Title: Peptide deformylase activated prodrugs
Abstract: The invention provides compounds and methods for using them to inhibit the growth of microorganism that expresses Peptide Deformylase, the compounds having the general formula: ##STR1##
Patent Number: 7,001,922 Issued on 02/21/2006 to Ballatore, et al.
| Inventors:
|
Ballatore; Carlo (San Diego, CA);
Sergeeva; Maria V. (San Diego, CA);
Doppalapudi; Venkata Ramana (San Diego, CA)
|
| Assignee:
|
Celmed Oncology (USA), Inc. (Saint-Laurent, CA)
|
| Appl. No.:
|
714255 |
| Filed:
|
November 14, 2003 |
| Current U.S. Class: |
514/565; 514/567; 564/15; 568/17 |
| Current Intern'l Class: |
A61K 31/19.2 (20060101); A61K 31/19.5 (20060101); C07F 9/02 (20060101); C07F 9/53.5 (20060101) |
| Field of Search: |
564/15
568/17
514/565,567
|
References Cited [Referenced By]
U.S. Patent Documents
| 4053638 | Oct., 1977 | Litchfield et al.
| |
| 4083974 | Apr., 1978 | Turi.
| |
| 4339440 | Jul., 1982 | Bajusz et al.
| |
| 5918568 | Jul., 1999 | Gjerløv.
| |
| 6110908 | Aug., 2000 | Guthery.
| |
| 6143790 | Nov., 2000 | Hallinan et al.
| |
| 6159706 | Dec., 2000 | Shepard.
| |
| 6245750 | Jun., 2001 | Shepard.
| |
| 6339151 | Jan., 2002 | Shepard et al.
| |
| 6448058 | Sep., 2002 | Patel et al.
| |
| 6613879 | Sep., 2003 | Firestone et al.
| |
| Foreign Patent Documents |
| WO 98/1305/9 | Apr., 1998 | WO.
| |
| WO 99/0811/0 | Feb., 1999 | WO.
| |
| WO 99/3775/3 | Jul., 1999 | WO.
| |
| WO 01/0745/4 | Feb., 2001 | WO.
| |
| WO 02/089739 | Nov., 2002 | WO.
| |
| WO 03/088913 | Oct., 2003 | WO.
| |
Other References
International Preliminary Examination Report dated Dec. 15, 2004 for PCT/US2002/036124.
Boduszek Bogdan, English Abstract Synthesis of novel Phosphonopeptides derived
from pyridylmethylphosphonate diphenyl esters.
Liu, et al. English Abstract "Synthesis and anticancer activities of new 5-fluorouracil-1-yl phosphonotripeptides".
Copy of Written Opinion for PCT/US02/14500.
Copy of International Search Report for PCT/US02/36124.
Copy of European Supplemental Search Report for EP Appl. No. 02741696.5.
Apfel, C. M. et al. "Hydroxamic acid derivatives as potent peptide deformylase
inhibitors and antibacterial agents" J. Med. Chem. (2000) 43:2324-2331.
Apfel, C. M. et al. "Peptide deformylase as an antibacterial drug target: Assays
for detection of its inhibition in Escherichia coli cell homogenates and
intact cells" Antimicrobial Agents and Chemotherapy. (Apr. 2001a) 45(4):1053-1057.
Apfel, C.M. et al. "Peptide deformylase as an antibacterial drug target: Target
validation and resistance development" Antimicrobial Agents and Chemotherapy
(Apr. 2001b) 45(4):1058-1064.
Becker, A. et al. "Iron center, substrate recognition and mechanism of peptide
deformylase" Nat. Struc. Biol. (Dec. 1998) 5(12):1053-1058.
Chan, M. K. et al. "Crystal structure of the Escherichia coli peotide
deformylase" Biochemistry (1997) 36:13904-13909.
Chen, D. Z. et al. "Actinonin , a naturally occurring antibacterial agent, is
a potent deformylase inhibitor" Biochemistry (2000) 39:1256-1262.
Clements, J. M. et al. "Antibiotic activity and characterization of BB-3497,
a novel peptide deformylase inhibitor" Antimicrobial Agents and Chemotherapy
(Feb., 2001) 45(2):563-570.
de Groot, F. M. H. et al. "Synthesis and biological evaluation of 2′-carbamate-linked
and 2′-carbonate-linked prodrugs of paclitaxel: selective activation by
the tumor-associated protease plasmin" J. Med. Chem. (2000) 43:3093-3102.
de Groot, F.M.H. et al. "Synthesis and Biological Evaluation of Novel Prodrugs
of Anthracyclines for Selective Activation by the Tumor-Associated Protease Plasmin"
J. Med. Chem. (1999) 42(25):5277-5283.
Dubowchik, G. M. and R. A. Firestone "Cathepsin B-sensitive depeptide prodrugs.
1. A model study of structural requirements for efficient release of doxorubicin"
Bioor. & Med. Chem. Letts. (1998) 8:3341-3346.
Durand, D. J. et al. "Peptide aldehyde inhibitors of bacterial peptide deformylases"
Archives of Biochemistry and Biophysics (Jul. 15, 1999) 367(2):297-302.
Giglione, C. et al. "Identification of eukaryotic peptide deformylases reveals
universality of N-terminal protein processing mechanisms" The EMBO Journal (2000) 19(21):5916-5929.
Giglione, C. et al. "Peptide deformylase as a target for new generation, broad
spectrum antimicrobial agents" Molecular Microbiology (2000) 36(6):1197-1205.
Hao, B. et al. "Structural basis for the design of antibiotics targeting peptide
deformylase" Biochemistry (1999) 38(15):4712-4719.
Hu, Y.-J. et al. "H-phosphonate derivatives as novel peptide deformylase inhibitors"
Bioor. & Med. Chem. Letts. (1998) 8(18):2479-2482.
Huntington, K. M. et al. "Synthesis and antibacterial activity of peptide deformylase
inhibitors" Biochemistry (2000) 39(15):4543-4551.
Jayasekera, M. M. K. et al. "Novel nonpeptidic inhibitors of peptide deformylase"
Archives of Biochem. and Biophy. (Sep. 15, 2000) 381(2):313-316.
Lackey, D. B. et al. "Enzyme-catalyzed therapeutic agent (ECTA) design: Activation
of the antitumor ECTA compound NB 1011 by thymidylate synthase" Biochem. Pharmocol.
(2001) 61:179-189.
Meinnel, T. "Vers une conception rationnelle de nouveaux agents antibactériens"
Path. Biol. (Oct. 1999) 47(8):780-783.
Meinnel, T. et al. "Methionine as translation start signal: A review of the enzymes
of the pathway in Escherichia coli" Biochemic (1993) 75(12):1061-1075.
Ragusa S. et al. "Control of peptide deformylase activity by metal cations" J.
Mol. Biol. (1998) 280:515-523.
Rajagopalan, P. T. R. and D. Pei "Oxygen-mediated inactivation of peptide deformylase"
Bio. Chem. (Aug. 28, 1998) 273(35):22305-22310.
Rajagopalan, P. T. R. et al. "Purification, characterization, and inhibition
of peptide deformylase from Escherichia coli" Biochem. (1997) 36(45):13910-13918.
Wei, Y. and D. Pei "Continuous spectrophotometric assay of peptide deformylase"
Analytical Biochem. (1997) 250(1):29-34.
Wei, Y. and D. Pei "Activation of antibacterial prodrugs by peptide deformylase"
Bioor. & Med. Chem. Letts. (2000a) 10(10):1073-1076.
Wei, Y. et al. "Identification of a potent peptide deformylase inhibitor from
a rationally designed combinatorial library" J. Comb. Chem. (2000b) 2(6):650-657.
|
Primary Examiner: Desai; Rita
Attorney, Agent or Firm: Foley & Lardner LLP
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional
Application No. 60/426,771, filed Nov. 14, 2002, the contents of which hereby incorporated
by reference into the present disclosure.
Claims
What is claimed:
1. A compound having the structure:
##STR10##
2. A composition comprising:
a compound of claim 1; and,
a pharmaceutically acceptable carrier.
Description
FIELD OF THE INVENTION
The present invention relates to the field of Enzyme Catalyzed Therapeutic Activation
(ECTA™) therapy and in particular, ECTA therapies specific for microorganisms
that express Peptide Deformylase ("PDF").
BACKGROUND
Throughout this disclosure, various publications are referenced by first
author and date, within parentheses, patent number or publication number. The complete
bibliographic reference is given at the end of the application. The disclosures
of these references are hereby incorporated by reference into this disclosure to
more fully describe the state of the art to which this application pertains.
Enzyme Catalyzed Therapeutic Activation (ECTA™) therapy is a novel
technology that provides unique prodrug substrates for target enzymes. Unlike conventional
therapies, ECTA prodrugs neither inhibit nor irreversibly inactivate the target
enzyme. U.S. Pat. Nos. 6,159,706; 6,245,750 and 6,339,151B1. See also PCT/US98/16607;
PCT/US99/01332; and PCT/US00/20008.
Target enzymes convert the ECTA prodrug into a toxin preferentially within
the target cell or in an environment wherein the target enzyme is expressed as
compared to an environment where it is absent, as in an infected cell. Because
the compounds do not require a targeting agent, they can be directly utilized,
topically or systemically.
ECTA molecules do not, in most instances, yield cytotoxic products spontaneously
(without target enzyme activation). They are not be appreciably activated by non-targeted
enzymes, as this may result in toxicity to non-diseased or non-infected tissue.
Table 1 summarizes the characteristics of ECTA molecules and enzyme activators.
| TABLE 1 |
|
| Characteristics of ECTA Target |
|
| Enzymes |
Characteristics of ECTA Prodrugs |
|
| Infectious Disease: Must be present |
Must be able to get into cells (by |
| only in target cells (including |
itself or as prodrug). |
| diseased cells, bacteria, fungi, |
| etc.). The enzyme should be |
| necessary for continued |
| viability or pathogenicity. |
| Must process a molecule that |
At least one of the products formed |
| resembles the natural substrate (an |
from the enzymatic reaction must |
| ECTA molecule) into cytotoxic |
be cytotoxic. However, the ECTA |
| species. The resemblance only has |
remain in inactive form until |
| to be significant with respect to the |
activated by the target enzyme. |
| specificity of the enzyme/substrate |
The compound must have a high |
| interaction and the ability of the |
degree of specificity for the targeted |
| enzyme to process the substrate |
enzyme, although conversion by a |
| intracellularly into toxic species. |
non-targeted enzyme is acceptable |
| |
if the product(s) are not cytotoxic. |
| Must not be inactivated by the |
Must not inhibit or deactivate the |
| ECTA molecule, intermediate(s), |
targeted enzyme. |
| or the product(s) of the reaction. |
|
In cases of bacterial, viral and fungal infections in plants, people or agriculturally
important animals, metabolic pathways being present in the pathogenic organisms,
but absent in the host are a source of potential ECTA target enzymes. For example,
some pathways, as well as the enzymes involved, have only been found in bacteria,
fungi and plants and not in mammalian cells. One example is the synthesis of "essential"
amino acids—amino acids that animals cannot synthesize and must ingest with
food. Nelson and Cox (1972).
Another example is Peptide Deformylase ("PDF", EC 3.5.1.88) which catalyses
deformylation of N-terminal N-formyl methionine in a growing polypeptide chain.
Meinnel (1999). The enzyme is present and active in bacteria (Meinnel et al, 1993),
but has not been reported to be active in mammalian cells. Sequences homologous
to bacterial PDF sequences have been recently found in mammals but their exact
function is unknown. Giglione (2000a) and (2000b).
Because the enzyme is not active in humans it has been used as a target for
antibacterial drugs, mostly PDF inhibitors. Dithiols can act as non-specific PDF
inhibitors by coordination of sulfhydryl groups with the active site metal ion.
Rajagopalan (1997). In case of 1,2- or 1,3-dithiols a slow extraction of the metal
ion from the active site takes place. The formation of stable 5- or 6-membered
rings, respectively, each containing two metal-sulfur bonds, accounts for this effect.
A rationally designed combinatorial library was used to select mechanism-based
PDF inhibitors of the general structure HS—CH
2—CH(R
a)—CONH—CH(R
b)—CONH—R
c.
Wei et al. (2000a). The optimal inhibitor selected from the library possesses an
n-Bu group as an R
a, R
b=—(CH
2)
3—NH—C(═NH)—NH
2,
and R
c is 2-naphthalene. This compound acts competitive PDF inhibitor
with a K
i of 15 nM.
Jayasekera et al. (2000) describes a series of non-peptidic compounds
structurally related to the known anticholesteremic thyropropic acid to inhibit
E. coli PDF. Actinonin is reported to be a potent PDF inhibitor with activity
in the subnanomolar K
i range. Chen (2000) and Guilloteau et al (2002).
Wei, et al. (2000a) describe that 5′-dipeptidyl derivatives of 5-fluorodeoxyuridine
release a small molecule (5-fluorodeoxyuridine (5-F-dUrd)) upon PDF catalyzed deformylation.
5-F-dUrd formation was monitored in the reaction of the substrate catalyzed by
purified PDF or
E. coli crude lysates. The compound was marginally cytotoxic
(IC
50>100 μM) when applied to
E. coli bacteria. Potency
was not increased by increased expression of PDF in bacteria (using a PDF-overexpressing
strain). The compound was slightly more effective (IC
50=50 μM)
against gram-positive microorgansims.
Additional inhibitors are described in Apfel et al. (2000), Apfel et al.
(2001a), Apfel et al. (2001b), Clements et al. (2001), Durand et al. (1999), Chen
et al. (2000), Yuan et al (2001), and Wise et al (2002)
However, a compound or agent that is not an inhibitor but rather selectively
and effectively activated by PDF to a toxin has not been described. This invention
satisfies this need and provides related advantages as well.
SUMMARY OF THE INVENTION
Thus, in one aspect, the present invention relates to a compound having the
structure:
##STR2##
wherein:
R1 is hydrogen or CH3;
R4 is selected from the group consisting of a substituted or
unsubstituted aryl group, a substituted or unsubstituted, saturated or unsaturated
C1-C6 alkyl group and CH2CH2XCH3,
wherein X is selected from the group consisting of oxygen, sulfur and CH2;
R5 and R6 are independently selected from the group
consisting of hydrogen, a substituted or unsubstituted C5-C14 aryl
group and a substituted or unsubstituted, saturated or unsaturated C1-C6
alkyl group;
=A1 is either absent, in which case the carbon to which it was
attached becomes a CH2 group, or A1 is selected from the
group consisting of oxygen, sulfur, NH, NOH, NR8, and CR9R10,
wherein R8, R9 and R10 are independently selected
from the group consisting of hydrogen and C1-C6 alkyl;
=A2 is either absent, in which case the carbon to which it was
attached becomes a CH2 group, or A2 is selected from the
group consisting of oxygen, sulfur, NH, NOH and NR11 wherein R11
is selected from the group consisting of hydrogen and C1-C6 alkyl;
B1 is either absent or is selected from the group consisting
of oxygen, sulfur, NR12 and CR13R14, wherein R12
R13 and R14 are independently selected from the group
consisting of hydrogen and a substituted or unsubstituted saturated or unsaturated alkyl;
B2 is selected from the group consisting of carbon, oxygen and
nitrogen, wherein, when B2 is oxygen, R5 and R6 are
absent and when B2 is nitrogen, one of R5 or R6 is absent;
B1 may be joined through R12, R13 or R14
to R5 or R6 to form a saturated or unsaturated, substituted
or unsubstituted ring which may contain 0-3 nitrogen atoms and/or 0-1 oxygen or
sulfur atoms;
B3 and B4 are independently the same or different
and are absent or are selected from the group consisting of oxygen, sulfur, NH,
CH2, NR17, CHR18, CR21R22 and,
when either linker1-B5-Toxin1 or linker2-B6-Toxin2
is absent, OR15, SR16, NR19R20 and
wherein R15-R25 are independently selected from the group
consisting of hydrogen, substituted or unsubstituted, saturated or unsaturated
alkyl and substituted or unsubstituted aryl;
B3 or B4 may be joined through one of R15-R25
to B1, R5 or R6 to form a saturated or unsaturated,
substituted or unsubstituted ring which contains one phosphorus atom and which
may contain 0-3 nitrogen atoms and/or 0-1 oxygen or sulfur atoms;
B5 and B6 are independently the same or different
and are absent or are selected from the group consisting of oxygen, sulfur, OC(═O),
SC(═S), OC(═O)NH, SC(═S)NH, OC(═S)NH, N(R26)
and C(R27)(R28), wherein R26, R27 and
R28 are independently selected from the group consisting of hydrogen
and a substituted or unsubstituted, saturated or unsaturated alkyl;
Linker1 and Linker2 are independently the same or
different and are absent or present and, if present, are traceless; and, one of
Toxin1 or Toxin2 may be absent.
In an aspect of this invention, R
1 is hydrogen and R
4 is
CH
2CH
2XCH
3, and X is selected from the group consisting
of CH
2, sulfur and oxygen.
In an aspect of this invention, X is sulfur.
In an aspect of this invention, A
1 and A
2 are both oxygen.
In an aspect of this invention, Linker
1-B
5-Toxin
1
is absent.
In an aspect of this invention, B
1 is absent.
In an aspect of this invention B
2 is carbon.
In an aspect of this invention, B
2 is nitrogen.
In an aspect of this invention, B
3 is selected from the group consisting
of OH, OCH
3, OCH
2CH
3 and OC
6H
5.
In an aspect of this invention, Linker
2 is selected from the group
consisting of
substituted or unsubstituted allyl;
substituted or unsubstituted benzyl;
C6H4CH2X1C(═X2),
wherein X1 and X2 are independently selected from the group
consisting of oxygen, sulfur and N(R29), wherein R29 is hydrogen
or C1-C6 alkyl; and,
(CH2)nN(R)C(═O), wherein n is 2 or 3 and R
is hydrogen or C1-C6 alkyl.
In an aspect of this invention B
6 is absent.
In an aspect of this invention, in any of the above compounds, Toxin
1
and Toxin
2 are independently selected from the group consisting of aminoglycosides,
mitomycin, CC-1065, ducarmycin, cyclopropyl indole, cyclopropyl benzoindole analogs,
anthracyclins, vinca alkaloids, mitomycins, bleomycins, penicillins, cephalosporins,
oxacillins, carbopenems, tetracyclins, chloramphenicols, macrolides, cycloserines,
fluoroquinolones (including, but not limited to, ciprofloxacin and norfloxacin),
glycopeptides, aminoglycosides, peptide antibiotics, oxazolidinones, quinolones,
sulfonamides, cytotoxic nucleosides, pteridine family, nitrogen mustards, polyhalogenated
biaryl ethers, diynenes, podophillotoxins, taxoids, doxorubicin, caminomycin, daunorubicin,
aminopterin, methotrexate, methopterin, dichloromethotrexate, mitomycin C, porfiromycin,
6-mercaptopurine, cytosine arabinoside, podophillotoxin, etoposide, etoposide phosphate,
melphalan, vindesine, vinblastine, vincristine, leurosidine, leurosine, bis-(2-chloroethyl)amine,
trichlorcarban, trichlorocarbanilide, triclosan, tribromosalicylanilide, sulphamethoxazole,
chloramphenicol, cycloserine, trimethoprim, chlorhexidine, hexachlorophene, fentichlor,
5-chloro-2-(2,4-dichlorophenoxy)phenol, 4-chloro-2-(2,4-dichlorophenoxy)phenol,
3-chloro-2-(2,4-dichlorophenoxy)phenol, 6-chloro-2-(2,4-dichlorophenoxy)phenol,
5-chloro-2-(3,4-dichlorophenoxy)phenol, 5-chloro-2-(2,5-dichlorophenoxy)phenol,
5-chloro-2-(3,5-dichlorophenoxy)phenol, 2,2′-dihydroxy biphenyl ether, halogenated
2-hydroxybenzophenones, 2-mercaptopyridine-N-oxide, combretastatin, camptothesin,
apoptolidene, cisplatin, epothilone, halichondrin, hemiasterlin, methioprim, thapsigargin,
chloroquine, 4-hydroxycyclophosphamide, etoposide, colchicine, melphalan, quercetin,
genistein, erbstatin, N-(4-aminobutyl)-5-chloro-2-naphtalen-sulfonamide, pyridinyloxazol-2-one,
isoquinolyloxazolone-2-one, verapamil, quinine, quinidine, and chloroquine.
In an aspect of this invention, Toxin
2 is a quinolone.
In an aspect of this invention, Toxin
2 is selected from the group
consisting
of triclosan, cyclopropylindole, cyclopropylbenzoindole and derivatives thereof.
In an aspect of this invention, B
1 is joined to R
5 or R
6
to form a ring.
An aspect of this invention is a compound having the structure:
##STR3##
An aspect of this invention is a composition comprising a compound hereof and
a pharmaceutically acceptable carrier.
An aspect of this invention is a method for the treatment of a disease caused
by a microorganism expressing a peptide deformylase enzyme, comprising administering
an effective amount of a compound of claim 1 to a subject in need thereof.
Also provided by this invention is a method for inhibiting the growth of a microorganism
that expresses PDF by contacting the microorganism with an effective amount of
the compound as describe above. This method inhibits the growth of a gram-positive
or gram-negative microorganism, e.g., of
S. aureus, S. epidermidis, K. pneumoniae,
E. aerogenes, E. cloacae, M. catarrhalis, E. coli, E. faecalis, H. influenzae or
P. aeruginosa. This method can be practiced in vitro, ex vivo and in vivo.
Further provided is a method for alleviating the symptoms of an infection by a
PDF expressing microorganism in a subject by administering or delivering to the
subject an effective amount of the compound described above. A "subject" is defined
herein and includes mammals such as human patients.
This invention also provides a composition comprising the prodrug compounds
as described above, alone or in combination with other compounds or other agents,
known or yet to be discovered, and a carrier. In one aspect, the carrier is another
molecule or an inert substance such as a plate or column. In an alternative embodiment,
the carrier is a pharmaceutically acceptable carrier. Pharmaceutically acceptable
carriers are known in the art and described briefly above.
DETAILED DESCRIPTION OF THE INVENTION
Brief Description of the Table
Table 1 shows a list of non-limiting charactertistics of ECTA target enzymes
and ECTA prodrugs.
Table 2 is a non-limiting list of bacteria that express PDF and therefore are
expected to be susceptible to the compounds of this invention.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is a proposed reaction scheme for PDF activation of the compounds of
this invention.
DISCUSSION
The practice of the present invention will employ, unless otherwise indicated,
conventional techniques of organic chemistry, pharmacology, molecular biology (including
recombinant techniques), cell biology, biochemistry, and immunology, which are
within the skill of the art. Such techniques are explained fully in the literature,
such as, "
MOLECULAR CLONING: A LABORATORY MANUAL" Second
Edition (Sambrook et al., 1989); "
OLIGONUCLEOTIDE SYNTHESIS"
(M. J. Gait, ed., 1984); "
ANIMAL CELL CULTURE" (R. I. Freshney,
ed., 1987); the series "
METHODS IN ENZYMOLOGY" (Academic
Press, Inc.); "
HANDBOOK OF EXPERIMENTAL IMMUNOLOGY" (D.
M. Weir & C. C. Blackwell, eds.); "
GENE TRANSFER VECTORS FOR MAMMALIAN
CELLS" (J. M. Miller & M. P. Calos, eds., 1987); "
CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY" (F. M. Ausubel et al., eds., 1987,
and periodic updates); "
PCR: THE POLYMERASE CHAIN REACTION"
(Mullis et al., eds., 1994); "
CURRENT PROTOCOLS IN IMMUNOLOGY"
(J. E. Coligan et al., eds., 1991); J. March, A
DVANCED O
RGANIC
C
HEMISTRY: R
EACTIONS,
M
ECHANISMS A
ND S
TRUCTURE,
4
th edition (John Wiley & Sons, NY (1992)).
As used herein, certain terms may have the following defined meanings.
The singular form "a," "an" and "the" include plural references unless the context
clearly dictates otherwise. For example, the term "a cell" includes a plurality
of cells, including mixtures thereof.
The term "comprising" is intended to mean that the compositions and methods include
the recited elements, but do not exclude others. "Consisting essentially of," when
used to define compositions and methods, shall mean excluding other elements of
any essential significance to the combination. Thus, a composition consisting essentially
of the elements as defined herein would not exclude trace contaminants from the
isolation and purification method and pharmaceutically acceptable carriers, such
as phosphate buffered saline, preservatives, and the like. "Consisting of" shall
mean excluding more than trace elements of other ingredients and substantial method
steps for administering the compositions of this invention. Embodiments defined
by each of these transition terms are within the scope of this invention.
As used herein, the term "peptidomimetic" refers to a compound containing non-peptidic
structural elements that is capable of mimicking or antagonizing the biological
action(s) of a natural parent peptide.
As used herein, the term "substituents" includes, but is not limited to halogen
atoms and halomethyl groups such as CF
3 and CCl
3; oxygen
containing groups such as oxo, hydroxy, carboxy, carboxyalkyl, alkoxy, alkoyl,
alkoyloxy, aryloxy, aryloyl, and aryloyloxy; nitrogen containing groups such as
amino, alkylamino, dialkylamino, cyano, azide and nitro; sulfur containing groups
such as thiol, alkylthiol, sulfonyl and sulfoxide; heterocyclic groups which may
themselves be substituted; alkyl groups which may themselves be substituted; and
aryl groups which may themselves be substituted.
The term "alkyl" refers to any branched or unbranched, cyclic or acyclic, saturated
(alkyl) or unsaturated (alkenyl or alkynyl) hydrocarbyl radical. This term is not
limited to a monovalent radical. Where cyclic, the alkyl group is C
3
to C
12. Where acyclic, the alkyl group is C
1 to C
16.
The term "alkoxy" and "aryloxy" means alkyl-O and aryl-O groups, respectively.
Reference to "alkoyl" and "aryloyl" groups means alkyl-CO and aryl-CO, respectively.
The term "aryl" refers to an aromatic group, such as phenyl or napthyl, or a
heteroatomic group containing one or more heteroatom, such as pyridyl, pyrrolyl,
furanyl and thiophenyl. This term is not limited to a monovalent radical. The term
"aromatic" refers to any compound characterized by the presence of at least one
benzene ring. Examples of aromatic groups include, but are not limited to, benzene
and naphthalene.
The alkyl and aryl groups may be substituted or unsubstituted. The term "aliphatic"
refers to any and all organic compounds of hydrogen and carbon characterized by
a straight chain of the carbon atoms. The subgroups of "aliphatic" compounds includes
alkanes, alkenes, and alkynes. The aliphatic groups may be unsubstituted or substituted.
See above for examples of substituents.
The term "alicyclic" refers to and covers any and all organic compounds of hydrogen
and carbon atoms joined to form one or more rings. The alicyclic groups may be
unsubstituted or substituted. See above for examples of substituents.
"Cycloalkyl" is intended to include saturated ring groups, such as cyclopropyl,
cyclobutyl, or cyclopentyl. This term is not limited to a monovalent radical. The
cycloalkyl groups may be unsubstituted or substituted. See above for examples of substituents.
The term "alkenyl" refers to normal alkenyl, branch chain alkenyl and cycloalkenyl
groups having one or more sites of unsaturation. "Alkynyl" is intended to include
hydrocarbon chains of either a straight or branched configuration and one or more
triple carbon-carbon bonds which may occur in any stable point along the chain,
such as ethynyl and propynyl. These terms are not limited to monovalent radicals.
The alkenyl or alkynyl groups may be unsubstituted or substituted. See above for
examples of substituents.
"Lower alkyl" means the above-defined broad definition of alkyl groups having
1 to 6 carbons in case of normal lower alkyl, and as applicable 3 to 6 carbons
for lower branch chained (e.g., 2-methyl-propane) and cycloalkyl groups. "Lower
alkenyl" is defined similarly having 2 to 6 carbons for normal lower alkenyl groups,
and 3 to 6 carbons for branch chained and cyclo-lower alkenyl groups. "Lower alkynyl"
is also defined similarly, having 2 to 6 carbons for normal lower allynyl groups,
and 4 to 6 carbons for branch chained lower alkynyl groups. The lower alkyl, alkenyl
and alkynyl groups may be unsubstituted or substituted. See above for examples
of substituents.
"Haloalkyl" is intended to include both branched and straight-chain saturated
aliphatic hydrocarbon groups having the specified number of carbon atoms, substituted
with 1 or more halogen (for example—C
vF
w where v=1
to 3 and w=1 to (2v+1)). The term haloalkyl is not limited to a monovalent radical.
"Halogen" includes fluorine, chlorine, bromine, iodine and astatine. Examples of
haloalkyl include, but are not limited to, trifluoromethyl, trichloromethyl, pentafluoroethyl,
and pentachloroethyl.
The term "heterocyclic" refers to any compound in which the ring structure is
a combination of more than one kind of atom. Examples of heterocyclic compounds
include, but are not limited to, pyridine, furan and pyrrole. This term is not
limited to a monovalent radical. The heterocyclic groups may be unsubstituted or
substituted. See above for definition of substituents.
As used herein the term "prodrug" means a precursor or derivative form of a pharmaceutically
active agent or substance that is less cytotoxic to a target cell as compared to
the drug metabolite and is capable of being enzymatically activated or converted
into the more active form.
A "composition" is intended to mean a combination of active agent and another
compound
or composition, inert (for example, a surface, a paint, a detectable agent or label
or a pharmaceutically acceptable carrier) or active, such as an adjuvant or disinfectant.
A "pharmaceutical composition" is intended to include the combination of an active
agent with a carrier, inert or active, making the composition suitable for diagnostic
or therapeutic use in vitro, in vivo or ex vivo.
The term "prophylactically effective amount" refers to an amount effective in
preventing infection in a subject or plant infestation.
The term "pharmaceutically acceptable carrier" and "biologically acceptable carrier"
refer to a carrier or adjuvant that is administered to a host or patient, together
with a compound of this invention, and which does not destroy the pharmacological
activity thereof and is non-toxic, when administered in doses sufficient to deliver
an effective amount of the compound. Examples of suitable carriers include liquid
phase carriers, such as sterile or aqueous solutions, as well as those described
below. Examples of pharmaceutically acceptable carrier include any of the standard
pharmaceutical carriers, such as a phosphate buffered saline solution, water, and
emulsions, such as an oil/water or water/oil emulsion, and various types of wetting
agents. The compositions also can include stabilizers and preservatives. For examples
of carriers, stabilizers and adjuvants, see Martin, REMINGTON'S PHARM. SCI., 15th
Ed. (Mack Publ. Co., Easton (1975)).
The term "treating" refers to any of the following: the alleviation of symptoms
of a particular disorder in a patient; the improvement of an ascertainable measurement
associated with a particular disorder; or a reduction in microbial number. One
of skill in the art can determine when a host has been "treated" by noting a reduction
in microbial load or an alleviation in symptoms associated with infection.
The term "pharmaceutically acceptable salt, prodrug or derivative" relates to
any pharmaceutically acceptable salt, ester, ether, salt of an ester, solvate,
such as ethanolate, or other derivative of a compound of the present invention
which, upon administration to a recipient, is capable of providing (directly or
indirectly) a compound of this invention or an active metabolite or residue thereof.
Particularly favored derivatives and prodrugs are those that increase the bioavailability
of the compounds of this invention when such compounds are administered to a mammal
(e.g., by allowing an orally administered compound to be more readily absorbed
into the blood) or which enhance delivery of the parent compound to a biological
compartment (e.g., the brain or lymphatic system).
Salts of the compounds of the present invention may be derived from inorganic
or organic acids and bases. Examples of acids include hydrochloric, hydrobromic,
sulfuric, nitric, perchloric, fumaric, maleic, phosphoric, glycolic, lactic, salicylic,
succinic, toluene-p-sulfonic, tartaric, acetic, citric, methanesulfonic, ethanesulfonic,
formic, benzoic, malonic, naphthalene-2-sulfonic and benzenesulfonic acids. Other
acids, such as oxalic, while not in themselves pharmaceutically acceptable, may
be employed in the preparation of salts useful as intermediates in obtaining the
compounds of the invention and their pharmaceutically acceptable acid addition
salts. Examples of bases include alkali metal (e.g., sodium) hydroxides, alkaline
earth metal (e.g., magnesium) hydroxides, ammonia, compounds of formula NW
4+,
wherein W is C
1-4 alkyl and THAM (2-amino-2-hydroxymethyl-1,3-propanediol).
Examples of salts include: acetate, adipate, alginate, aspartate, benzoate,
benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate,
digluconate, dodecylsulfate, ethanesulfonate, fumarate, flucoheptanoate, glycerophosphate,
hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate,
lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate,
palmoate, pectinate, persulfate, phenylproprionate, picrate, pivalate, propionate,
succinate, tartrate, thiocyanate, tosylate and undecanoate. Other examples of salts
include anions of the compounds of the present invention compounded with a suitable
cation such as Na
+, Li
+, NH
4+, and
NW
4+ (wherein W is a C
1-4 alkyl group).
For therapeutic use, salts of the compounds of the present invention will be
pharmaceutically acceptable. However, salts of acids and bases which are non-pharmaceutically
acceptable may also find use, for example, in the preparation or purification of
a pharmaceutically acceptable compound or for use to reduce microbial infestation
in plants.
The term "traceless linker" indicates a spacer or connector between two parts
of a single molecule such that when a particular bond is severed between the two
parts of the molecule, the connector which is still attached to the second part
of the molecule, eliminates leaving no trace of itself. This may most easily be
seen by examining the following non-limiting example:
##STR4##
Once the PDF has deformylated the prodrug to reveal the amine group, the amine
nitrogen attacks the phosphorus to form a six-member ring with the resultant elimination
of the oxygen-containing group, which in turn undergoes a semiquinone methide rearrangement
resulting in the release of the toxin with no remaining trace of the linker moiety
itself, the linker thus being "traceless." Other traceless linkers will be apparent
to those skilled in the art based on the disclosure herein and are within the scope
of this invention. See, for example, de Groot et al. (2000).
The term "effective amount" is to include therapeutically or prophylactically
effective amounts. The term refers to an amount effective in treating or preventing
an infection in a patient or an infestation in a plant either as monotherapy or
in combination with other agents.
"Inhibiting the growth" of a microorganism means reducing by contact
with an agent, the rate of proliferation of such a microorganism, in comparison
with a control microorganism of the same species not contacted with this agent.
A "subject" is any living entity, plant or animal, that is or can be a direct
or
indirect host to a PDF expressing microorganism. Thus, without limitation, crops
such as corn, soybeans, grains, ornamental plants, trees, etc. are subjects within
the scope of this invention as are animals such as, also without limitation, fish,
shellfish, birds, mammals, and humans. Mammals include, but are not limited to,
rodents, simians, farm animals, sport animals, pets and humans.
As used herein, "Toxin
1 and Toxin
2" are each an agent that
is toxic upon activation by an activating enzyme, and may be the same or different.
More generally, they each refer to any chemical entity useful to treat or prevent
harm to plants or animals caused by pathogenic organisms that express PDF. Such
chemical entities include, without limitation, anthelmintics, antibacterials including
both antibiotics and synthetic compounds, antifungals, antineoplastics, antiprotozoal
and the like. It is understood that the, in one aspect, either or both "Toxins"
may include B
3, B
4, B
5 or B
6 depending
on the structure of the prodrug and the mechanism of release of the active species
from it. An example of this is seen in FIG. 1, wherein the oxygen attached to the
"Tox" group is actually a component (as a hydroxyl group) of the actual Toxin,
triclosan. Some specific examples of chemical entities that can comprise Toxin
1
and Toxin
2 are aminoglycosides, mitomycin, CC-1065, ducarmycin,
cyclopropyl indole, cyclopropyl benzoindole analogs, anthracyclins, vinca alkaloids,
mitomycins, bleomycins, penicillins, cephalosporins, oxacillins, carbopenems, tetracyclins,
chloramphenicols, macrolides, cycloserines, fluoroquinolones (including, but not
limited to, ciprofloxacin and norfloxacin), glycopeptides, aminoglycosides, peptide
antibiotics, oxazolidinones, quinolones, sulfonamides, cytotoxic nucleosides, pteridine
family, nitrogen mustards, polyhalogenated biaryl ethers, diynenes, podophillotoxins,
taxoids, doxorubicin, carminomycin, daunorubicin, aminopterin, methotrexate, methopterin,
dichloromethotrexate, mitomycin C, porfiromycin, 6-mercaptopurine, cytosine arabinoside,
podophillotoxin, etoposide, etoposide phosphate, melphalan, vindesine, vinblastine,
vincristine, leurosidine, leurosine, bis-(2-chloroethyl)amine, trichlorcarban,
trichlorocarbanilide, triclosan, tribromosalicylanilide, sulphamethoxazole, chloramphenicol,
cycloserine, trimethoprim, chlorhexidine, hexachlorophene, fentichlor, 5-chloro-2-(2,4-dichlorophenoxy)phenol,
4-chloro-2-(2,4-dichlorophenoxy)phenol, 3-chloro-2-(2,4-dichlorophenoxy)phenol,
6-chloro-2-(2,4-dichlorophenoxy)phenol, 5-chloro-2-(3,4-dichlorophenoxy)phenol,
5-chloro-2-(2,5-dichlorophenoxy)phenol, 5-chloro-2-(3,5-dichlorophenoxy)phenol,
2,2′-dihydroxy biphenyl ether, halogenated 2-hydroxybenzophenones, 2-mercaptopyridine-N-oxide,
combretastatin, camptothesin, apoptolidene, cisplatin, epothilone, halichondrin,
hemiasterlin, methioprim, thapsigargin, chloroquine, 4-hydroxycyclophosphamide,
etoposide, colchicine, melphalan, quercetin, genistein, erbstatin, N-(4-aminobutyl)-5-chloro-2-naphtalen-sulfonamide,
pyridinyloxazol-2-one, isoquinolyloxazolone-2-one, verapamil, quinine, quinidine,
and chloroquine.
PDF is a well-studied enzyme whose crystallographic structure has been elucidated.
Chan et al. (1997). The enzyme has been expressed in
E. coli BL21(DE3) cells
Rajagopalan et al. (1997). The authors of the paper isolated the
E. coli def
gene by PCR using the primers designed based on the literature data on the sequence
of the gene. The purified enzyme is unstable due to fast oxidation of the catalytic
site Fe
2+ by atmospheric oxygen. Rajagopalan et al. (1998). The conditions
for proper handling of the enzyme to avoid inactivation have been reported. Rajagopalan
et al. (1997). Zn
2+ and Ni
2+ containing PDF's are stable
allowing for the in vitro evaluation of the enzyme catalytic properties. There
exists a simple continuous colorimetric assay for PDF. Wei et al. (1997). It utilizes
N-formylmethionylleucine p-nitroanilide as a substrate. A coupled aminopeptidase
reaction that follows the PDF reaction releases p-nitroaniline that can be monitored
spectrophotometrically at 405 nm.
PDF is a perfect ECTA target enzyme. It is active in bacteria and inactive in
human hosts. It has broad substrate specificity. Deformylation liberates a free
amino group of methionine (or another amino acid or an amino acid analog tolerated
as an R
3 of the substrate, such as norleucine) which can perform a subsequent
nucleophilic attack. With a rationally designed dipeptide or an equivalent peptidomimetic
fragment of the substrate the free amino group can attack an optimally positioned
phosphorus of the substrate thus forming a cyclic molecule and releasing a toxin.
The substrate can be optimized to enhance cyclization. The scheme of the proposed
reaction exemplifying a dipeptide-based substrate is given in FIG. 1. Here X can
be, for example, sulfur (methionine) or —CH
2— (norleucine).
R
1 and R
2 are aliphatic radicals that can be selected based
on the published SAR data for PDF. Hu et al. (1998).
This invention also provides a composition comprising the prodrug compounds
as described above, alone or in combination with other compounds or other agents,
known or yet to be discovered, and a carrier. In one aspect, the carrier is another
molecule or an inert substance such as a plate or column. In an alternative embodiment,
the carrier is a pharmaceutically acceptable carrier. Pharmaceutically acceptable
carriers are known in the art and described briefly above.
This invention also provides a composition comprising the prodrug compounds
as described above, alone or in combination with other compounds or other agents,
known or yet to be discovered, and a carrier. Any of the above noted compounds
can be combined with a carrier, such as a pharmaceutically acceptable carrier,
and/or an additional effective agent such as an antibiotic. These compounds and
compositions are also useful in combination with known or yet to be discovered
therapeutics and therapeutic methods to enhance the therapy or efficacy of the drug.
Also provided by this invention is a method for inhibiting the growth of a PDF
expressing microorganism by contacting the microorganism with an effective amount
of the compound as described above. Methods to detect PDF expression are known
in the art. See, for example, Wei, et al. (1997). This method is particularly useful
in inhibiting the growth of gram-positive and gram-negative microorganisms, e.g.,
S. aureus, S. epidermidis, K pneumoniae, E. aerogenes, E. cloacae, M. catarrhalis,
E. coli, E. faecalis, H. influenzae and
P. aeruginosa. and those identified
in Table 2, below. This method can be practiced in vitro, ex vivo and in vivo.
Further provided is a method for alleviating the symptoms of an infection in a
subject, wherein the infection is caused by a PDF expressing microorganism, by
administering or delivering to the subject an effective amount of the compound
described above. Also provided by this invention is a method for treating an infection
caused by a PDF expressing microorganism by administering or delivering to the
subject an effective amount of the compound described above. A "subject" is defined
above and includes mammals such as human patients. Examples of PDF expressing microorganims
and the corresponding diseases and symptoms caused by infection by these microorganisms,
are provided in Table 2, below.
| TABLE 2 |
|
| |
Disease or Symptom Caused by |
| PDF Expressing Microorganism |
Infection |
|
| Gram-Positive |
|
| Staphylococcus aureus |
major human pathogen, |
| |
bacteremia, pneumonia |
| Staphylococcus epidermidis and |
urinary tract infections, |
| other coagulase-negative |
osteomyelitis, bacteremia |
| staphylococci |
| Streptococcus pyogenes |
bacteremia, lymphagitis, |
| |
pneumonia |
| Streptococcus pneumoniae |
pneumonia, otitis media, |
| |
sinusitis |
| Streptococcus agalactiae |
primary bacteremia, |
| |
pneumonia, endocarditis, |
| |
osteomyelitis |
| Enterococcus species |
urinary tract infections, |
| |
bacteremia, endocarditis, intra- |
| |
abdominal and pelvic |
| |
infections, neonatal sepsis |
| Gram-Negative |
| Neisseria gonorrhoeae |
genital infection, perihepatitis |
| Moraxella catarrhalis |
otitis media, lower respiratory |
| |
tract infections, pneumonia, |
| |
bacteremia |
| Campylobacter jejuni |
acute enteritis, acute colitis, |
| |
bacteremia |
| Enterobacteriaceae (including |
enteric infections, urinary tract |
| Escherichia, Salmonella, Klebsiella, |
infections, respiratory |
| Enterobacter) |
infections, bacteremia |
| Pseudomonas aeruginosa |
endocarditis, respiratory |
| |
infections, bacteremia, central |
| |
nervous system infections |
| Acinetobacter species |
respiratory tract infections, |
| |
bacteremia, genitourinary |
| Haemophilus influenzae |
meningitis, epiglottitis, |
| |
pneumonia, bacteremia |
|
In the clinical use of the prodrug, antibiotics will likely follow well established
guidelines. Dosage will likely be similar to those already employed for most other
antibiotics. It is estimated that a dose of prodrug will be in the range of 100
μg to 100 mg to 1 gm, or given once every eight hours, or once a day, for
one or two weeks, or until the patient tests negative for infectious organisms.
The compounds of this invention are expected to be administered in doses that
reflect the literature doses for the active compounds of which they are prodrugs,
with the difference in molecular weight being taken into account. Thus, it is estimated
that the dosage for a prodrug hereof will typically be in the range of 100 μg
to 100 mg to 1 gm.
In one aspect, the invention encompasses a method of treating or protecting plants
from infections caused by PDF expressing microorganisms by applying an effective
amount of the substrate prodrug.
In order to achieve good dispersion and adhesion of the compounds to treat plants,
it may be advantageous to formulate the compounds with components that aid dispersion
and adhesion. Suitable formulations will be known to those skilled in the art.
This invention also provides a method for treating or protecting plants from
infection by microorganisms expressing PDF by applying an effective amount of the
prodrug compound to the foliage, roots or the soil surrounding the plants or roots.
These isolated compounds can be combined with known pesticides or insecticides.
Compounds within the present invention when used to treat or protect plants
from infections caused by PDF expressing microorganisms can be formulated as wettable
powders, granules and the like, or can be microencapsulated in a suitable medium
and the like. Examples of other formulations include, but are not limited to soluble
powders, wettable granules, dry flowables, aqueous flowables, wettable dispersible
granules, emulsifiable concentrates and aqueous suspensions. Other suitable formulations
will be known to those skilled in the art.
This invention further provides a method for administering the prodrug compound
to fish in an amount effective to either prevent or treat an infection caused by
PDF expressing microorganisms. The compound may be administered by incorporating
the compound into the food supply for the fish. Alternatively, the compound may
be added to the water in which fish live, or are contained within. Finally, the
compound may be administered to the fish as a suitable pharmaceutical preparation.
Other suitable formulations will be known to those skilled in the art. Further
provided is a process for producing the prodrugs of this invention. In general
the process requires the following steps:
##STR5##
With respect to the above diagram, X can be sulfur (methionine) or —CH
2—
(norleucine). R can be R
5 or R
6 as defined above. Reaction
conditions and full names for the abbreviations can be found in the experimental
examples infra.
This invention provides a method for identifying potential therapeutic agents
that inhibit the growth of an organism expressing PDF by contacting a sample containing
the PDF expressing microorganism with an effective amount of a candidate prodrug
compound. In a separate sample, the same microorganism is contacted with an effective
amount of a prodrug of this invention. If the agent has comparable anti-proliferative
ability as compared to a prodrug as described herein, the candidate is useful to
inhibit the growth of or kill a PDF-expressing microorganism.
The prodrug is contacted with the sample under conditions that favor the activation
of the prodrug by PDF and then the sample is assayed for growth inhibition or microbial
death. Alternatively, the sample can be tested for the presence of the byproducts
of the reaction of PDF on the substrate. Varying amounts of the substrate are contacted
with a microorganism that expresses PDF for an amount of time effective for PDF
to release the toxin from the cell, the bacteria is lysed and the analytes are
analyzed using methods known in the art (e.g., High Performance Liquid Chromatography
("HPLC")) to identify the reaction products.
Varying concentrations of the potential agent are contacted with the sample
to determine the optimal effective concentration of the agent. Thus, in one aspect,
this invention relates to the discovery, and use thereof, of agents that are selective
substrates for PDF.
Also provided by this invention are kits containing the prodrugs as described
herein and instructions necessary to perform the screen.
The methods of the invention can be practiced in vitro, ex vivo or in vivo. In
vivo practice of the invention in an animal such as a rat or mouse provides a convenient
animal model system that can be used prior to clinical testing of the therapeutic
agent or prodrug. In this system, a potential prodrug will be successful if microbial
load is reduced or the symptoms of the infection are ameliorated compared to an
untreated, infected animal. It also can be useful to have a separate negative control
group of cells or animals which has not been infected, which provides a basis for comparison.
When practiced in vivo, the candidate prodrug is administered or delivered to
the animal in effective amounts. As used herein, the term "administering" for in
vivo and ex vivo purposes means providing the subject with an effective amount
of the candidate prodrug effective to reduce microbial load. In these instances,
the agent or prodrug may be administered with a pharmaceutically acceptable carrier.
The agents, prodrugs and compositions of the present invention can be used in the
manufacture of medicaments and for the treatment of humans and other animals by
administration in accordance with conventional procedures, such as an active ingredient
in pharmaceutical compositions.
Methods of administering pharmaceutical compositions are known to those of
ordinary skill in the art and include, but are not limited to, microinjection,
intravenous or parenteral administration. The compositions are intended for topical,
oral, or local as well as intravenous, subcutaneous and intramuscular administration.
Administration can be effected continuously or intermittently throughout the course
of the treatment. Methods of determining the most effective means and dosage of
administration are known to those of skill in the art and will vary with the prodrug
used for therapy, the purpose of the therapy, the microorganism being treated,
the severity of the infection, and the subject being treated. Single or multiple
administrations can be carried out with the dose level and pattern being selected
by the treating physician. For example, the compositions can be administered to
a subject already suffering from an antibiotic resistant bacterial infection. In
this situation, an effective "therapeutic amount" of the composition is administered
to prevent continued and to at least partially arrest microbial growth and proliferation
and ameliorate the symptoms associated with an infection.
However, the prodrugs can be administered to subjects or individua
