker
molecule which links [CON] to A or B in a molecule; [CON] is a bond
or a connector molecule linking said linker molecule to A or B; and
Each n in a molecule is independently an integer from 1 to 15, Or a
pharmaceutically acceptable salt, solvate or polymorph thereof.
2. The compound according to claim 1 wherein A is an antibody
binding moiety according to the chemical formula: ##STR00093##
Where Y' is H or NO.sub.2; X is O, CH.sub.2, NR.sup.1, S(O),
S(O).sub.2, --S(O).sub.2O, --OS(O).sub.2, or OS(O).sub.2O; R.sup.1
is H, a C.sub.1-C.sub.3 alkyl group, or a --C(O)(C.sub.1-C.sub.3)
group; X' is CH.sub.2, O, N--R.sup.1, or S; R.sup.1'' is H or
C.sub.1-C.sub.3 alkyl; Z is a bond, a monosaccharide, disaccharide,
oligosaccharide, glycoprotein or glycolipid; X.sup.b is a bond, O,
CH.sub.2, NR.sup.1 or S; X'' is O, CH.sub.2, NR.sup.1; R.sup.1 is
H, a C.sub.1-C.sub.3 alkyl group or a --C(O)(C.sub.1-C.sub.3)
group; B is a cell binding moiety according to the chemical
formula: ##STR00094## Where X.sub.1 and X.sub.2 are each
independently CH.sub.2, O, NH or S; X.sub.3 is O, CH.sub.2,
NR.sup.1, S(O), S(O).sub.2, --S(O).sub.2O, --OS(O).sub.2, or
OS(O).sub.2O; R.sup.1 is H, a C.sub.1-C.sub.3 alkyl group, or a
--C(O)(C.sub.1-C.sub.3) group; k is an integer from 0 to 20, 8 to
12, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1, 2, 3, 4, 5 or 6; L is a
linker according to the chemical formula: ##STR00095## Or a
polypropylene glycol or polypropylene-co-polyethylene glycol linker
having between 1 and 100 glycol units; Where R.sub.a is H,
C.sub.1-C.sub.3 alkyl or alkanol or forms a cyclic ring with
R.sup.3 (proline) and R.sup.3 is a side chain derived from an amino
acid; and m is an integer from 1 to 100, 1 to 75, 1 to 60, 1 to 55,
1 to 50, 1 to 45, 1 to 40, 2 to 35, 3 to 30, 1 to 15, 1 to 10, 1 to
8, 1 to 6, 1, 2, 3, 4 or 5; or L is a linker according to the
chemical formula: ##STR00096## Where Z and Z' are each
independently a bond, --(CH.sub.2).sub.i--O, --(CH.sub.2).sub.i--S,
--(CH.sub.2).sub.i--N--R, ##STR00097## wherein said
--(CH.sub.2).sub.i group, if present in Z or Z', is bonded to [CON]
if present, ABT or CBT; Each R is independently H, or a
C.sub.1-C.sub.3 alkyl or alkanol group; Each R.sup.2 is
independently H or a C.sub.1-C.sub.3 alkyl group; Each Y is
independently a bond, O, S or N--R; Each i is independently 0 to
100; D is ##STR00098## or a bond, with the proviso that Z, Z' and D
are not each simultaneously bonds; j is 1 to 100; m' is 1 to 100;
n' is 1 to 100; and X.sup.1 is O, S or N--R, R is as defined above;
and The connector moiety [CON] is a bond or a moiety according to
the chemical structure: ##STR00099## Where X.sup.2 is O, S,
NR.sup.4, S(O), S(O).sub.2, --S(O).sub.2O, --OS(O).sub.2, or
OS(O).sub.2O; X.sup.3 is NR.sup.4, O or S; and R.sup.4 is H, a
C.sub.1-C.sub.3 alkyl or alkanol group, or a
--C(O)(C.sub.1-C.sub.3) group; or a pharmaceutically acceptable
salt, solvate or polymorph thereof.
3. (canceled)
4. (canceled)
5. (canceled)
6. (canceled)
7. (canceled)
8. (canceled)
9. (canceled)
10. (canceled)
11. (canceled)
12. (canceled)
13. (canceled)
14. (canceled)
15. (canceled)
16. (canceled)
17. (canceled)
18. A compound according to claim 1 according to the chemical
structure: ##STR00100## Where n is 0 to 12; and X is ##STR00101##
Where Y.sup.N, Y.sup.N1 and Y' is H or NO.sub.2; with the proviso
that at least one of Y.sup.N, Y.sup.N1 and Y' is NO.sub.2, or a
pharmaceutically acceptable salt, enantiomer, diastereomer, solvate
or polymorph thereof.
19. The compound according to claim 18 wherein X is ##STR00102##
and Y' is H or NO.sub.2.
20. The compound according to claim 19 wherein Y' is H.
21. The compound according to claim 18 wherein n is 0-6.
22. The compound according to claim 18 wherein n is 1-4.
23. A pharmaceutical composition comprising an effective amount of
a chimeric compound according to claim 18 in combination with a
pharmaceutically acceptable carrier, additive or excipient.
24. The composition according to claim 23 wherein said composition
further comprises an effective amount of an additional anticancer
agent.
25. The composition according to claim 24 wherein said additional
anticancer agent is an antimetabolite, an inhibitor of
topoisomerase I and II, an alkylating agent, a microtubule
inhibitor or mixtures thereof.
26. The composition according to claim 24 wherein said agent is
aldesleukin; aemtuzumab; alitretinoin; allopurinol; altretamine;
amifostine; anastrozole; arsenic trioxide; aparaginase; BCG Live;
bexarotene capsules; bexarotene gel; bleomycin; busulfan
intravenous; busulfan oral; calusterone; capecitabine; carboplatin;
carmustine; carmustine with poifeprosan 20 iplant; celecoxib;
chlorambucil; cisplatin; cladribine; cyclophosphamide; cytarabine;
cytarabine liposomal; dacarbazine; dactinomycin; actinomycin D;
dabepoetin alfa; daunorubicin liposomal; daunorubicin, daunomycin;
dnileukin diftitox, dexrazoxane; docetaxel; doxorubicin;
doxorubicin liposomal; domostanolone propionate; eliott's B
soution; epirubicin; eoetin alfa estramustine; etoposide phosphate;
etoposide (VP-16); exemestane; filgrastim; floxuridine
(intraarterial); fludarabine; fluorouracil (5-FU); fulvestrant;
gemtuzumab ozogamicin; goserelin acetate; hydroxyurea; Ibritumomab
Tiuxetan; idarubicin; ifosfamide; imatinib mesylate; Interferon
alfa-2a; Interferon alfa-2b; irinotecan; letrozole; leucovorin;
levamisole; lomustine (CCNU); meclorethamine (nitrogen mustard);
megestrol acetate; melphalan (L-PAM); mercaptopurine (6-MP); mesna;
methotrexate; methoxsalen; mitomycin C; mitotane; mitoxantrone;
nandrolone phenpropionate; nfetumomab; LOddC; orelvekin;
oxaliplatin; paclitaxel; pamidronate; pegademase; Pegaspargase;
Pegfilgrastim; pentostatin; pipobroman; plicamycin; mithramycin;
porfimer sodium; procarbazine; quinacrine; Rasburicase; Rituximab;
Sargramostim; streptozocin; talbuvidine (LDT); talc; tamoxifen;
temozolomide; teniposide (VM-26); testolactone; thioguanine (6-TG);
thiotepa; topotecan; toremifene; Tositumomab; Trastuzumab;
tretinoin (ATRA); Uracil Mustard; valrubicin; valtorcitabine
(monoval LDC); vinblastine; vinorelbine; zoledronate; and mixtures
thereof.
27. The composition according to claim 22 further comprising at
least one antiandrogen compound.
28. The composition according to claim 23 further comprising at
least one GNRh modulator.
29. The composition according to claim 23 further comprising at
least one agent selected from the group consisting of flutamide,
bicalutamide, nilutamide, cyproterone acetate, ketoconazole,
aminoglutethimide, abarelix, leuprolide, goserelin, triptorelin,
buserelin, abiraterone acetate, sorafenib and mixtures thereof.
30. The composition according to claim 23 further comprising at
least one agent selected from the group consisting of an enlarged
prostate agent, eulexin, flutamide, goserelin, leuprolide, lupron,
nilandron, nilutamide, zoladex and mixtures thereof.
31. The composition according to claim 23 in oral dosage form.
32. The composition according to claim 23 in parenteral dosage
form.
33. The composition according to claim 32 wherein said parenteral
dosage form is an intravenous dosage form.
34. The composition according to claim 23 in topical dosage
form.
35. A method of treating prostate cancer in a patient in need
thereof comprising administering to said patient an effective
amount of a compound according to claim 18.
36. The method according to claim 35 wherein said prostate cancer
is metastatic prostate cancer.
37. A method of treating prostate cancer in a patient in need
thereof comprising administering to said patient an effective
amount of a composition according to claim 23.
38. A method of inhibiting metastasis of prostate cancer in a
patient in need thereof comprising administering to said patient an
effective amount of a compound according to claim 18 to said
patient.
39. A method of treating cancer in a patient in need thereof
comprising administering to said patient a composition according to
claim 23.
40. The method according to claim 39 wherein said cancer is
stomach, colon, rectal, liver, pancreatic, lung, breast, cervix
uteri, corpus uteri, ovary, testis, bladder, renal, brain/CNS, head
and neck, throat, Hodgkin's disease, non-Hodgkin's lymphoma,
multiple myeloma, leukemia, melanoma, non-melanoma skin cancer,
acute lymphocytic leukemia, acute myelogenous leukemia, Ewing's
sarcoma, small cell lung cancer, choriocarcinoma, rhabdomyosarcoma,
Wilms' tumor, neuroblastoma, hairy cell leukemia, mouth/pharynx,
oesophagus, larynx, kidney cancer or lymphoma.
41. A method of treating prostate cancer in a patient wherein said
patient also has another form of cancer said method comprising
administering to said patient an effective amount of a composition
according to claim 23.
42. The method according to claim 41 wherein said other form of
cancer is stomach, colon, rectal, liver, pancreatic, lung, breast,
cervix uteri, corpus uteri, ovary, testis, bladder, renal,
brain/CNS, head and neck, throat, Hodgkin's disease, non-Hodgkin's
lymphoma, multiple myeloma, leukemia, melanoma, non-melanoma skin
cancer, acute lymphocytic leukemia, acute myelogenous leukemia,
Ewing's sarcoma, small cell lung cancer, choriocarcinoma,
rhabdomyosarcoma, Wilms' tumor, neuroblastoma, hairy cell leukemia,
mouth/pharynx, oesophagus, larynx, kidney cancer or lymphoma.
Description
RELATED APPLICATIONS
[0001] This application is a continuation in part application of
U.S. patent application Ser. No. 12/991,926 of identical title
filed Nov. 10, 2010, which is a United States national phase
application of PCT/US2009/002957 (published as WO2009/139863, filed
13 May 2009, which claims priority from U.S. provisional
application U.S. 61/127,539 of identical title filed May 13, 2008.
This application also claims the benefit of priority of U.S.
provisional application no. U.S. 61/360,732, filed Jul. 1, 2010 of
identical title. Each of the foregoing applications is incorporated
by reference in its entirety hereof.
FIELD OF THE INVENTION
[0003] The present invention relates to chemical compounds which
are used to recruit antibodies to cancer cells, in particular,
prostate cancer cells or metastasized prostate cancer cells. The
compounds according to the present invention comprise an antibody
binding terminus (ABT) moiety covalently bonded to a cell binding
terminus (CBT) through a linker and optionally and preferably, a
connector molecule. In addition, given that the protein target is
found on the neovasculature of most non-prostatic cancer cells, the
compounds in the present invention also serves as an antiangiogenic
therapy for other cancer types.
BACKGROUND OF THE INVENTION
[0004] It has been predicted that one out of every six American men
will develop prostate cancer in their lifetime. See, American
Cancer Society, Cancer Facts and Figures 2008. Atlanta: American
Cancer Society; 2008. Despite recent advances in both prostate
cancer detection and treatment, it remains one of the leading
causes of cancer-related death among the American male
population.
[0005] Anti-DNP antibodies are readily present in high
concentrations of the human bloodstream. See Ortega, E.;
Kostovetzky, M.; Larralde, C. Mol. Immun. 1984, 21, 883. A number
of cancer-related, antibody directing small molecules having been
synthesized. See, Lu, et al., Adv. Drug Deliv. Rev. 2004, 56, 1161;
Lu, et al., Mol. Pharmaceut. 2007, 4, 695; Carlson, et al., ACS
Chem. Bio. 2007, 2, 119; and Popkov, M.; Gonzalez, B.; Sinha, S.
C.; Barbas, C. F., III. Proc. Nat. Acad. Sci., 2009, 1.
[0006] The present invention is directed to the design and
synthesis of a new small-molecule capable of redirecting endogenous
anti-dinitrophenyl (DNP) antibodies selectively to prostate cancer
cells, and inducing antibody-directed, cell-mediated
cytotoxicity.
[0007] When prostate cancer is diagnosed prior to metastasis, the
patient has a greater then 99% chance of survival. The most
successful means for treating prostate cancer at this stage is a
radical prostatectomy. Unfortunately, this surgery carries with it
the risk of severing nerves and blood vessels associated with
sexual organs and the bladder, and can potentially result in
impotency or incontinency. Radiation therapy is yet another
commonly used procedure that carries the risk of impotency. Half
the patients who undergo radiation therapy for prostate cancer
become impotent within 2 years of treatment. In addition to the
adverse affects associated with these procedures, they are
significantly less effective in patients whose cancer has already
delocalized or metastasized on diagnosis. In these cases, patients
generally undergo even more invasive procedures such as hormonal
therapy or chemotherapy. Unfortunately, most patients eventually
stop responding to hormonal therapy and the most successful
chemotherapeutic, Taxotere, only prolongs the life of advanced
prostate cancer patients by 2.5 months on average.
[0008] As another alternative therapeutic, monoclonal antibody
(mAb)-based immunotherapy has proven clinically beneficial for
cancer patients while allowing them to maintain a good quality of
life. These antibodies can either regulate proliferation of cancer
cells through the manipulation of signal transduction, or promote
cytotoxicity. Two examples of FDA-approved mAb-based anticancer
drugs are Herceptin and Rituxan (Rituximab), which are currently
being used for the treatment of breast cancer and non-Hodgkin's
lymphoma, respectively. While there are no mAb-based therapeutics
currently available for prostate cancer patients, advanced clinical
studies on mAb-based immunotherapy has shown promise for the
treatment of prostate cancer including advanced prostate cancer.
Despite the major advantages of mAb-based immunotherapy, there are
significant pitfalls which may limit its potential. In general,
mAb-based therapeutics are highly costly ($70,000 for full course
of treatment of Herceptin), lack oral bioavailability, and can lead
to severe and often fatal side-effects. For example, Herceptin is
associated with heart problems and cannot be administered to
approximately 10% of cancer patients because of heart-related
complications. Rituxan can cause several side-effects which include
renal failure, infections and immune and pulmonary toxicity.
[0009] Although still in its infancy, the concept of using small
molecules to template the human immune response has shown realistic
potential. Recent reports have surfaced in which small molecules
have been used to direct antibodies to cancerous cells such as
breast carcinoma cells, melanoma cells, and nasopharyngeal
epidermal carcinoma cells. Animal studies have demonstrated that
these molecules can promote tumor rejection and antitumor immunity
in mice. Because this process allows for the direction of
endogenous antibodies selectively to the cell of interest, it has
the potential to harness the power of mAb-based therapeutics while
limiting the costs and side effects associated with administering
exogenous antibodies. By developing similar methods which recruit
anti-DNP antibodies to prostate cancer cells, the proposed research
will help broaden this field while creating a new therapy for all
forms of prostate cancer.
BRIEF DESCRIPTION OF THE FIGURES
[0010] FIG. 1 shows scheme 1, which is a schematic depiction of
small-molecule templated immunotherapy.
[0011] FIG. 2(A) shows computational modeling of cell-binding
terminus in the binding pocket of PSMA. FIG. 2(B) shows a model of
PSMA-small molecule-Anti-DNP antibody ternary complex.
[0012] FIG. 3 shows a prostate cancer antibody-recruiting molecule
of the present invention, PC-ARM (3).
[0013] FIG. 4 shows the synthesis of the prostate cancer
antibody-recruiting molecule of FIG. 3. The azide-functionalized
cell-binding terminus was synthesized in 3 steps by coupling
Cbz-protected lysine and t-butyl protected glutamic acid with
triphosgene, followed by Cbz deprotection and azide formation
(scheme 2). Heterobifunctional PEG 10 was synthesized in a five
step process from octaethylene glycol (scheme 2). These
intermediates were coupled via microwave assisted, copper-catalyzed
Huisgen cyloaddition, and deprotected using microwave assisted TFA
deprotection (scheme 3) afforded the prostate cancer
antibody-recruiting molecule of the present invention PC-ARM
(3).
[0014] FIG. 5 shows in (A) a representative flow cytometry
histogram illustrating small-molecule 3-dependant anti-DNP antibody
binding to PSMA-expressing LNCaP cells. LNCaP cells were
preincubated with 3 (50 nM), and subsequently incubated with Alexa
Fluor 488-conjugated anti-DNP antibodies. (B) Representative flow
cytometry histogram illustrating no small-molecule 3 dependant
anti-DNP antibody binding to PSMA-negative DU-145 cells. DU-145
cells were preincubated with 3 (50 nM), and subsequently incubated
with Alexa Fluor 488-conjugated anti-DNP antibodies.
[0015] FIG. 6 shows the change in % cell killing induced by PC-ARM
(3) (shown in the figure as P-ARM8). In short, LNCaP cells and
PBMCs were incubated in the presence of anti-DNP antibody IgG1,
IgG3 and a sample of normal human IgG known to have anti-DNP
antibodies for 24 hours at 37.degree. C. The change in %
cell-killing reflects the increase associated with addition of 50
nM P-ARM8 (3). The data was run in dodecaplets, and is reported as
the average .+-.SEM. In addition, each experiment was run in
parallel with 50 nM P-ARM8 alone to screen for inherent
small-molecule induced cell-killing.
[0016] FIG. 7 shows exemplary compounds according to the present
invention.
[0017] FIG. 8 shows additional exemplary compounds according to the
present invention.
[0018] FIG. 9 shows divergent synthetic scheme 1a which is used for
target synthesis.
[0019] FIG. 10 shows Scheme 2a--synthesis toward propargyl
intermediates used in synthesizing compounds according to the
invention.
[0020] FIG. 11 shows Scheme 3a which is directed to the synthesis
of intermediate 15, and proposed access to azide intermediate 16 to
be used in click chemistry.
[0021] FIG. 12 shows scheme 4a which describes a synthetic approach
to bis di-DNP lysine 21.
[0022] FIG. 13 shows scheme 5a which describes a synthetic approach
to the tris-azidylated analog which can be used to condense with
the bis-diDNP lysine 21 to produce compound 3 of FIG. 8 or similar
compounds.
[0023] FIG. 14 shows the structure and function of ARM-Ps
(antibody-recruiting molecules targeting prostate cancer). (A)
ARM-Ps recruit anti-DNP antibodies to PSMA-expressing prostate
cancer cells, and thereby bring about immune-mediated cytotoxicity.
(B) ARM-Ps are bifunctional and consist of an antibody-binding
terminus (ABT), a linker region, and a cell-binding terminus
(CBT).
[0024] FIG. 15 shows the correlation between K.sub.i values and
EHOMO of aromatic ring component of ARM-P analogues. Measured Ki
values (mean of triplicate experiments .+-.standard deviation) are
plotted versus HOMO energies calculated using density functional
theory (DFT). The hybrid functional B3LYP with a 6-31G*+basis set
was used.
[0025] FIG. 16 provides a close-up of PSMA active site bound to
bifunctional glutamate urea inhibitors ARM-P2 (gold), ARM-P4
(grey), and ARM-P8 (blue). Structures were superimposed on with
corresponding (or equivalent) C.alpha. atoms. Inhibitors are shown
in stick representation and protein residues are shown as lines.
Hydrogen bonding interactions are indicated by dashed lines. The
zinc ions and chloride ion in the active site are labeled as grey
and green dotted spheres, respectively, and water molecules are
depicted as red spheres. In both protein and inhibitor structures,
carbon atoms are colored as indicated above, and other atoms are
colored red (oxygen), and blue (nitrogen).
[0026] FIG. 17 shows that the PSMA/ARM-P2 complex reveals a
previously unreported arene-binding cleft. (A) Global view of PSMA
with a close-up of arene-binding site. Residues making up the
arene-binding cleft are labeled in cyan. The entrance lid (residues
542-548), which resides in an open conformation in the ARM-P2
complex, is indicated as a red loop. Overlaid on this complex is
the entrance lid in its closed conformation (colored blue), which
would come into steric conflict with the linker region of the
inhibitor. (B and C) Close-up images of the urea binding sites in
structures containing both open and closed entrance loops. In all
panels, structural data for PSMA with a the closed entrance lid
comes from the complex with the small urea-based inhibitor DCIBzL
(PDB ID-3IWW).30 The zinc ions in the active site are labeled as
orange spheres and the ARM-P2 carbons are colored gold. The DCIBzL
carbons in B and C are colored purple.
[0027] FIG. 18 provides a close-up view of the active site of PSMA
bound to MeO-P4. Hydrogen bonding interactions are indicated by
dashed lines. The zinc ions in the active site and adjacent
chloride ion are labeled as grey and green dotted spheres,
respectively, and water molecules are depicted as red/darker
spheres. In both protein and inhibitor structures, carbon atoms are
colored in olive, and other atoms are colored red (oxygen), and
blue (nitrogen).
[0028] FIG. 19 provides selected snapshots from the MD simulations
of PSMA/ARM-P complexes. The ligands are represented in varying
shaded sticks, Arg463, Arg511 and Trp541 are represented in lighter
gray sticks. Figure created with the program VMD..sup.37
OBJECTS OF THE INVENTION
[0029] It is an object of the invention to provide chimeric
compounds which can be used to treat virtually any cancer,
especially including prostate cancer and metastatic prostate
cancer.
[0030] It is an additional object of the invention to provide
chimeric compounds which can be used to provide pharmaceutical
compositions, including pharmaceutical compositions which include
additional bioactive agents or agents which assist in the treatment
of cancer, especially prostate cancer, including metastatic
prostate cancer.
[0031] It is still another object of the invention to provide
methods for treating cancer, including prostate cancer, including
metastatic prostate cancer.
[0032] Yet a further object of the invention is to provide methods
for inhibiting metastatis of cancer, especially including
metastatic prostate cancer.
[0033] These and/or other objects of the invention may be readily
gleaned from a review of the invention as described herein.
BRIEF DESCRIPTION OF THE INVENTION
[0034] It is an aspect of the invention to provide chimeric
antibody recruiting molecules which bind to prostate specific
membrane antigen (PMSA) and attract antibodies such that the
chimeric molecules will assist in immunotherapy of a patient with
virtually any cancer, especially including prostate cancer, and
further including metastatic prostate cancer.
[0035] In this first aspect of the invention, chimeric antibody
recruiting molecules are represented by the formula:
##STR00001##
Wherein A is an antibody binding moiety comprising a hapten which
is capable of binding to an antibody in a patient; B is a cell
binding moiety capable of binding to prostate specific membrane
antigen on the cell surface of cells in said patient; L is a linker
molecule which links [CON] to A or B in a molecule; [CON] is a bond
or a connector molecule linking said linker molecule to A or B; and
Each n in a molecule is independently an integer from 1 to 15, 1 to
10, 1 to 5, 1 to 3, 2 to 3, 2 to 5, Or a pharmaceutically
acceptable salt, solvate or polymorph thereof.
[0036] In an additional aspect of the invention, a pharmaceutical
composition comprises an effective amount of a chimeric compound as
described above, optionally and preferably in combination with a
pharmaceutically acceptable carrier, additive or excipient. In
alternative aspects, pharmaceutical combination compositions
comprise an effective amount of a chimeric compound as described
herein, in combination with at least one additional agent which is
used to treat cancer, including prostate cancer, especially
including metastatic prostate cancer or a secondary condition or
effect of cancer, especially prostate cancer (e.g., bone pain,
hyperplasia, osteoporosis, etc. as otherwise described herein).
[0037] In a further aspect of the invention, compounds according to
the present invention are used to treat cancer in a patient,
especially prostate cancer in male patients in need thereof. The
method of treating cancer comprises administering to a patient in
need an effective amount of a chimeric compound as otherwise
described herein in combination with a pharmaceutically acceptable
carrier, additive or excipient, optionally in further combination
with at least one additional agent which is effective in treating
cancer, especially including prostate cancer, metastatic cancer or
one or more of its secondary conditions or effects.
[0038] The present invention also relates to a method for
inhibiting prostate cancer to reduce or inhibit the spread or
metastasis of the cancer into other tissues of the patients' body,
especially including bones, the lymph (lymph nodes) system,
bladder, vas deferens, kidneys, liver, lungs and brain, among
others.
[0039] The present invention also relates to instances in which
destruction of non-cancerous cells which possess PSMA can be of
therapeutic use, especially in cancer therapy. For example, given
that PSMA is found on the neovasculare of many non-prostatic cancer
cells, but not on normal vasculature, the invention could be used
for antiangiogenic therapy for other forms of cancer by targeting
the neovasculature of those cancers.
DETAILED DESCRIPTION OF THE INVENTION
[0040] The following terms are used to describe the present
invention. In instances where a term is not specifically defined
herein, that term is given an art-recognized meaning by those of
ordinary skill applying that term in context to its use in
describing the present invention.
[0041] The term "compound", as used herein, unless otherwise
indicated, refers to any specific chemical compound disclosed
herein and includes tautomers, regioisomers, geometric isomers, and
where applicable, optical isomers (enantiomers) thereof, as well as
pharmaceutically acceptable salts and derivatives (including
prodrug forms) thereof. Within its use in context, the term
compound generally refers to a single compound, but also may
include other compounds such as stereoisomers, regioisomers and/or
optical isomers (including racemic mixtures) as well as specific
enantiomers or enantiomerically enriched mixtures of disclosed
compounds. The term also refers, in context to prodrug forms of
compounds which have been modified to facilitate the administration
and delivery of compounds to a site of activity. It is noted that
in describing the present compounds, numerous substituents, linkers
and connector molecules and variables associated with same, among
others, are described. It is understood by those of ordinary skill
that molecules which are described herein are stable compounds as
generally described hereunder.
[0042] The term "patient" or "subject" is used throughout the
specification within context to describe an animal, generally a
mammal and preferably a human, to whom treatment, including
prophylactic treatment (prophylaxis), with the compositions
according to the present invention is provided. For treatment of
those infections, conditions or disease states which are specific
for a specific animal such as a human patient or a patient of a
particular gender, such as a human male patient, the term patient
refers to that specific animal. Compounds according to the present
invention are useful for the treatment of cancer, especially
including prostate cancer and in particular, metastatic prostate
cancer.
[0043] The term "effective" is used herein, unless otherwise
indicated, to describe an amount of a compound or composition
which, in context, is used to produce or effect an intended result,
whether that result relates to the inhibition of the effects of a
toxicant on a subject or the treatment of a subject for secondary
conditions, disease states or manifestations of exposure to
toxicants as otherwise described herein. This term subsumes all
other effective amount or effective concentration terms (including
the term "therapeutically effective") which are otherwise described
in the present application.
[0044] The terms "treat", "treating", and "treatment", etc., as
used herein, refer to any action providing a benefit to a patient
at risk for prostate cancer or metastasis of prostate cancer,
including improvement in the condition through lessening or
suppression of at least one symptom, inhibition of cancer growth,
reduction in cancer cells or tissue, prevention or delay in
progression of metastasis of the cancer, prevention or delay in the
onset of disease states or conditions which occur secondary to
cancer or remission or cure of the cancer, among others. Treatment,
as used herein, encompasses both prophylactic and therapeutic
treatment. The term "prophylactic" when used, means to reduce the
likelihood of an occurrence or the severity of an occurrence within
the context of the treatment of cancer, including cancer metastasis
as otherwise described hereinabove.
[0045] The term "neoplasia" or "cancer" is used throughout the
specification to refer to the pathological process that results in
the formation and growth of a cancerous or malignant neoplasm,
i.e., abnormal tissue that grows by cellular proliferation, often
more rapidly than normal and continues to grow after the stimuli
that initiated the new growth cease. Malignant neoplasms show
partial or complete lack of structural organization and functional
coordination with the normal tissue and most invade surrounding
tissues, metastasize to several sites, and are likely to recur
after attempted removal and to cause the death of the patient
unless adequately treated. As used herein, the term neoplasia is
used to describe all cancerous disease states and embraces or
encompasses the pathological process associated with malignant
hematogenous, ascitic and solid tumors. Representative cancers
include, for example, prostate cancer, metastatic prostate cancer,
stomach, colon, rectal, liver, pancreatic, lung, breast, cervix
uteri, corpus uteri, ovary, testis, bladder, renal, brain/CNS, head
and neck, throat, Hodgkin's disease, non-Hodgkin's lymphoma,
multiple myeloma, leukemia, melanoma, non-melanoma skin cancer,
acute lymphocytic leukemia, acute myelogenous leukemia, Ewing's
sarcoma, small cell lung cancer, choriocarcinoma, rhabdomyosarcoma,
Wilms' tumor, neuroblastoma, hairy cell leukemia, mouth/pharynx,
oesophagus, larynx, kidney cancer and lymphoma, among others, which
may be treated by one or more compounds according to the present
invention. Because of the activity of the present compounds as
anti-angiogenic compounds, the present invention has general
applicability treating virtually any cancer in any tissue, thus the
compounds, compositions and methods of the present invention are
generally applicable to the treatment of cancer. Given that the
protein target is found on the neovasculature of most non-prostatic
cancer cells, the compounds in the present invention may also serve
as an antiangiogenic therapy for other cancer types.
[0046] In certain particular aspects of the present invention, the
cancer which is treated is prostate cancer or metastatic prostate
cancer. Separately, metastatic prostate cancer may be found in
virtually all tissues of a cancer patient in late stages of the
disease, typically metastatic prostate cancer is found in seminal
vesicles, lymph system/nodes (lymphoma), in bones, in bladder
tissue, in kidney tissue, liver tissue and in virtually any tissue,
including brain (brain cancer/tumor). Thus, the present invention
is generally applicable and may be used to treat any cancer in any
tissue, regardless of etiology.
[0047] The term "prostate cancer" is used to describe a disease in
which cancer develops in the prostate, a gland in the male
reproductive system. It occurs when cells of the prostate mutate
and begin to multiply uncontrollably. These cells may metastasize
(metastatic prostate cancer) from the prostate to virtually any
other part of the body, particularly the bones and lymph nodes, but
the kidney, bladder and even the brain, among other tissues.
Prostate cancer may cause pain, difficulty in urinating, problems
during sexual intercourse, erectile dysfunction. Other symptoms can
potentially develop during later stages of the disease.
[0048] Rates of detection of prostate cancers vary widely across
the world, with South and East Asia detecting less frequently than
in Europe, and especially the United States. Prostate cancer
develops most frequently in men over the age of fifty and is one of
the most prevalent types of cancer in men. However, many men who
develop prostate cancer never have symptoms, undergo no therapy,
and eventually die of other causes. This is because cancer of the
prostate is, in most cases, slow-growing, and because most of those
affected are over the age of 60. Hence, they often die of causes
unrelated to the prostate cancer. Many factors, including genetics
and diet, have been implicated in the development of prostate
cancer. The presence of prostate cancer may be indicated by
symptoms, physical examination, prostate specific antigen (PSA), or
biopsy. There is concern about the accuracy of the PSA test and its
usefulness in screening. Suspected prostate cancer is typically
confirmed by taking a biopsy of the prostate and examining it under
a microscope. Further tests, such as CT scans and bone scans, may
be performed to determine whether prostate cancer has spread.
[0049] Treatment options for prostate cancer with intent to cure
are primarily surgery and radiation therapy. Other treatments such
as hormonal therapy, chemotherapy, proton therapy, cryosurgery,
high intensity focused ultrasound (HIFU) also exist depending on
the clinical scenario and desired outcome.
[0050] The age and underlying health of the man, the extent of
metastasis, appearance under the microscope, and response of the
cancer to initial treatment are important in determining the
outcome of the disease. The decision whether or not to treat
localized prostate cancer (a tumor that is contained within the
prostate) with curative intent is a patient trade-off between the
expected beneficial and harmful effects in terms of patient
survival and quality of life.
[0051] An important part of evaluating prostate cancer is
determining the stage, or how far the cancer has spread. Knowing
the stage helps define prognosis and is useful when selecting
therapies. The most common system is the four-stage TNM system
(abbreviated from Tumor/Nodes/Metastases). Its components include
the size of the tumor, the number of involved lymph nodes, and the
presence of any other metastases.
[0052] The most important distinction made by any staging system is
whether or not the cancer is still confined to the prostate or is
metastatic. In the TNM system, clinical T1 and T2 cancers are found
only in the prostate, while T3 and T4 cancers have spread elsewhere
and metastasized into other tissue. Several tests can be used to
look for evidence of spread. These include computed tomography to
evaluate spread within the pelvis, bone scans to look for spread to
the bones, and endorectal coil magnetic resonance imaging to
closely evaluate the prostatic capsule and the seminal vesicles.
Bone scans often reveal osteoblastic appearance due to increased
bone density in the areas of bone metastasis--opposite to what is
found in many other cancers that metastasize. Computed tomography
(CT) and magnetic resonance imaging (MRI) currently do not add any
significant information in the assessment of possible lymph node
metastases in patients with prostate cancer according to a
meta-analysis.
[0053] Prostate cancer is relatively easy to treat if found early.
After a prostate biopsy, a pathologist looks at the samples under a
microscope. If cancer is present, the pathologist reports the grade
of the tumor. The grade tells how much the tumor tissue differs
from normal prostate tissue and suggests how fast the tumor is
likely to grow. The Gleason system is used to grade prostate tumors
from 2 to 10, where a Gleason score of 10 indicates the most
abnormalities. The pathologist assigns a number from 1 to 5 for the
most common pattern observed under the microscope, then does the
same for the second most common pattern. The sum of these two
numbers is the Gleason score. The Whitmore-Jewett stage is another
method sometimes used. Proper grading of the tumor is critical,
since the grade of the tumor is one of the major factors used to
determine the treatment recommendation.
[0054] Early prostate cancer usually causes no symptoms. Often it
is diagnosed during the workup for an elevated PSA noticed during a
routine checkup. Sometimes, however, prostate cancer does cause
symptoms, often similar to those of diseases such as benign
prostatic hypertrophy. These include frequent urination, increased
urination at night, difficulty starting and maintaining a steady
stream of urine, blood in the urine, and painful urination.
Prostate cancer is associated with urinary dysfunction as the
prostate gland surrounds the prostatic urethra. Changes within the
gland therefore directly affect urinary function. Because the vas
deferens deposits seminal fluid into the prostatic urethra, and
secretions from the prostate gland itself are included in semen
content, prostate cancer may also cause problems with sexual
function and performance, such as difficulty achieving erection or
painful ejaculation.
[0055] Advanced prostate cancer can spread to other parts of the
body and this may cause additional symptoms. The most common
symptom is bone pain, often in the vertebrae (bones of the spine),
pelvis or ribs. Spread of cancer into other bones such as the femur
is usually to the proximal part of the bone. Prostate cancer in the
spine can also compress the spinal cord, causing leg weakness and
urinary and fecal incontinence.
[0056] The specific causes of prostate cancer remain unknown. A
man's risk of developing prostate cancer is related to his age,
genetics, race, diet, lifestyle, medications, and other factors.
The primary risk factor is age. Prostate cancer is uncommon in men
less than 45, but becomes more common with advancing age. The
average age at the time of diagnosis is 70. However, many men never
know they have prostate cancer.
[0057] A man's genetic background contributes to his risk of
developing prostate cancer. This is suggested by an increased
incidence of prostate cancer found in certain racial groups, in
identical twins of men with prostate cancer, and in men with
certain genes. Men who have a brother or father with prostate
cancer have twice the usual risk of developing prostate cancer.
Studies of twins in Scandinavia suggest that forty percent of
prostate cancer risk can be explained by inherited factors.
However, no single gene is responsible for prostate cancer; many
different genes have been implicated. Two genes (BRCA1 and BRCA2)
that are important risk factors for ovarian cancer and breast
cancer in women have also been implicated in prostate cancer.
[0058] Dietary amounts of certain foods, vitamins, and minerals can
contribute to prostate cancer risk. Dietary factors that may
increase prostate cancer risk include low intake of vitamin E, the
mineral selenium, green tea and vitamin D. A large study has
implicated dairy, specifically low-fat milk and other dairy
products to which vitamin A palmitate has been added. This form of
synthetic vitamin A has been linked to prostate cancer because it
reacts with zinc and protein to form an unabsorbable complex.
Prostate cancer has also been linked to the inclusion of bovine
somatotropin hormone in certain dairy products.
[0059] There are also some links between prostate cancer and
medications, medical procedures, and medical conditions. Daily use
of anti-inflammatory medicines such as aspirin, ibuprofen, or
naproxen may decrease prostate cancer risk. Use of the
cholesterol-lowering drugs known as the statins may also decrease
prostate cancer risk. Infection or inflammation of the prostate
(prostatitis) may increase the chance for prostate cancer, and
infection with the sexually transmitted infections chlamydia,
gonorrhea, or syphilis seems to increase risk. Obesity and elevated
blood levels of testosterone may increase the risk for prostate
cancer.
[0060] Prostate cancer is classified as an adenocarcinoma, or
glandular cancer, that begins when normal semen-secreting prostate
gland cells mutate into cancer cells. The region of prostate gland
where the adenocarcinoma is most common is the peripheral zone.
Initially, small clumps of cancer cells remain confined to
otherwise normal prostate glands, a condition known as carcinoma in
situ or prostatic intraepithelial neoplasia (PIN). Although there
is no proof that PIN is a cancer precursor, it is closely
associated with cancer. Over time these cancer cells begin to
multiply and spread to the surrounding prostate tissue (the stroma)
forming a tumor. Eventually, the tumor may grow large enough to
invade nearby organs such as the seminal vesicles or the rectum, or
the tumor cells may develop the ability to travel in the
bloodstream and lymphatic system. Prostate cancer is considered a
malignant tumor because it is a mass of cells which can invade
other parts of the body. This invasion of other organs is called
metastasis. Prostate cancer most commonly metastasizes to the
bones, lymph nodes, rectum, and bladder.
[0061] In prostate cancer, the regular glands of the normal
prostate are replaced by irregular glands and clumps of cells. When
a man has symptoms of prostate cancer, or a screening test
indicates an increased risk for cancer, more invasive evaluation is
offered. The only test which can fully confirm the diagnosis of
prostate cancer is a biopsy, the removal of small pieces of the
prostate for microscopic examination. However, prior to a biopsy,
several other tools may be used to gather more information about
the prostate and the urinary tract. Cystoscopy shows the urinary
tract from inside the bladder, using a thin, flexible camera tube
inserted down the urethra. Transrectal ultrasonography creates a
picture of the prostate using sound waves from a probe in the
rectum.
[0062] After biopsy, the tissue samples are then examined under a
microscope to determine whether cancer cells are present, and to
evaluate the microscopic features (or Gleason score) of any cancer
found. In addition, tissue samples may be stained for the presence
of PSA and other tumor markers in order to determine the origin of
maligant cells that have metastasized. A number of other potential
approaches for diagnosis of prostate cancer are ongoing such as
early prostate cancer antigen-2 (EPCA-2), and prostasome
analysis.
[0063] In addition to therapy using the compounds according to the
present invention, therapy (including prophylactic therapy) for
prostate cancer supports roles in reducing prostate cancer for
dietary selenium, vitamin E, lycopene, soy foods, vitamin D, green
tea, omega-3 fatty acids and phytoestrogens. The selective estrogen
receptor modulator drug toremifene has shown promise in early
trials. Two medications which block the conversion of testosterone
to dihydrotestosterone (and reduce the tendency toward cell
growth), finasteride and dutasteride, are shown to be useful. The
phytochemicals indole-3-carbinol and diindolylmethane, found in
cruciferous vegetables (califlower and broccholi), have favorable
antiandrogenic and immune modulating properties. Prostate cancer
risk is decreased in a vegetarian diet.
[0064] Treatment for prostate cancer may involve active
surveillance, surgery (prostatecomy or orchiectomy), radiation
therapy including brachytherapy (prostate brachytherapy) and
external beam radiation as well as hormonal therapy. There are
several forms of hormonal therapy which include the following, each
of which may be combined with compounds according to the present
invention. [0065] Antiandrogens such as flutamide, bicalutamide,
nilutamide, and cyproterone acetate which directly block the
actions of testosterone and DHT within prostate cancer cells.
[0066] Medications such as ketoconazole and aminoglutethimide which
block the production of adrenal androgens such as DHEA. These
medications are generally used only in combination with other
methods that can block the 95% of androgens made by the testicles.
These combined methods are called total androgen blockade (TAB),
which can also be achieved using antiandrogens. [0067] GnRH
modulators, including agonists and antagonists. GnRH antagonists
suppress the production of LH directly, while GnRH agonists
suppress LH through the process of downregulation after an initial
stimulation effect. Abarelix is an example of a GnRH antagonist,
while the GnRH agonists include leuprolide, goserelin, triptorelin,
and buserelin. [0068] The use of abiraterone acetate can be used to
reduce PSA levels and tumor sizes in aggressive end-stage prostate
cancer for as high as 70% of patients. Sorafenib may also be used
to treat metastatic prostate cancer.
[0069] Each treatment described above has disadvantages which limit
its use in certain circumstances. GnRH agonists eventually cause
the same side effects as orchiectomy but may cause worse symptoms
at the beginning of treatment. When GnRH agonists are first used,
testosterone surges can lead to increased bone pain from metastatic
cancer, so antiandrogens or abarelix are often added to blunt these
side effects. Estrogens are not commonly used because they increase
the risk for cardiovascular disease and blood clots. The
antiandrogens do not generally cause impotence and usually cause
less loss of bone and muscle mass. Ketoconazole can cause liver
damage with prolonged use, and aminoglutethimide can cause skin
rashes.
[0070] Palliative care for advanced stage prostate cancer focuses
on extending life and relieving the symptoms of metastatic disease.
As noted above, abiraterone acetate shows some promise in treating
advance stage prostate cancer as does sorafenib. Chemotherapy may
be offered to slow disease progression and postpone symptoms. The
most commonly used regimen combines the chemotherapeutic drug
docetaxel with a corticosteroid such as prednisone. Bisphosphonates
such as zoledronic acid have been shown to delay skeletal
complications such as fractures or the need for radiation therapy
in patients with hormone-refractory metastatic prostate cancer.
Alpharadin may be used to target bone metastasis. The phase II
testing shows prolonged patient survival times, reduced pain and
improved quality of life.
[0071] Bone pain due to metastatic disease is treated with opioid
pain relievers such as morphine and oxycodone. External beam
radiation therapy directed at bone metastases may provide pain
relief. Injections of certain radioisotopes, such as strontium-89,
phosphorus-32, or samarium-153, also target bone metastases and may
help relieve pain.
[0072] As an alternative to active surveillance or definitive
treatments, alternative therapies may also be used for the
management of prostate cancer. PSA has been shown to be lowered in
men with apparent localized prostate cancer using a vegan diet
(fish allowed), regular exercise, and stress reduction. Many other
single agents have been shown to reduce PSA, slow PSA doubling
times, or have similar effects on secondary markers in men with
localized cancer in short term trials, such as pomegranate juice or
genistein, an isoflavone found in various legumes.
[0073] Manifestations or secondary conditions or effects of
metastatic and advanced prostate cancer may include anemia, bone
marrow suppression, weight loss, pathologic fractures, spinal cord
compression, pain, hematuria, ureteral and/or bladder outlet
obstruction, urinary retention, chronic renal failure, urinary
incontinence, and symptoms related to bony or soft-tissue
metastases, among others.
[0074] Additional prostate drugs which can be used in combination
with the chimeric antibody recruiting compounds according to the
present invention include, for example, the enlarged prostate
drugs/agents, as well as eulexin, flutamide, goserelin, leuprolide,
lupron, nilandron, nilutamide, zoladex and mixtures thereof.
Enlarged prostate drugs/agents as above, include for example,
ambenzyl, ambophen, amgenal, atrosept, bromanyl,
bromodiphenhydramine-codeine, bromotuss-codeine, cardura,
chlorpheniramine-hydrocodone, ciclopirox,
clotrimazole-betamethasone, dolsed, dutasteride, finasteride,
flomax, gecil, hexylol, lamisil, lanased, loprox, lotrisone,
methenamine, methen-bella-meth Bl-phen sal, meth-hyos-atrp-M
blue-BA-phsal, MHP-A, mybanil, prosed/DS, Ro-Sed, S-T Forte,
tamsulosin, terbinafine, trac, tussionex, ty-methate, uramine,
uratin, uretron, uridon, uro-ves, urstat, usept and mixtures
thereof.
[0075] The term "tumor" is used to describe a malignant or benign
growth or tumefacent.
[0076] The term "antibody binding terminal moiety", "antibody
binding terminus" or "antibody binding moiety" is use to described
that portion of a chimeric compound according to the present
invention which comprises at least one small molecule or hapten.
The term "hapten" is used to describe a small-molecular-weight
inorganic or organic molecule that alone is not antigenic but which
when linked to another molecule, such as a carrier protein
(albumin, etc.) or in the case of the present invention, a cell
binding terminal moiety of the present compounds is antigenic; and
an antibody raised against the hapten (generally, the hapten bonded
or complexed to the carrier) will react with the hapten alone.
[0077] It is preferred that the antibody binding terminal comprise
a hapten which is reactive (binds to) an endogenous antibody that
pre-exists in the patient prior to initialing therapy with the
compounds of the present invention and does not have to be
separately raised as part of a treatment regimen. Thus, haptens
which comprise a di- or trinitro phenyl group as depicted below, or
a digalactose hapten (Gal-Gal-Z, preferably Gal-Gal-sugar,
preferably Gal-Gal-Glu), are preferred. Additionally, a compound
according to the general structure:
##STR00002##
Where X'' is O, CH.sub.2, NR.sup.1, S; and
[0078] R.sup.1 is H, a C.sub.1-C.sub.3 alkyl group or a
--C(O)(C.sub.1-C.sub.3) group; May be used as haptens in the
present invention.
[0079] Further, a moiety according to the chemical structure:
##STR00003##
Where X.sup.b is a bond, O, CH.sub.2, NR.sup.1 or S may also be
used as a hapten (ABT) in the present invention.
[0080] The preferred di- or trinitro phenyl hapten (ABT) moiety for
use in the present invention may be represented by the following
formula:
##STR00004##
Where Y' is H or NO.sub.2;
X is O, CH.sub.2, NR.sup.1, S(O), S(O).sub.2, --S(O).sub.2O,
--OS(O).sub.2, or OS(O).sub.2O;
[0081] R.sup.1 is H, a C.sub.1-C.sub.3 alkyl group, or a
--C(O)(C.sub.1-C.sub.3) group; In alternative embodiments, in the
above dinitrosubstituted phenyl ABT moiety, one of the above nitro
groups (at the para or ortho position) may be replaced with a
hydrogen group H to provide a mononitrosubstituted phenyl ABT
moiety.
[0082] The (Gal-Gal-Z) hapten is represented by the chemical
formula:
##STR00005##
Where X' is CH.sub.2, O, N--R.sup.1, or S, preferably O; R.sup.1'
is H or C.sub.1-C.sub.3 alkyl; Where Z is a bond, a monosaccharide,
disaccharide, oligosaccharide, glycoprotein or glycolipid,
preferably a sugar group, more preferably a sugar group selected
from the monosaccharides, including aldoses and ketoses, and
disaccharides, including those disaccharides described herein.
Monosaccharide aldoses include monosaccharides such as aldotriose
(D-glyceraldehdye, among others), aldotetroses (D-erythrose and
D-Threose, among others), aldopentoses, (D-ribose, D-arabinose,
D-xylose, D-lyxose, among others), aldohexoses (D-allose,
D-altrose, D-Glucose, D-Mannose, D-gulose, D-idose, D-galactose and
D-Talose, among others), and the monosaccharide ketoses include
monosaccharides such as ketotriose (dihydroxyacetone, among
others), ketotetrose (D-erythrulose, among others), ketopentose
(D-ribulose and D-xylulose, among others), ketohexoses (D-Psicone,
D-Fructose, D-Sorbose, D-Tagatose, among others), aminosugars,
including galactoseamine, sialic acid, N-acetylglucosamine, among
others and sulfosugars, including sulfoquinovose, among others.
Exemplary disaccharides which find use in the present invention
include sucrose (which may have the glucose optionally
N-acetylated), lactose (which may have the galactose and/or the
glucose optionally N-acetylated), maltose (which may have one or
both of the glucose residues optionally N-acetylated), trehalose
(which may have one or both of the glucose residues optionally
N-acetylated), cellobiose (which may have one or both of the
glucose residues optionally N-acetylated), kojibiose (which may
have one or both of the glucose residues optionally N-acetylated),
nigerose (which may have one or both of the glucose residues
optionally N-acetylated), isomaltose (which may have one or both of
the glucose residues optionally N-acetylated),
.beta.,.beta.-trehalose (which may have one or both of the glucose
residues optionally N-acetylated), sophorose (which may have one or
both of the glucose residues optionally N-acetylated),
laminaribiose (which may have one or both of the glucose residues
optionally N-acetylated), gentiobiose (which may have one or both
of the glucose residues optionally N-acetylated), turanose (which
may have the glucose residue optionally N-acetylated), maltulose
(which may have the glucose residue optionally N-acetylated),
palatinose (which may have the glucose residue optionally
N-acetylated), gentiobiluose (which may have the glucose residue
optionally N-acetylated), mannobiose, melibiose (which may have the
glucose residue and/or the galactose residue optionally
N-acetylated), melibiulose (which may have the galactose residue
optionally N-acetylated), rutinose, (which may have the glucose
residue optionally N-acetylated), rutinulose and xylobiose, among
others. Oligosaccharides for use in the present invention as Z can
include any sugar of three or more (up to about 100) individual
sugar (saccharide) units as described above (i.e., any one or more
saccharide units described above, in any order, especially
including glucose and/or galactose units as set forth above), or
for example, fructo-oligosaccharides, galactooligosaccharides and
mannan-oligosaccharides ranging from three to about ten-fifteen
sugar units in size. Glycoproteins for use in the present invention
include, for example, N-glycosylated and O-glycosylated
glycoproteins, including the mucins, collagens, transferring,
ceruloplasmin, major histocompatability complex proteins (MHC),
enzymes, lectins and selectins, calnexin, calreticulin, and
integrin glycoprotein IIb/IIa, among others. Glycolipids for use in
the present invention include, for example, glyceroglycolipids
(galactolipids, sulfolipids), glycosphingolipids, such as
cerebrosides, galactocerebrosides, glucocerebrosides (including
glucobicaranateoets), gangliosides, globosides, sulfatides,
glycophosphphingolipids and glycocalyx, among others.
[0083] Preferably, Z is a bond (linking a Gal-Gal disaccharide to a
linker or connector molecule) or a glucose or glucosamine
(especially N-acetylglucosamine). It is noted that Z is linked to a
galactose residue through a hydroxyl group or an amine group on the
galactose of Gal-Gal, preferably a hydroxyl group. A preferred
hapten is Gal-Gal-Glu which is represented by the structure:
##STR00006##
[0084] The term "cell binding terminal moiety", "cell binding
terminus" or "cell binding moiety" is use to described that portion
of a chimeric compound according to the present invention which
comprises at least one small molecule or moiety which can bind
specifically to prostate specific membrane antigen (PSMA).
[0085] Preferred CBT groups for use in the present invention are
set forth below:
##STR00007##
Where X.sub.1 and X.sub.2 are each independently CH.sub.2, O, NH or
S;
X.sub.3 is O, CH.sub.2, NR.sup.1, S(O), S(O).sub.2, --S(O).sub.2O,
--OS(O).sub.2, or OS(O).sub.2O;
[0086] R.sup.1 is H, a C.sub.1-C.sub.3 alkyl group, or a
--C(O)(C.sub.1-C.sub.3) group; k is an integer from 0 to 20, 8 to
12, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1, 2, 3, 4, 5 or 6; or a salt
thereof.
[0087] The term "linker" refers to a chemical entity connecting an
antibody binding terminus (ABT) moiety to a cell binding terminus
(CBT) moiety, optionally through a connector moiety through
covalent bonds. The linker between the two active portions of the
molecule, that is the antibody binding terminus (ABT) and the cell
binding terminus (CBT) ranges from about 5 .ANG. to about 50 .ANG.
or more in length, about 6 .ANG. to about 45 .ANG. in length, about
7 .ANG. to about 40 .ANG. in length, about 8 .ANG. to about 35
.ANG. in length, about 9 .ANG. to about 30 .ANG. in length, about
10 .ANG. to about 25 .ANG. in length, about 7 .ANG. to about 20
.ANG. in length, about 5 .ANG. to about 16 .ANG. in length, about 5
.ANG. to about 15 .ANG. in length, about 6 .ANG. to about 14 .ANG.
in length, about 10 .ANG. to about 20 .ANG. in length, about 11
.ANG. to about 25 .ANG. in length, etc. Linkers which are based
upon ethylene glycol units and are between 8 and 12 glycol units in
length may be preferred. By having a linker with a length as
otherwise disclosed herein, the ABT moiety and the CBT moiety may
be situated to advantageously take advantage of the biological
activity of compounds according to the present invention which bind
to prostate specific membrane antigen (PSMA) and attractive
endogenous antibodies to the cell to which the compounds are bound,
resulting in the selective and targeted cell death of those cells,
in whatever tissues they may reside, which have PSMA. The selection
of a linker component is based on its documented properties of
biocompatibility, solubility in aqueous and organic media, and low
immunogenicity/antigenicity. Although numerous linkers may be used
as otherwise described herein, a linker based upon
polyethyleneglycol (PEG) linkages, polypropylene glycol linkages,
or polyethyleneglycol-co-polypropylene oligomers (up to about 100
units, about 1 to 100, about 1 to 75, about 1 to 60, about 1 to 50,
about 1 to 35, about 1 to 25, about 1 to 20, about 1 to 15, 1 to
10, about 8 to 12, about 1 to 8, etc.) may be favored as a linker
because of the chemical and biological characteristics of these
molecules. The use of polyethylene (PEG) linkages is preferred.
[0088] Preferred linkers include those according to the chemical
structures:
##STR00008##
Or a polypropylene glycol or polypropylene-co-polyethylene glycol
linker having between 1 and 100 glycol units; Where R.sub.a is H,
C.sub.1-C.sub.3 alkyl or alkanol or forms a cyclic ring with
R.sup.3 (proline) and R.sup.3 is a side chain derived of an amino
acid preferably selected from the group consisting of alanine
(methyl), arginine (propyleneguanidine), asparagine
(methylenecarboxyamide), aspartic acid (ethanoic acid), cysteine
(thiol, reduced or oxidized di-thiol), glutamine
(ethylcarboxyamide), glutamic acid (propanoic acid), glycine (H),
histidine (methyleneimidazole), isoleucine (1-methylpropane),
leucine (2-methylpropane), lysine (butyleneamine), methionine
(ethylmethylthioether), phenylalanine (benzyl), proline (R.sup.3
forms a cyclic ring with R.sub.a and the adjacent nitrogen group to
form a pyrrolidine group), serine (methanol), threonine (ethanol,
1-hydroxyethane), tryptophan (methyleneindole), tyrosine (methylene
phenol) or valine (isopropyl); m is an integer from 1 to 100, 1 to
75, 1 to 60, 1 to 55, 1 to 50, 1 to 45, 1 to 40, 2 to 35, 3 to 30,
1 to 15, 1 to 10, 1 to 8, 1 to 6, 1, 2, 3, 4 or 5; or A linker
according to the chemical formula:
##STR00009##
Where Z and Z' are each independently a bond,
--(CH.sub.2).sub.i--O, --(CH.sub.2).sub.i--S,
--(CH.sub.2).sub.i--N--R,
##STR00010##
wherein said --(CH.sub.2).sub.i group, if present in Z or Z', is
bonded to a connector, ABT or CBT; Each R is H, or a
C.sub.1-C.sub.3 alkyl or alkanol group; Each R.sup.2 is
independently H or a C.sub.1-C.sub.3 alkyl group; Each Y is
independently a bond, O, S or N--R; Each i is independently 1 to
100, 1 to 75, 1 to 60, 1 to 55, 1 to 50, 1 to 45, 1 to 40, 2 to 35,
3 to 30, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1, 2, 3, 4 or 5;
D is
##STR00011##
[0089] or a bond, with the proviso that Z, Z' and D are not each
simultaneously bonds; j is 1 to 100, 1 to 75, 1 to 60, 1 to 55, 1
to 50, 1 to 45, 1 to 40, 2 to 35, 3 to 30, 1 to 15, 1 to 10, 1 to
8, 1 to 6, 1, 2, 3, 4 or 5; m' is 1 to 100, 1 to 75, 1 to 60, 1 to
55, 1 to 50, 1 to 45, 1 to 40, 2 to 35, 3 to 30, 1 to 15, 1 to 10,
1 to 8, 1 to 6, 1, 2, 3, 4 or 5; n is 1 to 100, 1 to 75, 1 to 60, 1
to 55, 1 to 50, 1 to 45, 1 to 40, 2 to 35, 3 to 30, 1 to 15, 1 to
10, 1 to 8, 1 to 6, 1, 2, 3, 4 or 5;
X.sup.1 is O, S or N--R; and
[0090] R is as described above, or a pharmaceutical salt
thereof.
[0091] The term "connector", symbolized by [CON], is used to
describe a chemical moiety which is optionally included in chimeric
compounds according to the present invention which forms from the
reaction product of an activated ABT-linker with a CBT moiety
(which also is preferably activated) or an ABT moiety with an
activated linker-CBT as otherwise described herein. The connector
group is the resulting moiety which forms from the facile
condensation of two separate chemical fragments which contain
reactive groups which can provide connector groups as otherwise
described to produce chimeric compounds according to the present
invention. It is noted that a connector may be distinguishable from
a linker in that the connector is the result of a specific
chemistry which is used provide chimeric compounds according to the
present invention wherein the reaction product of these groups
results in an identifiable connector group which is distinguishable
from the linker group as otherwise described herein. It is noted
that there may be some overlap between the description of the
connector group and the linker group, especially with respect to
more common connector groups such as amide groups, oxygen (ether),
sulfur (thioether) or amine linkages, urea or carbonate
--OC(O)O--groups as otherwise described herein. It is further noted
that a connector (or linker) may be connected to ABT, a linker or
CBT at positions which are represented as being linked to another
group using the using the symbol
##STR00012##
Where two or more such groups are present in a linker or connector,
any of an ABT, a linker or a CBT may be bonded to such a group.
[0092] Common connector groups which are used in the present
invention include the following chemical groups:
##STR00013##
Where X.sup.2 is O, S, NR.sup.4, S(O), S(O).sub.2, --S(O).sub.2O,
--OS(O).sub.2, or OS(O).sub.2O;
X.sup.3 is O, S, NR.sup.4; and
[0093] R.sup.4 is H, a C.sub.1-C.sub.3 alkyl or alkanol group, or a
--C(O)(C.sub.1-C.sub.3) group.
[0094] The term "pharmaceutically acceptable salt" or "salt" is
used throughout the specification to describe a salt form of one or
more of the compositions herein which are presented to increase the
solubility of the compound in saline for parenteral delivery or in
the gastric juices of the patient's gastrointestinal tract in order
to promote dissolution and the bioavailability of the compounds.
Pharmaceutically acceptable salts include those derived from
pharmaceutically acceptable inorganic or organic bases and acids.
Suitable salts include those derived from alkali metals such as
potassium and sodium, alkaline earth metals such as calcium,
magnesium and ammonium salts, among numerous other acids well known
in the pharmaceutical art. Sodium and potassium salts may by
preferred as neutralization salts of carboxylic acids and free acid
phosphate containing compositions according to the present
invention. The term "salt" shall mean any salt consistent with the
use of the compounds according to the present invention. In the
case where the compounds are used in pharmaceutical indications,
including the treatment of prostate cancer, including metastatic
prostate cancer, the term "salt" shall mean a pharmaceutically
acceptable salt, consistent with the use of the compounds as
pharmaceutical agents.
[0095] The term "coadministration" 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, provided that effective concentrations
of all coadministered compounds or compositions are found in the
subject at a given time. Chimeric antibody-recruiting compounds
according to the present invention may be administered with one or
more additional anti-cancer agents or other agents which are used
to treat or ameliorate the symptoms of cancer, especially prostate
cancer, including metastatic prostate cancer. Exemplary anticancer
agents which may be coadministered in combination with one or more
chimeric compounds according to the present invention include, for
example, antimetabolites, inhibitors of topoisomerase I and II,
alkylating agents and microtubule inhibitors (e.g., taxol).
Specific anticancer compounds for use in the present invention
include, for example, Aldesleukin; Alemtuzumab; alitretinoin;
allopurinol; altretamine; amifostine; anastrozole; arsenic
trioxide; Asparaginase; BCG Live; bexarotene capsules; bexarotene
gel; bleomycin; busulfan intravenous; busulfan oral; calusterone;
capecitabine; carboplatin; carmustine; carmustine with Polifeprosan
20 Implant; celecoxib; chlorambucil; cisplatin; cladribine;
cyclophosphamide; cytarabine; cytarabine liposomal; dacarbazine;
dactinomycin; actinomycin D; Darbepoetin alfa; daunorubicin
liposomal; daunorubicin, daunomycin; Denileukin diftitox,
dexrazoxane; docetaxel; doxorubicin; doxorubicin liposomal;
Dromostanolone propionate; Elliott's B Solution; epirubicin;
Epoetin alfa estramustine; etoposide phosphate; etoposide (VP-16);
exemestane; Filgrastim; floxuridine (intraarterial); fludarabine;
fluorouracil (5-FU); fulvestrant; gemtuzumab ozogamicin; goserelin
acetate; hydroxyurea; Ibritumomab Tiuxetan; idarubicin; ifosfamide;
imatinib mesylate; Interferon alfa-2a; Interferon alfa-2b;
irinotecan; letrozole; leucovorin; levamisole; lomustine (CCNU);
meclorethamine (nitrogen mustard); megestrol acetate; melphalan
(L-PAM); mercaptopurine (6-MP); mesna; methotrexate; methoxsalen;
mitomycin C; mitotane; mitoxantrone; nandrolone phenpropionate;
Nofetumomab; LOddC; Oprelvekin; oxaliplatin; paclitaxel;
pamidronate; pegademase; Pegaspargase; Pegfilgrastim; pentostatin;
pipobroman; plicamycin; mithramycin; porfimer sodium; procarbazine;
quinacrine; Rasburicase; Rituximab; Sargramostim; streptozocin;
talbuvidine (LDT); talc; tamoxifen; temozolomide; teniposide
(VM-26); testolactone; thioguanine (6-TG); thiotepa; topotecan;
toremifene; Tositumomab; Trastuzumab; tretinoin (ATRA); Uracil
Mustard; valrubicin; valtorcitabine (monoval LDC); vinblastine;
vinorelbine; zoledronate; and mixtures thereof, among others.
[0096] In addition to anticancer agents, a number of other agents
may be coadministered with chimeric compounds according to the
present invention in the treatment of cancer, especially prostate
cancer, including metastatic prostate cancer. These include active
agents, minerals, vitamins and nutritional supplements which have
shown some efficacy in inhibiting prostate cancer tissue or its
growth or are otherwise useful in the treatment of prostate cancer.
For example, one or more of dietary selenium, vitamin E, lycopene,
soy foods, vitamin D, green tea, lycopene, omega-3 fatty acids and
phytoestrogens, including beta-sitosterol, may be utilized in
combination with the present compounds to treat prostate
cancer.
[0097] In addition, active agents, other than traditional
anticancer agents have shown some utility in treating prostate
cancer. The selective estrogen receptor modulator drug toremifene
may be used in combination with the present compounds to treat
cancer, especially prostate cancer, including metastatic prostate
cancer. In addition, two medications which block the conversion of
testosterone to dihydrotestosterone, finasteride and dutasteride,
are also useful in the treatment of prostate cancer when
coadministered with compounds according to the present invention.
The phytochemicals indole-3-carbinol and diindolylmethane, may also
be coadministered with the present compounds for their effects in
treating prostate cancer. Additional agents which may be combined
with compounds according to the present invention include
antiandrogens, for example, flutamide, bicalutamide, nilutamide,
and cyproterone acetate as well as agents which reduce the
production of adrenal androgens (e.g. DHEA), such as ketoconazole
and aminoglutethimide. Other active agents which may be combined
with compounds according to the present invention include, for
example, GnRH modulators, including agonists and antagonists. GnRH
antagonists suppress the production of LH directly, while GnRH
agonists suppress LH through the process of downregulation after an
initial stimulation effect. Abarelix is an example of a GnRH
antagonist, while the GnRH agonists include leuprolide, goserelin,
triptorelin, and buserelin, among others. These agents may be
combined with compounds according to the present invention in
effective amounts. In addition, abiraterone acetate may also be
combined with one or more compounds according to the present
invention in the treatment of prostate cancer, especially including
metastatic prostate cancer.
[0098] Other agents which may be combined with one or more
compounds according to the present invention, include the
bisphosphonates such as zoledronic acid, which have been shown to
delay skeletal complications such as fractures which occur with
patients having metastatic prostate cancer. Alpharadin, another
agent, may be combined with compounds according to the present
invention to target bone metastasis. In addition, bone pain due to
metastatic prostate cancer may be treated with opioid pain
relievers such as morphine and oxycodone, among others, which may
be combined with compounds according to the present invention.
[0099] The present invention preferably relates to compounds
according to the general chemical structure:
##STR00014##
Wherein A is an antibody binding moiety according to the chemical
formula:
##STR00015##
Where Y' is H or NO.sub.2;
X is O, CH.sub.2, NR.sup.1, S(O), S(O).sub.2, --S(O).sub.2O,
--OS(O).sub.2, or OS(O).sub.2O;
[0100] R.sup.1 is H, a C.sub.1-C.sub.3 alkyl group, or a
--C(O)(C.sub.1-C.sub.3) group; X' is CH.sub.2, O, N--R.sup.1, or S,
preferably O; R.sup.1' is H or C.sub.1-C.sub.3 alkyl; Z is a bond,
a monosaccharide, disaccharide, oligosaccharide, glycoprotein or
glycolipid; X.sup.b is a bond, O, CH.sub.2, NR.sup.1 or S;
X'' is O, CH.sub.2, NR.sup.1;
[0101] R.sup.1 is H, a C.sub.1-C.sub.3 alkyl group or a
--C(O)(C.sub.1-C.sub.3) group; B is a cell binding moiety according
to the chemical formula:
##STR00016##
Where X.sub.1 and X.sub.2 are each independently CH.sub.2, O, NH or
S;
X.sub.3 is O, CH.sub.2, NR.sup.1, S(O), S(O).sub.2, --S(O).sub.2O,
--OS(O).sub.2, or OS(O).sub.2O;
[0102] R.sup.1 is H, a C.sub.1-C.sub.3 alkyl group, or a
--C(O)(C.sub.1-C.sub.3) group; k is an integer from 0 to 20, 8 to
12, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1, 2, 3, 4, 5 or 6; L is a
linker according to the chemical formula:
##STR00017##
Or a polypropylene glycol or polypropylene-co-polyethylene glycol
linker having between 1 and 100 glycol units (1 to 75, 1 to 60, 1
to 55, 1 to 50, 1 to 45, 1 to 40, 2 to 35, 3 to 30, 1 to 15, 1 to
10, 1 to 8, 1 to 6, 1, 2, 3, 4 or 52 and 50, 3 and 45); Where
R.sub.a is H, C.sub.1-C.sub.3 alkyl or alkanol or forms a cyclic
ring with R.sup.3 (proline) and R.sup.3 is a side chain derived of
an amino acid preferably selected from the group consisting of
alanine (methyl), arginine (propyleneguanidine), asparagine
(methylenecarboxyamide), aspartic acid (ethanoic acid), cysteine
(thiol, reduced or oxidized di-thiol), glutamine
(ethylcarboxyamide), glutamic acid (propanoic acid), glycine (H),
histidine (methyleneimidazole), isoleucine (1-methylpropane),
leucine (2-methylpropane), lysine (butyleneamine), methionine
(ethylmethylthioether), phenylalanine (benzyl), proline (R.sup.3
forms a cyclic ring with R.sub.a and the adjacent nitrogen group to
form a pyrrolidine group), serine (methanol), threonine (ethanol,
1-hydroxyethane), tryptophan (methyleneindole), tyrosine (methylene
phenol) or valine (isopropyl); m is an integer from 1 to 100, 1 to
75, 1 to 60, 1 to 55, 1 to 50, 1 to 45, 1 to 40, 2 to 35, 3 to 30,
1 to 15, 1 to 10, 1 to 8, 1 to 6, 1, 2, 3, 4 or 5; or L is a linker
according to the chemical formula:
##STR00018##
Where Z and Z' are each independently a bond,
--(CH.sub.2).sub.i--O, --(CH.sub.2).sub.i--S,
--(CH.sub.2).sub.i--N--R,
##STR00019##
wherein said --(CH.sub.2).sub.i group, if present in Z or Z', is
bonded to [CON] if present, ABT or CBT; Each R is independently H,
or a C.sub.1-C.sub.3 alkyl or alkanol group; Each R.sup.2 is
independently H or a C.sub.1-C.sub.3 alkyl group; Each Y is
independently a bond, O, S or N--R; Each i is independently 0 to
100, 1 to 100, 1 to 75, 1 to 60, 1 to 55, 1 to 50, 1 to 45, 1 to
40, 2 to 35, 3 to 30, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1, 2, 3, 4
or 5;
D is
##STR00020##
[0103] or a bond, with the proviso that Z, Z' and D are not each
simultaneously bonds; j is 1 to 100, 1 to 75, 1 to 60, 1 to 55, 1
to 50, 1 to 45, 1 to 40, 2 to 35, 3 to 30, 1 to 15, 1 to 10, 1 to
8, 1 to 6, 1, 2, 3, 4 or 5; m' is 1 to 100, 1 to 75, 1 to 60, 1 to
55, 1 to 50, 1 to 45, 1 to 40, 2 to 35, 3 to 30, 1 to 15, 1 to 10,
1 to 8, 1 to 6, 1, 2, 3, 4 or 5; n' is 1 to 100, 1 to 75, 1 to 60,
1 to 55, 1 to 50, 1 to 45, 1 to 40, 2 to 35, 3 to 30, 1 to 15, 1 to
10, 1 to 8, 1 to 6, 1, 2, 3, 4 or 5; and
X.sup.1 is O, S or N--R,
[0104] R is as described above; and The connector moiety [CON] is a
bond or a moiety according to the chemical structure:
##STR00021##
Where X.sup.2 is O, S, NR.sup.4, S(O), S(O).sub.2, --S(O).sub.2O,
--OS(O).sub.2, or OS(O).sub.2O;
X.sup.3 is NR.sup.4, O or S; and
[0105] R.sup.4 is H, a C.sub.1-C.sub.3 alkyl or alkanol group, or a
--C(O)(C.sub.1-C.sub.3) group; or a pharmaceutically acceptable
salt, solvate or polymorph thereof.
[0106] In preferred aspects of the invention, the antibody binding
terminus (ABT) is
##STR00022##
Y' is NO.sub.2;
X' is O;
[0107] Z is a bond, a monosaccharide or a disaccharide.
[0108] In preferred aspects of the invention, CBT is
##STR00023##
Where k is an integer from 0 to 20, 1 to 20, more preferably 8 to
12.
[0109] In other preferred aspects the connector moiety [CON} is
a
##STR00024##
group which can be covalently bonded at
##STR00025##
with a ABT group, a CBT group or alternatively, a linker group to
provide compounds as otherwise described herein.
[0110] In still other preferred aspects the linker group is a oligo
or polyethyleneglycol moiety of the structure:
##STR00026##
Where m is from 1 to 100 or as otherwise described herein,
preferably about 8 to 12. Noted there is that polypropylene glycol
or polyethylene glycol-co-polypropylenen glycol linkers may be
substituted for PEG groups in the present compounds.
[0111] A number of preferred compounds, 20, 21, 22, 23, 24 and 25
are set forth in attached FIG. 7.
[0112] In certain preferred aspects, the compound is according to
the chemical structure:
##STR00027##
Where n is 0 to 12, 0 to 12, 0 to 8, 0 to 6, 1 to 4; and
X is
##STR00028##
[0113] Where Y.sup.N, Y.sup.N1 and Y' is H or NO.sub.2; with at
least one of Y.sup.N, Y.sup.N1 and Y' being NO.sub.2, Or a
pharmaceutically acceptable salt, enantiomer, diastereomer, solvate
or polymorph thereof.
[0114] In the above compounds, X is preferably
##STR00029##
where Y' is H or NO.sub.2, preferably H,
[0115] Pharmaceutical compositions comprising combinations of an
effective amount of at least one chimeric antibody-recruiting
compound according to the present invention, and one or more of the
compounds otherwise described herein, all in effective amounts, in
combination with a pharmaceutically effective amount of a carrier,
additive or excipient, represents a further aspect of the present
invention.
[0116] The compositions of the present invention may be formulated
in a conventional manner using one or more pharmaceutically
acceptable carriers and may also be administered in
controlled-release formulations. Pharmaceutically acceptable
carriers that may be used in these pharmaceutical compositions
include, but are not limited to, ion exchangers, alumina, aluminum
stearate, lecithin, serum proteins, such as human serum albumin,
buffer substances such as phosphates, glycine, sorbic acid,
potassium sorbate, partial glyceride mixtures of saturated
vegetable fatty acids, water, salts or electrolytes, such as
prolamine sulfate, disodium hydrogen phosphate, potassium hydrogen
phosphate, sodium chloride, zinc salts, colloidal silica, magnesium
trisilicate, polyvinyl pyrrolidone, cellulose-based substances,
polyethylene glycol, sodium carboxymethylcellulose, polyacrylates,
waxes, polyethylene-polyoxypropylene-block polymers, polyethylene
glycol and wool fat.
[0117] The compositions of the present invention may be
administered orally, parenterally, by inhalation spray, topically,
rectally, nasally, buccally, vaginally or via an implanted
reservoir. The term "parenteral" as used herein includes
subcutaneous, intravenous, intramuscular, intra-articular,
intra-synovial, intrasternal, intrathecal, intrahepatic,
intralesional and intracranial injection or infusion techniques.
Preferably, the compositions are administered orally,
intraperitoneally or intravenously.
[0118] Sterile injectable forms of the compositions of this
invention may be aqueous or oleaginous suspension. These
suspensions may be formulated according to techniques known in the
art using suitable dispersing or wetting agents and suspending
agents. The sterile injectable preparation may also be a sterile
injectable solution or suspension in a non-toxic
parenterally-acceptable diluent or solvent, for example as a
solution in 1,3-butanediol. Among the acceptable vehicles and
solvents that may be employed are water, Ringer's solution and
isotonic sodium chloride solution. In addition, sterile, fixed oils
are conventionally employed as a solvent or suspending medium. For
this purpose, any bland fixed oil may be employed including
synthetic mono- or di-glycerides. Fatty acids, such as oleic acid
and its glyceride derivatives are useful in the preparation of
injectables, as are natural pharmaceutically-acceptable oils, such
as olive oil or castor oil, especially in their polyoxyethylated
versions. These oil solutions or suspensions may also contain a
long-chain alcohol diluent or dispersant, such as Ph. Helv or
similar alcohol.
[0119] The pharmaceutical compositions of this invention may be
orally administered in any orally acceptable dosage form including,
but not limited to, capsules, tablets, aqueous suspensions or
solutions. In the case of tablets for oral use, carriers which are
commonly used include lactose and corn starch. Lubricating agents,
such as magnesium stearate, are also typically added. For oral
administration in a capsule form, useful diluents include lactose
and dried corn starch. When aqueous suspensions are required for
oral use, the active ingredient is combined with emulsifying and
suspending agents. If desired, certain sweetening, flavoring or
coloring agents may also be added.
[0120] Alternatively, the pharmaceutical compositions of this
invention may be administered in the form of suppositories for
rectal administration. These can be prepared by mixing the agent
with a suitable non-irritating excipient which is solid at room
temperature but liquid at rectal temperature and therefore will
melt in the rectum to release the drug. Such materials include
cocoa butter, beeswax and polyethylene glycols.
[0121] The pharmaceutical compositions of this invention may also
be administered topically, especially to treat skin cancers,
psoriasis or other diseases which occur in or on the skin. Suitable
topical formulations are readily prepared for each of these areas
or organs. Topical application for the lower intestinal tract can
be effected in a rectal suppository formulation (see above) or in a
suitable enema formulation. Topically-acceptable transdermal
patches may also be used.
[0122] For topical applications, the pharmaceutical compositions
may be formulated in a suitable ointment containing the active
component suspended or dissolved in one or more carriers. Carriers
for topical administration of the compounds of this invention
include, but are not limited to, mineral oil, liquid petrolatum,
white petrolatum, propylene glycol, polyoxyethylene,
polyoxypropylene compound, emulsifying wax and water.
[0123] Alternatively, the pharmaceutical compositions can be
formulated in a suitable lotion or cream containing the active
components suspended or dissolved in one or more pharmaceutically
acceptable carriers. Suitable carriers include, but are not limited
to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl
esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and
water.
[0124] For ophthalmic use, the pharmaceutical compositions may be
formulated as micronized suspensions in isotonic, pH adjusted
sterile saline, or, preferably, as solutions in isotonic, pH
adjusted sterile saline, either with our without a preservative
such as benzylalkonium chloride. Alternatively, for ophthalmic
uses, the pharmaceutical compositions may be formulated in an
ointment such as petrolatum.
[0125] The pharmaceutical compositions of this invention may also
be administered by nasal aerosol or inhalation. Such compositions
are prepared according to techniques well-known in the art of
pharmaceutical formulation and may be prepared as solutions in
saline, employing benzyl alcohol or other suitable preservatives,
absorption promoters to enhance bioavailability, fluorocarbons,
and/or other conventional solubilizing or dispersing agents.
[0126] The amount of compound in a pharmaceutical composition of
the instant invention that may be combined with the carrier
materials to produce a single dosage form will vary depending upon
the host and disease treated, the particular mode of
administration. Preferably, the compositions should be formulated
to contain between about 0.05 milligram to about 750 milligrams or
more, more preferably about 1 milligram to about 600 milligrams,
and even more preferably about 10 milligrams to about 500
milligrams of active ingredient, alone or in combination with at
least one additional non-antibody attracting compound which may be
used to treat cancer, prostate cancer or metastatic prostate cancer
or a secondary effect or condition thereof.
[0127] It should also be understood that a specific dosage and
treatment regimen for any particular patient will depend upon a
variety of factors, including the activity of the specific compound
employed, the age, body weight, general health, sex, diet, time of
administration, rate of excretion, drug combination, and the
judgment of the treating physician and the severity of the
particular disease or condition being treated.
[0128] A patient or subject (e.g. a male human) suffering from
cancer can be treated by administering to the patient (subject) an
effective amount of a chimeric antibody recruiting compound
according to the present invention including pharmaceutically
acceptable salts, solvates or polymorphs, thereof optionally in a
pharmaceutically acceptable carrier or diluent, either alone, or in
combination with other known anticancer or pharmaceutical agents,
preferably agents which can assist in treating prostate cancer,
including metastatic prostate cancer or ameliorate the secondary
effects and conditions associated with prostate cancer. This
treatment can also be administered in conjunction with other
conventional cancer therapies, such as radiation treatment or
surgery.
[0129] These compounds can be administered by any appropriate
route, for example, orally, parenterally, intravenously,
intradermally, subcutaneously, or topically, in liquid, cream, gel,
or solid form, or by aerosol form.
[0130] The active compound is included in the pharmaceutically
acceptable carrier or diluent in an amount sufficient to deliver to
a patient a therapeutically effective amount for the desired
indication, without causing serious toxic effects in the patient
treated. A preferred dose of the active compound for all of the
herein-mentioned conditions is in the range from about 10 ng/kg to
300 mg/kg, preferably 0.1 to 100 mg/kg per day, more generally 0.5
to about 25 mg per kilogram body weight of the recipient/patient
per day. A typical topical dosage will range from 0.01-3% wt/wt in
a suitable carrier.
[0131] The compound is conveniently administered in any suitable
unit dosage form, including but not limited to one containing less
than 1 mg, 1 mg to 3000 mg, preferably 5 to 500 mg of active
ingredient per unit dosage form. An oral dosage of about 25-250 mg
is often convenient.
[0132] The active ingredient is preferably administered to achieve
peak plasma concentrations of the active compound of about
0.00001-30 mM, preferably about 0.1-30 .mu.M. This may be achieved,
for example, by the intravenous injection of a solution or
formulation of the active ingredient, optionally in saline, or an
aqueous medium or administered as a bolus of the active ingredient.
Oral administration is also appropriate to generate effective
plasma concentrations of active agent.
[0133] The concentration of active compound in the drug composition
will depend on absorption, distribution, inactivation, and
excretion rates of the drug as well as other factors known to those
of skill in the art. It is to be noted that dosage values will also
vary with the severity of the condition to be alleviated. It is to
be further understood that for any particular subject, specific
dosage regimens should be adjusted over time according to the
individual need and the professional judgment of the person
administering or supervising the administration of the
compositions, and that the concentration ranges set forth herein
are exemplary only and are not intended to limit the scope or
practice of the claimed composition. The active ingredient may be
administered at once, or may be divided into a number of smaller
doses to be administered at varying intervals of time.
[0134] Oral compositions will generally include an inert diluent or
an edible carrier. They may be enclosed in gelatin capsules or
compressed into tablets. For the purpose of oral therapeutic
administration, the active compound or its prodrug derivative can
be incorporated with excipients and used in the form of tablets,
troches, or capsules. Pharmaceutically compatible binding agents,
and/or adjuvant materials can be included as part of the
composition.
[0135] The tablets, pills, capsules, troches and the like can
contain any of the following ingredients, or compounds of a similar
nature: a binder such as microcrystalline cellulose, gum tragacanth
or gelatin; an excipient such as starch or lactose, a dispersing
agent such as alginic acid, Primogel, or corn starch; a lubricant
such as magnesium stearate or Sterotes; a glidant such as colloidal
silicon dioxide; a sweetening agent such as sucrose or saccharin;
or a flavoring agent such as peppermint, methyl salicylate, or
orange flavoring. When the dosage unit form is a capsule, it can
contain, in addition to material of the above type, a liquid
carrier such as a fatty oil. In addition, dosage unit forms can
contain various other materials which modify the physical form of
the dosage unit, for example, coatings of sugar, shellac, or
enteric agents.
[0136] The active compound or pharmaceutically acceptable salt
thereof can be administered as a component of an elixir,
suspension, syrup, wafer, chewing gum or the like. A syrup may
contain, in addition to the active compounds, sucrose as a
sweetening agent and certain preservatives, dyes and colorings and
flavors.
[0137] The active compound or pharmaceutically acceptable salts
thereof can also be mixed with other active materials that do not
impair the desired action, or with materials that supplement the
desired action, such as other anticancer agents, antibiotics,
antifungals, antiinflammatories, or antiviral compounds. In certain
preferred aspects of the invention, one or more chimeric
antibody-recruiting compound according to the present invention is
coadministered with another anticancer agent and/or another
bioactive agent, as otherwise described herein.
[0138] Solutions or suspensions used for parenteral, intradermal,
subcutaneous, or topical application can include the following
components: a sterile diluent such as water for injection, saline
solution, fixed oils, polyethylene glycols, glycerine, propylene
glycol or other synthetic solvents; antibacterial agents such as
benzyl alcohol or methyl parabens; antioxidants such as ascorbic
acid or sodium bisulfite; chelating agents such as
ethylenediaminetetraacetic acid; buffers such as acetates, citrates
or phosphates and agents for the adjustment of tonicity such as
sodium chloride or dextrose. The parental preparation can be
enclosed in ampoules, disposable syringes or multiple dose vials
made of glass or plastic.
[0139] If administered intravenously, preferred carriers are
physiological saline or phosphate buffered saline (PBS).
[0140] In one embodiment, the active compounds are prepared with
carriers that will protect the compound against rapid elimination
from the body, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art.
[0141] Liposomal suspensions may also be pharmaceutically
acceptable carriers. These may be prepared according to methods
known to those skilled in the art, for example, as described in
U.S. Pat. No. 4,522,811 (which is incorporated herein by reference
in its entirety). For example, liposome formulations may be
prepared by dissolving appropriate lipid(s) (such as stearoyl
phosphatidyl ethanolamine, stearoyl phosphatidyl choline,
arachadoyl phosphatidyl choline, and cholesterol) in an inorganic
solvent that is then evaporated, leaving behind a thin film of
dried lipid on the surface of the container. An aqueous solution of
the active compound are then introduced into the container. The
container is then swirled by hand to free lipid material from the
sides of the container and to disperse lipid aggregates, thereby
forming the liposomal suspension.
General Chemical Synthesis
[0142] The chimeric antibody-recruiting compounds according to the
present invention may be synthesized readily using standard
chemical connectivity between the linker and cell binding terminus
(CBT) and the antibody binding terminus (ABT), along with
appropriate protecting groups when necessary. The approach uses
standard functional group chemistry in order to link a cell binding
moiety to an antibody binding moiety through a linker, which, in
preferred aspects, provides an optional connector moiety (between
the linker and the ABT or the linker and the CBT depending on
functional groups, reactions used, etc.) which forms when the CBT
is covalently bonded (connected) to the ABT antibody binding
through the linker. Noted here is the fact that the connector
moiety per se is not required and the linker, as otherwise
described herein, may be covalently bonded directly to a CBT and/or
an ABT without the formation of a specific connector moiety. In the
present invention, the connector moiety, which is preferably
included in chimeric antibody-recruiting compounds according to the
present invention, reflects its formation reflective of favorable
synthetic chemistries to provide chimeric compounds as otherwise
disclosed herein.
[0143] As depicted in the general scheme below, a carboxylic acid,
such L-A, could be coupled to either an amine or an alcohol, such
as C-A, to generate esters or amides through standard carbodiimide
conditions (DCC, EDC, DIC along with base and catalytic amine
(DMAP, imidazole), by conversion to the acid chloride through oxaly
chloride or thionyl chloride etc. followed by addition of
amine/alcohol.
[0144] Additionally, for example, an amine or an alcohol, such as
A-A, can be coupled to an isocyanate or an isothiocyanate, such as
C-E, to generate ureas, thioureas, or the corresponding carbonates
or thiocarbonates.
[0145] In yet another approach, a triazole may be synthesized
through a cycloaddition reaction between an azide, such as C-B, and
an alkyne, such as L-C. This can be catalyzed by copper, such as
copper sulfate along with ascorbic acid, to facilitate a clean
reaction.
[0146] Still, in a further approach, for example, a heterolinker
can be made through treating a nucleophile, such as A-A, with the
appropriate leaving group, such as L-E. Some leaving groups could
be halogens, such as bromine, or sulfonates, such as triflates or
tosylates.
TABLE-US-00001 ##STR00030## ##STR00031## ##STR00032## ##STR00033##
A-A ##STR00034## L-A ##STR00035## C-A ##STR00036## A-B ##STR00037##
L-B ##STR00038## C-B ##STR00039## A-C ##STR00040## L-C ##STR00041##
C-C ##STR00042## A-D ##STR00043## L-D ##STR00044## C-D ##STR00045##
A-E ##STR00046## L-E ##STR00047## C-E ##STR00048## A-F ##STR00049##
C-F LG = leaving group, such as Cl, Br, OTs, ect. PG = protecting
group, such as t-Bu, Bn, etc.
Compounds
[0147] Several high affinity ligands have been developed to target
PSMA selectively. See, Slusher, et al, Nature Medicine, 1999, 5,
1396. FIG. 1 depicts the small molecule templated immunotherapy
which is generally understood to represent the principal mechanism
of chimeric antibody-recruiting compounds according to the present
invention. PC-ARM (3, FIG. 3) was inspired by a urea-based,
tetrazole-containing ligand with exceptionally high affinity (2,
Ki=0.9 nM) {See, Kozikowski, J. Med. Chem., 2004, 47, 1729] and
refined with molecular modeling to accommodate a solvent-exposed
appendage (FIG. 2A). A model of this overall ternary complex (FIG.
2B) suggested that a sizeable tether length would be required (8-12
polyethylene glycol units).
[0148] The azide-functionalized cell-binding terminus was
synthesized in 3 steps by coupling Cbz-protected lysine and t-butyl
protected glutamic acid with triphosgene (See, Kozikowski, et al.,
J. Med. Chem., 2004, 47, 1729) followed by Cbz deprotection and
azide formation (Scheme 2, FIG. 4). Link, et al., J. Am. Chem.
Soc., 2004, 126, 10598. Heterobifunctional PEG 10 was synthesized
in a five step process from octaethylene glycol (Scheme 2, FIG. 4).
Natarajan, et al., J. Chem. Comm., 2007, 7, 695. These
intermediates were coupled via microwave assisted, copper-catalyzed
Huisgen cyloaddition, (Bouillon, et al., J. Org. Chem., 2006, 71,
4700) and deprotected using microwave assisted TFA deprotection
(Scheme 3, FIG. 4) afforded PC-ARM (3). This specific synthesis may
be genericized and applied to produce a large number of compounds
according to the present invention simply following the
experimental section set forth hereinbelow. Inhibition experiments
against a human recombinant PSMA (R&D Research) confirmed
indirectly that this long-tethered molecule could bind PSMA with
high affinity (Ki=0.9.+-.0.3 nM).
[0149] In order to confirm the antibody-recruiting capability of
our small-molecule, live-cell recruitment assays were performed
with PSMA-expressing LNCaP cells and Alexafluor488 conjugated
anti-DNP antibodies. In the presence of anti-DNP antibodies, only a
small shift was observed, likely due to non-specific binding.
However, when chimeric molecule 3 was added, an increase in
fluorescence was observed, indicating the formation of the desired
ternary complex formation (FIG. 5A). In fact, this increase in
fluorescence could be observed at concentrations well into the
picomolar concentration range, suggesting exceptional activity. An
observed decrease in fluorescence with the addition of either
2-phosphonomethyl pentanedioic acid (Slusher, et al., Nature
Medicine, 1999, 5, 1396) or di-DNP lysine confirmed that the
recruitment was the result of both the cell-binding and
antibody-binding termini. In addition, cells expressing no PSMA
showed no significant increase in fluorescence (FIG. 5B).
[0150] Further, the ability of our molecules to induce cell killing
was tested. While small-molecule (3) induced increases in
cell-killing were not seen, or were modest, (<20%) using a
complement-dependant cytotoxicity assays in the presence of
anti-DNP antibodies, significant increases in cell-death was
observed in preliminary antibody-dependant cell-mediated
cytotoxicity assay (ADCC). In the presence of anti-DNP antibodies
and human peripheral blood mononuclear cells, 3 is shown to mediate
cell killing of up to 40% on LNCaP cells. Furthermore, 3 alone does
not show cytotoxicity, and ADCC does not occur on PSMA-DU145 cells
(FIG. 6). These results are consistent with reports of naked
monoclonal antibodies to PSMA being capable inducing ADCC but not
CDC responses. Deo, et al., international patent publication
WO2003/064606. Current work is underway to confirm these results,
and better understand the mode of action, such as the specific
effector cells that mediate the killing.
Alternative Compounds
[0151] Alternatively, in relying on the above described general
approach, molecule (4) (FIG. 9) may be readily synthesized and used
as a synthon for the chemical synthesis of dimeric compounds
according to the present invention. Compound 4 or similar
synthetically manageable molecules that have a propargyl tether
that can be used for click chemistry (Scheme 1a, FIG. 9). See,
Sharpless and Manetsch, Expert Opinion on Drug Discovery 2006, 1,
525-538. Treating this molecule to azides 5 and 6 with variously
lengthened polyethylene glycol units allows rapid entry into a
group of chimeric recruiting molecules of different tether lengths.
Having the flexibility to alter the chain length is important to
assist in identifying an optimal CBT-ABT distance.
[0152] Efforts toward alkyne 4 have provided us with bromide
intermediate 12, which will generate our target through an Arbuzov
reaction with known phosphate 13 (Scheme 2a, FIG. 10). See,
Jackson, et al., J. Med. Chem. 2001, 44, 4170-4175. Sonogashira
coupling of 7 and 8 provides the carbon framework for the linker,
(Liu and Stahl, J. Am. Chem. Soc.; 2007; 129; 6328-6335) and LAH
reducing conditions generates 10 in the appropriate
trans-substituted orientation (Luo, et al., Chem. Comm. 2007,
2136-2138) and deprotects the acyl group. This newly deprotected
phenol is selectively deprotonated over the homoallylic alcohol
with potassium carbonate and trapped with propargyl bromide to
generate 11. The remaining alcohol can then be converted to an
alkyl bromide through the use of phosphorous tribromide.
[0153] The synthesis of the azide coupling partners is also
provided. See Scheme 3, FIG. 11. The focus was on azide 15 because
bis-DNP lysine attached to polyethylene glycol linkers has
demonstrated significant affinity toward anti-DNP antibodies. See,
Baird, et al. Biochem. 2003, 42, 12739-12748. This particular
azide, which possesses 3 polyethylene glycol units, was synthesized
in one-pot from commercially available
bis(2,4-dinitrophenyl)-lysine and
11-azido-3,6,9-trioxaundecan-1-amine. This was accomplished through
a Schotten-Bauman protocol to provide the desired product in an
unoptimized yield of 40%. Demko, et al., J. Org. Chem. 2001, 66,
7945-7950. Scheme 3a, FIG. 11. Alternatively, mono DNP azide 16 may
be readily prepared through nucleophilic aromatic substitution.
Synthesis of Polyvalent Derivatives
[0154] Polyvalent derivatives can be synthesized in a divergent
manner from the previously proposed intermediates. Synthetically
complementary bis-alkynyl and tris-azidyl compounds are known, and
can be used very effectively in this pursuit. Bis-alkynyl 20 can be
converted under click conditions with a longer PEG-derived
azido-DNP 19, to generate the desired bis-di DNP analog 21 (Scheme
4a, FIG. 12).
[0155] The tris-azidylated analog can be synthesized in a similar
manner from a known triazide 23. See Scheme 5a, FIG. 13. See, Kale,
et al., Biorg. Med. Chem. Lett. 2007, 17, 2459-2464. This triazide
can undergo click chemistry with protected intermediate 22 to
provide trimeric 2-PMPA analog 24. With these synthetic pieces
(synthons) in hand, the final molecule can be put together by
standard peptide coupling followed by TFA deprotection. The final
compound is presented in FIG. 8 (compound 3).
[0156] The experiments conducted and presented here demonstrate
that small-molecule antibody-recruiting molecules which bind
selectively to prostate-specific membrane antigen can recruit
antibodies to PSMA-expressing cells and induce cell killing. This
small-molecule mediated response represents a new treatment for
cancer.
[0157] The present invention is further described by way of the
presentation of the following examples. While these examples are to
be taken as exemplary of the present invention, they are not
limiting in any way.
Examples
General Information
[0158] Unless otherwise stated, all reactions are carried out in
flame-dried glassware under a nitrogen atmosphere. All reagents
were purchased from commercial suppliers and used without further
purification except the following: Triethylamine was distilled over
calcium hydride; CH.sub.2Cl.sub.2, PhMe, DMF, and THF were purified
using a solvent dispensing system; Water was purified using a
Milli-Q purification system.
[0159] Infrared (IR) spectra bands are characterized as broad (br),
strong (s), medium (m) and weak (w). .sup.1H NMR chemical shifts
are reported with the solvent residual peak as the internal
standard (CDCl.sub.3 .delta. 7.26 ppm or CD.sub.3OD .delta. 3.31
ppm). Data are reported as follows: chemical shift, integration,
multiplicity (s=singlet, d=doublet, t=triplet, q=quartet, br=broad,
m=multiplet), and coupling constants (Hz). .sup.13C NMR chemical
shifts are reported in ppm with the solvent as an internal
reference (CDCl.sub.3 .delta. 77.2 ppm
Synthesis
##STR00050##
[0160] 1-azido-3,6,9,12,15,18,21,24-octaoxaheptacos-26-yne
(azido-PEG8-yne): 23-Azido-3,6,9,12,15,18,21-heptaoxatricosan-1-ol
(azido-PEG8-ol)
##STR00051##
(1.1 g, 3.17 mmol, 1.0 equiv.) was dissolved in
N,N'-dimethylformamide (6 mL), and sodium hydride (152 mg, 6.34
mmol, 2.0 equiv.) was added, followed by propargyl bromide (80% in
PhMe, 683 .mu.L, 6.34 mmol, 2.0 equiv.). The reaction ran for 4 h
at rt, at which time it was found complete by NMR aliquot. The
reaction was taken up in CH.sub.2Cl.sub.2 (25 mL) and washed with a
saturated aqueous ammonium chloride solution (25 mL). The aqueous
solution was back-extracted with dichloromethane (2.times.10 mL),
and combined organics were dried over MgSO.sub.4 and concentrated
to a brown oil. Chromatography (3 cm.times.20 cm Silica gel, 3%
MeOH/CH.sub.2Cl.sub.2) yielded azido-PEG8-yne (960 mg, 78% yield).
IR (thin film/NaCl) 2874 (m), 2110 (m), 1160 (s), 1105 (s)
cm.sup.-1; .sup.1HNMR (400 MHz, CDCl.sub.3) .delta. 4.20 (d, J=2.4
Hz, 2H), 3.58 (m, 30H), 3.39 (t, J=5.1 Hz, 2H), 2.43 (t, J=2.4 Hz,
1H), 1.82 (s, 1H); .sup.13CNMR (125 MHz, CDCl.sub.3) .delta. 79.82,
74.72, 70.75, 7022, 68.27, 58.62, 50.84; HRMS (ES+) calc'd for
C.sub.19H.sub.35N.sub.3O.sub.8 (M+Na) m/z 456.231637. Found
456.23182. 3,6,9,12,15,18,21,24-oetaoxaheptacos-26-yn-1-amine (9):
23-Azido-3,6,9,12,15,18,21-heptaoxatricosan-1-ol
(azido-PEG8-yne)
##STR00052##
(960 mg, 2.52 mmol, 1 equiv.), triphenylphosphine (992 mg, 3.78
mmol, 1.5 equiv.), and water (68 .mu.L, 3.78 mmol, 1.5 equiv.) were
dissolved in THF (10 mL) and stirred for 12 h. Reaction was
concentrated and chromatographed (3 cm.times.20 cm Silica,
CH.sub.2Cl.sub.2 then ramp to 80:20:1
CH.sub.2Cl.sub.2:MeOH:Et.sub.3N) and concentrated to yield
3,6,9,12,15,18,21,24-oetaoxaheptacos-26-yn-1-amine (9) as a clear
oil (815 mg, 91% yield). IR (thin film, NaCl) 3105 (br), 2914 (m),
1781 (m), 1638 (m), 1169 (s) cm.sup.-1. .sup.1H-NMR (500 MHz,
CDCl.sub.3) .delta. 4.17 (d, 2H, J=2.4), 3.66-3.57 (m, 28H), 3.54
(t, 2H, J=5.3 Hz), 2.88 (t, 2H, J=5.0 Hz), 2.41 (t, 1H, J=2.4 Hz),
2.18 (br s, 2H). .sup.13C NMR (125 MHz, CDCl.sub.3) .delta. 79.52,
74.59, 72.54, 70.41, 70.38, 70.38, 70.35, 70.31, 70.20, 70.06,
68.90, 58.20, 41.44. HRMS (ES+) calc'd for C.sub.19H.sub.37NO.sub.8
(M+H) m/z 408.259194. Found 408.25712.
N-(2,4-dinitrophenyl)-3,6,9,12,15,18,21,24-oetaoxaheptacos-26-yn-1-amine
(10):
##STR00053##
3,6,9,12-tetraoxapentadec-14-yn-1-amine (815 mg, 2.27 mmol, 1
equiv.) was dissolved in EtOH (10 mL), and triethylamine (666
.mu.L, 0.726 mmol, 1.5 equiv.) and 1-chloro-2,4-dinitrobenzene (505
mg, 2.5 mmol, 1.5 equiv.) were added. The reaction flask was fitted
with a reflux condenser and the reaction was heated to reflux for
48 h, cooled, and concentrated to a yellow oil. The crude mixture
was purified by flash chromatography (3 cm.times.20 cm Silica, 3%
MeOH:CH.sub.2Cl.sub.2) to yield
N-(2,4-dinitrophenyl)-3,6,9,12,15,18,21,24-octaoxaheptacos-26-yn-1-amine
(10) as a yellow solid (1.15 g, >95% yield). IR (thin film/NaCl)
3363 (w), 2871 (s), 1621 (s), 1337 (m), 1103 (s) cm.sup.-1;
.sup.1H-NMR (500 MHz, CDCl.sub.3) .delta. 9.08 (d, 1H, J=2.6 Hz),
8.77 (bs, 1H), 8.21 (dd, 1H, J=2.6, J=9.5 Hz), 6.94 (d, 1H, J=9.5
Hz), 4.16 (6, 2H, J=2.4 Hz), 3.78 (t, 2H, J=5.0 Hz), 3.64 (m, 32H),
3.58 (q, 2H), 2.41 (t, 1H, 2.38 Hz); .sup.13C-NMR (125 MHz,
CDCl.sub.3) .delta. 148.5, 136.1, 130.5, 130.29, 124.3, 114.3,
79.8, 74.6, 70.7, 70.6, 70.5, 69.2, 68.7, 58.5, 43.3; HRMS (EI)
calc'd for C.sub.25H.sub.39N.sub.3O.sub.12 (M+H) m/z 574.260650.
Found 574.26106.
##STR00054##
(9S,13S)-tri-tert-butyl
3,11-dioxo-1-phenyl-2-oxa-4,10,12-triazapentadecane-9,13,15-tricarboxylat-
e (12):
##STR00055##
11(1.0 g, 3.38 mmol, 1.0 equiv.) and triethylamine (1.54 mL, 11.09
mmol, 3.28 equiv.) were dissolved in dichloromethane (30 mL) and
cooled to -78.degree. C. Triphosgene (341 mg, 1.15 mmol, 0.34
equiv.) in dichloromethane (10 mL) was added dropwise to the
reaction mixture. Upon complete addition, the reaction was allowed
to warm to room temperature and stirred for 30 minutes. 12 (757 mg,
2.03 mmol, 0.6 equiv) was added, followed by the addition of
triethylamine (283 .mu.L, 2.03 mmol, 0.6 equiv.). The reaction was
allowed to stir at room temperature overnight for 16 hours. The
reaction was then diluted with dichloromethane (50 mL), and washed
with water (100 mL.times.2). The crude mixture was dried over
Na.sub.2SO.sub.4 and concentrated under reduced pressure. Column
chromatography (Silica 1.5:1 hexane:ethyl acetate) yielded 4 (1.09
g, 86%) as a colorless oil with the following spectral
characteristics: IR (thin film/KBr) 3342, 2976, 1731, 1650, 1552,
1454, 1368, 1255, and 1153 cm.sup.-1; .sup.1H NMR (500 MHz,
CDCl.sub.3) .delta. 7.35 (d, J=3.75 Hz, 4H), 7.33-7.30 (m, 1H),
5.10 (d, J=4.55 Hz, 2H), 5.06-5.01 (m, 2H), 4.99 (s, 1H), 4.34-4.31
(m, 2H), 3.20-3.18 (m, 2H), 2.36-2.23 (m, 2H), 2.10-2.03 (m, 1H),
1.88-1.75 (m, 2H), 1.65-1.57 (m, 1H), 1.57-1.45 (m, 2H), 1.453 (s,
9H), 1.446 (s, 9H), 1.43 (s, 9H), 1.40-1.30 (m, 2H); .sup.13C NMR
(125 MHz, CDCl.sub.3) 172.6, 172.5, 172.2, 136.8, 128.6, 128.5,
128.2, 82.2, 82.0, 80.7, 66.7, 53.4, 53.2, 40.7, 32.8, 31.7, 29.4,
28.5, 28.2, 28.1, 22.3; HRMS (EI+) m/z 622.3695 [calc'd for
C.sub.32H.sub.51N.sub.3O.sub.9 (M+H)+622.3698]. (S)-di-tert-butyl
2-(3-((S)-6-amino-1-tert-butoxy-1-oxohexan-2-yl)ureido)pentanedioate
(12):
##STR00056##
X (2.35 g, 3.78 mmol, 1.0 equiv.) was dissolved in methanol (37.8
ml) and was added dropwise to a vigorously stirred reaction flask
containing dry 10% Pd/C (475 mg). H.sub.2 was bubbled through the
solution for 1-2 m, and then ran for 13 h under a balloon of
H.sub.2. The reaction was deemed complete by TLC (Rf=0.48 in 10%
MeOH/CH.sub.2Cl.sub.2), plugged through celite, and concentrated to
give a viscous oil, which was carried on without further
purification. (S)-di-tert-butyl
2-(3-((S)-6-azido-1-tert-butoxy-1-oxohexan-2-yl)ureido)pentanedioate
(14):
##STR00057##
Sodium azide (2.629 g, 40.75 mmol, 10.0 equiv.) was dissolved in
water (7.63 mL), and dichloromethane (12.91 mL) was added. The
reaction mixture was cooled to 0.degree. C. and triflic anhydride
(1.36 mL, 8.09 mmol, 2.0 equiv.) was added. The solution was
stirred for 3 h at rt, and the organic layer was separated from the
aqueous layer. The aqueous layer was extracted with dichloromethane
(3.times.4 mL). The organic layers were combined and washed with
aqueous Na.sub.2CO.sub.3(aq) to give 25 ml of 0.391 M TfN.sub.3,
Amine 13 (1.97 g, 4.04 mmol, 1.0 equiv.) was dissolved in water
(14.37 mL) and methanol (28.74). To this solution were added
CuSO.sub.4-5H.sub.2O (10.1 mg, 0.04 mmol, 0.01 equiv.) and
K.sub.2CO.sub.3 (837.5 mg, 6.06 mmol, 1.5 equiv.). The TfN.sub.3
solution (25 ml, 8.09 mmol, 2 equiv.) was added rapidly to the
stirring solution of 13, and the reaction stirred for 19 h at rt.
The organic layer was separated from the aqueous layer, and the
water/methanol layer was extracted once with dichloromethane. The
combined organic layers were dried over MgSO4, concentrated under
reduced pressure, and purified by column chromatography to yield 14
as a white solid (1.440 g, 71%). R.sub.f=0.68 in 10%
MeOH:CH.sub.2Cl.sub.2. IR (Thin film/NaCl) 3335, 2980, 2933, 2868,
2097, 1733, 1635, 1560, 1368, 1257, and 1155 cm.sup.-1; .sup.1HNMR
(500 MHz, CDCl.sub.3) .delta. 5.01 (d, J=8.25 Hz, 2H), 4.34 (m,
2H), 3.26 (t, J=7.4 Hz, 2H), 2.35-2.25 (m, 2H), 2.09-2.05 (m, 1H),
1.87-1.76 (m, 2H), 1.66-1.55 (m, 3H), 1.46 (s, 18H), 1.43 (s, 9H),
1.45-1.35 (m, 2H) ppm; .sup.13CNMR (125 MHz, CDCl.sub.3) .delta.
172.6, 172.4, 172.2, 156.8, 82.3, 82.1, 80.7, 53.4, 53.2, 51.3,
33.0, 31.7, 28.6, 28.5, 28.2, 28.1, 22.4 ppm; HRMS (EI+) m/z
514.3225 [calc'd for C.sub.24H.sub.43N.sub.5O.sub.7
(M+H)+514.3235].
##STR00058##
(S)-di-tert-butyl
2-(3-((S)-1-tert-butoxy-6-(4-(13-(2,4-dinitrophenylamino)-2,5,8,11-tetrao-
xatridecyl)-1H-1,2,3-triazol-1-yl)-1-oxohexan-2-yl)ureido)pentanedioate
(3):
##STR00059##
To a mixture of 10 (76 mg, 0.145 mmol, 1.0 equiv) and 14 (74.4 mg,
0.145 mmol, 1.0 equiv.) in water (1 mL) and tert-butanol (1 mL) in
a 5 ml .mu.wave reaction tube was added sodium ascorbate (7 mg,
0.036 mmol, 0.25 equiv.) and aqueous solution of 0.1 M copper (II)
sulfate (0.0725 ml, 0.00725 mmol, 0.05 equiv.). The tube was
capped, and subjected to microwave radiation for 10 minutes at
110.degree. C. The reaction was then concentrated and redissolved
in trifluoroacetic acid (2 mL) and dichloromethane (1 mL) in a 5 ml
microwave reaction tube. The tube was capped and subjected to
microwave radiation for 2 m at 70.degree. C. The resulting reaction
mixture was concentrated under reduced pressure, chromatographed
using HPLC, and concentrated to yield 3 (87 mg, 58% yield) as a
yellow oil. IR (thin film/NaCl) 3359 (w), 2925 (s), 1737 (s), 1622
(m), 1170 (s) cm.sup.-1; .sup.1HNMR (100 MHz, MeOD) .delta. 9.07
(d, J=2.7 Hz, 1H), 8.33 (dd, J=2.7, 9.6 Hz, 1H), 8.03 (s, 1H), 7.27
(d, J=9.6 Hz, 1H), 4.66 (s, 2H), 4.45 (t, J=7 Hz, 2H), 4.35-4.28
(m, 2H), 3.83 (t, J=7 Hz, 2H), 3.73-3.61 (m, 32H), 2.44-2.36 (m,
2H), 2.17-2.08 (m, 1H), 1.99-1.82 (m, 4H), 1.71-1.64 (m, 1H),
1.46-1.38 (m, 2H); .sup.13CNMR (125 MHz, MeOD) .delta. 176.4,
176.1, 175.7, 160.0, 149.9, 145.9, 137.0, 131.5, 131.0, 125.2,
124.7, 116.1, 71.6, 71.6, 71.5, 71.5, 70.9, 69.9, 64.8, 53.7, 53.5,
51.2, 44.1, 32.8, 31.1, 30.6, 28.8, 23.4 ppm; HRMS (ES+) calc'd for
C.sub.37H.sub.58N.sub.8O.sub.19 (M+H) m/z 919.389098. Found
919.38801.
NAALADase Inhibition Experiments
[0161] A 10 mM stock solution of N-acetyl-aspartyl-glutamate (NAAG)
in 40 mM NaOH was diluted to 40 .mu.M in Tris buffer (0.1M
Tris-HCl, pH=7.5), and was added to 384 well plate (25 .mu.l per
well). For Km measurements and controls, 2.times. dilution (40
.mu.M-312 nM) series of NAAG were made and added to separate wells.
For IC.sub.50 measurements, solutions of recruiting molecule 3 in
water (2 .mu.L per well, dilution series) were added to wells. For
all other wells, 2 .mu.L of water was added. To initiate reactions,
rhPSMA (R&D research) diluted in Tris buffer (20 pg/mL), was
added to each well (25 .mu.l per well). For negative controls, Tris
buffer was added (25 .mu.l per well). The plate was covered and
incubated for 15 minutes, at which time the protein was deactivated
by heating the plate to 95.degree. C. for 3 minutes. After plate
was allowed to cool, glutamic acid release was visualized using an
Amplex.RTM.-Red glutamic acid/glutamate oxidase assay kit
(Invitrogen). Km and IC.sub.50 values were calculated using
graphpad prism software, and Ki was calculated from these values
using the Cheng-Prusoff Equation. This process was run in
triplicate, and is reported in the manuscript as the average of
three runs .+-.standard deviation.
Flow Cytometry Recruiting Experiments
[0162] Antibody Recruitment Flow Cytometry: LNCaP and DU145 cells
were detached, counted, washed, and resuspended with flow cytometry
buffer (25 mM Tris-HCl, 150 mM NaCl, 1.5% BSA, 5 mM Glucose, 1.5 mM
MgCl.sub.2, pH 7.2) to a density of 2.times.10.sup.5 cells
mL.sup.-1 of buffer, and 1 mL was added to each epindorf tube per
experiment. Solutions of 3 in water (2 .mu.L, variable
concentration per experiment) in flow cytometry buffer were added
to the cells, and the cells were incubated at 4.degree. C. for 60
minutes. For cell-binding termini competition experiments,
solutions of PMPA in water (2 .mu.L, variable concentrations) were
added prior to incubation. Following incubation, the cells were
washed three times with flow cytometry buffer. 20 .mu.L of 1 mg
ml.sup.-1 human IgG in mouse serum were added to each tube and the
tubes were incubated for 5 minutes at room temperature to allow
blocking of Fc receptors. 200 .mu.L of flow cytometry buffer were
added, and to that was added 2 .mu.L of 2 mg ml.sup.-1
AlexaFluor488 conjugated rabbit anti-dinitrophenyl IgG--fraction
KLH. For antibody-binding terminus competition experiments, a
solution of di-DNP Lysine (2 .mu.L of 5 mM solution in water) was
added prior to incubation. The tubes were incubated at 4.degree. C.
for 60 minutes and taken up with 850 .mu.L of flow cytometry
buffer. The cells were spun down and washed with flow cytometry
buffer (2.times.1 mL). The cells were taken up with 1 mL of Tris
buffered saline (25 mM TrisHCl, 150 mM NaCl, pH 7.2) and 2 .mu.L of
500 .mu.g mL.sup.-1 of propidium iodide was added, and samples were
analyzed immediately on FACSCalibur instrument (Becton Dickinson).
The data was analyzed using FlowJo (Tree Star Inc.), gating for
live cells on FL-3. An experiment omitting 3 was done as a control.
The experiment was repeated in triplicate to ensure
reproducibility.
Further Examples
[0163] The following experiments relate to a number of additional
compounds, i.e., antibody-recruiting molecules targeting prostate
cancer (ARM-Ps) which were synthesized and tested for binding
affinity and/or inhibition of PMSA. ARM-Ps belong to a class of
glutamate urea compounds capable of inhibiting PSMA with high
potency. See FIG. 14.
[0164] During the course of developing ARM-Ps, the inventors
observed that bifunctional DNP-containing conjugates were
strikingly more potent than the parent glutamate urea compounds
from which they were derived. Furthermore, we also noted that
potency increases were correlated to the length of the linker
regions connecting the two poles of the molecule. Here we provide a
molecular basis for these findings, which involves the disclosure
of a previously unreported arene-binding site on PSMA. These
conclusions are supported by extensive biochemical,
crystallographic, and computational studies.
[0165] Synthesis: All starting materials and reagents were
purchased from commercially available sources and used without
further purification. .sup.1H NMR shifts are measured using the
solvent residual peak as the internal standard (CDCl.sub.3
.quadrature.7.26, MeOD .quadrature.3.31), and reported as follows:
chemical shift, multiplicity (s=singlet, bs=broad singlet,
d=doublet, t=triplet, dd=doublet of doublet, q=quartet,
m=multiplet), coupling constant (Hz), integration. .sup.13C NMR
shifts are measured using the solvent residual peak as the internal
standard (CDCl.sub.3 .quadrature.77.20 or MeOD .quadrature.49.00 or
DMSO-d.sub.6 .quadrature.39.01), and reported as chemical shifts.
Infrared (IR) spectral bands are characterized as broad (br),
strong (s), medium (m), and weak (w).
Chemical Synthesis
##STR00060##
[0166] 2,4-dinitro-N-(2-(prop-2-ynyloxy)ethyl)aniline (s-2a).
##STR00061##
2-(2,4-dinitrophenylamino)ethanol (s-1a) (410 mg, 1.80 mmol, 1.0
equiv.) was dissolved in 3 mL of DMF and slowly added to a slurry
of NaH (86.4 mg, 3.6 mmol, 2 equiv.) in 5 mL of DMF in a flame
dried flask pre-cooled to 0.degree. C. To the resulting slurry, 80%
propargyl bromide (0.240 mL, 2.16 mmol, 1.2 equiv.) in toluene,
cooled to 0.degree. C., was added slowly. The ice bath was removed
and the reaction was allowed to stir at room temperature for an
additional 15 hours. The reaction was then re-cooled to 0.degree.
C., quenched with saturated NH.sub.4Cl, and extracted with diethyl
ether (3.times.150 mL). The organic layers were combined, dried,
concentrated under reduced pressure, and chromatographed (silica
gel, 1.times.25 cm, 0% CH.sub.3OH in CHCl.sub.3, then 2.5%
CH.sub.3OH in CHCl.sub.3) to yield
2,4-dinitro-N-(2-(prop-2-ynyloxy)ethyl)aniline (s-2a) as a dark
yellow solid (310 mg, 64.8%). IR (thin film) 3356 (m), 3285 (m),
3105 (w), 2871 (w), 2117 (w), 1616 (s), 1584 (s), 1521 (s), 1499
(m), 1423 (m), 1274 (s), 1089 (s), 920 (m); .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 9.16 (d, J=2.6 Hz, 1H), 8.76 (bs, 1H), 8.28
(dd, J=9.5, 2.7 Hz, 1H), 6.96 (d, J=9.5 Hz, 1H), 4.25 (d, J=2.4 Hz,
2H), 3.91-3.84 (m, 2H), 3.65 (d, J=5.3 Hz, 1H), 2.50 (t, J=2.4 Hz,
1H). .sup.13C NMR (125 MHz, CDCl.sub.3) .delta. 149.5, 136.3,
130.7, 130.4, 124.4, 114.1, 78.9, 75.5, 67.3, 58.7, 43.3. HRMS
(ES+) calc'd for C.sub.11H.sub.11N.sub.3O.sub.5 (M+H) m/z 266.0732
Found 266.0771.
2,4-dinitro-N-(2-(2-(prop-2-ynyloxy)ethoxy)ethyl)aniline
(s-2b).
##STR00062##
2-(2-(2,4-dinitrophenylamino)ethoxy)ethanol (s-1b) (100 mg, 0.369
mmol, 1.0 equiv.) was dissolved in 0.81 mL of DMF and slowly added
to a slurry of NaH (17.71 mg, 0.738 mmol, 2.0 equiv.) in 0.81 mL of
DMF in a flame dried flask pre-cooled to 0.degree. C. An 80%
solution of propargyl bromide in toluene (0.0490 .quadrature.L,
0.443 mmol, 1.2 equiv.) was added slowly. The ice bath was then
removed and the reaction was allowed to stir at room temperature
for an additional 2 hours. The reaction was then re-cooled to
0.degree. C., quenched with saturated NH.sub.4Cl, and then
extracted with diethyl ether (3.times.50 mL). The organic layers
were combined, dried with Na.sub.2SO.sub.4, concentrated under
reduced pressure, and chromatographed (silica gel, 3.times.25 cm,
0% EtOAc in hexanes, then 10% EtOAc in hexanes, then 20% EtOAc in
hexanes, then 30% EtOAc in hexanes) to yield
2,4-dinitro-N-(2-(2-(prop-2-ynyloxy)ethoxy)ethyl)aniline (s-2b) as
a dark yellow solid (50 mg, 45% yield). IR (thin film) 3360 (m),
3285 (m), 3107 (w), 2872 (m), 1621 (s), 1588 (m), 1524 (m), 1425
(w), 1335 (s), 1305 (m), 1133 (m), 1101 (m), 920 (w), 832 (w);
.sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 9.15 (d, J=2.6 Hz, 1H),
8.81 (bs, 1H), 8.27 (dd, J=9.4, 2.3 Hz, 1H), 6.96 (d, J=9.5 Hz,
1H), 4.21 (d, J=2.3 Hz, 2H), 3.84 (t, J=5.2 Hz, 2H), 3.74 (m, 4H),
3.61 (q, J=5.2 Hz, 2H), 2.44 (t, J=2.3 Hz, 1H). .sup.13C NMR (125
MHz, CDCl.sub.3) .delta. 148.6, 136.3, 130.7, 130.4, 124.4, 114.2,
79.6, 74.9, 70.7, 69.3, 68.9, 58.7, 43.4. HRMS (ES+) calc'd for
C.sub.13H.sub.15N.sub.3O.sub.6 (M+H) m/z 310.0994 Found
310.1033.
##STR00063##
3-(2-(2-azidoethoxy)ethoxy)prop-1-yne (s-4).
##STR00064##
2-(2-azidoethoxy)ethanol.sup.1 (s-3) (3 g, 22.89 mmol, 1.0 equiv.)
was slowly added to a slurry of NaH (1.10 g, 45.78 mmol, 2 equiv.)
in 102.64 mL of DMF in a flame dried flask pre-cooled to 0.degree.
C. To the resulting slurry, 80% propargyl bromide in toluene (3.06
mL, 27.47 mmol, 1.2 equiv.), cooled to 0.degree. C., was added
slowly. The ice bath was removed and the reaction was allowed to
stir at room temperature for an additional 3 hours. The reaction
was then re-cooled to 0.degree. C., and 3 mL of cold H.sub.2O was
added to quench the reaction, after which the reaction was
concentrated under reduced pressure and taken up with saturated
NH.sub.4Cl. The reaction was extracted with diethyl ether, dried
with Na.sub.2SO.sub.4, concentrated under reduced pressure, and
chromatographed (silica gel, 3.times.25 cm, 0% CH.sub.3OH in
CHCl.sub.3, then 10% MeOH in CHCl.sub.3) to yield
3-(2-(2-azidoethoxy)ethoxy)prop-1-yne (s-4) as a dark brown oil
(2.79 g, 72.1%). IR (thin film) 3291 (m), 2867 (m), 2099 (s), 1442
(w), 1347 (w), 1285 (m), 1101 (s), 1032 (w), 920 (w), 942 (w), 646
(m); .sup.1H NMR (125 MHz, CDCl.sub.3) .delta. 4.21 (d, J=2.4 Hz,
1H), 3.74-3.65 (m, 3H), 3.40 (t, J=5.1 Hz, 1H), 2.43 (t, J=2.4 Hz,
0H). .sup.13C NMR (125 MHz, CDCl.sub.3) .delta. 79.5, 74.6, 70.5,
70.0, 69.1, 58.5, 50.6. HRMS (ES+) calc'd for
C.sub.7H.sub.11N.sub.3O.sub.2 (M+H) m/z 170.0885 Found 170.0924.
2-(2-(prop-2-ynyloxy)ethoxy)ethanamine (s-5).
##STR00065##
3-(2-(2-azidoethoxy)ethoxy)prop-1-yne (s-4) (2 g, 11.82 mmol, 1.0
equiv) was dissolved in THF (30.5 mL). Triphenylphosphine (3.72 g,
14.20 mmol, 1.2 equiv.) and water (0.21 mL, 11.83 mol, 1.0 equiv.)
were added to the solution and the reaction was allowed to stir at
room temperature for 10 hours. The reaction was concentrated under
reduced pressure and chromatographed (5% CH.sub.3OH in CHCl.sub.3,
then 5% CH.sub.3OH in CHCl.sub.3+5% Et.sub.3N) to yield
2-(2-(prop-2-ynyloxy)ethoxy)ethanamine (s-5) as a pale green oil
(1.35 g, 80%). IR (thin film) 3250 (m), 2863 (m), 2112 (w), 1589
(w), 1443 (w), 1349 (m), 1291 (w), 1093 (s), 1037 (m), 918 (w), 840
(w), 673 (m); .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 4.21 (d,
J=2.3 Hz, 2H), 3.73-3.68 (m, 2H), 3.68-3.62 (m, 2H), 3.51 (t, J=5.2
Hz, 2H), 2.87 (t, J=5.2 Hz, 2H), 2.43 (dd, J=2.4 Hz, 1H). .sup.13C
NMR (125 MHz, CDCl.sub.3) .delta. 79.7, 74.7, 73.7, 70.3, 69.2,
58.6, 41.9. HRMS (ES+) calc'd for C.sub.7H.sub.13NO.sub.2 (M+H) m/z
144.0980 Found 144.1019.
2-nitro-N-(2-(2-(prop-2-ynyloxy)ethoxy)ethyl)aniline (s-6).
##STR00066##
To 1-chloro-2-nitrobenzene (152 mg, 0.97 mmol, 1.0 equiv.) was
added neat 2-(2-(prop-2-ynyloxy)ethoxy)ethanamine (s-5) (900 mg,
6.31 mmol, 6.5 equiv.), and the resulting slurry was heated to
100.degree. C. for 6 hours during which time the solid dissolved.
At the end of this period, the heating bath was removed, the
reaction content was mixed with water (50 mL), and then extracted
with CH.sub.2Cl.sub.2 (3.times.50 mL). The organic layers were
combined, dried with Na.sub.2SO.sub.4, concentrated under reduced
pressure, and chromatographed (silica gel, 1.times.25 cm, 0%
CH.sub.3OH in CH.sub.2Cl.sub.2, then 10% CH.sub.3OH in
CH.sub.2Cl.sub.2) to yield
2-nitro-N-(2-(2-(prop-2-ynyloxy)ethoxy)ethyl)aniline (s-6) as a
dark yellow oil (76 mg, 30%). IR (thin film) 3378 (m), 3286 (m),
3085 (w), 2875 (m), 2114 (w), 1616 (s), 1570 (s), 1508 (s), 1417
(m), 1349 (m), 1228 (s), 1093 (s), 1034 (m), 740 (m), 670 (s);
.sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 8.23 (bs, 1H), 8.18 (dd,
J=8.6, 1.6 Hz, 1H), 7.43 (ddd, J=8.6, 7.0, 1.6 Hz, 1H), 6.86 (d,
J=8.6 Hz, 1H), 6.65 (ddd, J=8.3, 7.0, 1.2 Hz, 1H), 4.22 (d, J=2.4
Hz, 2H), 3.80 (t, J=5.5 Hz, 2H), 3.73 (d, J=2.4 Hz, 4H), 3.52 (q,
J=5.4 Hz, 2H), 2.43 (t, J=2.4 Hz, 1H). .sup.13C NMR (125 MHz,
CDCl.sub.3) .delta. 145.45, 136.13, 132.22, 126.94, 115.39, 113.78,
79.56, 74.61, 70.50, 69.20, 69.19, 58.52, 42.75; HRMS (ES+) calc'd
for C.sub.13H.sub.16N.sub.2O.sub.4 (M+H) m/z 265.1144 Found
265.1181. 4-nitro-N-(2-(2-(prop-2-ynyloxy)ethoxy)ethyl)aniline
(s-7).
##STR00067##
To 1-chloro-4-nitrobenzene (152 mg, 0.97 mmol, 1.0 equiv.) was
added neat 2-(2-(prop-2-ynyloxy)ethoxy)ethanamine (s-5) (900 mg,
6.31 mmol, 6.5 equiv.), and the resulting slurry was heated to
100.degree. C. for 19 hours. At the end of this period, the heating
bath was removed, and the reaction content was mixed with water (50
mL) and then extracted with CH.sub.2Cl.sub.2 (3.times.50 mL). The
organic layers were combined, dried with Na.sub.2SO.sub.4,
concentrated under reduced pressure, and chromatographed (silica
gel, 1.times.25 cm, 0% EtOAc in CH.sub.2Cl.sub.2, then 30% EtOAc in
CH.sub.2Cl.sub.2 to yield
4-nitro-N-(2-(2-(prop-2-ynyloxy)ethoxy)ethyl)aniline (s-7) as a
dark yellow oil (40 mg, 16%). IR (thin film) 3351 (m), 3239 (m),
2858 (w), 2112 (w), 1599 (s), 1535 (w), 1500 (w), 1467 (s), 1284
(s), 1086 (s), 1034 (w); .sup.1H NMR (400 MHz, CDCl.sub.3) .delta.
8.06 (d, J=9.2 Hz, 2H), 6.53 (d, J=9.2 Hz, 2H), 5.05 (bs, 1H), 4.19
(d, J=2.4 Hz, 2H), 3.76-3.70 (m, 2H), 3.70-3.64 (m, 4H), 3.38 (q,
J=5.3 Hz, 2H), 2.45 (t, J=2.4 Hz, 1H). .sup.13C NMR (125 MHz,
CDCl.sub.3) .quadrature.153.4, 138.0, 126.4, 111.2, 79.5, 74.9,
70.3, 69.1, 69.0, 58.5, 42.9. HRMS (ES+) calc'd for
C.sub.13H.sub.16N.sub.2O.sub.4 (M+H) m/z 265.1144 Found
265.1177.
##STR00068##
N-(2-(2-(prop-2-ynyloxy)ethoxy)ethyl)aniline (s-9).
##STR00069##
2-(2-(prop-2-ynyloxy)ethoxy)ethyl 4-methylbenzenesulfonate
(s-8).sup.2 (100 mg, 0.335 mmol, 0.31 equiv.) was dissolved in
aniline (102 mg, 1.10 mmol, 1 equiv.). The reaction was allowed to
proceed at 100.degree. C. in a sealed reaction vessel for 5 hours,
after which time it was chromatographed (Silica Gel, 25 g RediSep
pre-packed column, 0% EtOAc:Hexanes.fwdarw.20% EtOAc:Hexanes) to
yield N-(2-(2-(prop-2-ynyloxy)ethoxy)ethyl)aniline (s-9) as a brown
oil (39.0 mg, 53.1%). IR (thin film) 3393 (m), 3278 (m), 3052 (w),
2865 (m), 2115 (w), 1603 (s), 1506 (s), 1461 (w), 1320 (w), 1277
(m), 1099 (s), 1030 (w), 750 (s), 693 (s); .sup.1H NMR (500 MHz,
CDCl.sub.3) .delta. 7.22-7.16 (m, 2H), 6.72 (t, J=7.3 Hz, 1H),
6.67-6.63 (m, 2H), 4.22 (d, J=2.4 Hz, 2H), 4.12 (s, 1H), 3.74-3.70
(m, 4H), 3.70-3.66 (m, 2H), 3.32 (t, J=5.2 Hz, 2H), 2.46 (t, J=2.4
Hz, 1H). .sup.13C NMR (125 MHz, CDCl.sub.3) 148.7, 129.6, 117.9,
113.5, 80.0, 75.1, 70.5, 70.1, 69.5, 58.9, 43.9. HRMS (ES+) calc'd
for C.sub.13H.sub.17NO.sub.2 (M+H) m/z 220.1293 Found 220.1327.
4-methoxy-N-(2-(2-(prop-2-ynyloxy)ethoxy)ethyl)aniline (s-10).
##STR00070##
K.sub.2CO.sub.3 (380 mg, 2.76 mmol, 2.5 equiv.) was added to a
solution of 4-methoxyaniline (136 mg, 1.1 mmol, 1 equiv.) in 1 mL
of DMF, and the resulting slurry was heated to 100.degree. C.
2-(2-(prop-2-ynyloxy)ethoxy)ethyl 4-methylbenzenesulfonate (s-8)
(100 mg, 0.276 mmol, 0.25 equiv.), dissolved in DMF (1 mL), was
then added to the reaction via syringe-pump over 5 hours. The
reaction was stirred for an additional 12 hours, after which time
it was concentrated under reduced pressure and partially purified
(Silica Gel, 12 g RediSep pre-packed column, 0%
EtOAc:Hexanes.fwdarw.20% EtOAc:Hexanes.fwdarw.50% EtOAc:Hexanes,
followed by EtOAc flush). The material obtained after
chromatography was carried directly on to the next step without
further purification.
N-(2-(2-(prop-2-ynyloxy)ethoxy)ethyl)cyclohexanamine (s-11).
##STR00071##
Cyclohexylamine (150 mg, 1.5 mmol, 1 equiv.) and
2-(2-(prop-2-ynyloxy)ethoxy)ethyl 4-methylbenzenesulfonate (s-8)
(100 mg, 0.335 mmol, 0.2 equiv.) were dissolved in 1 mL of ethanol.
The reaction was allowed to proceed under microwave irradiation at
80.degree. C. for 10 minutes, after which it was concentrated under
reduced pressure and chromatographed (Silica Gel, 25 g RediSep
pre-packed column, 10% EtOAc:Hexanes.fwdarw.50% EtOAc:Hexanes,
followed by EtOAc flush) to give
N-(2-(2-(prop-2-ynyloxy)ethoxy)ethyl)cyclohexanamine (s-10) as a
clear oil (28 mg, 37%). IR (thin film) 3253 (w), 2924 (s), 2852
(s), 2113 (w), 1449 (m), 1349 (m), 1263 (w), 1102 (s), 919 (w), 839
(w); .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 4.19 (d, J=2.4 Hz,
2H), 3.70-3.65 (m, 2H), 3.65-3.60 (m, 2H), 3.59 (t, J=5.4 Hz, 2H),
2.80 (t, J=5.4 Hz, 2H), 2.42 (t, J=2.3 Hz, 1H), 2.40-2.34 (m, 1H),
1.90-1.84 (m, 2H), 1.72-1.69 (m, 2H), 1.64-1.54 (m, 1H), 1.30-1.15
(m, 3H), 1.15-0.99 (m, 2H), 0.92-0.81 (m, 1H). .sup.13C NMR (100
MHz, CDCl.sub.3) .delta. 79.6, 74.6, 70.9, 70.1, 69.0, 58.4, 56.8,
42.3, 33.4, 26.2, 25.1. HRMS (ES+) calc'd for
C.sub.13H.sub.23NO.sub.2 (M+H) m/z 226.1762 Found 226.1797.
##STR00072##
(S)-2-(3-((S)-1-carboxy-5-(4-((2,4-dinitrophenylamino)methyl)-1H-1,2,3-tr-
iazol-1-yl)pentyl)ureido)pentanedioic acid (1).
##STR00073##
2,4-dinitro-N-(prop-2-ynyl)aniline.sup.3 (48.75 mg, 0.220 mmol, 1.1
equiv.) and azide s-12.sup.4 (100 mg, 0.194 mmol, 1 equiv.) were
added to a mixture of water (0.694 mL) and t-BuOH (0.694 mL). The
slurry was placed in a microwave reaction tube, to which a 0.1 M
solution of sodium ascorbate in water (0.388 mL, 0.039 mmol, 0.2
equiv.) and a 0.1 M solution of copper (II) sulfate in water (0.078
mL, 0.008 mmol, 0.04 equiv.) were added. The tube was capped and
subjected to microwave irradiation at 110.degree. C. for 20
minutes. The crude mixture was concentrated under reduced pressure,
and taken up in 67% trifluoroacetic acid in CH.sub.2Cl.sub.2 (3
mL). The tube was capped and subjected to microwave irradiation at
70.degree. C. for 2 minutes. The crude mixture was concentrated
under reduced pressure, purified via HPLC, and the pure fractions
were collected and concentrated under reduced pressure to yield 1
(47.7 mg, 43.6% over two steps) as a yellow solid. IR (thin film)
3367 (br), 2946 (br), 1720 (m), 1619 (s), 1589 (m), 1524 (w), 1425
(w), 1338 (m), 1203 (m), 1137 (m); .sup.1H NMR (400 MHz, MeOD)
.delta. 9.05 (d, J=2.6 Hz, 1H), 8.29 (dd, J=9.5, 2.6 Hz, 1H), 8.00
(s, 1H), 7.24 (d, J=9.6 Hz, 1H), 4.82 (s, 2H), 4.41 (t, J=7.0 Hz,
2H), 4.29 (ddd, J=18.6, 8.5, 4.9 Hz, 2H), 2.50-2.33 (m, 2H),
2.20-2.10 (m, 1H), 2.03-1.80 (m, 4H), 1.72-1.62 (m, 1H), 1.48-1.36
(m, 2H). .sup.13CNMR (125 MHz, DMSO-d.sub.6) 174.3, 174.1, 173.7,
157.2, 147.8, 143.1, 135.2, 130.1, 130.0, 123.5, 123.0, 115.7,
52.0, 51.5, 49.3, 38.4, 29.8, 29.4, 27.4, 22.1. HRMS (ES+) calc'd
for C.sub.21H.sub.26N.sub.8O.sub.11 (M+H) m/z 567.1755 Found
567.1796.
(S)-2-(3-((S)-1-carboxy-5-(4-((2-(2,4-dinitrophenylamino)ethoxy-
)methyl)-1H-1,2,3-triazol-1-yl)pentyl)ureido)pentanedioic acid
(2).
##STR00074##
2,4-dinitro-N-(2-(prop-2-ynyloxy)ethyl)aniline (s-2a) (51.4 mg,
0.194 mmol, 1 equiv.) and azide s-12 (100 mg, 0.194 mmol, 1 equiv.)
were added to a mixture of water (0.694 mL) and t-BuOH (0.694 mL).
This slurry was placed in a microwave reaction tube, to which a 0.1
M solution of sodium ascorbate in water (0.388 mL, 0.039 mmol, 0.2
equiv.) and 0.1 M solution of copper (II) sulfate in water (0.078
mL, 0.008 mmol, 0.04 equiv.) were added. The tube was capped and
subjected to microwave irradiation at 110.degree. C. for 20
minutes. The crude mixture was concentrated under reduced pressure,
and taken up in 67% trifluoroacetic acid in CH.sub.2Cl.sub.2 (3
mL). The tube was capped and subjected to microwave irradiation at
70.degree. C. for 2 minutes. The crude mixture was concentrated
under reduced pressure, purified via HPLC, and the pure fractions
were collected and concentrated under reduced pressure to yield 2
(38.0 mg, 32.2% over two steps), as a yellow oil. IR (thin film)
3356 (m), 2938 (m), 1731 (s), 1621 (s), 1586 (m), 1525 (m), 1426
(w), 1336 (s), 1306 (w), 1137 (w), 1087 (m), 833 (w); .sup.1H NMR
(500 MHz, MeOD) .delta. 9.00 (d, J=2.2 Hz, 1H), 8.25 (dd, J=9.6,
2.3 Hz, 1H), 7.98 (s, 1H), 7.18 (d, J=9.6 Hz, 1H), 4.68 (s, 2H),
4.41 (t, J=6.9 Hz, 2H), 4.28 (ddd, J=18.1, 8.2, 5.0 Hz, 2H), 3.82
(t, J=5.0 Hz, 2H), 3.67 (t, J=5.0 Hz, 2H), 2.48-2.33 (m, 2H),
2.17-2.10 (m, 1H), 2.01-1.81 (m, 4H), 1.71-1.64 (m, 1H), 1.46-1.35
(m, 2H). .sup.13CNMR (125 MHz, DMSO-d.sub.6) 174.4, 174.1, 173.7,
157.2, 148.3, 143.6, 134.9, 129.9, 129.7, 123.8, 123.6, 115.6,
67.3, 63.4, 52.1, 51.6, 49.1, 42.6, 31.5, 29.9, 29.4, 27.5, 22.1.
HRMS (ES+) calc'd for C.sub.23H.sub.30N.sub.8O.sub.12 (M+H) m/z
611.2017 Found 611.2074.
(S)-2-(3-((S)-1-carboxy-5-(4-((2-(2-(2,4-dinitrophenylamino)ethoxy)ethoxy-
)methyl)-1H-1,2,3-triazol-1-yl)pentyl)ureido)pentanedioic acid
(3).
##STR00075##
2,4-dinitro-N-(2-(2-(prop-2-ynyloxy)ethoxy)ethyl)aniline (s-2b) (60
mg, 0.194 mmol, 1 equiv.) and azide s-12 (100 mg, 0.194 mmol, 1
equiv.) were added to a mixture of water (0.694 mL) and t-BuOH
(0.694 mL). This slurry was placed in a microwave reaction tube, to
which a 0.1 M solution of sodium ascorbate in water (0.388 mL,
0.039 mmol, 0.2 equiv.) and 0.1 M solution of copper (II) sulfate
in water (0.078 mL, 0.008 mmol, 0.04 equiv.) were added. The tube
was capped and subjected to microwave irradiation at 110.degree. C.
for 20 minutes. The crude mixture was concentrated under reduced
pressure, and taken up in 67% trifluoroacetic acid in
CH.sub.2Cl.sub.2 (3 mL). The tube was capped and subjected to
microwave irradiation at 70.degree. C. for 2 minutes. The crude
mixture was concentrated under reduced pressure, purified via HPLC,
and the pure fractions were collected and concentrated under
reduced pressure to yield 3(31.0 mg, 24.6% over two steps) as a
yellow oil. IR (thin film) 3360 (m), 2933 (m), 1726 (s), 1621 (s),
1587 (m), 1525 (w), 1425 (w), 1337 (s), 1306 m), 1136 (m); .sup.1H
NMR (400 MHz, MeOD) .quadrature. 9.01 (d, J=2.7 Hz, 1H), 8.26 (dd,
J=9.6 Hz, 2.7 Hz, 1H), 7.97 (s, 1H), 7.20 (d, J=9.6 Hz, 1H), 4.63
(s, 2H), 4.41 (t, J=7.0 Hz, 2H), 4.33-4.23 (m, 2H), 3.80 (t, J=5.2
Hz, 2H), 3.72-3.68 (m, 4H), 3.66 (t, J=5.2, 2H), 2.48-2.32 (m, 2H),
2.19-2.08 (m, 1H), 2.00-1.80 (m, 4H), 1.73-163 (m, 1H), 1.46-1.35
(m, 2H). .sup.13CNMR (125 MHz, DMSO-d.sub.6) .quadrature. 174.3,
174.1, 173.6, 157.2, 148.2, 143.8, 134.9, 129.9, 129.6, 123.6,
115.6, 69.7, 68.9, 68.2, 63.6, 52.0, 51.5, 49.1, 42.5, 31.4, 29.8,
29.4, 27.4, 22.1. HRMS (ES+) calc'd for
C.sub.25H.sub.34N.sub.8O.sub.13 (M+H) m/z 655.2279 Found
655.2275.
##STR00076##
2,5,8,11,14,17,20,23,26,29,32,35,38-tridecaoxahentetracont-40-yne
(s-13e).
##STR00077##
2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaheptatriacontan-37-ol
(150 mg, 22.89 mmol, 1.0 equiv.) was slowly added to a slurry of
NaH (48 mg, 45.78 mol, 8 equiv.) in DMF (1.4 mL) in a flame dried
flask pre-cooled to 0.degree. C. 80% propargyl bromide in toluene
(36 .quadrature.L, 27.47 mmol, 1.2 equiv.), cooled to 0.degree. C.,
was added slowly. The ice bath was removed and the reaction was
allowed to stir at room temperature for an additional 2 hours. The
reaction was then re-cooled to 0.degree. C., 0.5 mL of ice cold
water was added, and the reaction was concentrated under reduced
pressure. The remaining residue was redissolved in dichloromethane,
then partially purified via silica gel chromatography (10% MeOH in
DCM) to remove all the salts. After partial purification,
2,5,8,11,14,17,20,23,26,29,32,35,38-tridecaoxahentetracont-40-yne
(s-13e) was obtained as a brown oil that was taken on to the next
step without full purification.
(S)-2-(3-((S)-1-carboxy-5-(4-(methoxymethyl)-1H-1,2,3-triazol-1-yl)pentyl-
)ureido)pentanedioic acid (8).
##STR00078##
Methyl propargyl ether (s-13a) (81.8 mg, 1.16 mmol, 6 equiv.) and
azide s-12 (100 mg, 0.194 mmol, 1 equiv.) were added to a mixture
of water (0.694 mL) and t-BuOH (0.694 mL). This slurry was placed
in a microwave reaction tube to which 0.1 M solution of sodium
ascorbate in water (0.388 mL, 0.039 mmol, 0.2 equiv.) and 0.1 M
solution of copper (II) sulfate in water (0.078 mL, 0.008 mmol,
0.04 equiv.) were added. The tube was capped and subjected to
microwave irradiation at 110.degree. C. for 20 minutes. The crude
mixture was concentrated under reduced pressure, and taken up in
67% trifluoroacetic acid in CH.sub.2Cl.sub.2 (3 mL). The tube was
capped and subjected to microwave irradiation at 70.degree. C. for
2 minutes. The crude mixture was concentrated under reduced
pressure, purified via HPLC, and the pure fractions were collected
and concentrated under reduced pressure to yield 8(32.9 mg, 41.4%
over two steps) as a clear oil. IR (thin film) 3368 (br), 2936 (m),
1670 (s), 1564 (m), 1437 (w), 1193 (s), 1139 (s), 1064 (m), 839
(w), 800 (w), 723 (w). .sup.1H NMR (500 MHz, MeOD) .delta. 8.00 (s,
1H), 4.55 (s, 2H), 4.43 (t, J=7.0 Hz, 2H), 4.36-4.25 (m, 2H), 3.38
(s, 3H), 3.18 (s, 0H), 2.48-2.35 (m, 2H), 2.19-2.12 (m, 1H),
2.02-1.82 (m, 4H), 1.75-1.64 (m, 1H), 1.48-1.36 (m, 2H).
.sup.13CNMR (125 MHz, MeOD) 176.5, 176.2, 175.8, 160.1, 145.6,
125.2, 66.2, 58.4, 53.7, 53.5, 51.3, 32.8, 31.1, 30.7, 28.8, 23.4.
HRMS (ES+) calc'd for C.sub.16H.sub.25N.sub.5O.sub.8 (M+H) m/z
416.1737 Found 415.2019.
(S)-2-(3-((S)-1-carboxy-5-(4-((2-(2-methoxyethoxy)ethoxy)methyl-
)-1H-1,2,3-triazol-1-yl)pentyl)ureido)pentanedioic acid (9).
##STR00079##
3-(2-(2-methoxyethoxy)ethoxy)prop-1-yne (s-13b).sup.5 (30.7 mg,
0.194 mmol, 1 equiv.) and azide s-12 (100 mg, 0.194 mmol, 1 equiv.)
were added to a mixture of water (0.694 mL) and t-BuOH (0.694 mL).
This slurry was placed in a microwave reaction tube, to which a 0.1
M solution of sodium ascorbate in water (0.388 mL, 0.039 mmol, 0.2
equiv.) and 0.1 M solution of copper (II) sulfate in water (0.078
mL, 0.008 mmol, 0.04 equiv.) were added. The tube was capped and
subjected to microwave irradiation at 110.degree. C. for 20
minutes. The crude mixture was concentrated under reduced pressure,
and taken up in 67% trifluoroacetic acid in CH.sub.2Cl.sub.2 (3
mL). The tube was capped and subjected to microwave irradiation at
70.degree. C. for 2 minutes. The crude mixture was concentrated
under reduced pressure, purified via HPLC, and the pure fractions
were collected and concentrated under reduced pressure to yield 9
(30.6 mg, 31.5% over two steps) as a clear oil, IR (thin film) 3346
(br), 2931 (m), 1734 (s), 1642 (m), 1562 (s), 1451 (w), 1201 (s),
1087 (s); .sup.1H NMR (400 MHz, MeOD) .delta. 8.01 (s, 1H), 4.65
(s, 2H), 4.43 (t, J=7.0 Hz, 2H), 4.33-4.24 (m, 2H), 3.69-3.64 (m,
4H), 3.64-3.60 (m, 2H), 3.56-3.49 (m, 2H), 3.33 (s, 3H), 2.46-2.34
(m, 2H), 2.16-2.11 (m, 1H), 2.00-1.82 (m, 4H), 1.71-1.64 (m, 1H),
1.48-1.31 (m, 2H). .sup.13CNMR (125 MHz, CD.sub.3OD) .quadrature.
176.4, 176.1, 175.8, 160.1, 145.8, 125.3, 72.9, 71.5, 71.3, 70.8,
64.8, 59.1, 53.7, 53.5, 51.3, 32.8, 31.1, 30.7, 28.8, 23.4. HRMS
(ES+) calc'd for C.sub.20H.sub.33N.sub.5O.sub.10 (M+H) m/z 504.2261
Found 504.2590.
(S)-2-(3-((S)-5-(4-2,5,8,11,14-pentaoxapentadecyl-1H-1,2,3-triazol-1-yl)--
1-carboxypentyl)ureido)pentanedioic acid (10).
##STR00080##
2,5,8,11,14-pentaoxaheptadec-16-yne (s-13d).sup.5 (52 mg, 0.194
mmol, 1 equiv.) and azide s-12 (100 mg, 0.194 mmol, 1 equiv.) were
added to a mixture of water (0.694 mL) and t-BuOH (0.694 mL). This
slurry was placed in a microwave reaction tube, to which a 0.1 M
solution of sodium ascorbate in water (0.388 mL, 0.039 mmol, 0.2
equiv.) and 0.1 M solution of copper (II) sulfate in water (0.078
mL, 0.008 mmol, 0.04 equiv.) were added. The tube was capped and
subjected to microwave irradiation at 110.degree. C. for 20
minutes. The crude mixture was concentrated under reduced pressure,
and taken up in 67% trifluoroacetic acid in CH.sub.2Cl.sub.2 (3
mL). The tube was capped and subjected to microwave irradiation at
70.degree. C. for 2 minutes. The crude mixture was concentrated
under reduced pressure, purified via HPLC, and the pure fractions
were collected and concentrated under reduced pressure to yield 10
(19.8 mg, 15.9% over two steps) as a clear oil. IR (thin film) 3323
(br), 2921 (m), 1734 (s), 1642 (w), 1562 (m), 1452 (w), 1201 (m),
1089 (m), 845 (w); .sup.1H NMR (500 MHz, MeOD) .delta. 8.04 (s,
1H), 4.65 (s, 2H), 4.44 (t, J=7.0 Hz, 2H), 4.34-4.24 (m, 2H),
3.70-3.65 (m, 4H), 3.65-3.58 (m, 12H), 3.52 (dd, J=5.6, 3.6 Hz,
2H), 3.34 (s, 3H), 2.47-2.35 (m, 2H), 2.19-2.09 (m, 1H), 2.02-1.82
(m, 4H), 1.72-1.65 (m, 1H), 1.45-1.36 (m, 2H). .sup.13CNMR (125
MHz, DMSO-d.sub.6) 176.4, 176.1, 160.1, 145.7, 125.3, 72.9, 71.5,
71.3, 70.8, 64.8, 59.1, 53.7, 53.5, 51.3, 32.8, 31.1, 30.7, 28.8,
23.4. HRMS (ES+) calc'd for C.sub.24H.sub.41N.sub.5O.sub.12 (M+H)
m/z 592.2785 Found 592.2783.
(S)-2-(3-((S)-5-(4-2,5,8,11,14,17,20,23,26-nonaoxaheptacosyl-1H-1,2,3-tri-
azol-1-yl)-1-carboxypentyl)ureido)pentanedioic acid (11).
##STR00081##
2,5,8,11,14,17,20,23,26-nonaoxanonacos-28-yne (s-13d).sup.6 (41 mg,
0.097 mmol, 1 equiv.) and azide s-12 (50 mg, 0.097 mmol, 1 equiv.)
were added to a mixture of water (0.350 mL) and t-BuOH (0.350 mL).
This slurry was placed in a microwave reaction tube, to which a 0.1
M solution of sodium ascorbate in water (0.194 mL, 0.019 mmol, 0.2
equiv.) and 0.1 M solution of copper (II) sulfate in water (0.039
mL, 0.004 mmol, 0.04 equiv.) were added. The tube was capped and
subjected to microwave irradiation at 110.degree. C. for 20
minutes. The crude mixture was concentrated under reduced pressure,
and taken up in 67% trifluoroacetic acid in CH.sub.2Cl.sub.2 (3
mL). The tube was capped and subjected to microwave irradiation at
70.degree. C. for 2 minutes. The crude mixture was concentrated
under reduced pressure, purified via HPLC, and the pure fractions
were collected and concentrated under reduced pressure to yield 11
(9.6 mg, 21.4% over two steps) as a clear oil. IR (thin film) 3332
(br), 2919 (m), 2875 (m), 1734 (s), 1557 (m), 1452 (w), 1203 (s),
1088 (s), 946 (w), 850 (w); .sup.1H NMR (400 MHz, MeOD) .delta.
8.00 (s, 1H), 4.64 (s, 2H), 4.42 (t, J=6.9 Hz, 2H), 4.35-4.23 (m,
2H), 3.66 (s, 4H), 3.62 (m, J=5.5, 1.1 Hz, 26H), 3.56-3.52 (m, 2H),
3.36 (d, J=1.1 Hz, 3H), 2.45-2.41 (m, 2H), 2.22-2.07 (m, 1H),
1.98-1.82 (m, 4H), 1.72-1.64 (m, 1H), 1.44-1.35 (m, 2H),
.sup.13CNMR (125 MHz, MeOD) 176.4, 176.1, 175.8, 160.1, 145.9,
125.2, 73.0, 71.5, 71.3, 70.8, 64.9, 59.1, 53.7, 53.5, 51.2, 32.8,
31.1, 30.7, 28.8, 23.4. HRMS (ES+) calc'd for
C.sub.32H.sub.57N.sub.5O.sub.16 (M+H) m/z 768.3834 Found 768.3865.
(S)-2-(3-((S)-5-(4-2,5,8,11,14,17,20,23,26,29,32,35,38-tridecaoxanonatria-
contyl-1H-1,2,3-triazol-1-yl)-1-carboxypentyl)ureido)pentanedioic
acid (12).
##STR00082##
Crude
2,5,8,11,14,17,20,23,26,29,32,35,38-tridecaoxahentetracont-40-yne
(s-13e) (58 mg, 0.097 mmol, 1 equiv.) and azide s-12 (50 mg, 0.097
mmol, 1 equiv.) were added to a mixture of water (0.350 mL) and
t-BuOH (0.350 mL). This slurry was placed in a microwave reaction
tube, to which a 0.1 M solution of sodium ascorbate in water (0.194
mL, 0.019 mmol, 0.2 equiv.) and 0.1 M solution of copper (II)
sulfate in water (0.039 mL, 0.004 mmol, 0.04 equiv.) were added.
The tube was capped and subjected to microwave irradiation at
110.degree. C. for 20 minutes. The crude mixture was concentrated
under reduced pressure, and taken up in 67% trifluoroacetic acid in
CH.sub.2Cl.sub.2 (3 mL). The tube was capped and subjected to
microwave irradiation at 70.degree. C. for 2 minutes. The crude
mixture was concentrated under reduced pressure, purified via HPLC,
and the pure fractions were collected and concentrated under
reduced pressure to yield 12 (13.0 mg, 9.6%, 3 steps) as a clear
oil. IR (thin film) 3369 (br), 2878 (s), 1673 (s), 1561 (w), 1456
(w), 1351 (w), 1200 (s), 1105 (s), 950 (w), 836 (w), 800 (w), 721
(w); .sup.1H NMR (500 MHz, MeOD) .delta. 8.00 (s, 1H), 4.64 (s,
2H), 4.42 (t, J=7.0 Hz, 2H), 4.33-4.24 (m, 2H), 3.69-3.59 (m, 46H),
3.56-3.52 (m, 2H), 2.48-2.34 (m, 2H), 2.19-2.09 (m, 1H), 2.02-1.81
(m, 4H), 1.71-1.64 (m, 1H), 1.45-1.33 (m, 2H). .sup.13CNMR (125
MHz, MeOD) 176.4, 176.1, 175.8, 160.1, 145.9, 125.2, 72.9, 71.5,
71.3, 70.7, 65.0, 59.1, 53.7, 53.5, 51.1, 32.9, 31.1, 30.8, 28.9,
23.4. LCMS (ES+) calc'd for C.sub.40H.sub.73N.sub.5O.sub.20 (M+H)
m/z 944.49 Found 944.72,
##STR00083##
(S)-2-(3-((S)-1-carboxy-5-(4-((2-(2-(2-nitrophenylamino)ethoxy)ethoxy)met-
hyl)-1H-1,2,3-triazol-1-yl)pentyl)ureido)pentanedioic acid
(13).
##STR00084##
2-nitro-N-(2-(2-(prop-2-ynyloxy)ethoxy)ethyl)aniline (s-6) (51.2
mg, 0.194 mmol, 1 equiv.) and azide s-12 (100 mg, 0.194 mmol, 1
equiv.) were added to a mixture of water (0.694 mL) and t-BuOH
(0.694 mL). This slurry was placed in a microwave reaction tube, to
which a 0.1 M solution of sodium ascorbate in water (0.388 mL,
0.039 mmol, 0.2 equiv.) and 0.1 M solution of copper (II) sulfate
in water (0.078 mL, 0.008 mmol, 0.04 equiv.) were added. The tube
was capped and subjected to microwave irradiation at 110.degree. C.
for 20 minutes. The crude mixture was concentrated under reduced
pressure, and taken up in 67% trifluoroacetic acid in
CH.sub.2Cl.sub.2 (3 mL). The tube was capped and subjected to
microwave irradiation at 70.degree. C. for 2 minutes. The crude
mixture was concentrated under reduced pressure, purified via HPLC,
and the pure fractions were collected and concentrated under
reduced pressure to yield 13 (36.5 mg, 31.0% over two steps) as a
dark yellow oil. IR (thin film) 3335 (br), 2923 (m), 1722 (s), 1668
(m), 1602 (s), 1561 (w), 1470 (w), 1308 (s), 1187 (m), 1111 (w),
998 (w), 836 (w); .sup.1H NMR (500 MHz, MeOD) .delta. 8.12 (dd,
J=8.6, 1.6 Hz, 1H), 7.98 (s, 1H), 7.49 (ddd, J=8.6, 6.9, 1.6 Hz,
1H), 7.02 (dd, J=8.7, 0.9 Hz, 1H), 6.67 (ddd, J=8.3, 6.9, 1.2 Hz,
1H), 4.65 (s, 2H), 4.41 (t, J=7.1 Hz, 2H), 4.30 (ddd, J=17.8, 8.4,
5.0 Hz, 2H), 3.78 (t, J=5.3 Hz, 2H), 3.71 (m, 4H), 3.53 (t, J=5.3
Hz, 2H), 2.44-2.40 (m, 2H), 2.21-2.10 (m, 1H), 2.00-1.81 (m, 4H),
1.72-1.65 (m, 1H), 1.47-1.35 (m, 2H). .sup.13CNMR (125 MHz,
DMSO-d.sub.6) .quadrature. 174.4, 174.1, 173.7, 157.2, 145.2,
143.9, 136.6, 131.0, 126.2, 123.6, 115.4, 114.7, 69.6, 68.9, 68.4,
63.6, 52.1, 51.6, 49.1, 42.0, 31.5, 29.9, 29.4, 27.5, 22.1. HRMS
(ES+) calc'd for C.sub.25H.sub.35N.sub.7O.sub.11 (M+H) m/z 610.2428
Found 610.2471.
(S)-2-(3-((S)-1-carboxy-5-(4-((2-(2-(4-nitrophenylamino)ethoxy)ethoxy)met-
hyl)-1H-1,2,3-triazol-1-yl)pentyl)ureido)pentanedioic acid
(14).
##STR00085##
4-nitro-N-(2-(2-(prop-2-ynyloxy)ethoxy)ethyl)aniline (s-7) (34.2
mg, 0.129 mmol, 1 equiv.) and azide s-12 (66.7 mg, 0.129 mmol, 1
equiv.) were added to a mixture of water (0.600 mL) and t-BuOH
(0.600 mL). This slurry was placed in a microwave reaction tube, to
which a 0.1 M solution of sodium ascorbate in water (0.260 mL,
0.026 mmol, 0.2 equiv.) and a 0.1 M solution of copper (II) sulfate
in water (0.051 mL, 0.005 mmol, 0.04 equiv.) were added. The tube
was capped and subjected to microwave irradiation at 110.degree. C.
for 20 minutes. The crude mixture was concentrated under reduced
pressure, and taken up in 67% trifluoroacetic acid in
CH.sub.2Cl.sub.2 (3 mL). The tube was capped and subjected to
microwave radiation at 70.degree. C. for 2 minutes. The crude
mixture was concentrated under reduced pressure, purified via HPLC,
and the pure fractions were collected and concentrated under
reduced pressure to yield 14 (21.7 mg, 27.6% over two steps) as a
yellow oil. IR (thin film) 3335 (br), 2924 (m), 2870 (m), 1722 (s),
1668 (m), 1602 (s), 1561 (m), 1505 (w), 1470 (w), 1308 (s), 1187
(m), 1118 (m), 837 (w); .sup.1H NMR (500 MHz, MeOD) .delta. 8.01
(d, J=10.4 Hz, 1H), 7.95 (s, 1H), 6.64 (d, J=10.4 Hz, 1H) 4.63 (s,
2H), 4.39 (t, J=7.0 Hz, 2H), 4.29 (ddd, J=16.4, 8.4, 5.0 Hz, 2H),
3.71-3.62 (m, 6H), 3.38 (t, J=5.4 Hz, 2H), 2.47-2.34 (m, 2H),
2.19-2.08 (m, 1H), 1.99-1.80 (m, 4H), 1.71-1.64 (m, 1H), 1.42-1.36
(m, 2H). .sup.13CNMR (125 MHz, DMSO-d.sub.6) .quadrature. 174.4,
174.1, 173.7, 157.3, 154.6, 143.8, 135.6, 126.2, 123.7, 110.8,
69.6, 68.9, 68.6, 63.5, 52.1, 51.6, 49.1, 42.3, 31.5, 29.9, 29.4,
27.5, 22.1. HRMS (ES+) calc'd for C.sub.25H.sub.35N.sub.7O.sub.11
(M+H) m/z 610.2428 Found 610.2468.
(S)-2-(3-((S)-1-carboxy-5-(4-((2-(2-(phenylamino)ethoxy)ethoxy)-
methyl)-1H-1,2,3-triazol-1-yl)pentyl)ureido)pentanedioic acid
(15).
##STR00086##
N-(2-(2-(prop-2-ynyloxy)ethoxy)ethyl)aniline (s-9) (35 mg, 0.160
mmol, 1 equiv.) and azide 31 (82 mg, 0.160 mmol, 1 equiv.) were
added to a mixture of water (1 mL) and t-BuOH (1 mL). This slurry
was placed in a microwave reaction tube, to which a 0.1 M solution
of sodium ascorbate in water (7.8 mg, 0.04 mmol, 0.25 equiv.) and a
0.1 M solution of copper (II) sulfate in water (0.080 mL, 0.008
mmol, 0.05 equiv.) were added. The tube was capped and subjected to
microwave irradiation at 110.degree. C. for 20 minutes. The crude
mixture was concentrated under reduced pressure, and taken up in
67% trifluoroacetic acid in CH.sub.2Cl.sub.2 (3 mL). The tube was
capped and subjected to microwave irradiation at 70.degree. C. for
2 minutes. The crude mixture was concentrated under reduced
pressure, purified via HPLC, and the pure fractions were collected
and concentrated under reduced pressure to yield 15 (13.7 mg, 15.3%
over two steps) as a light brown oil. IR (thin film) 3369 (br),
2939 (m), 1664 (s), 1563 (m), 1497 (w), 1438 (w), 1188 (s), 1134
(s), 837 (w), 798 (w), 753 (w), 721 (w); .sup.1H NMR (400 MHz,
MeOD) .delta. 8.02 (s, 1H), 7.58-7.49 (m, 5H), 4.64 (s, 2H), 4.37
(t, J=7.0 Hz, 2H), 4.33-4.24 (m, 2H), 3.70-3.63 (m, 6H), 3.27 (t,
J=5.4 Hz, 2H), 2.46-2.34 (m, 2H), 2.19-2.08 (m, 1H), 1.98-1.79 (m,
4H), 1.71-1.62 (m, 1H), 1.44-1.32 (m, 2H). .sup.13CNMR (125 MHz,
DMSO-d.sub.6) .quadrature. 174.4, 174.1, 173.7, 157.3, 146.2,
143.8, 129.1, 123.7, 118.3, 114.2, 69.6, 68.9, 68.2, 63.6, 52.1,
51.7, 49.1, 44.1, 31.5, 29.9, 29.4, 27.5, 22.1, HRMS (ES+) calc'd
for C.sub.25H.sub.36N.sub.6O.sub.9 (M+H) m/z 565.2577 Found
565.2621.
(S)-2-(3-((S)-1-carboxy-5-(4-((2-(2-(4-methoxyphenylamino)ethoxy)ethoxy)m-
ethyl)-1H-1,2,3-triazol-1-yl)pentyl)ureido)pentanedioic acid
(16).
##STR00087##
4-methoxy-N-(2-(2-(prop-2-ynyloxy)ethoxy)ethyl)aniline (s-10) (34
mg, 0.137 mmol, 1 equiv.) and azide s-12 (70 mg, 0.137 mmol, 1
equiv.) were added to a mixture of water (1 mL) and t-BuOH (1 mL).
This slurry was placed in a microwave reaction tube, to which a 0.1
M solution of sodium ascorbate (6 mg, 0.034 mmol, 0.25 equiv.) and
a 0.1 M solution of copper (II) sulfate in water (0.068 mL, 0.007
mmol, 0.05 equiv.) were added. The tube was capped and subjected to
microwave irradiation at 110.degree. C. for 20 minutes. The crude
mixture was concentrated under reduced pressure, and taken up in
67% trifluoroacetic acid in CH.sub.2Cl.sub.2 (3 mL). The tube was
capped and subjected to microwave irradiation at 70.degree. C. for
2 minutes. The crude mixture was concentrated under reduced
pressure, purified via HPLC, and the pure fractions were collected
and concentrated under reduced pressure to yield 16 (9.3 mg, 11.8%
over three steps) as a light brown oil. IR (thin film) 3347 (br),
2956 (m), 1728 (s), 1670 (s), 1564 (m), 1513 (m), 1443 (w), 1259
(w), 1200 (s), 1137 (m), 1029 (w), 837 (w); .sup.1H NMR (400 MHz,
MeOD) .delta. 8.01 (s, 1H), 7.45-7.39 (m, 2H), 7.09-7.04 (m, 2H),
4.68 (s, 2H), 4.42 (t, J=7.0 Hz, 2H), 4.30 (dd, J=8.6, 5.0 Hz, 1H),
4.22 (dd, J=8.5, 4.9 Hz, 1H), 3.87 (s, 3H), 3.75-3.73 (m, 2H),
3.72-3.68 (m, 2H), 3.68-3.64 (m, 2H), 3.57-3.50 (m, 2H), 2.49-2.33
(m, 2H), 2.17-2.12 (m, 1H), 2.01-1.79 (m, 4H), 1.72-1.57 (m, 1H),
1.45-1.32 (m, 2H). .sup.13CNMR (125 MHz, DMSO-d.sub.6) 174.4,
174.1, 173.7, 157.2, 143.7, 126.5, 123.7, 121.6, 117.4, 114.9,
69.7, 68.8, 65.9, 63.5, 55.4, 52.1, 51.6, 49.1, 48.4, 31.5, 29.9,
29.4, 27.5, 22.{tilde over (1)} HRMS (ES+) calc'd for
C.sub.26H.sub.38N.sub.6O.sub.10 (M+H) m/z 595.2683 Found 595.2722.
(S)-2-(3-((S)-1-carboxy-5-(4-((2-(2-(cyclohexylamino)ethoxy)ethoxy)methyl-
)-1H-1,2,3-triazol-1-yl)pentyl)ureido)pentanedioic acid (17).
##STR00088##
N-(2-(2-(prop-2-ynyloxy)ethoxy)ethyl)cyclohexanamine (s-11) (16 mg,
0.071 mmol, 1 equiv.) and azide s-12 (16 mg, 0.071 mmol, 1 equiv.)
were added to a mixture of water (0.500 mL) and t-BuOH (0.500 mL).
This slurry was placed in a microwave reaction tube, to which a 0.1
M solution of sodium ascorbate in water (3.4 mg, 0.018 mol, 0.25
equiv.) and a 0.1 M solution of copper (II) sulfate in water
(0.0355 mL, 0.00355 mmol, 0.05 equiv.) were added. The tube was
capped and subjected to microwave irradiation at 110.degree. C. for
20 minutes. The crude mixture was concentrated, and taken up in 67%
trifluoroacetic acid in CH.sub.2Cl.sub.2 (3 mL). The tube was
capped and subjected to microwave irradiation at 70.degree. C. for
2 minutes. The crude mixture was concentrated under reduced
pressure, purified via HPLC, and the pure fractions were collected
and concentrated under reduced pressure to yield 17 (7.9 mg, 19.7%
over two steps) as a clear oil. IR (thin film) 3344 (w), 2939 (m),
2865 (w), 1732 (m), 1670 (s), 1453 (w), 1201 (s), 1136 (m), 1088
(w), 799 (w); .sup.1H NMR (400 MHz, MeOD) .delta. 8.02 (s, 1H),
4.65 (s, 2H), 4.43 (t, J=7.0 Hz, 2H), 4.34-4.21 (m, 3H), 3.76-3.64
(m, 7H), 3.26-3.19 (m, 2H), 3.08 (s, 1H), 2.50-2.34 (m, 2H),
2.16-2.10 (m, 3H), 2.03-1.81 (m, 6H), 1.70-1.63 (m, 2H), 1.46-1.28
(m, 7H), 1.28-1.14 (m, 1H). .sup.13CNMR (125 MHz, DMSO-d.sub.6)
174.2, 173.9, 173.5, 157.1, 143.7, 123.7, 69.7, 69.5, 68.7, 68.3,
65.8, 63.4, 56.0, 49.1, 43.1, 31.4, 29.8, 29.3, 28.3, 27.4, 24.6,
23.8, 22.1. HRMS (ES+) calc'd for C.sub.25H.sub.42N.sub.6O.sub.9
(M+H) m/z 571.3047 Found 571.3091.
Biological Assays and Crystallographic Data
Measurement of PSMA K.sub.m:
[0167] A 10 mM solution of N-acetyl-aspartylglutamate (NAAG) in 40
mM NaOH, and was then diluted in Reaction Buffer (100 mM Tris-HCl,
pH 7.5) to a final NAAG concentration of 40 .quadrature.M. The
solution was added to a 384 well plate (20 .quadrature.L per well).
For K.sub.m measurements and controls the NAAG solution was
serially diluted 2-fold in Reaction Buffer to obtain final NAAG
concentrations ranging from 40 .quadrature.M-312.5 nM. rhPSMA (20
ng/mL in Reaction Buffer, 20 .quadrature.L, R&D Research) was
then added to each well. Reaction Buffer (20 .quadrature.L) was
added to the K.sub.m control series. The plate was incubated at
room temperature for 15 min, and then heated to 95.degree. C. for 3
minutes. The plate was allowed to cool to room temperature, and
glutamic acid levels were measured using a commercially available
Amplex.RTM.-Red Glutamic Acid/Glutamate Oxidase Assay Kit
(Invitrogen). Fluorescence intensities were measured using a
Synergy 2 multiwell plate reader (Biotek), fitted with excitation
and emission filters of 545 nm and 590 nm, respectively. The
K.sub.m was calculated using nonlinear least-squares regression
algorithms contained in the GraphPad Prism software package to
provide an average K.sub.m value for this enzymatic reaction of
0.925 .mu.M. This value is consistent with that reported in the
literature.sup.7 and was employed in subsequent K.sub.i
calculations (see below).
PSMA Inhibition Assay:
[0168] For IC.sub.50 measurements, inhibitors were dissolved in
Reaction Buffer containing 40 .mu.M NAAG to a final volume of 100
.mu.L. Then, 25 .mu.L of this solution was transferred to each of
three wells in a microtiter plate, and 5 .mu.L aliquots were
serially diluted into 20 .mu.L of solution containing 40 .mu.M NAAG
over 10 wells (5-fold dilutions). Inhibitor concentration therefore
ranged over 6 orders of magnitude in these experiments. rhPSMA (20
ng/mL in Reaction Buffer, 20 .mu.L, R&D Research) was then
added to each well. The plate was incubated at room temperature for
15 min, and then heated to 95.degree. C. for 3 minutes. The plate
was allowed to cool to room temperature, and glutamic acid levels
were measured using a commercially available Amplex.RTM.-Red
Glutamic Acid/Glutamate Oxidase Assay Kit (Invitrogen).
Fluorescence intensities were measured using a Synergy 2 multiwell
plate reader (Biotek), fitted with excitation and emission filters
of 545 nm and 590 nm, respectively. The concentration of inhibitors
giving 50% inhibition of enzyme activity (IC.sub.50) was calculated
from the least-squares regression line of the residual enzymatic
activity plotted as a function of logarithmic inhibitor
concentrations using algorithms contained in the GraphPad Prism
software package. K.sub.i values were obtained from IC.sub.50
values using the Cheng-Prusoff equation, a substrate concentration
of 20 .mu.M, and a K.sub.m value of 0.925 .mu.M. All reported
values in Tables 1 and 2 represent the average of at least three
replicates .+-.standard deviation.
rhPSMA Expression and Purification
[0169] The extracellular domain of human PSMA (amino acids 44-750)
was expressed and purified as described previously and we designate
this construct rhPSMA.sup.8. For crystallization experiments,
rhPSMA was dialyzed against 20 mM MOPS, 20 mM NaCl, pH 7.4, and
concentrated to 10 mg/mL.
Crystallization, Data Collection and Processing
[0170] The stock solutions of individual inhibitors at 50 mM were
prepared in 25% (v/v) acetonitrile in water. Diffraction quality
crystals of rhPSMA/ARMs complexes were grown at 293 K by vapor
diffusion in hanging drops. The stock solution of rhPSMA was mixed
in a 10:1 ratio with an ARM and hanging drops formed by mixing
equal volumes of the protein and reservoir solutions (33% (v/v)
pentaerythritol propoxylate PO/OH 5/4 (Hampton Research), 0.5%
(w/v) PEG 3350, and 100 mM Tris-HCl, pH 8.0). Prior to the data
collection, the crystals were flash-frozen in liquid nitrogen
directly from the hanging drop. Each of the four datasets was
collected from a single crystal at 100 K using synchrotron
radiation at the SER-CAT sector 22 beamlines of the Advanced Photon
Source (Argonne, Ill., USA) equipped with MAR225 or MAR300CCD
detectors. Data were integrated and scaled with the HKL2000
package.sup.9.
Electron Map Density Fit
[0171] Individual compounds were fit into the positive electron
density in the final stages of the refinement. For all four
inhibitors, clear interpretable densities were observed for the
C-terminal part encompassing the P1' glutarate, the urea linkage,
the lysine linker, and the triazole ring
Structure Solution and Refinement
[0172] Structure determination of rhPSMA/ARMs complexes was carried
out using difference Fourier methods with the ligand-free rhPSMA
(PDB code 2OOT;.sup.10) as a starting model. Calculations were
performed with the program Refmac 5.1.sup.11, and the refinement
protocol was interspersed with manual corrections to the model
employing the program Coot.sup.12. Library and PDB-format files of
individual inhibitors were prepared using the PRODRG server.sup.13
and the inhibitors were fitted into the positive electron density
map in the final stages of the refinement. During the refinement
process, .about.1% of the randomly selected reflections were kept
aside for cross-validation (R.sub.free). The quality of the final
model was evaluated using the MOLPROBITY server.sup.14. The data
collection and refinement statistics are summarized in Table
S1.
PDB Accession Numbers
[0173] Atomic coordinates of the present structures together with
the experimental structure factor amplitudes will be deposited in
the RCSB Protein Data Bank.
TABLE-US-00002 TABLE S1 Data calculations and refinement statistics
GCPII/ARM-P2 GCPII/ARM-P4 GCPII/ARM-P8 GCPIIIARM-M4 PDB code TBD
TBD TBD TBD Data Collection Statistics Wavelength ({acute over
(.ANG.)}) 1.0000 1.0000 1.0000 1.0000 Temperature (K) 100 Space
group I222 Unit-cell parameters: a = 101.5; a = 101.7; a = 101.8;
a=101.5; a, b, c ({acute over (.ANG.)}) b = 130.0; b = 130.0 b =
130.0; b = 130.0; c = 158.6 c = 159.0 c = 158.8 c = 159.2
Resolution limits ({acute over (.ANG.)}) 30.0-1.69 30.0-1.59
30.0-1.59 30.0-1.78 (1.75-1.69)* (1.65-1.59)* (1.63-1.59)*
(1.84-1.78)* Number of unique 114,649 137,271 137,748 100,565
reflections (9,759) (11,088) (10,972) (9,717) Redundancy 5.8 (2.5)
7.0 (5.0) 6.6 (3.8) 7.1 (5.6) Completeness (%) 97.8 (84.1) 97.6
(79.9) 96.9 (77.9) 99.7 (97.3) I/.sigma.(I) 18.4 (2.1) 27.8 (2.0)
15.4 (2.6) 21.3 (2.5) R.sub.merge 0.086 (0.492) 0.058 (0.501) 0.078
(0.438) 0.086 (0.520) Refinement Statistics Resolution limits
({acute over (.ANG.)}) 30.0-1.69 30.0-1.59 30.0-1.59 20.0-1.78
(1.73-1.69)* (1.63-1.59)* (1.63-1.59)* (1.82-1.78)* Total number of
112,878 (6,994) 135,823 (7,887) 135,631 (7,827) 99,006 (6,972)
reflections Number of 111,178 (6,872) 134,450 (7,815) 133,594
(7,726) 97,519 (6,871) reflections in working set Number of 1,700
(122) 1,373 (72) 2,037 (101) 1,487 (101) reflections in test set R
factor 16.0 (23.6) 16.8 (25.3) 16.1 (24.6) 15.7 (26.6) Free-R 18.5
(29.3) 19.1 (33.0) 18.3 (28.7) 18.5 (29.1) Total number of non-
6,491 6,618 6,687 6,546 H atoms Number of inhibitor 92.sup.# 52
128.sup.# 41 atoms Number of ions 4 4 4 4 Number of water 505 612
581 563 molecules Average B factor ({acute over (.ANG.)}.sup.2)
Protein atoms 28.0 25.9 24.5 18.7 Water molecules 38.4 36.3 37.4
27.4 Ligand atoms 40.3 48.5 51.8 48.8 r.m.s.d. Bond lengths ({acute
over (.ANG.)}) 0.021 0.018 0.017 0.021 Bond angles (.degree.) 1.85
1.71 1.69 1.72 Planarity ({acute over (.ANG.)}) 0.011 0.010 0.011
0.010 Chiral centers ({acute over (.ANG.)}.sup.3) 0.14 0.13 0.12
0.14 Ramachandran plot (%)** Most favored 97.7 97.7 97.7 97.8
Allowed 2.3 2.3 2.3 2.2 Disallowed 0 0 0 0 Missing residues 44-54;
654-655 44-54; 654-655 44-54; 654-655 44-54; 654-655 *Values in
parentheses correspond to the highest resolution shells
**Calculated with MOLPROBITY.sup.14 .sup.#inhibitor modeled in two
conformations
Computational Studies
Quantum Chemical Computations
[0174] Models of substituted methyl-amino-phenyls were constructed
using the software Maestro..sup.15 All calculations were carried
out using the Jaguar suite of electronic structure programs..sup.16
Geometry optimization was performed using Density functional theory
with a 6-41G*+basis set and the hybrid B3LYP functional..sup.17
Molecular Dynamics Simulations
[0175] The crystal structure of ARM-P2-DNP in complex with PSMA
(695 residues) was used to setup all the protein-ligand complexes.
MeO-P0, ARM-P0, ARM-P2, ARM-P4 and ARM-P8 were modeled in the same
protein structure on the basis of available experimental data. The
LigPrep module of the software Maestro was used to add missing
hydrogen atoms, choose the protonation state of protein side
chains, and minimize the energy of the protein-ligand
complex..sup.18 The 2005 update of the OPLS force field was used
throughout..sup.19 The resulting structure was then embedded in a
triclinic box of circa 13300 TIP3P water molecules,.sup.20 the
dimension of the box was circa 96.times.87.times.94 .ANG.. The net
charge of the system was neutralized by addition of one sodium ion
to the solvent box. The total number of atoms was circa 53,000
atoms. The simulations were performed with the Desmond molecular
dynamics package.sup.21. All bond lengths to hydrogen atoms were
constrained using MM-SHAKE..sup.22 Van der Waals and short range
electrostatic interactions were cut off at 9 .ANG.. Long range
electrostatic interactions were computed using the particle mesh
Ewald method using a 32.times.32.times.32 grid with .sigma.=2.18
.ANG. and fifth-order B-splines for interpolation..sup.23 A RESPA
integrator was used with a time-step of 2 fs, and the long range
electrostatic interactions were computed every 6 fs..sup.24 Each
system was initially energy minimized with steepest decent and then
subjected to the following equilibration protocol: 12 ps of
dynamics at 10 K in the NVT ensemble (Berendsen thermostat).sup.25
and harmonic restraints (50 kcal/mol/A.sup.2) on the solutes heavy
atoms, followed by 12 ps in the NPT ensemble (Berendsen thermostat
and barostat) at 10 K and retaining harmonic restraints on the
solutes heavy atoms, followed by 24 ps in the NPT ensemble
(Berendsen thermostat and barostat) at 300 K and retaining harmonic
restraints on the solutes heavy atoms, followed by 24 ps in the NPT
ensemble (Berendsen thermostat and barostat) at 300 K without
harmonic restraints on the solutes heavy atoms, followed by 100 ps
of dynamics at 300 K in the NPT ensemble (Martyna-Tobias-Klein
barostat and Nose-Hoover thermostat)..sup.26,27 The production
simulations were run for 50 nanoseconds in the NPT ensemble (300 K,
1 bar, Martyna-Tobias-Klein barostat and Nose-Hoover thermostat).
Coordinates were saved every 10 ps and analyzed using the software
Visual Molecular Dynamics..sup.28
Results and Discussion
Dependence of Linker Length on Binding Affinity
TABLE-US-00003 [0176] TABLE 1 Linker length dependence on PSMA
inhibitory potency. ##STR00089## Compound X n IC.sub.50 (nM).sup.b
K.sub.i (nM).sup.c 1, ARM-P0 DNP.sup.a 0 1.76 .+-. 0.41 0-078 .+-.
0.018 2, ARM-P1 DNP 1 1.05 .+-. 0.11 0.047 .+-. 0.005 3, ARM-P2 DNP
2 0.54 .+-. 0.18 0.024 .+-. 0.008 4, ARM-P4 DNP 4 0.46 .+-. 0.18
0.020 .+-. 0.008 5, ARM-P6 DNP 5 2.29 .+-. 0.50 0.101 .+-. 0.027 6,
ARM-P8 DNP 8 3.29 .+-. 1.14 0.145 .+-. 0.050 7, ARM-P12 DNP 12 37.3
.+-. 15.2 1.65 .+-. 0.72 8, MeO-P0 OMe 0 30.5 .+-. 12.1 1.35 .+-.
0.54 9, MeO-P2 OMe 2 40.8 .+-. 9.4 1.81 .+-. 0.42 10, MeO-P4 OMe 4
30.2 .+-. 14.4 1.34 .+-. 0.64 11, MeO-P8 OMe 8 131.0 .+-. 57.6 5.79
.+-. 2.54 12, MeO-P12 OMe 12 165.2 .+-. 58.9 7.30 .+-. 2.60
##STR00090## .sup.bIC.sub.50 values represent the mean of
triplicate experiments. .sup.cK.sub.i values were calculated from
IC.sub.50 and K.sub.m values using the Cheng-Prusoff equation as
described in the supporting information.
[0177] To evaluate in detail the effect of linker length on PSMA
binding affinity, we prepared various derivatives of ARM-P (Table
1, 1-12). These compounds consist of glutamate ureas linked to DNP
or methoxy groups by oxyethylene moieties of varying lengths. They
are named ARM-Px and MeO-Px, respectively, wherein "x" corresponds
to the number of oxyethylene units in the linker, Evaluation of
these compounds for their ability to inhibit PSMA activity proved
quite revealing. In all cases, ARM-P derivatives were found to
possess Ki values lower in magnitude than their counterparts
lacking DNP (compounds I-7 versus 8-12). In some cases, the
affinity difference was up to two orders of magnitude (compound 3
versus 9). This result indicated to us that perhaps the DNP
function itself might be playing a role in binding PSMA. Such a
hydrophobic interaction involving an aromatic ring and PSMA was not
completely unexpected given the proximity of the glutamate-urea
binding site to a known hydrophobic pocket in PSMA..sup.17,18
Indeed, inhibitors containing hydrophobic functionality distal to
the glutamic acid moiety have exhibited high potency against
PSMA..sup.12,19,20
[0178] A model involving binding of the DNP moiety to the
hydrophobic pocket adjacent to the S1 site did not explain the
decrease in affinity between ARM-P2 and derivatives with shorter
oxyethylene linkers (i.e., ARM-P0 and ARM-P1). Indeed, one would
expect ARM-P0 and ARM-P1 to exhibit enhanced potency versus ARM-P2
because of the close proximity of the accessory hydrophobic pocket
to the P1 glutamate binding cavity. The opposite trend suggested
perhaps the presence of an alternative hydrophobic binding site,
situated at a substantial distance away from this cavity. This
hypothesis is supported by observations in related systems in which
bifunctional ligands bind proteins at two remote sites. In such
systems, an ideal linker length between binding poles is required
for maximum affinity; linkers that are too short to access
secondary binding sites experience suboptimal enthalpic benefit
from bivalent binding, while linkers that are too long experience
high entropic costs upon bivalent binding..sup.21-23
[0179] Notably, compounds within the MeO-P series (8-12) containing
relatively short linkers all bind PSMA with comparable affinity.
The presence of linkers consisting of 8 oxyethylene groups or
longer appears to inhibit compound binding, a trend that can be
explained on steric grounds..sup.24 The increased sensitivity of
ARM-P derivatives to changes in linker length versus MeO-P
compounds suggests that factors other than simple steric bulk are
operating for the ARM-Ps.
Structure--Activity Studies: Effect of Varying Aromatic Groups on
Binding Affinity
TABLE-US-00004 [0180] TABLE 2 Dependence of K.sub.i on substituents
and electronics of aromatic ring. ##STR00091## Com- E.sub.HOMO
pound X (eV).sup.a IC.sub.50 (nM).sup.b K.sub.i (nM).sup.c 3, DNP
-0.251 0.54 .+-. 0.18 0.024 .+-. 0.008 ARM-P2 13 o-NO.sub.2-Ph
-0.228 1.78 .+-. 0.15 0.078 .+-. 0.007 14 p-NO.sub.2-Ph -0.231 1.36
.+-. 0.18 0.060 .+-. 0.008 15 Ph -0.201 16.6 .+-. 6.3 0.73 .+-.
0.28 16 p-MeO-Ph -0.181 21.9 .+-. 9.9 0.97 .+-. 0.44 17 Cyclohexyl
N/A 342.3 .+-. 170.5 15.1 .+-. 7.5 .sup.aE.sub.HOMO of the aryl
ring (X) was calculated using Density Functional Theory, and
reported in electron-volts. The hybrid functional B3LYP with a
6-31G*+ basis set was used. .sup.bIC.sub.50 values represent the
mean of triplicate experiments. .sup.cK.sub.i values were
calculated using IC.sub.50 and K.sub.m values via the Cheng-Prusoff
equation as outlined in the supporting information. A K.sub.m value
of 925 nM was using in these calculations.
[0181] To test further our model for bidentate binding, we set out
to probe the impact of the phenyl ring substituent on inhibitor
potency. We therefore synthesized analogues of ARM-P2 replacing the
DNP moiety with a range of electronically distinct aromatic species
(3, 13-17, Table 2). As shown, these analogues included nitrophenyl
(ortho and para to the linker), p-methoxyphenyl, phenyl, and
cyclohexyl derivatives. Consistent with the hypothesis for
multisite interaction, profound changes in affinity were observed
in this series. Interestingly, the parent dinitrophenyl-containing
compound (ARM-P2) possessed the highest potency of all the
analogues tested (Ki=24 pM), and removal of nitro groups led to
three-fold decreases in affinity in the p-nitrophenyl (13) and
o-nitrophenyl (14) analogues (Ki=60 and 78 pM, respectively). The
similarities between these analogues suggests that inhibitor
potency is dictated by electronic rather than steric effects.
Phenyl (15) and methoxyphenyl (16) analogues proved an additional
order of magnitude less potent than mononitrated derivatives
(Ki=730 and 970 pM, respectively), and the cyclohexyl-substituted
derivative (17) proved yet another order of magnitude worse than
the least potent aryl compounds (Ki=15.1 nM). This may result from
the enhanced steric bulk of the cyclohexyl substituent versus
planar arenes.
[0182] To quantify electronic effects in this system, we performed
density functional theory (DFT) calculations to relate the electron
density of the aromatic ring to PSMA inhibitory potency..sub.25 For
substituted arenes this can be conveniently quantified by
calculating the arene HOMO energy. An excellent correlation was
observed between the HOMO energies of aromatic substituents and
experimentally determined K.sub.i values (FIG. 15). Electron poor
aromatic rings are expected to experience strong .pi.-stacking
interactions with electron-rich arenes,.sup.26,27 suggesting
perhaps that such interactions may be dominant in dictating binding
affinity in this system. These results are strongly indicative of
multisite binding in the ARM-P series, and led us to test this
hypothesis further using X-ray crystallography.
Crystallographic Studies
[0183] Initial refinement and analysis. Crystal structures were
determined for PSMA in complex with ARM-P ligands containing 2, 4,
and 8 oxyethylene units in the linker region (3, 4, and 6) and with
MeO-P4 (10), which lacks the DNP moiety, and were refined at the
resolution of 1.69 .ANG., 1.59 .ANG., 1.59 .ANG., and 1.78 .ANG.,
respectively. Individual compounds were fit into the positive peaks
on the difference F.sub.o-Fc electron density map in the final
stages of refinement. For all four inhibitors, clear interpretable
densities were observed for the C-terminal part encompassing the
P1' glutarate, the urea linkage, the lysine linker and the triazole
ring. Although density corresponding to the DNP phenyl ring is
defined in all ARM-P complexes, density corresponding to the nitro
groups is absent suggesting that the DNP moiety is present in at
least two different conformations. Also, electron density peaks
corresponding to the poly-oxyethylene linker were absent from all
complexes, consistent with a lack of intermolecular contacts
between this flexible element and the protein.
[0184] Structures of ARM-P2, ARM-P4, and ARM-P8 in complex with
PSMA are depicted in FIG. 16. Despite the attachment of large
oxyethylene linkers, the glutamate urea portions of all inhibitors
interact with the protein active site in a fashion reminiscent of
previously reported complexes with urea12,13,17 and phosphonate28
inhibitors, and the substrate N-acetyl-aspartyl-glutamate (NAAG).29
In all structures, positioning of the P1' glutarate is enforced by
H-bonds (indicated as dashed lines) with Arg210, Asn257, Tyr552,
Lys699, Tyr700, and active-site water molecules, and hydrophobic
interactions with the side chains of Phe209 and Leu428. The ureido
nitrogen atoms serve as H-bond donors in interactions with Glu424
and the Gly518 main chain carbonyl, and the carbonyl oxygen makes
contacts with both the catalytic zinc atom and Tyr552, and His553.
The P1 .alpha.-carboxylate in all inhibitors structurally overlaps
with the equivalent groups of previously reported complexes, and is
held in place by interactions with an arginine-rich patch (Arg463,
Arg534, Arg536) along with H-bonding contacts to Asn519, the Ser517
main-chain carbonyl, and water molecules,.sup.17,18
[0185] Discovery of an arene-binding cleft. A key site of
interaction between PSMA and all ARM-P derivatives is the triazole
ring, which was observed to pack against the side chains of Tyr552
and Tyr700 in all complexes (FIG. 16). The steric hindrance caused
by the oxyethylene linker emanating from the triazole ring prevents
closure of the enzyme's entrance lid (amino acids Trp541-Gly548),
as observed for PSMA complexes with smaller ligands..sup.18 A key
consequence of the entrance lid's open conformation is the
revelation of a previously unreported binding cleft for the DNP
ring (FIG. 17)..sup.18
[0186] The arene-binding region, formed from the indole group of
Trp541 and the guanidinium group of Arg511 holds the DNP ring in
close contact with these groups at distances of 3.1 .ANG. and 3.9
.ANG., respectively. The bottom of the cleft is lined by the Arg463
side chain. Positioning of the phenyl ring creates a plane
virtually parallel to both indole and guanidinium functionalities,
suggesting that simultaneous .pi.-cation (DNP-Arg511) and
.pi.-stacking (DNP-Trp541) interactions may both contribute to
inhibitor binding..sub.31,32 Critically, the arene-binding region
is only revealed upon opening of the entrance lid (FIG. 17B);
closure of the entrance lid, as in the overlaid complex between
PSMA and the small urea-based inhibitor DCIBzL,.sub.30 would lead
to significant steric overlap with the triazole moiety as well as
closure of the arene-binding site (FIG. 17C). Thus, the protein is
capable of adopting two separate conformations, each suited to
accommodate high-affinity binding interactions with distinct
classes of glutamate-urea inhibitors.
[0187] A key structural feature was observed in the MeO-P4 complex
(FIG. 18). Here, unlike in the ARMP complexes, Trp541 exists in two
distinct conformations. The non-stacking conformation is rotated
approximately 4 .ANG. from what is seen in ARM-P complexes, and
blocks the arene-binding groove. The conformational flexibility
exhibited by Trp541 in the PSMA/MeO-P4 complex suggests that when
present, the dinitroarene stabilizes the side chain indole moiety
via .pi.-stacking, as implied by the ARMP structures depicted
above. Taken together, these data provide strong support for a
model in which ARM-Ps bind PSMA through interactions at both the
enzyme active site and at a newly reported arenebinding cleft.
Notably, the complex between PSMA and MPE,.sub.33 a
methotrexate-derived phosphonate, was also shown to possess an open
entrance lid like the complexes disclosed herein..sup.18 It was
concluded from the PSMA/MPE complex that the protein's ability to
adopt an open conformation serves to enable its binding to
relatively large substrates, such as folyl-poly-.gamma.-glutamates.
One might imagine that the revelation of an arene-binding site upon
opening of the entrance lid might serve to enhance affinity for
these arene-containing enzyme substrates. Interestingly, however,
the pendant pteroyl ring in the MTE complex was not observed to
interact with the PSMA arene-binding cleft, perhaps due to its
relatively short linkage to the zinc-binding phosphonate region.
The observations reported herein suggest that perhaps larger
natural poly-.gamma.-glutamate substrates are able to make use of
the arene-binding site, however further studies are necessary to
test this possibility.
Molecular Dynamics (MD) Simulations
[0188] To clarify the nature of the protein-ligand interactions in
the ARM-P complexes, explicit solvent molecular dynamics (MD)
simulations were carried out using crystallographic data for PSMA
complexes with ARM-P0, ARMP-P2, ARM-P4, ARM-P8 and MeO-P0. Each
protein-ligand complex was modeled with the OPLS-AA force field,34
embedded in a triclinic box of TIP3P water molecules.35 Dynamics
were simulated for 50 ns using the Desmond software package..sup.36
These simulations revealed a number of noteworthy features (see
Supporting Information for video files for all simulations).
Although the PSMA active site and glutamate urea moieties are
fairly rigid throughout the timescale of MD simulations, distal
protein-ligand interactions exhibit highly dynamic behavior. For
example, the simulation of the MeO-P0-PSMA complex revealed that
the arene-binding site is unstable in the absence of DNP; Trp541
tends to rotate toward Arg511, thus obscuring the arenebinding site
(FIG. 19, panels a-c). This observation directly correlates with
the disorder in Trp541 observed in the MeO-P4-PSMA crystal
structure (FIG. 16). Furthermore, the PEG moieties in all complexes
are highly dynamic and do not seem to form specific interactions
with PSMA, suggesting that these make minimal enthalpic
contributions to binding affinity. These observations also explain
the absence of electron density corresponding to linker regions in
all crystal structures.
[0189] By far the most stable intermolecular contact in the
arene-binding site in ARM-P-PSMA complexes is the stacking
interaction between DNP and Trp541. For all ARMs, the DNP moieties
participate in face-to-face interactions with Trp541 side chain
indole moieties for significant time periods throughout MD
simulations. Simulations of the ARM-P0-PSMA complex revealed a
remarkable level of flexibility in the triazole-alkyl region, which
enables .pi.-stacking contacts in the arene-binding site to remain
intact even in the absence of an oxyethylene linker (FIG. 19,
panels d-f). When stacked with the Trp541 side chain indole, the
DNP ring is observed to rotate in-plane, supporting the hypothesis
that the lack of well defined electron density corresponding to
nitro groups in crystal structures is due to the presence of
multiple arene conformations. However, in all ARM-P complexes, the
nitro groups in the DNP ring are frequently observed pointing
toward the Arg463 side chain guanidinium group, suggesting possible
hydrogen bonding or electrostatic interactions between these
groups. Furthermore, although crystallographic data support a role
for .pi.-cation interactions with Arg511 in the arene-binding site,
this residue is highly disordered in MD simulations, and does not
form long-lived contacts with the ligand. This observation is
consistent with the data presented in Table 2 and FIG. 15, which
suggest that cation-.pi. interactions play a relatively minor role
versus .pi.-stacking interactions in stabilizing these systems.
Notably, during the course of MD simulations for both ARM-P2
(panels g-i) and ARM-P8 (panels m-o), the DNP ring dissociates from
the arene-binding cleft, whereas this interaction remains intact in
the ARM-P4 simulation (panels j-l). Taken together, these data
suggest that the DNP-Trp541 interactions are relatively weak.
Interestingly, the contact with Trp541 reforms rapidly during the
simulations of ARM-P2, but not ARM-P8; this likely reflects both
the high entropic penalty associated with bivalent binding in
ARM-P8 as well as the tendency for the molecule's lengthy PEG
linker to occupy the arene-binding site, thus preventing the DNP
group's return to Trp541. From a functional standpoint, the
propensity of ARM-P8 to disengage from the PSMA arene-binding site
enables it to form ternary complexes with prostate cancer cells and
antibodies, which is critical to its cytotoxic activity..sup.3
However, this functionality comes at the expense of PSMA binding
affinity. This model suggests the possibility of ultra
high-affinity ARM-P analogues capable of interacting simultaneously
with the PSMA arene-binding site and anti-DNP antibodies.
CONCLUSION
[0190] In the present application we have detailed the discovery of
an arene-binding site on pro state-specific membrane antigen
(PSMA), which gives rise to unusually high affinity binding
interactions with designed bifunctional antibody-recruiting small
molecules (ARMs). The conclusions presented herein are supported by
extensive crystallographic, biochemical, and computational data,
which, taken together, strongly suggests a model in which bidentate
binding of ARM-Ps to PSMA leads to substantial increases in
inhibitor potency. The serendipitous nature of the discovery
reported herein along with the relative simplicity of the PSMA
arene-binding site--which consists merely of three amino acids only
one of which (Trp541) is responsible for affinity
enhancement--suggest that low-affinity binding sites for arenes
could be quite prevalent among proteins. Along these lines, it is
well-documented that a large proportion of circulating
immunoglobulin possess high-affinity binding activity against
nitroarene ligands,38 and between 1 and 10% of myeloma proteins
bind nitrophenyl ligands..sup.39 The possibility that such binding
sites arise from conserved folds within immunoglobulin domains has
been suggested,.sup.40 however, this trend may also result from the
unique immunogenicity of nitroarenes,.sup.41,42 a property that has
also been attributed to their propensity to form hydrophobic
contacts with proteins..sub.41 In either case, although structural
data exists demonstrating the unique propensity of nitroarenes to
engage in .pi.-stacking interactions with aromatic amino acid side
chains,.sub.43,44 the proteomic prevalence of nitroarene-binding
motifs has not been systematically explored. The widespread
existence of such binding sites could enable facile optimization of
small molecule ligands for proteins identified through
high-throughput screening, and could find ready utility in
fragment-based approaches to inhibitor design..sup.45
[0191] Although underexplored, strategies that utilize small
molecules to enhance recognition of pathogens by the human immune
system promise to leverage the strengths of both antibody- and
small-moleculebased therapeutic approaches. The results reported
herein suggest the possibility for improving such technologies for
treating prostate cancer. For example, ultra-high-affinity ARM-Ps
could be constructed by exploiting the presence of the
arene-binding site in PSMA and converting the highly flexible first
generation ARM-Ps into more rigid scaffolds. More broadly, the
high-level expression of PSMA (GCPII) on prostate cancer cell
surfaces and on tumor neovasculature,.sup.46 as well as its
putative role in the pathophysiology of schizophrenia,.sup.47 have
rendered it an extremely useful and popular target for inhibitor
design. The results presented herein therefore could substantially
impact the development of effective diagnostic and therapeutic
approaches for patients suffering from cancer and other
diseases.
[0192] The complete disclosure of all patents, patent applications,
and publications, and electronically available material (including,
for instance, nucleotide sequence submissions in, e.g., GenBank and
RefSeq, and amino acid sequence submissions in, e.g., SwissProt,
PIR, PRF, PDB, and translations from annotated coding regions in
GenBank and RefSeq) cited herein are incorporated by reference. Any
inconsistency between the material incorporated by reference and
the material set for in the specification as originally filed shall
be resolved in favor of the specification as originally filed. The
foregoing detailed description and examples have been given for
clarity of understanding only. No unnecessary limitations are to be
understood therefrom. The invention is not limited to the exact
details shown and described, for variations obvious to one skilled
in the art will be included within the invention defined by the
following claims.
[0193] All headings are for the convenience of the reader and
should not be used to limit the meaning of the text that follows
the heading, unless so specified.
* * * * *