U.S. patent application number 09/530307 was filed with the patent office on 2001-08-16 for novel metal complexes.
Invention is credited to GLEASON, JOHN G., LUENGO, JUAN I..
Application Number | 20010014454 09/530307 |
Document ID | / |
Family ID | 22052594 |
Filed Date | 2001-08-16 |
United States Patent
Application |
20010014454 |
Kind Code |
A1 |
GLEASON, JOHN G. ; et
al. |
August 16, 2001 |
NOVEL METAL COMPLEXES
Abstract
Invented are zinc chelated dimeric cell-surface receptor
ligands, pharmaceutical compositions containing these compounds,
and methods of using these compounds as agonist of dimeric
cell-surface receptors. Also invented are novel processes used in
preparing these compounds. Also invented are novel receptor binding
moieties of the invented zinc chelated cell-surface receptor
ligands.
Inventors: |
GLEASON, JOHN G.;
(DOWNINGTOWN, PA) ; LUENGO, JUAN I.; (AUDUBON,
PA) |
Correspondence
Address: |
SMITHKLINE BEECHAM CORPORATION
CORPORATE INTELLECTUAL PROPERTY-US, UW2220
P. O. BOX 1539
KING OF PRUSSIA
PA
19406-0939
US
|
Family ID: |
22052594 |
Appl. No.: |
09/530307 |
Filed: |
April 27, 2000 |
PCT Filed: |
October 30, 1998 |
PCT NO: |
PCT/US98/23186 |
Current U.S.
Class: |
435/7.1 |
Current CPC
Class: |
A61P 7/04 20180101; A61P
31/12 20180101; A61P 43/00 20180101; G01N 2333/705 20130101; A61P
31/10 20180101; C07D 487/04 20130101; C07F 3/003 20130101; A61P
21/02 20180101; A61K 31/555 20130101; G01N 33/566 20130101; A61P
31/04 20180101; A61K 31/315 20130101; A61P 3/04 20180101; A61P 3/10
20180101; A61P 1/16 20180101 |
Class at
Publication: |
435/7.1 |
International
Class: |
G01N 033/53 |
Claims
What is claimed is:
1. A method for agonizing dimeric cell-surface receptors in a
subject in need thereof which comprises administering to the
subject a therapeutically effective amount of a zinc chelated
dimeric cell-surface receptor ligand.
2. A method for agonizing dimeric cell-surface receptors in a
subject in need thereof which comprises administering to the
subject a therapeutically effective amount of an organic molecule
having a molecular weight from about 100 to about 850, containing
of from 1 to 4 zinc binding motifs and provided that each zinc
binding motif forms at least two coordinate bonds to a zinc
ion.
3. A method for identifying agonists of dimeric cell-surface
receptors which comprises contacting the receptor with dimeric
cell-surface receptor ligand candidates in the presence of a zinc
ion source, and selecting ligand candidates which bind to the
receptor.
4. A process for the preparation of a zinc chelated dimeric
cell-surface receptor ligand which comprises reacting one or more
receptor binding moieties and a zinc ion source followed by
optional isolation of the zinc chelated dimeric cell-surface
receptor ligand.
5. A zinc chelated cell-surface receptor ligand prepared by the
process of claim 4.
6. A zinc chelated cell-surface receptor ligand.
7. A pharmaceutical composition comprising a pharmaceutically
acceptable carrier and a compound of claim 5.
8. A pharmaceutical composition comprising a pharmaceutically
acceptable carrier and a compound of claim 6.
9. A process for preparing a pharmaceutical composition containing
a pharmaceutically acceptable carrier or diluent and an effective
amount of a compound of claim 5 which process comprises bringing
the compound of claim 5 into association with the pharmaceutically
acceptable carrier or diluent.
10. A process for preparing a pharmaceutical composition containing
a pharmaceutically acceptable carrier or diluent and an effective
amount of a compound of claim 6 which process comprises bringing
the compound of claim 6 into association with the pharmaceutically
acceptable carrier or diluent.
11. An isolated dimeric cell-surface receptor binding moiety of a
zinc chelated cell-surface receptor ligand.
12. A dimeric cell-surface receptor binding moiety.
13. The method of claim 1 wherein the cell-surface receptor is the
EPO receptor.
14. The method of claim 2 wherein the cell-surface receptor is the
EPO receptor.
15. A method for agonizing the EPO receptor in a subject in need
thereof which comprises administering to the subject a
therapeutically effective amount of a compound of claim 11.
16. A method for agonizing the EPO receptor in a subject in need
thereof which comprises administering to the subject a
therapeutically effective amount of a compound of claim 12.
17. A pharmaceutical composition comprising a pharmaceutically
acceptable carrier and a compound of claim 11.
18. A pharmaceutical composition comprising a pharmaceutically
acceptable carrier and a compound of claim 12.
19. A process for preparing a pharmaceutical composition containing
a pharmaceutically acceptable carrier or diluent and an effective
amount of a compound of claim 11 which process comprises bringing
the compound of claim 11 into association with the pharmaceutically
acceptable carrier or diluent.
20. A process for preparing a pharmaceutical composition containing
a pharmaceutically acceptable carrier or diluent and an effective
amount of a compound of a compound of claim 12 which process
comprises bringing the compound of claim 12 into association with
the pharmaceutically acceptable carrier or diluent.
21. A method for agonizing dimeric cell-surface receptors in a
subject in need thereof which comprises administering to the
subject a therapeutically effective amount of a compound of claim
11.
22. A method for agonizing dimeric cell-surface receptors in a
subject in need thereof which comprises administering to the
subject a therapeutically effective amount of a compound of claim
12.
23. A compound of claim 11 for use as an active therapeutic
substance.
24. A compound of claim 12 for use as an active therapeutic
substance.
25. Use of a compound of claim 11 in the manufacture of a
medicament for use in therapy.
26. Use of a compound of claim 12 in the manufacture of a
medicament for use in therapy.
27. The method of claim 1 wherein the dimeric cell-surface receptor
is the M-CSF receptor.
28. The method of claim 2 wherein the dimeric cell-surface receptor
is the M-CSF receptor.
29. A method for agonizing the M-CSF receptor in a subject in need
thereof which comprises administering to the subject a
therapeutically effective amount of a compound of claim 11.
30. A method for agonizing the M-CSF receptor in a subject in need
thereof which comprises administering to the subject a
therapeutically effective amount of a compound of claim 12.
31. The method of claim 1 wherein the dimeric cell-surface receptor
is the GRH receptor.
32. The method of claim 2 wherein the dimeric cell-surface receptor
is the GRH receptor.
33. A method for agonizing the GRH receptor in a subject in need
thereof which comprises administering to the subject a
therapeutically effective amount of a compound of claim 1.
34. A method for agonizing the GRH receptor in a subject in need
thereof which comprises administering to the subject a
therapeutically effective amount of a compound of claim 12.
35. The method of claim 1 wherein the dimeric cell-surface receptor
is the TPO receptor.
36. The method of claim 2 wherein the dimeric cell-surface receptor
is the TPO receptor.
37. A method for agonizing the TPO receptor in a subject in need
thereof which comprises administering to the subject a
therapeutically effective amount of a compound of claim 1.
38. A method for agonizing the TPO receptor in a subject in need
thereof which comprises administering to the subject a
therapeutically effective amount of a compound of claim 12.
39. The method of claim 1 wherein the dimeric cell-surface receptor
is the leptin receptor.
40. The method of claim 2 wherein the dimeric cell-surface receptor
is the leptin receptor.
41. A method for agonizing the leptin receptor in a subject in need
thereof which comprises administering to the subject a
therapeutically effective amount of a compound of claim 1.
42. A method for agonizing the leptin receptor in a subject in need
thereof which comprises administering to the subject a
therapeutically effective amount of a compound of claim 12.
43. The method of claim 1 wherein the dimeric cell-surface receptor
is the IFN receptor.
44. The method of claim 2 wherein the dimeric cell-surface receptor
is the IFN receptor.
45. A method for agonizing the IFN receptor in a subject in need
thereof which comprises administering to the subject a
therapeutically effective amount of a compound of claim 11.
46. A method for agonizing the IFN receptor in a subject in need
thereof which comprises administering to the subject a
therapeutically effective amount of a compound of claim 12.
47. The method of claim 1 wherein the dimeric cell-surface receptor
is the insulin receptor.
48. The method of claim 2 wherein the dimeric cell-surface receptor
is the insulin receptor.
49. A method for agonizing the insulin receptor in a subject in
need thereof which comprises administering to the subject a
therapeutically effective amount of a compound of claim 1.
50. A method for agonizing the insulin receptor in a subject in
need thereof which comprises administering to the subject a
therapeutically effective amount of a compound of claim 12.
51. The method of claim 1 wherein the dimeric cell-surface receptor
is selected from the TRK receptors.
52. The method of claim 2 wherein the dimeric cell-surface receptor
is selected from the TRK receptors.
53. A method for agonizing a selected TRK receptor in a subject in
need thereof which comprises administering to the subject a
therapeutically effective amount of a compound of claim 11.
54. A method for agonizing a selected TRK receptor in a subject in
need thereof which comprises administering to the subject a
therapeutically effective amount of a compound of claim 12.
55. The method of claim 1 wherein said metal chelated dimeric
cell-surface receptor ligand comprises a symmetrical multimer of a
receptor binding moiety.
Description
FIELD OF THE INVENTION
[0001] This invention relates to metal complexed receptor ligands,
methods for making and identifying them and their use as agonist of
dimeric receptors. More specifically, the invention describes a
method to promote the oligomerization of dimeric receptors.
BACKGROUND OF THE INVENTION
[0002] Many soluble proteins, such as cytokines, hormones and
growth factors, exert their functions by binding and activating
cell-surface receptors (Arai, K. -I. et al. Annu. Rev. Biochem.
1990, 59, 783; Bazan, J. F. Proc. Natl. Acad. Sci. U.S.A. 1990, 87,
6934; Ullrich A. and Schlessinger, J. Cell, 1990, 61, 203-212).
These receptors are comprised of three distinct domain, an
extracellular ligand-binding domain, a transmembrane domain and a
cytoplasmic domain, which is responsible for signal transduction
within the cell. Some receptors, such as those for erythropoietin
(EPO), thrombopoietin (TPO), and granulocyte-colony stimulating
factor (G-CSF), contain the ligand-binding and signal-transduction
domains within the same polypeptide subunit. Others, such as
receptors for interleukin-2 (IL-2), IL-3 and IL-6 have separate
components for ligand-binding and signal transduction. Although the
mechanism of receptor activation varies for specific
receptor-ligand pairs, a common feature of many
single-transmembrane receptors appears to be their aggregation on
the cell membrane in response to binding of their specific ligands.
This aggregation event can be in the form of homodimerization, in
the case of receptors with a single subunit, or heterodimerization,
in the case of receptors with different subunits. It has become
clear that receptor aggregation is part of the biological signal by
which the target cell responds to the presence of specific hormones
and growth factors (Young, P. R. "Protein hormones and their
receptors", Curr. Opin. Biotech. 1992. 3, 408-421; Heldin. C. H.,
"Dimerization of cell surface receptors in signal transduction).
Typical examples of such receptors are growth factor receptors with
tyrosine kinase activity as well as cytokine receptors.
[0003] Monoclonal antibodies have been discovered which have
agonist activity to the dimeric receptors such as those from
epidermal growth factor (EGF, Fernandez-Pol, J. J. Biol. Chem.
1985, 260, 5003-11; Serrero, G. U.S. Pat. No. 5,723,115), G-CSF
(Takahashi, T. et al. J. Biol. Chem. 1996, 271, 17555-17560), tumor
necrosis factor (TNF, Fine, S. M. et al. J. Biol. Chem. 1996,
27126, 15303-15306.), growth hormone receptor (Rowlinson, S. W. et
al. J. Biol. Chem. 1998, 2739, 5307-5314, EPO (Young, P. R. and
Erickson-Miller, C. L. WO 9640231; Chaovapong, W. L. et al. WO
9748729.) and gp 130, the common chain for members of the IL-6
family (Fourcin, M. et al. J. Biol. Chem. 1996, 271, 11756-11760).
Ability of the monoclonal antibodies to activate the receptors is
believed to be due to the presence of the two antigen binding
sites, which can bridge the two receptor subunits and facilitate
aggregatior.
[0004] More recently peptides with agonist activity were identified
by screening of phage display libraries against the EPO (Wrighton,
N. C. et al. Science 1996, 273, 458-463; Wrighton, N. C. et al.
U.S. Pat. No. 5,773,569) and TPO receptors (Cwirla, S. E. et al.
Science 1997, 276, 1696-1699; Dower, W. J. et al. WO 9640750). The
peptides ranged from 14 to 20 residues and activated the receptors
by promoting their dimerization on the cell surface. These agonist
peptides are unrelated to EPO and TPO and appear to act as dimeric
agents, as demonstrated in the crystal structure of the EMP1/EBP
complex (Livnah, O. et al. Science 1996, 273, 454-471).
[0005] Despite the success of monoclonal antibodies and dimeric
peptides in eliciting agonist response in certain dimeric
receptors, they are not generally considered desirable candidates
for development of pharmaceutical compositions. Lack of oral
bioavailability and a limited serum half-life limit the
desirability and efficacy of monoclonal antibodies and polypeptides
as pharmaceutical agents. Consequently, a need exists for
non-antibody ligands which have agonist properties towards dimeric
cell-surface receptors.
[0006] Notwithstanding the fact that these receptors have been the
subject of such research efforts for over a decade, only one
application (PCT/US97/08864) describes small organic molecules
which exhibit agonist activity towards dimeric cell-surface
receptors. This application does not mention zinc chelated small
organic molecules.
[0007] As disclosed herein it has unexpectedly been discovered that
zinc chelated receptor ligands have agonist properties towards
dimeric cell-surface receptors.
SUMMARY OF THE INVENTION
[0008] Accordingly, one aspect of the present invention is a method
for agonizing dimeric cell-surface receptors which comprises
contacting the receptor with a zinc chelated receptor ligand.
[0009] Another aspect of the invention is a method for identifying
agonists of dimeric cell-surface receptors.
[0010] A third aspect of the invention relates to zinc chelated
dimeric cell-surface receptor ligands.
[0011] A fourth aspect of the invention relates to an isolated
receptor binding moiety of a zinc chelated dimeric cell-surface
receptor ligand.
[0012] A fifth aspect of the invention is a method for making zinc
chelated dimeric cell-surface receptor ligands.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 shows the activity of two different samples of
compound 1a (from Example 1) on the murine myeloid cell line NFS60
that contained a G-CSF-responsive element linked to a minimal
promoter and the gene for luciferase. Activity of compound 1a is
below the threshold of 150% over background. The study was
performed as a Luciferase assay configured on the G-CSF-responsive
NFS60 cell line as described in Tian et al., Science 281, 257-259
(1998). The experiments shown in FIGS. 2-8 used the same NFS60 cell
line.
[0014] FIG. 2 shows the same type of experiment in NFS60 cells, but
run in the presence of 1 uM zinc (II). The activity of compound 1a
is about 350% over control, which indicates that zinc(II)
potentiates the activity of compound 1a.
[0015] FIG. 3 is an analysis of the effect of
ethylenediaminetetraacetic acid (as used herein--EDTA) on the
activity of Compound 1a (from Example 1) in NFS60 cells. Shown are
luciferase response curves of Compound 1a at the indicated
concentrations and in the presence of various concentrations of
EDTA. EDTA at 1.2 millimolar concentration antagonized the activity
of compound 1a. The media in this assay contained a small amount
(1-5 uM) of zinc(II).
[0016] FIG. 4 is an analysis of the effect of EDTA on the activity
of recombinant G-CSF on NFS60 cells. Shown are luciferase response
curves of recombinant G-CSF at the indicated concentrations and in
the presents of various concentrations of EDTA. EDTA at both 1.2
and 5 millimolar has little effect on the activity of recombinant
G-CSF in the assay.
[0017] FIGS. 5 and 6 depict an analysis of the activity of metal
chlorides alone on the basal luciferase level of NFS60 cells. Shown
are luciferase response curves of the indicated metal chlorides at
various concentrations. None of the metals have a meaningful effect
on the basal luciferase levels at concentrations equal or less than
10 micromolar.
[0018] FIGS. 7 and 8 show an analysis of the effect of metal
chlorides on the EDTA depleted activity of Compound 1a on NFS60
cells. Shown are luciferase response curves as effected by the
indicated metal chlorides. Only zinc (II) at concentrations 0.5-10
micromolar can overcome the inhibition in luciferase activity
caused by 50 micromolar concentration of the metal chelator EDTA.
None of the other metals tested could overcome the inhibitory
effect of EDTA.
DETAILED DESCRIPTION OF THE INVENTION
[0019] All publications, including but not limited to patents and
patent applications, cited in this specification are herein
incorporated by reference as though fully set forth.
[0020] By the term "heteroatom(s)", as used herein is meant
nitrogen, oxygen or sulfur, preferably nitrogen.
[0021] By the term "treating" and derivatives thereof as used
herein, is meant prophylatic or therapeutic therapy.
[0022] By the term "organic molecule" and derivatives thereof as
used herein, is meant the standard usage in the art to the ordinary
organic chemist and as such excludes inorganic molecules and
peptide molecules.
[0023] The zinc chelated receptor ligands of this invention that
have agonist properties towards dimeric cell-surface receptors are
compounds that consist of one or more receptor binding moieties,
preferably 1 to 4 moieties, most preferably 1 or 2 moieties,
wherein each receptor binding moiety forms at least two coordinate
bonds to each of one or more zinc ions, preferably each moiety will
form two or three coordinate bonds to each of one or two zinc
ions.
[0024] By the term "receptor binding moiety", and derivatives
thereof, as used herein means a small organic molecule having a
molecular weight from about 100 to about 850, preferably having a
molecular weight from about 200 to about 750, most preferably
having a molecular weight from about 300 to about 650 and having
from 1 to 4 zinc binding motifs, preferably having one or two zinc
binding motifs. In one embodiment, metal chelation forms a
symmetrical multimer, such as a dimer, of the receptor binding
moiety.
[0025] By the term "zinc binding motif", and derivatives thereof,
as used herein means a continuation of atoms within a receptor
binding moiety that have the following characteristics:
[0026] 1) each continuation consist of 3 to 10 atoms, preferably 4
to 8 atoms, most preferably 4 or 5 atoms,
[0027] 2) each continuation further consisting of two or more
heteroatoms, preferably from 2 to 4 heteroatoms, most preferably 2
to 3 heteroatoms, preferably at least one of the heteroatoms is
nitrogen, wherein the heteroatoms are separated from each other by
one to four additional atoms selected from the group consisting of
carbon, nitrogen, sulfur and oxygen, preferably carbon or nitrogen,
preferably by 2 to 4 additional atoms, most preferably by 2 or 3
additional atoms, and
[0028] 3) the configuration of heteroatoms within the zinc binding
motif allows for chelate coordination to a zinc (II) ion by
providing for the formation of at least two coordinate bonds,
preferably two or three coordinate bonds, simultaneously to a zinc
ion.
[0029] Examples of zinc binding motifs for use in the present
invention include but are not limited to the following:
--N--C--C--N--, --N--C.dbd.C--N--, --N--C--C.dbd.N--,
--N.dbd.C--C.dbd.N--, --O--C--C--N--, -- O--C.dbd.C--N--,
--O--C--C.dbd.N--, --O.dbd.C--C.dbd.N--, --S--C--C--N--,
--S--C.dbd.C--N--, --S--C--C.dbd.N--, --S.dbd.C--C.dbd.N--, --S--
C--C--S--, --N.dbd.C--N--N--, --N--C--N--N--, --O.dbd.C--N--N--,
--S.dbd.C--N--N--, --O--C--C.dbd.O--, --O--N--C.dbd.O--,
--N.dbd.C-- N--C.dbd.N--, --O.dbd.C--N--C.dbd.N--,
--N.dbd.C--C--C.dbd.N--, --O--C.dbd.C--C.dbd.O--- ,
--N--C--C--C--N--, --N--C--C.dbd.C--N--, -- N.dbd.C--C.dbd.C--N--,
--N.dbd.C--C.dbd.C--O--, --N.dbd.C--C.dbd.C--S--,
--S.dbd.C--C.dbd.C--S--- , --O.dbd.C--N--C.dbd.N--,
--N--N--C--C.dbd.N--, --N--N--C--N--N--, --N--C.dbd.N--C.dbd.N--,
--N.dbd.C--N--C.dbd.N--C--C--N-- and
--N.dbd.C--N--C.dbd.N--C--C.dbd.N--.
[0030] Preferred receptor binding moieties of the present invention
comprise one or more of the following functional groups, preferably
one or two of the following functional groups:
2-guanidinobenzimidazoles, 2-guanidinobenzoxazoles,
2-guanidionbenzothiazole, 2-mercaptomethylpyridines, acylacetones,
acylhydrazines, 2-aminoethanethiols, 2-(imidazol-4-yl)ethylamines,
2-(imidazol-2-yl)ethylamines, 2-(imidazol-4-yl)ethylimines,
2-(imidazol-2-yl)ethylimines, 2-picolylamine, 8-hydroxyquinolines,
8-aminoquinolines, 8-mercaptoquinolines, ethylenediamines,
pyridine-2-carboxaldimines, 2,2'-bipyridyls, 2-thiobenzaldimines,
2-hydroxybenzaldimines and
2,5-diimino-3a6a-diaryl-1,2,3,3a,4,5,6,6a-octa-
hydroimidazo[4,5-d]imidazoles
[0031] The above functional groups will generally form part of a
larger molecule and may be further substituted in the formation of
a receptor binding moiety. Preferred substituents for optional use
on the above functional groups consist of one or more groups
selected from the following: alkyl, aryl, hydroxy, alkoxy, acyloxy,
carbamoyl, amino, N-acylamino, ketone, halogen, cyano, thio,
carboxy and carboxamido.
[0032] As noted from the depiction of
bis{2,5-bis[2-benzimidazolylimino]-3-
a,6a-bis(2-pyridyl)-1,2,3,3a,4,5,6,6a-octahydroimidazo[4,5-d]imidazole-N,N-
'}-zinc(II) in Example 1 below, an 8 atom zinc binding motif
(specifically the --N.dbd.C--N--C.dbd.N--C--C.dbd.N--) is
essentially an overlap of a 5 atom zinc binding motif (that is
--N.dbd.C--N--C.dbd.N--) and a 4 atom zinc binding motif (that is
--N--C--C.dbd.N--) in a continuation. As such, preferred zinc
binding motifs of the instant invention consist of a continuation
of 4 or 5 atoms either individually or as part of a combination.
Further, each atom of a zinc binding motif of the present invention
may be further substituted, may be saturated or contain various
degrees of unsaturation or may form part of a larger linear system
or an aromatic or nonaromatic ring system.
[0033] The zinc chelated receptor ligands of this invention are
included in the pharmaceutical compositions of the invention and
used in the methods of the invention. The receptor binding moieties
of this invention are included in the pharmaceutical compositions
of the invention and used in the methods of the invention.
[0034] By the term "co-administering" and derivatives thereof as
used herein is meant either simultaneous administration or any
manner of separate sequential administration of a zinc chelated
receptor ligand, as described herein, and a further active
ingredient or ingredients. For example, antibacterial agents or
antifungal agents. Preferably, if the administration is not
simultaneous, the agents are administered in a close time proximity
to each other. Furthermore, it does not matter if the agents are
administered in the same dosage form, e.g. one agent may be
administered subcutaneously and another agent may be administered
orally.
[0035] The zinc chelated receptor ligands of this invention are
prepared by reacting one or more receptor binding moieties and a
zinc ion source, such as Zn(NO.sub.3).sub.2, in a solvent, followed
by optional isolation of the zinc chelated receptor ligand. The
order in which the indicated ingredients are utilized in the
presently invented process is not critical. All orders of addition
of the indicated ingredients are within the scope of the invention.
Further, the zinc chelated receptor ligands of this invention can
be prepared in vivo by the administration of a receptor binding
moiety to a subject and utilization of naturally occurring zinc
ions in the body of the subject.
[0036] Pharmaceutically acceptable salts, hydrates and solvates are
formed when appropriate by methods well known to those of skill in
the art.
[0037] Because the pharmaceutically active compounds of the present
invention are active as agonist of dimeric cell-surface receptors
they exhibit therapeutic utility in treating disease states
associated with compromised function of such dimeric cell-surface
receptors. For example, a zinc chelated G-CSF receptor agonist
would exhibit efficacy in treating bacterial infections, fungal
infections, neutropenia, including chemotherapy-induced neutropenia
and bone marrow transplantation and in mobilizing peripheral blood
stem cells and other conditions with depressed leukocyte
production.
[0038] In determining the potency of the presently invented
compounds as agonist of dimeric cell-surface receptors, the
following assays are employed:
[0039] Luciferase Assay
[0040] Compounds of the present invention are tested for potency as
agonist of a dimeric cell-surface receptor in a Luciferase reporter
gene assay such as described in Tian et al., Science 281. 257-259
(1998). For example, for G-CSF NFS60 cells (Holmes, et al., Proc.
Natl. Acad. Sci. USA 82: 6687-6691 (1985)) are selected because
they express endogenous G-CSF receptors closely matching the
pattern of STAT (signal transducers and activators of
transcription) activation observed in primary murine and human bone
marrow cells.
[0041] Luciferase Assay and EDTA
[0042] In order to determine the requisiteness of zinc chelation of
small organic molecules to agonist activity at dimeric cell-surface
receptors, the above luciferase assay was performed on the G-CSF
receptor in the presence of EDTA. EDTA is a strong metal chelator
and had as it only effect, the removal of zinc from the
ligand-receptor interaction.
[0043] CFU-G Assay
[0044] Compounds of this invention are also tested for activity in
the following assays: CFU-G assay (an example of which is described
in King A G, Talmadge J., Badger A M, Pelus L M. Regulation of
colony stimulating activity production from bone marrow stromal
cells by the hematoregulatory peptide, HP-5. Exp. Hematol.
20:223-228, 1992) and in vivo evaluation of peripheral blood
neutrophil and monocyte count in the mouse (an example of which is
described in Pelus, L. M.; King, A. G.; Broxmeyer, H. E.; DeMarsh,
P. L.; Petteway, S. R.; Bhatnagar, P. K., In vivo modulation of
hematopoiesis by a novel hematoregulatory peptide Exp-Hematol. 1994
22(3): 239-47). In order to confirm the requirement for zinc(II)
chelation, the above CFU-G assay was also conducted in the presence
of EDTA.
[0045] Isothermal Titration Microcalorimetry
[0046] Zinc-mediated affinity of compounds from this invention for
dimeric cell-surface receptors (specifically the G-CSF receptor)
was measured by isothermal titration microcalorimetry experiments.
Titration microcalorimetry detects binding as heat originating from
the intrinsic bond forming enthalpy change. In this assay, the
compounds were titrated first against zinc(II) alone. In a separate
experiment, the compounds were then assayed in the presence of zinc
and a dimeric cell-surface receptor/Fc fusion protein (a G-CSF/Fc
fusion protein), which contained the extracellular domain of the
receptor presented in a dimeric form due to the Fc component.
Interaction with the fusion protein construct was confirmed from
the binding enthalpy change, which was substantially increased over
that of zinc alone. When the latter experiment was carried out in
the absence of zinc, no heat of binding was detected, indicating
that no interaction with the fusion protein construct occurred
under those conditions.
[0047] Compounds a and 3a bind to the fusion protein construct with
high, submicromolar affinity only in the presence of zinc.
Compounds 1, 1a, 2a, and 3a showed activation above 150% of control
between the concentration range of 1 to 100 micromolar in the
luciferase assay. Further, compound 1a and 3a showed activation
above 150% of control between the concentration range of 1 to 100
micromolar in the murine assay. Compounds 1a and 3a showed
elevation of peripheral blood neutrophil and monocyte count in the
mouse.
[0048] As demonstrated by the results depicted in FIG. 1, the
agonist activity of Compound 1a in the absence of zinc is below the
activity threshold of 150% over background. However, as shown in
FIG. 2, the presence of 1 uM zinc(II) activates compound 1a, so
that it becomes an agonist of the dimeric cell-surface receptor
with an efficacy of 350% over background at 1 uM. This is an
indication that zinc(II) mediates the activity of compound 1a.
[0049] As demonstrated by the results depicted in FIG. 3, the
agonist activity of Compound 1a was abrogated in the presence of
EDTA, confirming that a metal ion mediates the activity.
[0050] Conversely, the results depicted in FIG. 4 indicate that the
agonist activity of the natural ligand (i.e. G-CSF or recombinant
G-CSF as demonstrated herein) is not mediated by metal ions.
[0051] The results depicted in FIGS. 5 and 6 indicate that metal
ions alone are insufficient to trigger an agonist response at a
dimeric cell-surface receptor.
[0052] The results depicted in FIGS. 7 and 8 indicate that
chelation of a small molecule to zinc ions (and not ions of
manganese, iron, copper or cobalt) is a requirement for activation
of the dimeric cell-surface receptor by organic molecules.
[0053] The results depicted in FIGS. 1 through 8 demonstrate, for
the first time, that zinc chelated small molecules, or zinc
chelated receptor ligands as used herein, are necessary for
activation of dimeric cell-surface receptors by organic
molecules.
[0054] Based on the description in the specification and in the
Examples one of skill in the art can readily design and prepare a
zinc chelated dimeric cell-surface receptor ligands. Further, one
of skill in the art can readily determine if a dimeric cell-surface
receptor ligand candidate is acting as an agonist of the receptor
by using the assays described herein and then repeating the
experiments of FIGS. 1 through 8.
[0055] The pharmaceutically active compounds within the scope of
this invention are useful as dimeric cell-surface receptor agonist
in mammals, including humans, in need thereof.
[0056] The present invention therefor provides a method of treating
disease states associated with compromised function of dimeric
cell-surface receptors, which comprises administering a zinc
chelated receptor ligand in a quantity effective to enhance
receptor activation. For example, a zinc chelated G-CSF receptor
agonist would exhibit efficacy in treating bacterial infections,
fungal infections, neutropenia, including chemotherapy-induced
neutropenia and bone marrow transplantation and in mobilizing
peripheral blood stem cells and other conditions with depressed
leukocyte production, through the administration of a zinc chelated
G-CSF receptor ligand in a quantity effective to enhance leukocyte
production. The zinc chelated receptor ligands of the present
invention also provide for a method of treating the above indicated
disease states because of their demonstrated ability to act as
agonist of dimeric cell-surface receptors. The drug may be
administered to a patient in need thereof by any conventional route
of administration, including, but not limited to, intravenous,
intramuscular, oral, subcutaneous, intradermal, and parenteral.
Also, the drug may be formed in vivo by the administration of a
dimeric cell-surface receptor binding moiety (or receptor binding
moiety as used herein) by the same methods of administration
described herein and in about the same amounts as described herein
for zinc chelated receptor ligands. Further, the possibility exists
that solubility and bioavailability concerns will be associated
with the zinc chelated receptor ligands of the present invention.
Thus, depending on the particular moiety in question, it will often
be preferable to administer a receptor binding moiety of the
present invention and therein by subsequently form a zinc chelated
receptor ligand in vivo using plasma as the solvent and naturally
occurring zinc ions. It is also contemplated herein that a receptor
binding moiety of the present invention be administered with a zinc
source so as to facilitate the in vivo formation of a zinc chelated
receptor ligand.
[0057] The pharmaceutically active zinc chelated receptor ligands
of the present invention or, when desired and appropriate, the
receptor binding moieties of the present invention are incorporated
into convenient dosage forms such as capsules, tablets, or
injectable preparations. Solid or liquid pharmaceutical carriers
are employed. Solid carriers include, starch, lactose, calcium
sulfate dihydrate, terra alba, sucrose, talc, gelatin. agar,
pectin, acacia, magnesium stearate, and stearic acid. Liquid
carriers include syrup, peanut oil, olive oil, saline, and water.
Similarly, the carrier or diluent may include any prolonged release
material, such as glyceryl monostearate or glyceryl distearate,
alone or with a wax. The amount of solid carrier varies widely but,
preferably, will be from about 25 mg to about 1 g per dosage unit.
When a liquid carrier is used, the preparation will be in the form
of a syrup, elixir, emulsion, soft gelatin capsule, sterile
injectable liquid such as an ampoule, or an aqueous or nonaqueous
liquid suspension.
[0058] The pharmaceutical preparations are made following
conventional techniques of a pharmaceutical chemist involving
mixing, granulating, and compressing, when necessary, for tablet
forms, or mixing, filling and dissolving the ingredients, as
appropriate, to give the desired oral or parenteral products.
[0059] Doses of the presently invented pharmaceutically active zinc
chelated receptor ligands of the present invention or, when desired
and appropriate, the receptor binding moieties of the present
invention, in a pharmaceutical dosage unit as described above will
be an efficacious, nontoxic quantity preferably selected from the
range of 0.001-125 mg/kg of active compound, preferably 0.001-60
mg/kg. When treating a human patient in need of an agonist of a
dimeric cell-surface receptor, the selected dose is administered
preferably from 1-6 times daily, orally or parenterally. Preferred
forms of parenteral administration include topically, rectally,
transdermally, by injection and continuously by infusion. Oral
dosage units for human administration preferably contain from 0.05
to 3500 mg of active compound. Oral administration, which uses
lower dosages is preferred. Parenteral administration, at high
dosages, however, also can be used when safe and convenient for the
patient.
[0060] Optimal dosages to be administered may be readily determined
by those skilled in the art, and will vary with the particular zinc
chelated receptor ligand or receptor binding moiety in use, the
strength of the preparation, the mode of administration, and the
advancement of the disease condition. Additional factors depending
on the particular patient being treated will result in a need to
adjust dosages, including patient age, weight, diet, and time of
administration.
[0061] Another aspect of the present invention is a method for
identifying agonists of dimeric cell-surface receptors and receptor
ligands identified thereby. In the method, the dimeric cell-surface
receptor is contacted with receptor ligand candidates in the
presence of a micromolar concentration of zinc(II). Ligand
candidates which bind to the dimeric cell-surface receptor are
selected by receptor binding assays well known to those skilled in
the art, such as competitive and non-competitive binding
measurements (Immobilized Affinity Ligand Techniques, G. T.
Hermanson, A. K. Mallia, P. K. Smith Eds., Academic Press Inc. San
Diego, Calif. 1992), isothermal microcalorimetry (Rapid Measurement
of Binding Constants and Heats of Binding Using a New Titration
Calorimeter T. Wiseman, S. Williston, J. F. Brandts, and L. -N. Lin
(1989) Analytical Biochemistry 179, 131-137.), sedimentation
equilibrium (T. Horan et al. Biochemistry 1996, 35, 4886-4896),
ELISA. RIA methodologies (An Introduction to Radioimmunoassays and
Related Techniques, T. Chard, Elsevier Science Publishers,
Amsterdam, The Netherlands, 1990), BIAcore.RTM. (BIAtechnology
Handbook, Pharmacia Biosensor AB, Uppsala, Sweden, 1994),
fluorescence anysotiopy methodology (Luminesct Spectroscopy of
Proteins, E. A. Permyakov, CRC Press Inc., Boca Raton, Fla. 1992),
flow cytometry technology (Flow Cytometry and Cell Sorting, A.
Radbruch, Springer-Verlag, New York, N.Y. 1992).
[0062] In general, the dimeric cell-surface receptor in isolated,
immobilized or cell-bound form is contacted with a plurality of
zinc chelated receptor ligand candidates and those candidates which
bind to and interact with the receptor are selected. Optionally,
the isolated, immobilized or cell-bound receptor is contacted with
a variety of metal-chelating receptor ligand candidates in the
presence of zinc(II). Binding interaction can be measured directly
by using radioactively labeled ligand candidates or indirectly, by
using cells expressing the dimeric cell-surface receptor and
measuring the occurrence of an event mediated by the formation of a
dimeric cell-surface receptor--ligand complex. Alternatively, the
ligand candidates can be subjected to competitive binding assays in
which the known receptor ligand, labeled preferably with an
analytically detectable reagent, most preferably radioactivity, is
included with the ligand candidates and a candidate's ability to
inhibit the binding of the labeled ligand is measured.
[0063] Positive receptor ligand candidates are screened for
biological function by any one of the receptor function assays well
known to those skilled in the art. It is expected that a positive
ligand binding candidate will exhibit agonist activity in receptor
function assays.
[0064] An example of an appropriate competitive binding assay for
the G-CSF receptor involves the immobilization of the G-CSF
receptor and incubation with compounds of interest with I.sup.125
radiolabeled G-CSF following the general procedure already
described for other cytokine receptors (C .L. Martens et al. J.
Biol. Chem. 1995, 270, 21129, E. Whitehorn et al. Biotechnology
1995, 13, 1215, S. D. Yanofsky et al. Proc. Nati. Acad. Sci. U.S.A.
1996, 93, 7381, N. C. Wrighton et al. Science, 1996, 273, 458, S.
E. Cwirla, Science, 1997, 276, (1996).
[0065] The method of this invention of inducing agonist activity at
a dimeric cell-surface receptor in mammals, including humans,
comprises administering to a subject in need of such activity an
effective amount of a pharmaceutically active zinc chelated
receptor ligand of the present invention or, when desired and
appropriate, a receptor binding moiety of the present
invention.
[0066] The invention also provides for the use of a presently
invented zinc chelated receptor ligand or a presently invented
receptor binding moiety in the manufacture of a medicament for use
as an agonist of a dimeric cell-surface receptor.
[0067] The invention also provides for the use of a zinc chelated
receptor ligand or a receptor binding moiety in the manufacture of
a medicament for use in therapy.
[0068] The invention also provides for the use of a zinc chelated
receptor ligand or a receptor binding moiety in the manufacture of
a medicament for use in enhancing the activity of a dimeric
cell-surface receptor.
[0069] The invention also provides for the use of a zinc chelated
receptor ligand or a receptor binding moiety in the manufacture of
a medicament for use in treating disease states associated with
compromised dimeric cell-surface receptor activity. For example,
bacterial and fungal infections.
[0070] The invention also provides for a pharmaceutical composition
for use as an agonist of a dimeric cell-surface receptor which
comprises a zinc chelated receptor ligand or a receptor binding
moiety and a pharmaceutically acceptable carrier.
[0071] The invention also provides for a pharmaceutical composition
for use in treating bacterial infections which comprises a zinc
chelated receptor ligand or a receptor binding moiety and a
pharmaceutically acceptable carrier.
[0072] The invention also provides for a pharmaceutical composition
for use in treating fungal infections which comprises a zinc
chelated receptor ligand or a receptor binding moiety and a
pharmaceutically acceptable carrier.
[0073] The invention also provides for a process for preparing a
pharmaceutical composition containing a pharmaceutically acceptable
carrier or diluent and a zinc chelated receptor ligand or a
receptor binding moiety which comprises bringing the zinc chelated
receptor ligand or the receptor binding moiety into association
with the pharmaceutically acceptable carrier or diluent.
[0074] No unacceptable toxicological effects are expected when
compounds of the invention are administered in accordance with the
present invention.
[0075] In addition, the pharmaceutically active compounds of the
present invention can be co-administered with further active
ingredients, such as other compounds known to treat disease states
associated with compromised dimeric cell-surface receptor activity.
For example, compounds to treat bacterial infections and fungal
infections.
[0076] As indicated above, the zinc chelated compounds of the
invention are utilized as agonists of cell-surface receptors whose
signal transduction mechanism involves receptor dimerization or
oligomerization. These receptors are divided in five superfamilies
(reviewed by Heldin, C. H. Dimerization of Cell Surface Receptors
in Signal Transduction, Cell 1995, 80, 213) as follows:
protein-tyrosine kinase receptors (PDGFR-.alpha., PDGFR-.beta.,
SCFR, CSF-R, Flk-2, EGFR, Erb2,Erb3, Erb4, FGFR-1, FGFR-2, FGFR-3,
FGFR-4, insuline R, IGF-1R, HGFR, MSPR, Flt-1, Flk-1, Trk, TrkB,
TrkC, Eph, Elk, Eck. Cck5, Sek, Eck, Erk), cytokine receptors (GHR,
TPOR, EPOR. PRLR, G-CSFR. leptin R. IL-3R. GM-CSFR, IL-5R, IL,-6R,
LIFR, CNTRFR, IL-11R, IL-2R, IL-4R, IL-7R, IFN-.alpha., IFN-.beta.,
IFN-.gamma., IL-10R), TNF receptors (TNFR, LGNFR, CD40, OX-40, Fas,
CD27, CD30), antigen receptors (TCR, BCR) and serine/threonine
kinase receptors (TGF-.beta.R, ActR-II).
[0077] With regards to the presently invented subject matter, the
term dimeric cell-surface receptor(s) refers to the receptors of
the five superfamilies as listed above, with the exception of the
G-CSF receptor.
[0078] In a further aspect of the invention, the zinc chelated
compounds of the invention are utilized as agonist of the
erythropoietin (EPO) receptor. Additionally, a therapeutically
effective amount of a receptor binding moiety of the invention is
administered to a subject in need of treatment for anemia.
[0079] In a further aspect of the invention, the zinc chelated
compounds of the invention are utilized as agonist of the
macrophage-colony-stimula- ting factor (M-CSF) receptor.
Additionally, a therapeutically effective amount of a receptor
binding moiety of the invention is administered to a subject in
need of treatment for neutropenia.
[0080] In a further aspect of the invention, the zinc chelated
compounds of the invention are utilized as agonist of the growth
hormone (GRH) receptor. Additionally, a therapeutically effective
amount of a receptor binding moiety of the invention is
administered to a subject in need of treatment for growth hormone
deficiency.
[0081] In a further aspect of the invention, the zinc chelated
compounds of the invention are utilized as agonist of the
thrombopoietin (TPO) receptor. Additionally, a therapeutically
effective amount of a receptor binding moiety of the invention is
administered to a subject in need of treatment for
thrombocytopenia.
[0082] In a further aspect of the invention, the zinc chelated
compounds of the invention are utilized as agonist of the leptin
receptor. Additionally, a therapeutically effective amount of a
receptor binding moiety of the invention is administered to a
subject in need of treatment for obesity.
[0083] In a further aspect of the invention, the zinc chelated
compounds of the invention are utilized as agonist of the
interferon (IFN) alpha receptor. Additionally, a therapeutically
effective amount of a receptor binding moiety of the invention is
administered to a subject in need of treatment for hepatitis C.
[0084] In a further aspect of the invention, the zinc chelated
compounds of the invention are utilized as agonist of the
interferon (IFN) beta receptor. Additionally, a therapeutically
effective amount of a receptor binding moiety of the invention is
administered to a subject in need of treatment for multiple
sclerosis.
[0085] In a further aspect of the invention, the zinc chelated
compounds of the invention are utilized as agonist of the insulin
receptor. Additionally, a therapeutically effective amount of a
receptor binding moiety of the invention is administered to a
subject in need of treatment for diabetes
[0086] In a further aspect of the invention, the zinc chelated
compounds of the invention are utilized as agonist of the tyrosine
kinase (TRK) receptors. Additionally, a therapeutically effective
amount of a receptor binding moiety of the invention is
administered to a subject in need of treatment for CNS
diseases.
[0087] In a further aspect of the invention, the zinc chelated
compounds of the invention are utilized as dimeric cell-surface
receptor agonist.
[0088] Without further elaboration, it is believed that one skilled
in the art can using the preceding description, utilize the present
invention to its fullest extent. The following Examples are,
therefore to be construed as merely illustrative and not a
limitation of the scope of the present invention in any way.
Experimental Details
EXAMPLE 1
Preparation of Compound 1
Bis{2,5-bis[2-benzimidazolylimino]-3a,6a-bis(2-p-
yridyl)-1,2,3,3a,4,5,6,6a-octaliydroimidazo[4,5-d]imidazole-N,N'}-zinc(II)
[0089] 1
[0090] a)- Preparation of Compound
1a-2,5-Bis[2-benzimidazolylimino]-3a,6a-
-bis(2-pyridyl)-1,2,3,3a,4,5,6,6a-octahydroimidazo[4,5-d]imidazole.
[0091] A mixture of 2,2'-pyridil (15.8 g, 74.4 mmol) and
2-guanidinobenzimidazole (19.5 g, 111.7 mmol) in methanol (440 mL)
was treated with a solution of sodium hydroxide (2.97 g, 74.4 mmol)
in 74 mL and the resulting mixture was left standing at room
temperature for 4 days. The crystalline material was filtered and
dried under vacuum to yield 21.1 g of the title compound as
off-white crystals (72%). mp: 305-307.degree. C. (dec); HPLC
retention time 4.5 min (reversed phase, Beckman ultrasphere ODS 4.6
mm.times.25 cm column, 20 min gradient elution with 20:80 to 60:40
acetonitrile: water containing 0.1% TFA@2 mL/min); .sup.1H NMR (300
MHz, d.sub.6-DMSO) d 11.5 (br s, NH, 2 H), 10.0 (br s, NH, 2 H),
8.6 (br s, NH. 2 H), 8.38 (d, J=4.2 Hz, 2 H), 7.55 (t, J=7.8 Hz, 2
H), 7.29 (d, J=7.8 Hz, 2 H), 7.27-7.21 (m, 4 H), 7.14 (br s, 2 H),
6.98 (dd, J=5.8, 3.2 Hz, 4 H); MS (ESI) m/z 527 [M+H].sup.+: Anal.
Calcd. for C.sub.28H.sub.22N.sub.12. 2/3H.sub.2O: C, 62.44; H,
4.37. N, 31.21; Found: C, 62.72; H, 4.08; N, 30.86.
[0092] b)- Preparation of Compound 1-
Bis{2,5-bis[2-benzimidazolylimino]-3-
a,6a-bis(2-pyridyl)-1,2,3,3a,4,5.6,6a-octalydroimidazo[4,5-d]imidazole-N,N-
'}-zinc(II).
[0093] A solution of compound from Example 1a (40 mg, 0.076 mmol)
in 2 mL of 10% aqueous acetic acid was treated with a solution of
zinc nitrate hexahydrate (24.9 mg, 0.0836 mmol) in water (1 mL).
The mixture was left standing at room temperature for 6 h, and was
then centrifuged, decanted and rinsed with water three times. The
title compound was obtained as a white powder (13 mg). HPLC
retention time 10.4 min (reversed phase, Beckman ultrasphere ODS
4.6 mm.times.25 cm column, 20 min gradient elution with 20:80 to
60:40 acetonitrile:water containing 0.1% TFA@2 mL/min); MS (ESI)
m/z 1182 [M].sup.+, 591 [M].sup.++.
EXAMPLE 2
Preparation of Compound
2-Bis{2,5-bis[2-benzimidazolylimino]-3a,6a-dipheny-
l-1,2,3,3a,4,5,6,6a-octahydroimidazo[4,5-d]imidazole-N,N'}-zinc(II).
[0094] 2
[0095] a)- Preparation of Compound
2a-2,5-Bis[2-benzimidazolylimino]-3a,6a-
-diphenyl-1,2,3,3a,4,5,6,6a-octakhydroimidazo[4,5-d]imidazole.
[0096] A mixture of benzil (1.05 g, 5.0 mmol) and
2-guanidinobenzimidazole (1.57 g, 9.0 mmol) in benzene (25 mL) was
refluxed in pyridine (10 mL) for 1 h. After evaporating most of the
pyridine under reduced pressure, the residue was treated with hot
toluene and the resulting precipitate was filtered. The precipitate
was then dissolved in 9:1 water:acetic acid (30 mL); the solution
was filtered and the filtrate was neutralized to pH 7 with
phosphate buffer. A precipitate formed, that was then collected and
triturated with water to afford the title compound (0.42 g, 16%).
.sup.1H NMR (300 MHz, d.sub.6-DMSO) d 11.5 (br s, NH, 2 H). 10.0
(br s. NH, 2 H), 8.6 (br s, NH, 2 H), 7.28-7.10 (m, 14 H). 6.97
(dd. J=6.0, 3.0 Hz. 4 H); MS (ESI) m/z 525 [M+H].sup.+; Anal.
Calcd. for C.sub.30H.sub.24N.sub.10. 1/2CH.sub.3CO.sub.2H.
3/4H.sub.2O: C. 65.37: H, 4.88; N, 24.65. Found: C. 65.36; H, 4.79;
N, 24.48.
[0097] b)- Preparation of Compound
2-Bis{2,5-bis[2-benzimidazolylimino]-3a-
,6a-diphenyl-1,2,3,3a,4,5,6,6a-octahydroimidazo[4,5-d]imidazole-N,N'}-zinc-
(II).
[0098] A solution of compound from Example 2a (50 mg, 0.095 mmol)
in 2 mL of methanol containing a drop of formic acid was treated
with a solution of zinc nitrate hexahydrate (31.0 mg, 0.104 mmol)
in methanol (1 mL). The mixture was left standing at room
temperature for 18 h, and was then centrifuged, decanted and rinsed
with water three times to yield the title compound as a white
powder (35 mg). MS (ESI) m/z 1178 [M].sup.+, 589 [M].sup.++.
EXAMPLE 3
Preparation of Compound 3
Bis{5-(2-benzimidazolylimino)-2-[(5-methyl-2-ben-
zimidazolyl)imino]-3a,6a-bis(2-pyridyl)-1,2,3,3a,4,5,6,6a-octahydroimidazo-
[ 4,5-d]imidazole-N,N'}-zinc(II)
[0099] 3
[0100] a) Preparation of Compound 3a
-5-(2-benzimidazolylimino)-2-[(5-meth-
yl-2-benzimidazolyl)imino]-3a,6a-bis(2-pyridyl)-1,2,3,3a,4,5,6,6a-octahydr-
oimidazo[ 4,5-d]imidazole bis(trifluoroacetate) salt.
[0101] A mixture of 2,2'-pyridil (135 mg, 0.636 mmol),
2-guanidinobenzimidazole (92.8 mg, 0.530 mmol) and
5-methyl-2-guanidinobenzimidazole (100 mg, 0.530 mmol) in methanol
(3 mL) was treated with a solution of sodium hydroxide (38 mg, 0.95
mmol) in 0.5 mL of water and the resulting mixture was left
standing at room temperature for 2 days. The crystalline material
was filtered and purified by reversed phase preparative HPLC
(Rainin Dynamax, 5 .mu.M C18 column: 21.4 mm.times.25 cm, elution
with gradient acetonitrile-water containing 0.1% trifluoroacetic
acid) to yield the title compound as a white powder (88 mg, 18%).
.sup.1H NMR (300 MHz, d.sub.6-DMSO) .delta. 13.0 (br s, NH, 4 H),
9.8 (br s, NH. 4 H), 8.39 (d, J=4.3 Hz, 2 H), 7.64 (td, J =7.8, 1.7
Hz, 2 H), 7.57 (d, J =7.8 Hz, 2 H), 7.49-7.46 (m, 2 H), 7.38-7.33
(m, 3 H), 7.27 (s, 1 H), 7.21-7.13 (m, 3 H), 2.44 (s, 3 H); MS
(ESI) m/z 541 [M+H].sup.+
[0102] b)- Preparation of Compound
3-Bis{5-(2-benzimidazolylimino)-2-[(5-m-
ethyl-2-benzimidazolyl)imino]-3a,6a-bis(2-pyridyl)-1,2,3,3a,4,5,6,6a-octah-
ydroimidazo[ 4,5-d]imidazole-N,N'}-zinc(II)
[0103] A solution of compound from Example 3a (70 mg, 0.091 mmol)
in 10 mL of water was treated with a solution of zinc nitrate
hexahydrate (40 mg, 0.136 mmol) in water (1 mL). The mixture was
left standing at room temperature for 1 d, and was then
centrifuged, decanted and rinsed with water three times. The title
compound was obtained as a white powder (29 mg). HPLC retention
time 11.8 min (reversed phase, Beckman ultrasphere ODS 4.6
mm.times.25 cm column, 20 min gradient elution with 20:80 to 60:40
acetonitrile:water containing 0.1% TFA@2 mL/min); MS (ESI) m/z 1210
[M].sup.+, 605 [M].sup.++.
EXAMPLE 4
Preparation of Compound 4 -
Bis{2,5-bis[(5-methyl-2-benzimidazolyl)imino]--
3a,6a-bis(2-pyridyl)-1,2,3,3a,4,5,6,6a-octahydroimidazo[4,5-d]imidazole-N,-
N'}-zinc(II)
[0104] 4
[0105] a) Preparation of Compound 4a
-2,5-bis[(5-methyl-2-benzimidazolyl)i-
mino]-3a,6a-bis(2-pyridyl)-1,2,3,3a,4,5,6,6a-octahydroimidazo[4,5-d]imidaz-
ole.
[0106] A mixture of 2,2'-pyridil (603 mg, 2.84 mmol) and
5-methyl-2-guanidinobenzimidazole (489 mg, 2.58 mmol) in methanol
(17 mL) was treated with a solution of sodium hydroxide (113.6 mg,
2.84 mmol) in 2.8 mL of water. The title compound was isolated as a
grey powder (450 mg, 63% yield). mp: 290-291.degree. C. (dec); HPLC
retention time 7.1 min (reversed phase, Beckman ultrasphere ODS 4.6
mm.times.25 cm column, 20 min gradient elution with 20:80 to 60:40
acetonitrile: water containing 0.1% TFA@2 mL/min); .sup.1H NMR (300
MHz, d.sub.6-DMSO) .delta. 11.3 (br s, NH, 2 H), 10.0 (br s, NH, 2
H), 8.5 (br s, NH, 2 H), 8.32 (d,J=4.2 Hz 2 H), 7.54 (t, J=7.6 Hz,
2 H), 7.31 (d, J=7.6 Hz, 2 H), 7.16 (d, J= 7.8 Hz, 2 H), 7.16-7.09
(m, 2 H), 7.09 (s, 2 H), 7.14 (br s, 2 H), 6.86 (d, J=7.8, Hz, 2
H), 2.34 (s, 6 H); MS (ESI) m/z 555 [M+H].sup.+.
[0107] b)- Preparation of Compound
4-Bis{2,5-bis[(5-methyl-2-benzimidazoly-
l)imino]-3a,6a-bis(2-pyridyl)-1,2,3,3a,4,5,6,6a-octahydroimidazo[4,5-d]imi-
dazole-N,N'}-zinc(II)
[0108] A solution of compound from Example 4a (50 mg, 0.091 mmol)
in 10 mL of water containing a few drops of formic acid was treated
with a solution of zinc nitrate hexahydrate (40 mg, 0.136 mmol) in
water (1 mL). The mixture was left standing at room temperature for
1 d, and was then centrifuged, decanted and rinsed with water three
times. The title compound was obtained as a white powder (19 mg).
HPLC retention time 13.3 min (reversed phase, Beckman ultrasphere
ODS 4.6 mm.times.25 cm column, 20 min gradient elution with 20:80
to 60:40 acetonitrile: water containing 0.1% TFA@2 mL/min); MS
(ESI) m/z 1238 [M].sup.+, 619 [M].sup.++.
EXAMPLE 5--CANSULE COMPOSITION
[0109] An oral dosage form for administering a presently invented
agonist of the G-CSF receptor is produced by filing a standard two
piece hard gelatin capsule with the ingredients in the proportions
shown in Table I, below.
1TABLE I INGREDIENTS AMOUNTS
Bis{2,5-bis[2-benzimidazolylimino]-3a,6a-bis(2-pyridyl)- 25 mg
1,2,3,3a,4,5,6,6a-octahydroimidazo[4,5-d]imidazole-N,N'}- zinc(II)
(Compound.) Lactose 55 mg Talc 16 mg Magnesium Stearate 4 mg
EXAMPLE 6--INJECTABLE PARENTERAL COMPOSITION
[0110] An injectable form for administering a presently invented
agonist of the G-CSF receptor is produced by stirring 1.5% by
weight of
2,5-Bis[2-benzimidazolylimino]-3a,6a-diphenyl-1,2,3,3a,4,5,6,6a-octahydro-
imidazo[4,5-d]imidazole (Compound 2a) in 10% by volume propylene
glycol in water.
EXAMPLE 7--TABLET COMPOSITION
[0111] The sucrose, calcium sulfate dihydrate and a presently
invented agonist of the G-CSF receptor, as shown in Table II below,
are mixed and granulated in the proportions shown with a 10%
gelatin solution. The wet granules are screened, dried, mixed with
the starch, talc and stearic acid, screened and compressed into a
tablet.
2 TABLE II INGREDIENTS AMOUNTS
2,5-Bis[2-benzimidazolylimino]-3a,6a-bis(2-pyridyl)- 20 mg
1,2,3,3a,4,5,6,6a-octahydroimidazo[4,5-d]imidazole (Compound 1a)
calcium sulfate dihydrate 30 mg sucrose 4 mg starch 2 mg talc 1 mg
stearic acid 0.5 mg
[0112] While the preferred embodiments of the invention are
illustrated by the above, it is to be understood that the invention
is not limited to the precise instructions herein disclosed and
that the right to all modifications coming within the scope of the
following claims is reserved.
* * * * *