JP2003508049A - Methods for identifying inhibitors of Cdc25 - Google Patents

Abstract

  (57) [Summary] The present invention relates to crystalline polypeptides comprising the catalytic domain of Cdc25B or Cdc25C protein, including the catalytic domain of human Cdc25B. The present invention also relates to a method for determining the three-dimensional structure of the catalytic region of Cdc25 using these crystals. The present invention also provides a method of using a three-dimensional structure of a Cdc25 catalytic domain in a method for designing and / or identifying a potential inhibitor of Cdc25 activity, for example, a compound that inhibits binding of a native substrate to the Cdc25 catalytic domain. Also relevant.

Description

Detailed Description of the Invention

RELATED APPLICATION This application claims priority based on US Provisional Application 60 / 172,215 filed Aug. 31, 1999, the entire contents of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION The application of modern molecular genetics to the study of cancer has demonstrated that certain DNA sequence alterations can be responsible for tumor development, growth and progression. Many of these changes either direct cell proliferation (growth-stimulating factors / receptors such as Ras, ErbB2, bce / abl) or indirectly (transcribed proteins such as Rb, myc and p53).
It happens in the changing gene. Recently, it has become clear that many of these oncogenic changes are associated with changes in cell cycle regulation. For example, many oncogenes encode components of the pathway by which growth factor signals are transmitted during the cell cycle to induce cell differentiation. Changes in cell cycle proteins such as cyclins D1 and E have also been shown to be tumorigenic in some cell types.

Cdc25 is a family of dual-specificity phosphatases that dephosphorylate both phosphorylated tyrosine and phosphorylated threonine residues of proteins. Cdc
25 regulates cell cycle progression by suppressing the phosphorylation status of these cyclin-dependent kinases. When Tyr ¹⁵ and Thr ¹⁴ are phosphorylated, these cyclin-dependent kinases (cdk) are inactive and thus block cell cycle progression. As a result of dephosphorylation by Cdc25, cdk is activated and the cell cycle proceeds. It is clear that the activity of Cdc25 phosphatase is required for cell cycle transition and this enzyme acts as a rate limiting cell division activator. For example, mutation of Cdc25 in yeast results in cells that arrest in the G2 phase. Mutation of the Cdc25 homologue in Drosophila causes a G2 arrest in early embryogenesis.

Mammalian Cdc25 has three distinct homologues, Cdc25A and Cdc2.
It has been confirmed that there are 5b and Cdc25C. Each of these is believed to target a particular cdk / cyclin complex. C in mammalian cells
Microinjection of antibodies against dc25A or Cdc25C resulted in cell S phase (Ho
ffmann et al., EMBO J. 13: 4302-10 (1994))
And M phase (Millar et al., Proc. Nat. Acad, Sci.
USA 88: 10500-4 (1991)), respectively. Recently published papers have shown that Cdc25C plays an important role in arrest at DNA damage checkpoints, such as arrest by cytotoxic agents. The injury activates the serine kinase Chk-1 and thus Cd
Serine 216 of c25C is phosphorylated. This phosphorylation renders Cdc25C a binding partner for the family of 14-3-3 binding proteins. 14-3-3 binding to the protein is carried out by the cdk25C cdk / cyclin complex cdc2 / cycB
It is believed that the G2 cell cycle is arrested (Fur
Nari et al., Science 277: 1495-1497 (1997);
Sanchez et al., Science 277: 1497-1501 (1
997), Peng et al., Science 277: 1501-1505.
(1997)). There are also studies showing that both Cdc25A and Cdc25B act as oncogenes and can transform overexpressed cells (Galaktionov et al., Science 269: 1575-15).
77 (1995)).

Due to its role in cell cycle regulation, Cdc25 is considered to be applicable to therapeutic methods for the purpose of suppressing proliferative diseases such as cancer. C
Advances in dc25 biochemical assays have enabled high-throughput screening of compound libraries and testing of compounds that mimic substrate structure to identify Cdc25 inhibitors; however, rational Structure-based design is not possible at this time due to lack of accurate 3D structure data.

SUMMARY OF THE INVENTION The present invention provides a plurality of polypeptides having a ligand binding domain of Cdc25,
Crystal forms of these polypeptides and Cd using these crystal forms of polypeptides
It relates to investigating the three-dimensional structure of the catalytic domain of c25. The present invention is Cdc2
The three-dimensional structure of the catalytic domain of 5 is also related to methods of designing and / or identifying potential inhibitors of Cdc activity, such as compounds that inhibit the binding of the native substrate of the catalytic domain of Cdc25.

In one embodiment, the invention provides a plurality of polypeptides having a ligand binding domain of Cdc25, a crystalline form of these polypeptides optionally conjugated to a ligand, and a three-dimensional structure of the Cdc catalytic domain. The three-dimensional structure of these polypeptides is provided including. It is desirable that the polypeptide has a catalytic activity of Cdc25.

In another embodiment, the present invention provides a method for examining the three-dimensional structure of a crystalline polypeptide having the catalytic domain of Cdc25. This method includes (1) Cd
obtaining a polypeptide crystal having a catalytic domain of c25; (2) obtaining X-ray diffraction data of the crystal; (3) the X-ray diffraction data, and an atom of the Cdc binding domain of the second polypeptide. Determining the structure of the crystal using the coordinates and. The method may optionally include the further step of obtaining the polypeptide prior to obtaining the crystals.

The present invention further relates to methods of identifying compounds that are potential inhibitors of Cdc25. This method comprises: (1) obtaining a crystal of a polypeptide having a Cdc25 catalytic domain; (2) obtaining the atomic coordinates of the polypeptide crystal; (3) using the atomic coordinates to catalyze Cdc25 Defining a domain; and (4) identifying compounds that match the catalytic domain. This method comprises obtaining or synthesizing the compound identified in step 4, and assessing the ability of the identified compound to inhibit at least one biological activity of Cdc25, such as an enzymatic activity. May be further included.

In another embodiment, a method of identifying a potential inhibitor of Cdc25 comprises the step of adding one or more of the functional groups and / or components of the compound to or in the catalytic domain of Cdc25. The step of examining the ability of the Cdc25 to interact with one or more of the subsites of the catalytic domain when bound is included. Generally, the catalytic domain of Cdc25 is defined by the atomic coordinates of the polypeptide having the catalytic domain of Cdc25. If the compound is capable of interacting with a preselected number or combination of subsites, or its calculated interaction energy is within the desired or preselected range, the compound is Cdc.
Identified as 25 potential inhibitors.

The present invention further provides methods for designing compounds that are potential inhibitors of Cdc25. The method comprises (1) identifying one or more functional groups capable of interacting with one or more of the subsites of the Cdc25 catalytic domain; (2) one or more identified in step 1. Identifying a scaffold for providing the functional groups of Cdc25 in an orientation suitable for interaction with one or more subsites of the catalytic domain of Cdc25. The compound produced by the binding of the identified functional group or moiety to the scaffold is a potential inhibitor of Cdc25. Cdc
The 25 catalytic domain is generally defined by the atomic coordinates of the polypeptide having the Cdc25 catalytic domain.

In yet another embodiment, the invention provides a compound that is an inhibitor of Cdc25 and that conforms to or binds to the catalytic domain of Cdc25.
Such compounds often have one or more functional groups that interact with one or more of the subsites of the catalytic domain when the compound is bound to the catalytic domain of Cdc25. . Generally, the catalytic domain of Cdc25 is defined by the atomic coordinates of the polypeptide having the catalytic domain of Cdc25. In one example, the Cdc25 inhibitor is a compound identified or designed by the methods of the invention.

The invention further provides methods for treating a Cdc25-mediated condition in a patient. This method includes, for example, a C of the present invention, such as a compound identified as a Cdc25 inhibitor by the method of the present invention, or a compound designed to inhibit Cdc25.
It includes administering to a patient a therapeutically or prophylactically effective amount of a Cdc inhibitor, such as a dc inhibitor.

The present invention provides several advantages. For example, the invention provides the three-dimensional structure of the catalytic domain of the Cdc25 protein, which was first detailed. This structure is Cd
Allowing the rational development of Cdc25 inhibitors by allowing the design and / or identification of molecular structures with properties that facilitate binding of c to the catalytic domain. The methods using this structure disclosed herein therefore allow for more rapid discovery of compounds that may be useful in the treatment of conditions mediated, at least in part, by Cdc.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to X-ray crystallographic studies of polypeptides having the catalytic domain of Cdc25. The atomic coordinates obtained in this study fit the catalytic domain and are therefore C
It is useful for identifying compounds that are potential inhibitors of dc25. These Cdc2
5 inhibitors are useful in the treatment of patients with conditions modulated by Cdc25 activity, such as conditions characterized by excessive, inappropriate or undesired cell proliferation.

Recent evidence indicates that Cdc25 plays a role in the development of cancer. For example, studies have suggested that transgenic mice under the control of the MMTV promoter are predisposed to DMBA-induced breast cancer when Cdc25B is overexpressed (Slosberg et al., Proc.
． Am. Assoc. Cancer Res. 39: 255 (19
98)). Both Cdc25A and Cdc25B are associated with breast cancer (Galaktio).
Nov et al., Science 269: 1575-1577 (1995)).
, Head and Neck Cancer (Gasparotto et al., Cancer Res 57:23.
66-2368 (1997)), gastric cancer (Kudo et al., Jpn J. Can.
cer Res 88: 947-952 (1997)) and non-Hodgkin's lymphoma (Hernandez et al., Cancer Res 58: 1762-).
1767 (1998)).

As a result of a study on a cell line in which the cdk inhibitor p15 is absent, TGF-β is
It has been shown that it is possible to inhibit cell development by modulating the level of dc25A (Iavarone et al., Nature 387: 417-42).
2 (1997)). Similarly, the levels of growth of Cdc25A and tumor cell lines have been shown to be modulated by α-interferon (Tiefe.
nbrun et al., Mol Cell Biol 16: 3934-3944.
(1996)). Moreover, antisense oligonucleotides against Cdc25B have recently been shown to inhibit the growth of certain tumor cell lines (Garn.
erhamrick et al., Int. J. Cancer 76: 729-7
28 1998). These results support the notion that Cdc25 inhibitors can block one or more pathways involved in cell transformation.

The X-ray crystal structure of human Cdc25A was reported by Saper et al. In 1998 (Saper et al., Cell 93: 617-625 (1998)). This structure does not detail the catalytic loop or amino acid residues at the carboxyl terminus at the atomic level. Furthermore, this structure does not include the inhibitor bound to Cdc25A.

The examples describe the production of polypeptides having the catalytic domain of human Cdc25A, Cdc25B and Cdc25C and the crystallization of Cdc25A and Cdc25B polypeptides. The term "catalytic domain" as used herein refers to Cd
Any or all of the following sites of c25: a substrate binding site; a site to which a pentapeptide inhibitor described below binds, and a site from which a substrate is cleaved. Cdc2
For 5A, the catalytic domain is defined by amino acid residues from approximately residue 336 to approximately residue 523 of SEQ ID NO: 1. Cd
For c25B, the catalytic domain is approximately 378 of SEQ ID NO: 2.
Specified by the amino acid residues from the 1st residue to about the 566th residue (
Xu et al., J. Biol. Chem. , 271: 5118-5124
(1996)).

The prepared polypeptide is shown in Table 9 together with its N-terminal and C-terminal amino acid residues.
It is shown as a list in. The amino acid sequences of these polypeptides are shown in Figures 4-14. The residue numbers in Table 9 represent the appropriate residue in the amino acid sequence of the corresponding native protein shown in Figures 1, 2 and 3 (SEQ ID NO: 1, 2 or 3). The amino acid sequence of the natural protein (SEQ ID NO: 1, 2 or 3) is SWISS-PR.
OT ((Bairoch et al., Nucleic Acid Res. 22: 3.
578 (1994)). As described in the examples, some of these crystals were examined by X-ray crystallography to obtain the atomic coordinates of the peptide. In some cases, the polypeptide was not ligated, ie not complexed with the ligand. In other cases, the polypeptide was complexed with the ligand and the atomic coordinates of the ligand bound to the catalytic domain of Cdc25 were also obtained.

The atomic coordinates of the five crystals examined by X-ray crystal structure analysis are shown in FIGS. 15A-15PPP,
16A-16I, 17A-17EE, 18A-18X and 19A-19I. The term "atomic coordinates" (or "structural coordinates") is a mathematical formula derived from a mathematical equation for a pattern obtained by diffraction of X-rays from atoms (center of scattering) of a crystalline polypeptide having a Cdc25 catalytic domain molecule. Points to the target coordinates. An electron density map of the repeating unit of the crystal is calculated using the diffraction data. This electron density map is used to locate individual atoms within the unit cell of the crystal. As is well known in the art, atomic coordinates may be transformed into different coordinate systems without affecting the relative position of the atoms.

In detail, a high resolution crystal structure of the polypeptide represented as Cdc25B (ΔN8B) in Table 9 was obtained, complexed with the following pentapeptide inhibitor, represented herein as “cdc1249”.

[0023]

[Chemical 10]

The polypeptide Cdc25B (ΔN8B) includes human Cdc25B (SEQ I
Residues Leu 368 to Arg 562 of D NO: 2) are included. Crystals of a complex of this polypeptide and cdc1249 had the space group P4 ₃ 2 ₁ 2, a = 70.29, c = 130.59. The term "space group" refers to the set of symmetrical elements of a unit cell of a crystal. X-ray crystallographic analysis of Cdc25B (ΔN1B) showed that this unit cell contained 8 polypeptide molecules. This protein; inhibitor in active site; second molecule of this inhibitor (terminal side of catalytic domain); water molecule; and atomic coordinates of non-hydrogen atoms in sodium and chlorine counterions, FIGS. 15A-15PPP. Shown in. For the inhibitor molecule in the active site, the second Glu residue after CB and the C-terminal Glu-
All heavy (non-hydrogen) atoms except amide were observed. For the second inhibitor molecule, all non-hydrogen atoms were observed. The carbon atom in the amino acid side chain is
In the present application, it is represented as CB, CG, CD, etc. for convenience, where CB is a carbon atom bonded to α-carbon and CG is a side chain carbon atom bonded to CB or the like. Letters representing carbon atoms are ordered according to the corresponding Greek letter.

When the structure of the polypeptide having the catalytic domain of Cdc25B complexed with the ligand is examined, it is significantly different from the structure investigated by Saper et al. For Cdc25A. The most important point is that in the protein structure of Cdc25B, the ligand is bound to the catalytic site, and all the protein atoms near this ligand can be clearly distinguished. In the Cdc25A structure of Saper et al., There is no bound ligand and the resolution of the catalytic loop is also very low (the residues that make up the catalytic loop are irregular). One residue of this catalytic loop, Arg479, appears to be misplaced within Cdc25A when compared to the structures examined for polypeptides with the catalytic domain of Cdc25B or other known phosphatase structures.
Therefore, it is not possible to directly obtain reliable information on the ligand binding from this Cdc25A protein structure, and the low atomic resolution around the binding site means that the ligand binding mode of the ligand binding mode can be determined using molecular modeling techniques. It means that the reliability is low even if prediction or ligand design is performed. Cdc25 mentioned here
Another major difference between the B catalytic domain structure and the Cdc25A structure of Saper et al. Is the C-terminal region (residues 531-547, Cdc25B number). C
This region of dc25B, which is well resolved in the structure of the invention, has an alpha helix pointing away from the body of the protein, where multiple residues of this helix, eg Met531 and Arg544, It interacts with the bound ligand. In contrast, in the Cdc25A structure of Saper et al., This region is not clear except for Asp492 (Cdc25A numbering system), and the positions of the few residues observed are also likely to be incorrect. For example, the sequence is moving away from the body of the protein towards a symmetrical molecule. Therefore, the position of this C-terminus in the Cdc25A structure seems to be determined by the packing force of this crystal.

[0026]   The structure of the Cdc25B (ΔN8B) / cdc1249 complex is the ligand (HO _Three SCH_Two) The phenyl group of the Phe residue is: Phe 475, Se in the catalytic loop.
r 477 and Glu 478; C-terminal Met 531; naphtha of said ligand
Cyl group; Pro 4 of a symmetric polypeptide molecule (where the crystals are in contact)
Shown to be completely surrounded by hydrophobic groups including 57 and Ile 458
It Also, PheCH_TwoSO_ThreeLigand between H and 2-OMe-naphth
There is also a hydrogen bond from NH to the carbonyl oxygen atom of Pro 457. 2-OM
The naphthyl ring of the e-naphth group has the above symmetry relationship in Pro 457.
Molecules and also in Lys 455 and Ser 456
Nder Waals are in contact. The methyl group of the 2-OMe-naphth group is
Located within the globe of polypeptide molecules. This naphthyl group has a C-terminal M
also in contact with et 531 and Leu 540 and the Nal residue of this ligand
There is. Between the inhibitor Nal and Arg 544, probably a cation -π,
An important interaction works, which is π-π or van der Waals interaction
(The side chain of Arg 544 is hydrogen-bonded to Tyr 428 to give CB-CG.
Have an undesired twist angle). This Nal residue is a Glu in the catalytic loop.
Van der with side chains of 478 and Arg 479, and C-terminal Met 531
It is in contact with Wals.

This pentapeptide inhibitor has a helical conformation in which a plurality of 3 ₁₀ / α characteristics are mixed. This inhibitor has two intermolecular H bonds: PheC
From the amide O between H ₂ SO ₃ H and 2-OMe-naphth, two Glu
There is up to the backbone NH between the residues and up to the backbone NH between the second Glu and Nal: These hydrophobic groups of this ligand are close to each other, and this state can be called hydrophobic contraction.

As mentioned above, no electron density was observed for the terminal carboxylic acid of this ligand. The results indicate that there may be multiple binding modes for this part of the ligand.

It was observed that in the crystal structure, the second molecule of the inhibitor is bound to the protein at the site distal to the catalytic site. It is believed that this molecule stabilizes the crystal by making many favorable interactions at the interface of two symmetrical protein molecules. The conformation of the ligand molecule at this terminal site is very similar to that of the ligand at the binding site. This indicates that this ligand is a low-energy conformation that does not cause a large bias when interacting with proteins. This result has been confirmed by molecular modeling and conformational analysis.

The C-terminal region of the catalytic domain of Cdc25B is helical and plays an important role in ligand binding. This region was not observed in the structure of Saper et al. The shape of this part of the protein can be determined, for example, by salt concentration, constituent length, protein-protein interaction (CDK / cyclin), binding ligand and especially pH.
It may be highly flexible due to factors such as

Analysis of the three-dimensional structure of the catalytic domain of Cdc25B reveals the presence of multiple subsites, each with a molecular functional group capable of interacting with the complementary component of the inhibitor. Subsites 1-16 of the catalytic domain of Cdc25B are defined below. This catalytic domain is composed of a catalytic loop and a region surrounding it. Sixteen subsites have been defined; subsites 1-9 correspond to pockets, clefts, grooves, etc., and the remaining seven are bumps, the tips of solvent exposed amino acid side chains. FIG. 20 (top view) and FIG. 21 (side view) illustrate binding site regions with subsite labeling.

Subsites are characterized as follows based on the properties of the chemical constituents with which they are complementary or with which they can interact. Such components include hydroxyl, amino and carbonyl groups, halogen atoms such as fluorine, chlorine, bromine and iodine; and groups containing trivalent phosphorus, divalent and tetravalent sulfur, oxygen and nitrogen atoms and the like, Such as other groups containing a heteroatom with at least one lone paired electron,
Hydrogen bond acceptor (“HA”); hydroxyl, amino, carboxyl groups, and any group to which a hydrogen atom is attached which is mentioned as a hydrogen atom acceptor,
Hydrogen bond donors (“HD”); linear, branched and cyclic alkyl groups; linear, branched and cyclic alkenyl groups; linear, branched and cyclic alkynyl groups; monocyclic and polycyclic aromatic hydrocarbyls. Groups and hydrophobic groups (“H”) such as aryl groups such as monocyclic and polycyclic heteroaryl groups; primary, secondary, tertiary and quaternary ammonium groups and substituted or unsubstituted guanidinium groups, sulfonium groups And a positively charged group (“P”) such as phosphonium group; and a negatively charged group such as carboxylate group, sulfonamide group, sulfamate group, boronate group, vanadate group, sulfonate group, sulfinate group and phosphonate group (“N”) may be included. Any chemical moiety may include one or more of these groups.

Subsite 1: catalytic loop; interacting chemical components: HA, H, N; related residues: Cys 473; Glu 474; Phe 475; Ser 476
Ser 477; Glu 478; Arg 479; Non-hydrogen atoms interacting with HA and N: Cys 473 S; Glu 474.
N; Phe 475 N; Ser 476 N; Ser 477 N; Glu
478 N; Arg 479 N, NE, non-hydrogen atom interacting with NH 2 H: Glu 474 CA, CB, CG, CD; Ph
e 475 CB, CG, CD1, CD2, CE1, CE2, CZ; Ser 4
77 CB; Glu 478 CB, CG, CD

Subsite 2: Swimming pool interacting chemical components: HA, HD, H, N, P Related residues: Cys 426; Tyr 428; Pro 444; Leu 445
Glu 446; Glu 478; Arg 479; Arg 482; Met
483; Arg 544; Thr 547; Arg 548 Non-hydrogen atoms interacting with HA and N: Tyr 428 OH; Arg 482.
NH1 or NH2; non-hydrogen atoms that interact with Arg 544 NH1 HD and P: Cys 426 O; Tyr 428
OH; Pro 444 O; Glu 446 OE1, OE2; Thr 547
Non-hydrogen atoms interacting with OG1 H: Leu 445 CA, CB, CD1; Glu
446 CA, CB, CG, CD; Arg 479 CA, CB, CG, CD;
Met 483 CA, CB, CG, SD, CE; Thr 547 CB, CG
2; Arg 548 CA, CB, CG, CD, CE

Subsite 3: Anion binding site Chemical components interacting: HA, N Related residues: Arg 482; Arg 544 Non-hydrogen atoms interacting with HA and N: Arg 482 NH1, NH2; Ar
g 544 NH1, NH2

Subsite 4: Group interacting chemical components between catalytic loop and swimming pool: H Related residues: Glu 478; Arg 479; Met 531; Arg 544
Non-hydrogen atoms that interact with H: Glu 478 CA, CB, CG, CD; Ar
g 479 CA, CB, CG, CD, CZ; Met 531 CB, CG, S
D, CE; Arg 544 CG, CD, CZ.

Subsite 5: Nal binding domain Chemical components interacting: HA, HD, H, N Related residues: Tyr 428; Glu 478; Arg 479; Met 531
Leu 540; Arg 544 Non-hydrogen atoms interacting with HA and HD: Tyr 428 OH Non-hydrogen atoms interacting with H: Tyr 428 CD1, CE1; Glu 47
8 CA, CB, CG, CD; Arg 479 CA, CB, CG, CD, CZ
Met 531 CG, SD, CE; Leu 540 CB, CG, CD1,
CD2; non-hydrogen atom that interacts with Arg 544 CG, CD, CZ N: Arg 544 NE, NH1, NH2

Subsite 6: 2-MeO-Nal binding domain Chemical components interacting: H Related residues: Phe 475; Met 531; Asn 532; Leu 540
Non-hydrogen atoms that interact with H: Phe 475 CG, CD1, CD2, CE1
, CE2, CZ; Met 531 CB, CG, SD, CE; Asn 532
CB, CG; Leu 540 CB, CG, CD1, CD2

Subsite 7: Interactions related to Ile 458 of the polypeptide in symmetrical relationship Chemical components interacting: H Related residues: Phe 475; Non-hydrogen atoms interacting with Ser 477 H: Phe 475 C, CB, CG, CD1, CD
2, CE1, CE2, CZ; Ser 477 CB

Subsite 8: Pro 457-related interaction of polypeptides in symmetry Interaction chemical components: HA, HD, H Related residues: Glu 474; Phe 475; Met 531; Asn 532
Non-hydrogen atoms interacting with HA: Asn 532 ND2 HD Non-hydrogen atoms interacting with HD: Glu 474 OE1, OE2; Asn 5
32 Non-hydrogen atoms interacting with OD1 H: Glu 474 CB, CG, CD; Phe 4
75 CG, CD1, CD2, CE1, CE2, CZ; Met 531 C, C
A, CB, CG, SD, CE; Asn 532 CA, CB, CG

Subsite 9: Region around Leu 540 Chemical components interacting: H Related residues: Tyr 428; Met 531; Lys 537; Leu 540
Lys 541; non-hydrogen atom interacting with Arg 544 H: Tyr 428 CD1, CE1; Met 53;
1 CB, CG, SD, CE; Lys 537 CA, CB, CG, CD, CE
Leu 540 CB, CG, CD1, CD2; Lys 541 CA, CB
, CG, CD, CE; Arg 544 CB, CG, CD

[0042] Subsite 10: Ser 477 Interacting chemical components: HA, HD Related residues: Ser 477 Non-hydrogen atoms interacting with HA and HD: Ser 477 OG

[0043] Subsite 11: Glu 478 Interacting chemical components: HD, P Related residues: Glu 478; Non-hydrogen atoms interacting with HD and P: Glu 478 OE1, OE2

[0044] Subsite 12: Lys 394 Interacting chemical components: HA, N Non-hydrogen atoms interacting with HA and N: Lys 394 NZ

Subsite 13: Arg 482 interacting chemical components: HA, N HA and non-hydrogen atoms interacting with N: Arg 482 NE, NH 1 or NH.
Two

[0046] Subsite 14: Arg 544 Interacting chemical components: HA, N Non-hydrogen atoms interacting with HA and N: Arg 544 NE, NH2

Subsite 15: Phe 475 Interacting Chemical Component: Non-Hydrogen Atom Interacting with H H: Phe 475 CB, CG, CD1, CD2,
CE1, CE2, CZ

[0048] Subsite 16: Asn 532 Interacting chemical components: HA, HD, H Non-hydrogen atoms that interact with HA: Asn 532 ND2 Non-hydrogen atoms interacting with HD: Asn 532 OD1 Non-hydrogen atoms that interact with H: Asn 532 CB, CG

20-28 are various diagrams showing the structure of the catalytic domain of Cdc25 and the interaction of cdc1249 with the polypeptide. For example, FIG. 20 shows that the ligand was bound by the catalytic loop (thick bond line) and the ligand was at the terminal site (thin bond line).
FIG. 4 is a diagram showing the complex of Cdc25B and cdc1249 bound by the method in the form of secondary structure of protein. FIG. 21 illustrates this complex in the form of two symmetrical protein molecules interacting with a ligand bound to the catalytic site. Water molecules and ions are not shown. FIG. 22 is a top view of the molecular surface around the ligand binding region. The terminal atom of Arg 482 has been excluded so that the swimming pool is clearly visible. Water molecules and ions are not shown. The composite illustrated in FIG. 23 is a side view of the view illustrated in FIG. FIG. 24 is a top view of a complex of Cdc25B and cdc1249 in which a protein residue is labeled. Water molecules and ions are not shown. 25 is a side view of the composite shown in FIG. FIG. 26 is a top view of this complex, showing the molecular surface around the ligand binding region, labeled at each subsite. Arg 482
The terminal atoms of are excluded so that the swimming pool is clearly visible.
Water molecules and ions are not shown. FIG. 27 is a side view of the complex illustrated in FIG. 26, with only subsites 1-6 labeled. The catalytic loop of Cdc25A is not shown, and the well-defined catalytic loop of Cdc25B is shown in violet. FIG. 28 is a side view of a potential strong binding inhibitor complexed with Cdc25B. This engineered ligand binds in the catalytic loop and the swimming pool and spans the glove between the two.

In one embodiment, the invention provides a plurality of polypeptides having a catalytic domain of Cdc25 and a crystalline form of said plurality of polypeptides, optionally bound to a ligand,
And the three-dimensional structure of the plurality of polypeptides, including the three-dimensional structure of the catalytic domain of Cdc25. Generally, these three-dimensional structures are defined by atomic coordinates derived from X-ray crystallographic analysis of the plurality of polypeptides. The plurality of polypeptides may include the catalytic domain of Cdc25 from any species, such as yeast or other unicellular organisms, invertebrates or vertebrates. Suitably said polypeptide comprises a mammalian Cdc25A, Cdc25B or Cd
It includes the catalytic domain of mammalian Cdc25, such as c25C. More preferably,
The polypeptide comprises the catalytic domain of human Cdc25A, Cdc25B or Cdc25C. In one embodiment, the polypeptide comprises SEQ
Amino acids Leu 336 to Thr 506 of ID NO: 1, SEQ I
Amino acids Leu 378 to Arg 548 of D NO: 2, or SEQ
Includes amino acids Leu 282 to Val 453 of ID NO: 3.
In some embodiments, the polypeptide comprises the amino acids Leu 336 to Leu 523 of SEQ ID NO: 1; Gly 323 to Leu 52.
3; Glu 326 to Arg 519; or Glu 326 to Thr 506; Leu 378 to Gln 5 of SEQ ID NO: 2.
66; Asp 365 to Gln 566; Glu 368 to Arg
562; Glu 368 to Ser 549 or Glu 368
To Arg 548; or the amino acid Leu 2 of SEQ ID NO: 3
82 to Pro 473 or Gly 280 to Val 453,
May be included.

The crystalline form of the polypeptide preferably further comprises a ligand bound to the catalytic domain of Cdc25. The ligand is preferably a small (less than about 1500 molecular weight) organic molecule, such as a peptide such as a pentapeptide.

In one embodiment, the present invention relates to a method for examining the three-dimensional structure of a first polypeptide having the catalytic domain of the CdC25 protein. The methods include (
1) obtaining a crystal having the first polypeptide; (2) obtaining X-ray diffraction data of the crystal; (3) the X-ray diffraction data, and a second having a catalytic domain of a Cdc25 protein. And the atomic coordinates of the polypeptide to determine the crystal structure of the first polypeptide to examine the three-dimensional structure of the first polypeptide. The second polypeptide may include the same Cdc25 catalytic domain as the first polypeptide, or may include a different Cdc25 catalytic domain. Either or both of the first and second polypeptides may optionally be conjugated to a ligand. That is, the crystal of the first polypeptide may have a complex of the first polypeptide and a ligand. The atomic coordinates of the second polypeptide may include the atomic coordinates of the ligand molecule bound to the second polypeptide. The atomic coordinates of the second polypeptide are generally such that, for example, a crystal having the second polypeptide or the second polypeptide.
It has already been revealed by X-ray crystal structure analysis of the complex between the polypeptide and the ligand. This atomic coordinate of the second polypeptide may be used to determine the crystal structure by methods well known in the art, such as molecular replacement or isomorphous replacement. Said second polypeptide preferably has the catalytic domain of mammalian Cdc25, more preferably mammalian Cdc25B, most preferably human Cdc25B. For example, the available atomic coordinates include the atomic coordinates shown here, preferably Figures 15A-15P.
The atomic coordinates shown up to PP are included.

The present invention also provides methods for identifying compounds that are potential inhibitors of Cdc25. This method comprises: (1) obtaining a crystal of a polypeptide having a Cdc25 catalytic domain; (2) obtaining the atomic coordinates of the polypeptide by X-ray diffraction using the crystal; and (3) the above. Defining the catalytic domain of Cdc25 using atomic coordinates; and (4) identifying a compound that matches the catalytic domain. The method further comprises obtaining the compound identified in step 4, eg from a compound library or by synthesis, and assessing the ability of the identified compound to inhibit the enzymatic activity of Cdc25. Good.

Said polypeptide is preferably eg a mammalian Cdc25A, Cdc25B
Alternatively, it has the catalytic domain of mammalian Cdc25, such as Cdc25C. More preferably, said polypeptide is human Cdc25A, Cdc25B or Cdc25C.
Have. In a preferred embodiment, said polypeptide is a polypeptide of the invention as described above.

The polypeptide may be crystallized by methods well known in the art, such as, for example, the methods mentioned by way of example, to give polypeptide crystals suitable for X-ray diffraction. The obtained crystalline polypeptide may be dipped in a solution containing a ligand to prepare a crystalline polypeptide / ligand complex. Preferably, the ligand solution is a solvent in which the polypeptide cannot be dissolved.

The atomic coordinates of the polypeptide (and ligand) may be determined, for example, by X-ray crystallography using methods well known in the art. When the atoms of the polypeptide and the ligand are present, the atomic coordinates of these atoms may be determined using the data obtained by the crystal structure analysis. Detailed elucidation of the X-ray crystal structure, as is well known in the art, makes it possible to study the coordinates of some or all of these non-hydrogen atoms. These atomic coordinates may be used to determine the three-dimensional structure of the Cdc25 catalytic domain, as is well known in the art. This structure may then be used to assess the ability of any compound to fit into this catalytic domain, preferably by computer-assisted methods.

A compound is of a size and shape that allows it to be physically present within the catalytic domain, ie its shape is complementary to said catalytic domain and is not associated with undesirable steric interactions or van der Waals. If it is possible for it to be present in the catalytic domain without a Waals interaction, it is compatible with that catalytic domain. Suitably, the compound comprises one or more functional groups and / or moieties capable of interacting with one or more subsites within the catalytic domain. Computer-aided methods for assessing the ability of a compound to fit into the catalytic domain defined by the atomic coordinates of the polypeptide are well known in the art, but representative examples are given below.

In another embodiment, the method for identifying a potential inhibitor of Cdc25 comprises the step of functionalizing one or more functional groups of the compound when the compound is present within the catalytic domain of Cdc25. And / or include determining the ability of the component to interact with one or more subsites of the catalytic domain of Cdc25. Suitably, the catalytic domain of Cdc25 is defined by the atomic coordinates of the polypeptide having the catalytic domain of Cdc25. A compound is considered to be a potential inhibitor of Cdc25 if it is capable of interacting with a preselected number or combination of subsites.

When a functional group or moiety of a compound participates in an energetically favorable or stable interaction with one or more complementary moieties within a subsite of the catalytic domain of Cdc25, It is said to "interact" with this subsite. Two chemical moieties are “complementary” if they are capable of engaging in attractive or stabilizing interactions, such as electrostatic or van der Waals interactions, when their positional relationship is favorable. Is. Generally, the attractive interaction is an ion-ion (or salt bridge), ion-dipole, dipole-dipole, hydrogen bond, π-π or hydrophobic interaction. For example, the negatively charged component and the positively charged component are complementary since they can form a salt bridge when their positional relationship is suitable. Similarly, the hydrogen bond donor and hydrogen bond acceptor are complementary if their positional relationship is preferred.

In general, evaluation of the interaction between a test compound and the catalytic domain of Cdc25 uses computer-aided methods, such as are well known in the art. In such a method, calculating the strength of the expected interaction between a compound and the protein defined by atomic coordinates, and the interaction energy during the binding of the compound with the protein. Evaluate by. The compound most likely to be a Cdc inhibitor, calculated as having an interaction energy within a preselected range, or in the opinion of a computational chemist employing this method, , Provided from, for example, a compound library or synthetically, and assayed for their ability to inhibit Cdc25. The interaction energy of a compound generally depends on its ability to interact with one or more subsites of the catalytic domain of the protein.

In one embodiment, the atomic coordinates used in the method of the present invention are shown in Figures 15A-15PP.
It is the atomic coordinates shown in P, 16A-16I, 17A-17EE or 18A-18X. Preferably, the atomic coordinates are the atomic coordinates shown in Figures 15A-15PPP.
Of course, FIGS. 15A-15PPP, 16A-16I, 17A-17EE
It is also possible to replace the atomic coordinates shown in 18A-18X and 18A-18X with, for example, different atomic coordinates by a method well known to those skilled in the art without significantly changing the three-dimensional structure represented therein.

In some cases, a component of the compound may interact with a subsite via two or more individual interactions. When one component of the compound and a certain sub-site have complementary properties, are in close proximity to each other, and are in an orientation suitable for causing a stabilizing interaction. And can interact with each other. The range of the distance that the component of the compound and the subsite can take depends on the distance dependence of the interaction, as is well known in the art. For example, hydrogen bonding often occurs when the hydrogen bond donor atom bearing the hydrogen atom and the hydrogen bond acceptor atom are separated from about 2.5Å to about 3.5Å. Hydrogen bonding is well known in the art (Pimentel et al., The Hydrogen Bond,
San Francisco: Freeman (1960)). In general, the interaction or binding between the compound and the Cdc25 catalytic domain will generally depend on the number and strength of these individual interactions.

[0063] The ability of a test compound to interact with one or more of the subsites of the catalytic domain of Cdc25B is determined by the functional groups or components of said test compound, with specific protein subsites such as subsites 1-16 above. Interactions with one or more of the amino acid side chains in may be investigated by computer evaluation. Generally, a compound capable of participating in a stabilizing interaction with a predetermined number of subsites, preferably without simultaneously participating in a strong destabilizing interaction, is a Cdc25 It is said to be a potential inhibitor. Such compounds will interact with one or more subsites, preferably two or more subsites, more preferably three or more subsites.

The invention further provides methods for designing compounds that are potential inhibitors of Cdc25. This method comprises the steps of (1) identifying one or more functional groups capable of interacting with one or more subsites of the catalytic domain of Cdc25; (2) one identified in step 1 or Identifying a scaffold that imparts a plurality of functional groups in an orientation suitable for interaction with one or more of the Cdc25 catalytic domain subsites. The compound obtained by adhering the identified functional group or component to the identified scaffold is a potential inhibitor of Cdc25. The catalytic domain of Cdc25 is generally that of a polypeptide having the catalytic domain of Cdc25, eg, Figures 15A-15PPP, 16A-16I, 17A-17.
It is defined by the atomic coordinates shown in EE, 18A-18X or 19A-19I. Suitably, the catalytic domain of Cdc25 is defined by the atomic coordinates shown in Figures 15A-15PPP.

Suitable methods well known in the art may also be used to identify chemical moieties, fragments or functional groups that are capable of interacting favorably with one or more particular subsite combinations. Good. These methods include, but are not limited to: interactive molecular graphics; molecular mechanics; conformational analysis; energy evaluation; docking; database searching; pharmacophore modeling; de novo design and characterization. . These methods may be used to combine multiple chemical components, fragments and functional groups into a single inhibitor molecule. These same methods may also be used to determine whether a chemical moiety, fragment or functional group is able to interact favorably with a combination of one or more particular subsites. .

In one example, in designing a potential inhibitor of human Cdc25, first an overview and electrostatic complementarity of the three-dimensional structure of the catalytic domain, including subsites 1-16, is determined. One skilled in the art may then use interaction molecular modeling techniques to visually inspect the fit of the modeled candidate inhibitor to the binding site. Suitable visualization programs include INSIGHT2 (Molecular Simulations, Inc., San Diego, CA), QUANTA (Molecular Simulations, Inc., San Diego, CA), SYBYL (Tripos, Inc., St. Louis, MO), RASMOL (Roger Sayle
Et al., Trends Biochem. Sci. 20: 374-376 (
1995)), GRASP (Nicholls et al., Proteins 11:
281-289 (1991)) and MIDAS (Ferrin et al., J. M.
ol. Graphics 6: 13-27 (1988)).

In a further embodiment of the invention, a database search program is used which searches a database of known small molecules of three-dimensional structure for possible candidates for the target protein site. Suitable software programs include CATALLYST (Molecular Simulations, Inc., San Diego, Calif.), UNITY (Trypos, Inc., St. Louis, Mo.), FLEXX (Rarey et al., J. Mol. Biol. 261: 47).
0-489 (1996)), CHEM-3DBS (Oxford Molecular Group, Oxford, UK), DOCK (Kuntz et al., J.
． Mol. Biol 161: 269-288 (1982)) and MA.
CCS-3D (MDL Information Systems Incorporated, San Leandro, CA). The molecules found in this search are not necessarily expected to be examples of themselves. Because, during the initial search, all interactions will necessarily be fully evaluated. Rather, candidates such as these are expected to serve as a framework for further design and provide a molecular scaffold on which appropriate atomic substitutions can be made. As a matter of course,
The chemical complementarity of these molecules may be assessed, but scaffolds, functional groups, linkers and / or monomers may be modified to maximize electrostatic, hydrogen bonding and hydrophobic interactions with the enzyme. Is expected to be. Goodford (Goodford J Med Chem 28: 849-857 (1985)) has created a computer program GRID for multiple different chemical groups (called probes) on the molecular surface of the binding site. To investigate the regions with high affinity. This makes GRID a tool for devising alterations that could facilitate the binding of known ligands.

Electrostatic interaction, hydrogen bonding, hydrophobic interaction, desolvation, conformational distortion, and
All of a range of factors, including the cooperative movement of ligands and enzymes, influence the binding effect and should be taken into account when trying to design bioactive inhibitors.

Yet another example of a computer-aided molecular design method for identifying inhibitors includes searching for fragments that fit within subsites of the binding region and that bind to a predefined scaffold. . The scaffold itself may be identified in this way. Suitable programs for searching for such functional groups and monomers include LUDI (Boehm,
J Comp. Aid. Mol. Des. 6: 61-78 (199
2)), CAVEAT ("Molecular Recognition in
Bartlett et al., In The Chemical and Biological Problems, The Royal Chem. Soc., 78.
: 182-196 (1989)) and MCSS (Miranker et al. Proteins 11: 29-34 (1991)).

Yet another embodiment of a computer-aided molecular design method for identifying inhibitors of a phosphatase of interest is to exert the desired structural and electrostatic complementarity with the active site of this enzyme. It involves de novo synthesis of potential inhibitors by algorismically connecting multiple small molecule fragments. in this way,
A large number of small molecules are repeatedly spliced with each other as a template to model the Cdc active site. A program suitable for this technique is GROW (Moon et al. Prote
ins 11: 314-328 (1991)) and SPROUT (Gille.
t et al. J Comp. Aid. Mol. Des. 7: 127 (199
3)) is included.

In yet another embodiment, the suitability of candidate inhibitors may be examined using an empirical scoring function. In this way, it is possible to grade the binding affinity of a set of inhibitors. An example of such a method is described by Muegge et al. And references incorporated therein (Muegge et al., J Med. Chem. 4).
2: 791-804 (1999)).

For example, Cohen et al. (J. Med. Chem. 33: 883-894.
(1994)); Navia et al. (Current Opinions in
Structural Biology 2: 202-210 (1992).
); Baldwin et al. (J. Med. Chem. 32: 2510-25.
13 (1989)); Appelt et al. (J. Med. Chem. 34:
1925-1934 (1991)); and Ealick et al. (Proc.
Nat. Acad. Sci. USA 88: 11540-11544.
Other modeling techniques described in (1991)) may be used in accordance with the present invention.

A compound identified as a potential inhibitor of Cdc25 from any one of the above methods is then made, eg synthetically or from a compound library, which compound inhibits Cdc25 in vitro. You may evaluate your ability. Such in vitro assays may be performed by methods well known in the art, such as contacting Cdc25 with the test compound in the presence of a substrate for Cdc25, eg, in solution.
The rate of substrate change may be measured in the presence of the test compound and compared to the rate in the absence of the test compound. A preferred assay for Cdc25 biological activity is:
US Pat. Nos. 5,861,249; 5,695,950; 5,443,962; and 5,294,538, each of which is incorporated by reference herein in its entirety. is there.

The inhibitor identified or designed by the method of the present invention may be a competitive inhibitor, a non-competitive inhibitor or a non-competitive inhibitor. "Competitive" inhibitors are Cd
It is a substance that inhibits the activity of Cdc25 by binding to the same kinetic type as the substrate of c25 and directly competing with the substrate of the active site of Cdc25. Competitive inhibition can be completely reversed by increasing the substrate concentration. An "uncompetitive" inhibitor is C
It inhibits the activity of this enzyme by binding to a kinetic type different from the substrate of dc25. Such an inhibitor binds to Cdc25 already bound to the substrate,
It does not bind to this released enzyme. Uncompetitive inhibition cannot be completely reversed with increasing substrate concentration. A "non-competitive" inhibitor is a substance that can bind both free Cdc25 and substrate-bound Cdc25.

In another embodiment, the present invention has been identified, for example, as an inhibitor of at least one biological activity of Cdc25, or designed in the above-described manner to inhibit at least one biological activity of Cdc25. Provided are Cdc25 inhibitors, such as compounds, capable of binding the catalytic domain of Cdc25, and methods of use thereof. For example, the present invention includes compounds that interact with one or more, preferably two or more, more preferably three or more of subsites 1 to 16 of Cdc25.

In one embodiment, the Cdc25 inhibitors of the present invention are positions that interact with subsite 1, subsite 2 and at least one other subsite when present within the catalytic domain of Cdc25. With one or more components in. For example, the functional group capable of interacting with subsite 1 may be a hydrogen bond acceptor, a hydrophobic moiety or a negatively charged group. Suitably, the functional groups include both negatively charged groups and hydrophobic groups. The functional group capable of interacting with subsite 2 may be a hydrogen bond donor, a hydrophobic moiety, a negatively charged group or a positively charged group.

In another embodiment, the Cdc25 inhibitor of the invention comprises subsites 1, 2
And 3, and optionally multiple functional groups in positions to interact with one or more additional subsites.

The Cdc25 inhibitors of the present invention include compounds having a plurality of functional groups arranged to interact with subsites 1, 2 and 3 and optionally one or more further subsites. Is also included. In another embodiment, the inhibitor has a plurality of functional groups at positions that interact with subsite 1, subsite 3, subsite 4, and optionally one or more additional subsites. .

In another embodiment, the Cdc25 inhibitors of the present invention include compounds that have a functional group at a position that interacts with the following group of subsites. Each of these groups may optionally include one or more further subsites: subsites 1 and 5; subsites 1, 4 and 5; subsites 1, 5 and 6; subsites, respectively. 1, 7 and / or 8; subsites 1, 2 and 9; subsites 1, 2, 4 and 9; subsites 1, 3 and 9; subsites 1, 3, 4 and 9.

One component of the inhibitory compound is located on the appropriate amino acid side chain within a given subsite when located within the Cdc25 catalytic domain defined by the atomic coordinates shown in FIGS. 15A to 15EE. It is "positioned to interact" with this subsite when it is sufficiently close and properly oriented thereto.

As indicated in the description of subsites above, it is presumed that a plurality of subsites 1-16 are capable of interacting with more than one type of component. For each subsite in the list below, the preferred type of interacting component that the potential inhibitor has is indicated.

Subsite 1: Negatively charged component (Arg 479) and hydrophobic component (Glu
474, Phe 475, Ser 477, Glu 478).

[0083] Subsite 3: negatively charged component (Arg 482; Arg 544)

Subsite 5: Hydrophobic, preferably aromatic component (Tyr 428; Glu 478.
Arg 479; Met 531; Leu 540; Arg 544).

Subsite 8: Hydrophobic, preferably alkyl component (Glu 474; Phe 47
5; Met 531; Asn 532).

[0086] Subsite 11: Positively charged component (Glu 478)

[0087] Subsite 12: negatively charged component (Lys 394)

[0088] Subsite 13: negatively charged component (Arg 482)

[0089] Subsite 14: negatively charged component (Arg 544)

[0090] Subsite 16: Hydrophobic and hydrogen donor / acceptor (Asn 532)

The K i value at which the preferred Cdc25 inhibitor of the present invention inhibits the enzymatic activity of Cdc25 is at least about 1 mM, preferably at least about 100 μM, more preferably at least about 10 μM.

In a preferred embodiment, the Cdc25 inhibitors of the present invention, when present in or associated with the catalytic domain of Cdc25, have two or more of the following: (a) human Cdc25B is in a position that interacts with Arg 479,
Negatively charged functional group; (b) Cys 426, Tyr 42 of human Cdc25B.
8, Pro 444, Glu 446 and Thr 547 in positions to interact with one or more hydrogen bond donors or positively charged functional groups; (c) Tyr 428, Arg 482 and Arg of human Cdc25B. (D) Leu 445, Glu 446, Arg 479, Met 483, hydrogen bond acceptors or negatively charged functional groups in positions to interact with one or more of 544.
, A hydrophobic component at a position that interacts with one or more of Thr 547 and Arg 548; (e) Arg 482 and Arg 54 of human Cdc25B.
A negatively charged functional group in a position to interact with one or more of 4; (f)
Human Cdc25B Glu 478, Arg 479, Met 531 and Ar
a hydrophobic component positioned to interact with one or more of g 544; (g
) Human Cdc25B Tyr 428, Glu 478, Arg 479, Me
a hydrophobic component in a position to interact with one or more of t531, Leu540 and Arg544; (h) Phe 475, Me of human Cdc25B
a hydrophobic component at a position that interacts with one or more of t531, Asn 532 and Leu 540; (i) Phe 475 and S of human Cdc25B.
hydrophobic component in a position to interact with one or more of er 477; (j
) Human Cdc25B Glu 474, Phe 475, Met 531 and A
a hydrophobic component at one or more interacting positions of sn 532; (k
) Human Cdc25B Tyr 428, Met 531, Lys 537, Ly
s 541, Leu 540 and Arg 544 at a position that interacts with a hydrophobic component; (l) Human Cdc25B at a position that interacts with Ser 477, a hydrogen bond donor or hydrogen bond acceptor; Hydrogen bond donor or positively charged functional group at the position to interact with Glu 478; human Cdc2
5B, a negatively charged functional group at a position that interacts with Lys 394; (n) a negatively charged functional group at a position that interacts with Lys 394 of human Cdc25B; (o) interacts with Arg 482 of human Cdc25B. Negatively charged functional group at a position to act; (p) Negatively charged functional group at a position to interact with Arg 544 of human Cdc25B; and (q) At a position to interact with Asn 532 of human Cdc25B. Negatively charged functional group.

In preferred embodiments, the Cdc25 inhibitor of the invention comprises (a) and (e); (a) and at least one of (b), (c) and (d); (A
), (E) and at least one of (b), (c) and (d); (a)
, (E) and (f); (a) and (g); (a), (f) and (g); (a),
(G) and (h); (a) and at least one of (i) and (j); (
a), (k) and at least one of (b), (c) and (d).

Suitable Cdc25 inhibitors of the present invention include peptides, peptidomimetics or other molecular scaffolds to which components and / or functional groups that interact with Cdc25 subsites are attached, either directly or via intervening components. It has a skeleton. The scaffold may be, for example, a peptide or peptidomimetic backbone, for example a cyclic or polycyclic moiety, such as a monocyclic, bicyclic or tricyclic moiety, and a single hydrocarbyl or heterocycle. Alternatively, a plurality of them may be included. The molecular scaffolds shape the attached interacting components into a shape or orientation suitable for interaction with the appropriate residue of Cdc25.

The information derived from the Cdc25B (ΔN8B) / cdc1249 crystal structure described above was effectively used to generate a potential inhibitor of Cdc25A. Compound cd shown below
c1671 inhibits Cdc25A with an IC ₅₀ of 5 μM.

[0096]

[Chemical 11]

As mentioned above, the cdc1249 molecule in the catalytic domain of Cdc25B is
It adopts a "turn" conformation, where the backbone of this peptide has a helical turn. Furthermore, this structure shows that the interaction between the two internal glutamyl residues of cdc1249 and the residues of the catalytic domain is not strong. Therefore, replacing one glutamyl residue with one substituted or unsubstituted prolyl or dehydroprolyl residue, which appears to stabilize this "turn" conformation, results in a more effective inhibitor. Was supposed to be. To test this conjecture, the compound cdc1763 shown below was synthesized. This compound inhibits Cdc25A with an IC ₅₀ of 2.2 μM and is twice as effective as cdc1671.

[0098]

[Chemical 12]

Furthermore, replacing the first glutamyl residue with a neutral amino acid such as the t-butyl ester of glutamic acid itself or a neutral amino acid such as norvaline or norleucine alters the physicochemical properties of the peptide. You may let me.
The following pentapeptides, cdc1719, cdc1748 and cdc1749 were made. They have IC ₅₀ values comparable to or higher than cdc1679 (1.1 μM, 2.5 μM, 1.5 μM for Cdc25A, respectively).

[0100]

[Chemical 13]

[0101]

[Chemical 14]

[0102]

[Chemical 15]

[0103]   In a preferred embodiment, the Cdc inhibitor of the present invention is

[0104]

[Chemical 1]

[0105] Represented by the formula shown below, or a pharmaceutically acceptable salt or a prodrug thereof, or
A combination of these. Where R₁Is R_Three-CO, R_FourR₅N-CO, R₆−
SO_Two, R₇R₈NSO_TwoAnd where R_Three, R_Four, R₅, R₆, R₇And R ₈ Are each individually hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkyl.
Reel, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloheteroar
Kill, E- or Z-aryl-C_Two-C_Four-Alkenyl or aryl-C_Two−
C_Four-Is alkynyl.

[0106] Suitable alkyl substituents, hydrogen, _{hydroxy, C 1-6} alkoxy, phenoxy, benzyloxy, halogen, _{amino, C 1-6} alkylamino, di _{-C 1 -6 alkylamino, C 1-6} Included are alkyl-CO-NH, substituted and unsubstituted cycloalkyl, substituted and unsubstituted heterocycloalkyl and substituted or unsubstituted aryl.

[0107]   Aryl groups are, for example, phenyl, naphthyl, anthracenyl, phenanthre.
Nyl, fluorenyl, pyridyl, pyridazinyl, pyridinonyl, furanyl, thie
Nil, thiazolyl, isothiazolyl, imidazolyl, triazolyl, pyrrolyl,
Tetrazolyl, benzimidazolyl, pyrazinyl, pyrimidyl, quinolyl, iso
Quinolyl, benzofuranyl, benzodihydrofuranyl, benzothienyl, pyrazo
Ril, indolyl, purinyl, isoxazolyl, oxazolyl or dibenzof
It may be a lanyl group. Substituted aryl groups are, for example, mono-, di- or tri-substituted.
And a suitable substituent is C_1-6Alkyl, halo, hydroxy, C_{1 -6} Alkylamino, di-C_1-6Alkyl amino, C_1-6Alkoxy,
C_1-6Alkylthio, C_1-6Alkyl carbonyl, phenyl carbonyl,
Benzyl carbonyl, C_1-6Alkyl-sulfonyl, C_1-6Alkyl-sulfo
Nyl-amino, C_1-6Alkyl-carbonyl-amino, carboxyl, OC _1-6 Alkyl carboxyl, carboxyl alkenyl, OC_1-6Archi
Le Carboxyl alkenyl, C_1-6Alkylcarbamoyl, cyano, nit
B, trifluoromethyl and oxytrifluoromethyl may be individually selected.
Yes.

Suitable cycloalkyl groups include substituted and unsubstituted C3-8-cycloalkyl,
Includes adamantyl and bicyclooct [3.3.0] -yl. Examples of suitable heterocycloalkyl groups include substituted and unsubstituted pyrrolidinyl, piperazinyl, tetrahydropyranyl, tetrahydrofuranyl, pyrrolidinonyl and morpholinyl. Suitable substituents for substituting a cycloalkyl or heterocycloalkyl group include, for example, C _1-6 alkyl, halo, hydroxy, C _1-6 alkylamino, di-C _1-6 alkylamino, C _1-6 alkoxy, Included is one or more of C _1-6 alkylthio and C _1-6 alkylcarbonyl.

R ₄ and R ₅ or R ₇ and R ₈ can also work together to form a 4- to 7-membered ring. R ₃ -CO further may be an amino acid residue of the formula _R 9 -CO-G, wherein _{R 9} is _{hydrogen, C 1-6} alkyl, phenyl, benzyl, naphthyl,
Benzyloxy or C _1-6 alkoxy, G is Asp, Asn, Pro,
Ala, Val, Lys, Gly, Arg, Ile, Ser, Thr, Leu,
Trp, Cys, Tyr, Met, Gln, Glu, Phe or His residues.

[0110]   A1 is

[0111]

[Chemical 2]

[0112] It is an amino acid residue represented by the formula shown in. Where R₁₀Is hydrogen or C_1-6A
Rukiru; R₁₁Is hydrogen or C_1-6Alkyl; n is 0, 1 or 2
Yes; X is SO_ThreeH, SO_TwoNR₁₂R_Thirteen, CH_Two-SO_ThreeH, CF_Two-SO _Three H, CH_Two-SO_TwoNR₁₂R_Thirteen, CF_Two-SO_TwoNR₁₂R_Thirteen, And
, Where R₁₂And R_ThirteenAre hydrogen and C, respectively_1-6Alkyl or substituted or
Unsubstituted phenyl, benzyl, furanyl, thiophenyl, thiazolyl, isothia
Zolyl, pyrazolyl, isoxazolyl or oxazolyl; or where R ₁₂ Is hydrogen and R_ThirteenIs hydroxy, C_1-6-Alkoxy, C_1-6-A
It may be alklylcarbonyl or substituted or unsubstituted benzoyl; or X
Is PO_ThreeH_Two, CH_TwoPO_ThreeH_Two, CF_Two-PO_ThreeH_Two, OCH_TwoPO_ThreeH_Two,
COOH, CH_TwoCOOH, CF_TwoCO_TwoH, OCH_TwoCO_TwoH, OCF_TwoCO _Two H, OCH (CO_TwoH)_Two, O-CF (CO_TwoH)_Two, NH-SO_Two-R₁₄ And where R₁₄Is C_1-6Alkyl, benzyl or phenyl; or
, X is NH-CO-COO-R₁₅And where R₁₅Is C_1-6Alkyl,
Benzyl or phenyl. Suitably X is at the 3 or 4 position of the phenyl ring.
is there. Z is hydrogen, C_1-6Alkyl, halo, hydroxy, C_1-6Alkyrrha
Mino, Di-C_1-6Alkylamino, C_1-6Alkoxy, C_1-6Alkylchi
Oh, C_1-6Alkylcarbonyl, halogen-substituted C_1-6Alkyl carbonyl
, Formyl, phenylcarbonyl, benzylcarbonyl, C_1-6Alkyl-su
Lefonil, C_1-6Alkyl-sulfonyl-amino, carboxyl, OC_{1- 6} Alkyl carboxyl, carboxyl alkenyl, OC_1-6Alkylcal
Boxylalkenyl, C_1-6Alkylcarbamoyl, cyano, nitro, trif
Luoromethyl, oxytrifluoromethyl, or-(CH_Two)_m-NR₁₆R_{1 7} And_,Where m is 0, 1 or 2 and R₁₆And R₁₇Are each individually
, Hydrogen, C_1-6Alkyl, C_1-6Alkyl-carbonyl, amino-C_{2- 6} Alkyl, C_1-6Alkyl-amino-C_2-6Alkyl, di-C_1-6Al
Kill-Amino-C_2-6Alkyl, hydroxy-C_2-6Alkyl, C_1-6A
Lucoxy-C_1-6Alkyl, aryl-C_0-6Alkyl, C_3-8Cycloa
Rukiru-C_0-6Alkyl and heterocycloalkyl-C_0-6Select from alkyl
Is selected. Aryl, cycloalkyl and heterocycloalkyl are R_Three, R_Four And R₅Is as described above.

[0113]   A2 is

[0114]

[Chemical 3]

An amino acid residue of the formula: where R ₁₈ is hydrogen or C _1-6 alkyl; R ₁₉ is hydrogen or C _1-6 alkyl; R ₂₀ is the amino acid Gly,
Ala, Val, Leu, Ile, Nva, Nle, Asp, Glu, Lys,
Asn, Gln, Phe, His, homoleucine, Glu (C _1-6 alkyl)
, Asp (C _1-6 alkyl), Lys (Boc). R ₂₀ is-
(CH ₂ ) _o —COOR ₂₁ where o = 3-5 and R ₂₁ is hydrogen or C _1-6 alkyl. R ₁₉ and R ₂₀ are also 3 with an α-carbon.
May form a cyclic carbon system of from 1 to 7 members. R ₁₈ and R ₂₀ may also form a 4- to 7-membered heterocyclic ring system with the nitrogen atom and the α-carbon.
A2 is, for example, thioprolyl, dehydroprolyl or a substituted or unsubstituted prolyl, such as mono- or di-substituted prolyl, wherein these substituents are each individually hydrogen, C _1-6 alkyl, phenyl, hydroxy and _{C 1-6} alkoxy. R ₁₈ and R ₂₀ may also form an 8- to 12-membered nitrogen-containing heterocyclic ring system such as isoindolinyl, octahydroindolyl or dihydroindolyl.

In preferred embodiments, A2 is aspartyl or an ester thereof; glutamyl or an ester thereof; α-aminoadipic acid or an ester thereof; valyl,
Norvalyl or leucyl.

[0117]   A3 is

[0118]

[Chemical 4]

An amino acid represented by the general formula shown in, wherein R ₂₂ has the meaning described above for R ₁₈ of Chemical formula 3, R ₂₃ has the meaning described above for R ₁₉ of Chemical formula 3, R ₂₄ has the meaning described above for R ₂₀ in Chemical formula 3.

In preferred embodiments, A3 is aspartyl or an ester thereof; glutamyl or an ester thereof; α-aminoadipic acid or an ester thereof; valyl, norvalyl or leucyl; or R ₂₃ and R ₂₄ are It forms a 3- to 7-membered ring; or R ₂₂ and R ₂₄ together with the nitrogen atom form a substituted or unsubstituted heterocycle. For example, R ₃ is prolyl or substituted prolyl such as 2-methylprolyl, 3-methylprolyl, 5-phenylprolyl, 3-hydroxyprolyl, 3-t-butoxyprolyl, 3,3-dimethylprolyl; dehydroprolyl. It may be ril, isoindolyl, octahydroindolyl or dihydroindolyl.

[0121]   A4 is

[0122]

[Chemical 5]

[0123] It is an amino acid represented by the general formula shown in. Where R₂₅Is hydrogen or C_1-6A
Rukiru; R₂₆Is hydrogen or C_1-6Is alkyl; R₂₇Is-(CH _Two ) P- (CH (R₂₈)) Q-aryl; p is 0, 1 or 2;
q is 0, 1 or 2, and R₂₈Is hydrogen or methyl.

Suitable aryl groups include substituted and unsubstituted phenyl, naphthyl, anthracenyl, phenanthrenyl, fluorenyl, pyridyl, pyridazinyl, pyridinonyl, furanyl, thienyl, thiazolyl, isothiazolyl, imidazolyl, triazolyl, pyrrolyl, tetrazolyl, benzyl. It includes imidazolyl, pyrazinyl, pyrimidyl, quinolyl, isoquinolyl, benzofuranyl, tetrahydronaphthyl, benzodihydrofuranyl, quinazoline, benzothienyl, pyrazolyl, indolyl, purinyl, isoxazolyl, oxazolyl, dibenzofuranyl.

Suitable substituents on said plurality of aryl groups include hydrogen, C _1-6 alkyl, halo,
_{Hydroxy, C 1-6} alkylamino, di _{-C 1-6 alkylamino, C 1 -6 alkoxy, C 1-6 alkylthio, C 1-6} alkyl - carbonyl, halogen - substituted _{C 1-6} alkylcarbonyl, formyl - , Phenylcarbonyl, benzylcarbonyl, C _1-6 alkyl-sulfonyl, C _1-6 alkyl-sulfonyl-amino, carboxyl, O—C _1-6 alkylcarboxyl, carboxylalkenyl, O—C _1-6 alkylcarboxylalkenyl, C _1-6 alkylcarbamoyl, cyano, nitro, trifluoromethyl, oxytrifluoromethyl, aryl, Y is O, S, NH and s is 0, 1, 2 or 3 Y-
_{(CH 2)} s _{-C 3-8} - cycloalkyl, Y- _{(CH 2) s} - aryl; Y is is O, NH, or S, is from u is 2 to _{6, R 29} is OH, CH _2- O
H, is a NH ₂ or _{NH (C = NH) NH 2} , Y- (CH 2) u -R 29; Y
Is O, NH or S, v is _1-6 , R ₃₀ is COC _1-6 alkyl,
It is COOH or _{CONH 2, Y- (CH 2)} v -R 30; R 31 is _{COC 1 -6} alkyl, COOH, CONH ₂ or phenyl, Y- (CH = CH)
-R ₃₁ is included. Suitable aryl groups within the above substituents include substituted and unsubstituted phenyl, pyridyl, furanyl, thienyl, thiazolyl, isothiazolyl, imidazolyl, pyrazinyl, pyrimidyl, pyrazolyl, isoxazolyl and oxazolyl, each of which is 1 One or more of hydroxy, amino, carboxyl, carboxamido, halo, _{hydroxy, C 1-6} alkylamino, di _{-C 1-6 alkylamino, C 1-6 alkoxy, C 1-6 alkylthio, C 1 -6} alkylcarbonyl , Y is O, S or NH, t is 0, 1, 2 or 3, and the heterocycloalkyl group is morpholinyl, pyrrolidinyl, piperazinyl, N-substituted piperazinyl, piperidinyl, tetrahydropyranyl, tetrahydrofuranyl and pyrrolidinonyl. Consists of It is selected from, Y- _{(CH 2) t} - can be substituted with heterocycloalkyl. In one preferred embodiment R ₂₇ is
An aryl group selected from substituted or unsubstituted phenyl, naphthyl such as 1-naphthyl, and benzothienyl such as 3-benzothienyl.

R ₂ is NR ₃₂ R ₃₃ , where R ₃₂ is hydrogen or C _1-6 alkyl; R ₃₃ is (CH ₂ ) _w —W— (CH ₂ ) _x —V, where And W is a single bond, wherein the sum of w and x is 1 to 6, or w is 0, 1, 2 or 3 and x is 0, 1, 2 or 3 And W may be aryl or aryl-T, where T is O, S or NH. Suitable aryl groups include substituted and unsubstituted phenyl, naphthyl, pyridyl, furanyl, thienyl and pyrimidyl. When w is 0, 1, 2 or 3 and x is 0, 1, 2 or 3, W may be C _3-8 cycloalkyl. V is COOR ₃₄ , where R ₃₄ is hydrogen or C _1-6 alkyl; or, V is COC _1-6 alkyl, CONH ₂ , SO ₃ H or NO ₂ .

R ₂ is

[0128]

[Chemical 6]

[0129] It may be amino acid A5 represented by the formula: Where R₃₅Is hydrogen or C _1-6 Is alkyl; R₃₆Is hydrogen or C_1-6Is alkyl; R₃₇A
Minoic acids Asp, Asn, Glu, Gln, Asp (C_1-6Alkyl), Glu
(C_1-6Alkyl) or y is 3, 4 or 5 and R₄₂Is hydrogen or C_{1- 6} Is alkyl (CH_Two)_y-COOR₄₂Is a side chain of R; or R₃₇Is (
CH_Two)_z-CONR₄₀R₄₁Where z is from 1 to 5 and R_{Four 0} And R₄₁And are hydrogen or C, respectively_1-6-Alkyl or R₄₀,
R₄₁And a nitrogen atom forms a 5- to 8-membered heterocycle; or R₃₇Is (
CH_Two) A-SO_ThreeH, where a is 1, 2, 3, 4 or 5; or (
CH_Two)_b-Tetrazolyl, where b is 1, 2, 3, 4 or 5;
Is R₃₇Is (CH_Two)_d-Phenyl- (CH_Two)_e-COOR₄₃And
Where d is 0 to 2, e is 0 to 2 and R₄₃Is hydrogen or C_{1 -6} Alkyl; or R₃₇Is (CH_Two)_d-Phenyl- (CH_Two)_e−
CONR₄₄R₄₅Where d is 0 to 2 and e is 0 to 2
And R₄₄And R₄₅And are hydrogen and C, respectively._1-6Alkyl, or
R₄₄And R₄₅And the nitrogen atom form a 5- to 8-membered heterocycle. Suitable
Has R₃₇Is aspartic acid or glutamic acid; y is 3 to 5, R ₄₂ Is hydrogen or C_1-6Is alkyl (CH_Two)_y-COOR₄₂; Or e
0, 1 or 2 and R₄₃Is hydrogen or C_1-6Alkyl-phenyl- (
CH_Two)_e-COOR₄₃Is the side chain of. U is hydroxy, C_1-6Alkoxy
Or NR₃₈R₃₉And where R₃₈And R₃₉And each individually, water
Elementary; substituted or unsubstituted C_1-10-Alkyl, substituted or unsubstituted aryl or
It is a substituted or unsubstituted cycloalkyl or bicycloalkyl. Multiple suitable
The alkyl substituents include hydrogen, hydroxy, halogen, substituted and unsubstituted aryl groups.
And substituted and unsubstituted cycloalkyl. The aryl group is substituted and
Unsubstituted phenyl, naphthyl, anthracenyl, phenanthrenyl, fluorenyl
, Pyridyl, pyridazinyl, pyridinonyl, furanyl, thienyl, thiazolyl
, Isothiazolyl, imidazolyl, triazolyl, pyrrolyl, tetrazolyl, beta
Imidazolyl, pyrazinyl, pyrimidyl, quinolyl, isoquinolyl, benzo
Furanyl, benzothienyl, pyrazolyl, indolyl, purinyl, isoxazol
It can be selected from ril, oxazolyl, dibenzofuranyl. Multiple suitable ants
And the aryl groups are C_1-6Alkyl, halo, hydroxy, C_1-6Alkyl
  Amino, di-C_1-6Alkyl amino, C_1-6Alkoxy, C_1-6Al
Kircio, C_1-6Alkylcarbonyl, phenylcarbonyl, benzylcarbo
Nil, C_1-6Alkyl-sulfonyl, C_1-6Alkyl-sulfonyl-amino
, C_1-6Alkyl-carbonyl-amino, carboxyl, OC_1-6Archi
Lecarboxyl, carboxyl alkenyl, OC_1-6Alkyl carboxyl
Alkenyl, C_1-6Alkylcarbamoyl, cyano, nitro, trifluorome
Tyl and oxytrifluoromethyl. Multiple suitable cycloalkyl groups include
, C_3-8-Including cycloalkyl, adamantyl and bicyclooctyl.
Suitably U is OH or NHR₃₈And where R₃₈Is t-butyl, isop
Ropil, 2,4-dimethylpent-3-yl, cyclopentyl, cyclohexyl
Or bicyclooct [3.3.0] yl; or R₃₈And R₃₉And
Form a pyrrolidinyl or piperazinyl ring with a nitrogen atom.

In a subset of compounds of the formula shown in Formula 1, R ₂ is (CH ₂ ) _w —W—
_{(CH 2)} a x -COOR _34, wherein W is a single bond, phenyl, or _{C 6} - cycloalkyl.

The terms “amino acid residue” and “peptide residue” refer to the carboxyl group (
By an amino acid or peptide molecule that does not have -OH (attached to the C-terminus) or the proton of its amino group (attached to the N-terminus). Generally, the abbreviations used in this application to mean amino acids and protecting groups are IUPAC-
IUB Biochemistry Nomenclature Committee (Biochemistry (1972) 11: 1
726-1732)). For example, Met, Ile
, Leu, Ala and Gly represent “residues” of methionine, isoleucine, leucine, alanine and glycine, respectively. A residue means a free radical derived by removing the OH moiety from the carboxyl group of the corresponding α-amino acid and the H moiety from the α-amino group. The term "amino acid side chain" is a portion of an amino acid excluding the _-CH (NH 2) COOH portion, K. D. Kople, "
Peptides and Amino Acids ", WA Benjamin's Incorporated, New York and Amsterdam, 1966.
, Page 2 and 33 are defined on the page; Such examples of side chains of the common amino _acids, -CH ₂ CH 2 SCH ₃ (the side chain of _{methionine), - CH (CH 3)} -
CH ₂ CH ₃ (side chain of _{isoleucine), - CH 2 CH (CH} 3) 2 ( side chain of leucine) or H- is (the side chain of glycine).

To a large extent, the amino acids used in the present invention are the naturally occurring amino acids found in proteins, or the naturally occurring anabolic or catabolic activities of such amino acids containing amino and carboxyl groups. It is a product of action. Particularly preferred multiple amino acid side chains include side chains selected from the following amino acid side chains:
Glycine, alanine, valine, cysteine, leucine, isoleucine, serine,
Threonine, methionine, glutamic acid, aspartic acid, glutamine, asparagine, lysine, arginine, proline, histidine, phenylalanine, tyrosine and tryptophan.

However, the term amino acid residue refers to an analog of any of the amino acids mentioned herein,
Derivatives and homologues are also included. For example, the present invention provides amino acid analogs with longer or shorter side chains while still possessing a carboxyl, amino or other reactive precursor functional group for cyclization and a plurality of suitable amino acids. The use of amino acid analogs with multiple mutated side chains with functional groups is contemplated. For example, an amino acid analog of, for example, α-cyanoalanine, canavanine, diencoric acid, norleucine, 3-phosphoserine, homoserine, dihydroxyphenylalanine, 5-hydroxytryptophan, 1-methylhistidine, or 3-methylhistidine to the desired peptidomimetic The body may be included. Metabolites or precursors of other naturally occurring amino acids that have suitable side chains in the present invention will be understood by those of skill in the art. Such materials are included within the scope of this invention.

Further, D and L stereoisomers of such amino acids are also included in the scope of the present invention when the structures of the amino acids can take stereoisomeric forms. The shapes of amino acids and amino acid residues are represented by the appropriate symbols D, L or DL in the present application, but even when the shapes are not represented, the amino acids or amino acid residues are represented by the shapes D, L or D.
Can take L. Asymmetric carbon atoms may be contained in a plurality of structures of the compound of the present invention. Therefore, it should be understood that the isomers resulting from such asymmetry are included in the scope of the present invention. Isomers such as these are made in substantially pure form by conventional separation techniques and stereochemically controlled synthetic methods, such as isomer # 1.
Or, it is called by an arbitrary name such as # 2. As used herein, an amino acid having a unique name is understood to include both the D or L stereoisomers, preferably the L stereoisomer, except where otherwise indicated. Should be.

The term “protecting group” as used herein refers to temporary substituents which protect a potentially reactive functional group from undesired chemical alterations. Examples of such protecting groups include esters of carboxylic acids, silyl esters of alcohols, and acetals and ketals of aldehydes and ketones, respectively. A review of the field of protecting group chemistry has been made (Greene, TW; Wuts, PG.
M. Protective Groups in Organic Synt
hesis, 3rd edition; Wiley: New York, 1999; and Koci.
enski, P.P. J. Protecting Groups, Chime Publisher:
New York, 1994).

The term “N-terminal protecting group” or “amino protecting group” as used herein can be used to protect the N-terminus of an amino acid or peptide from unwanted reactions during synthetic procedures. , Refers to various amino protecting groups. Examples of suitable groups include, for example, acyl protecting groups such as formyl, dansyl, acetyl, benzoyl, trifluoroacetyl, succinyl and methoxysuccinyl; aromatic urethane protecting groups such as carbonylbenzyloxy (Cbz); Included are aliphatic urethane protecting groups such as t-butyloxycarbonyl (Boc) or 9-fluorenylmethoxycarbonyl (FMOC). A peptidomimetic of the invention having a side chain containing an amino group or an azepine ring substituent, such as where R3 is lysine or arginine, or R8, R1, R2 or Y has a free amino group, It may optionally have a suitable N-terminal protecting group attached to this side chain.

The term “C-terminal protecting group” or “carboxyl protecting group” as used herein refers to a group intended to protect a carboxylic acid group, such as the C-terminus of an amino acid or peptide. Benzyl or other suitable esters or ethers are examples of C-terminal protecting groups well known in the art.

In addition to the various side chain substitutions that can be made to produce the desired peptidomimetics, the present invention provides for conformationally-fixed mimics or secondary conformations of peptides that are not cleaved by hydrolases. It is specifically intended for use with mimetics. Various surrogates of the amide bond of peptides have been developed. Commonly used amide bond surrogates include the following groups (i) trans-olefins, (ii) fluoroalkenes, (iii) methylene-oxy, (iv) methylene-amino, (v).
Included are methylene-thio, (vi) dihydrooxyethylene, (vii) phosphonamide, (viii) sulfonamide and (ix) ketomethylene.

[0139]

[Chemical 16]

Furthermore, a peptidomimetic in which the backbone of the peptide is changed to a larger extent may be used. Peptidomimetics belonging to this category include (i) retro-inverso (original retro-inverso) analogues and (ii) N-alkylglycine analogues (so-called peptoids).

Furthermore, for example, G.L. of Harvard University is used. L. The method of combinatorial chemistry by Verdine has contributed to the development of new peptidomimetics. For example, the so-called "peptide morphing"
The method focuses on randomly generating a library of peptide analogs with a wide range of peptide binding substituents.

In one exemplary embodiment, the peptidomimetic may be obtained as a retro-inverso analog of the peptide. Such retro-inverso analogs may be made based on methods well known in the art such as those described in US Pat. No. 4,522,752 to Sisto et al.

Such retro-enantio analogs may be synthesized from commercially available D-amino acids (or analogs thereof) using standard solid or solid phase peptide synthesis techniques. . For example, in one suitable solid phase synthesis method, it is preferably amino protected (fluorenyl-methoxycarbonyl, Fmoc).
The D- _A1 residue (or analog thereof) is covalently attached to a solid support, such as chlorotrityl chloride resin. The Fmoc protecting group is removed by washing the resin with dimethylformamide, dichloromethane (DCM) and methanol and treating with piperidine in DCM. This resin is washed and neutralized, and the following F
The moc-protected D-amino acid (D-A2) is introduced by coupling with diisopropylcarbodiimide. The resin is washed again and this cycle is repeated for each of the remaining amino acids (D-A3, D-A4, etc.).

Upon completion of synthesis of the protected retro-enantiopeptide, treatment with trifluoroacetic acid removes the protecting group and separates the peptide from the solid support. The final product is purified by HPLC to give pure retro-enantio analog.

In yet another exemplary embodiment, trans-olefin derivatives of the polypeptide of interest may be made. For example, an example of an olefin analog derives an example of the following pentapeptide:

2-EtO-naphthyl-CO-Smp-NH-CH (CH3) -CH = CH-
CH (CH3) -CO-Phe-Glu-NHtBu

The trans-olefin analog of this pentapeptide was prepared as described in Y. K. Shue et al. (Tetrahedron Letters, 28: 3225 (1987).
) May be used for the synthesis. For the above illustrative example, Boc
-Amino L-Ala is converted to the corresponding α-amino aldehyde, which is treated with vinyl cuprate (original vinylcuprate) to give a mixture of different diastereoisomeric alcohols. This allyl alcohol (
The original allyl alcohol was acetylated with acetic anhydride in pyridine and the olefin was cleaved with osmic acid / sodium periodate to form the aldehyde, which was concentrated with a Wittig reagent derived from a protected alanine precursor. , Allyl-type acetate (original word allylic acetate) is produced. The allylic acetate is selectively hydrolyzed with sodium carbonate in methanol, and the allylic alcohol is treated with triphenylphosphine and carbon tetrabromide to produce the allylic bromide (original allyllic bromide). This compound is reduced with zinc in acetic acid to give the rearranged trans-olefin as a mixture of diastereoisomers to the newly formed core. These diastereoisomers are separated by performing a selective transfer hydrogenolysis to expose the free carboxylic acid to give a pseudodipeptide (pseudod).
ipeptide).

Other synthetic methods for trans-olefin building blocks are described in J. S.
Wai et al. (Tetrahedron Letters 36: 3461 (1
995)), T.S. Ikuba et al. (J. Org. Chem. 56: 4.
370 (1991)) and J. A. McKinney (Tetrahed
ron Letters 35: 5985 (1994)).

This Fmoc-protected pseudodipeptide is then attached in place of A2 and A3 in the sequence. Other pseudodipeptides can also be made by the methods described above by simply replacing the appropriate Boc amino acid with Wittig reagent.

Depending on the nature of the reagents used, it will be necessary to vary the processing method, but any such variation is purely conventional and will be apparent to those skilled in the art.

The pseudodipeptide synthesized by the above method may be coupled with another pseudodipeptide to create a peptide analog having multiple olefin functional groups instead of the multiple amide functional groups.

For example, Fmoc-protected Glu-Ala or Tyr-Glu was prepared,
These may then be coupled by standard methods to give analogs of the pentapeptide with altered inter-residue olefinic linkages.

Yet another class of peptidomimetic derivative is the phosphonate derivative.
The synthesis of such a phosphonate derivative may be performed by a known synthetic method.

For example, Peptides: Chemistry and Biology
(Escom Science Publishers, Leiden, 1988, p. 11
8) Roots et al .; Peptides: Structure and
See Petrillo et al. In Function (9th American Peptide Symposium Bulletin, Pierce Chemical Company Rockland, Illinois, 1985).

Other peptidomimetic structures are well known in the art and can be readily adapted to use the peptidomimetic of interest. These are the general formula 2
Substituting two adjacent amino acids, preferably the amino acid sequence A2-A3. The synthetic treatment method for producing this peptide mimetic is the same as the ordinary peptide synthesis method. In constructing the sequence from the C-terminus, the Fmoc-protected form of this peptidomimetic is used in place of the corresponding two amino acids. R1-A1- (PM-x) -A4-R in which x is 1 to 18 is bonded to the peptide mimetics PM-1 to PM-18 shown below under ordinary peptide bonding conditions.
A structure such as 2 may be obtained.

In one embodiment, the invention provides that A2 and A3 cooperate to provide (a) 6-amino-5-oxoperhydropyrido [2,1-b] [1,3] thiazole- 3-carboxylic acid, preferably (R, S, S) -6-amino-5-oxoperhydropyrido [2,1-b] [1,3] thiazole-3-carboxylic acid (PM-1) ;
(B) 6-amino-5-oxoperhydro-3-indolizinecarboxylic acid,
Suitably (S, S, S) -6-amino-5-oxoperhydro-3-indolizinecarboxylic acid (PM-2); (c) (S, R) -6-amino-5-oxoperoxide. Hydro-8a-indolizinecarboxylic acid or (R, R) -6-amino-
5-oxoperhydro-8a-indolizinecarboxylic acid (PM-3); (
d) (R, S) -6-amino-5-oxoperhydro-8a-indolizinecarboxylic acid or (S, S) -6-amino-5-oxoperhydro-8a-indolizinecarboxylic acid (PM- 4); (e) 2- (3-amino-2-oxo-1)
, 2-Dihydro-1-pyridinyl) acetic acid (PM-5); (f) 2- (3-amino-2-oxo-6-phenyl-1,2-dihydro-1-pyridinyl) acetic acid (PM
-6); (g) 3-Aminobenzoic acid (PM-7); (h) 4-Aminobenzoic acid (
PM-8); (i) 3-aminomethylbenzoic acid (PM-9-1); (j) (S).
-3- (1-Aminoethyl) benzoic acid or (R) -3- (1-aminoethyl) benzoic acid (PM-9-2); (k) (S) -3- (1-aminopropyl) benzoic acid Acid or (R) -3- (1-aminopropyl) benzoic acid PM-9-3); (l) (S
) -3- (1-Aminobutyl) benzoic acid or (R) -3- (1-aminobutyl)
Benzoic acid (PM-9-4); (m) 2- (3-amino-2-oxo-1-azepinyl) acetic acid, preferably (S) -2- (3-amino-2-oxo-1-). Azepinyl) acetic acid (PM-10); (n) 2- [8- (aminomethyl) -3,6-dimethyl-9,10,10-trioxo-9,10-dihydro-10 [lambda] ^6- thioxanthene-1- Yl] acetic acid (PM-11); (o) 2- (2-oxopiperazino) acetic acid (PM-12); (p) 2- [8- (aminomethyl) -2-oxo-5-phenyl-2,3 -Dihydro-1H-1,4-benzodiazepin-1-yl] acetic acid (P
M-13-1); (q) 2- [8- (aminomethyl) -2-oxo-5-methyl-2,3-dihydro-1H-1,4-benzodiazepin-1-yl] acetic acid (PM
-13-2); (r) 3-Aminopropionic acid (PM-14); (s) 4-Aminobutyric acid (PM-15); (t) 5-Aminopentanoic acid (PM-16); (u) Two
-[2- (2-aminoethoxy) ethoxy] acetic acid (PM-17); and (v) 2
-(3-Amino-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepin-1-yl) acetic acid, preferably (S) -2- (3-amino-2-oxo- Provided is a compound of the formula shown in Formula 1, which forms a peptidomimetic residue selected from 2,3,4,5-tetrahydro-1H-1-benzazepin-1-yl) acetic acid.

[0157]

[Chemical 17]

Peptidomimetic residues are shown herein in a manner similar to that defined above for "amino acid residue" or "peptide residue". "Peptidomimetic residues"
The term means that there is no -OH on the carboxyl group (attached to the C-terminus) or there is no proton on the amino group (attached to the N-terminus). Some peptidomimetics are, for example, PM-1, PM-7, PM-8, PM-10,
Amino groups such as PM-14, 15 and 16 are commercially available in protected or unprotected acid form. The synthesis of these various peptidomimetics
For PMs 5 and 6, see P. D. Edwards et al. (J. Med. Che
m. 1996, 39, 1112) and F.I. J. Brown et al. (J. Med.
． Chem. 1994, 37, 1259), PM 3 and 4 are described in J.
D. Gramberg et al. (Tetrahedron Letters 1
994, 35, 861) and PM2 is described in H.264. -G. Lombart and others (
J. Org. Chem. 61: 9437 (1996)), PM-12.
Regarding A. Pohlmann et al. (J. Org. Chem. 62: 1.
016 (1997)), regarding PM-9, L.M. Chen et al. (Tetra
Hedron Letters 36: 8715 (1995)), PM-.
For A.17, M. P. Koskinen (Biorg. & Med
． Chem. Lett. 5: 573 (1995)), regarding PM-13, W. C. Ripka et al. (Tetrahedron 49: 3593)
(1993)), PM. Sato and others (Biochem
． Biophys. Res. Commun. 187: 199 (19
92)). Nagai (Tetrahedron
Letters 26: 647 (1985)). For peptide mimetics, 1-azabicyclo [4.3.0] nonane surrogate (Kim
J. J. et al. Org. Chem. 62: 2847 (1997)), or N-acyl piperazic acid (original piperatic acid).
d) (Xi et al., J. Am. Chem. Soc. 120: 80 (19
98)), or a 2-substituted piperazine component as a fixed amino acid analog (Williams et al., J. Med. Chem. 39: 13).
45-1348 (1996)). In yet another embodiment, some amino acid residues are, for example, monocyclic or bicyclic aromatic or heteroaromatic nuclei, or diaromatic, aromatic-heteroaromatic or diheterocyclic. It may be substituted with aryl and biaryl moieties such as aromatic nuclei.

Peptidomimetics of interest may be optimized, for example, by combining combinatorial synthetic techniques with high-throughput screening described herein.

Further, other examples of mimetope include protein utilizing compounds, carbohydrate utilizing compounds, lipid utilizing compounds, nucleic acid utilizing compounds, natural organic compounds, synthetic organic compounds, anti-idiotypic antibodies and / or catalytic antibodies or fragments thereof. But is not limited to these.

Many efficient methods have been published in the literature for the synthesis of racemic unnatural amino acids. Strecker synthesis involves reacting an aldehyde with ammonia or with a substituted primary amine with a hydrogen cyanide to produce an alpha-amino nitrile, which is hydrolyzed to the corresponding amino acid (Houben-Wey.
1, Methoden der organischen Chemise V
ol. 11/2, p. 305 (1958)). Concentration of amine R ₁ -NH ₂ , aldehyde R ₂ CHO, acid R ₃ -COOH and isocyanide R ₄ -NC (Ug
i reaction) is a method commonly used to make racemic amino acids of the type (R ₃ —CO) NR ₁ —CHR ₂ —CO—NHR ₃ (Ugi et al., Lie).
bigs Ann. Chem. 1967, 709, 1; Ugi and others A
ngew ,. Chem. Int. ed. 1962, 1, 8). The case of carrying out the Ugi reaction in the solid phase is also described (Strocker et al., Te.
trahedron Letters 37: 1149 (1996); Zh
ang et al., Tetrahedron Letters 37: 751 (19).
96); Tempest et al., Angew. Chem. Int. Ed. Three
5: 640 (1996); Sutherlin et al., J. Am. Org. Che
m. 61: 8350 (1996); Short et al., Tetrahedro.
n Letters 37: 7489 (1996)). In addition, deprotonation of the (N-diphenylmethylene) glycine derivative with a strong acid such as sodium hydroxide or lithium diisopropylamide and the corresponding anion with the corresponding bromomethylphenyl derivative or bromomethylnaphthyl derivative, for example. Racemic amino acids may be made by reaction with an alkylating agent. Protection (Fmoc, Cbz, Boc, Alloc) or acylation of the amino component followed by hydrolysis gives the corresponding amino acid derivative. Building blocks _{(R 1 -CO- (2-Br} ) -Smp-OH and _R 1 -CO- (2-CHO
) -Smp-OH) is shown in Chemical Formula 21:

[0162]

[Chemical 21]

Another method for producing amino acids consists in obtaining a racemic amino acid by hydrolyzing a dehydroamino acid obtained by reacting the anion of a (N-diphenylmethylene) -glycine derivative with an aldehyde. Further, a hydroxynaphthylalanine derivative is described by Vela et al. (J. Org. Chem. 55: 2
913 (1990)).

Using the Wittig reaction of an alpha-phosphoryl-glycine derivative with an aldehyde or its Horner-Emmons variant, Ciattini (Ciaattin
i et al., Synthesis 2: 140 (1988)) and Shin (S.
hin et al., Tetrahedron Lett. 28: 3827 (198
7); Shin et al., Chem. Pharm. Bull. 38: 2020
The corresponding dehydroamino acid described by (1990)) may be synthesized.

The racemic amino acid derivative is generated by hydrogenolysis of the dehydroamino acid derivative with a metal catalyst such as palladium on carbon or a phosphine or amine complex of rhodium, ruthenium or palladium. I. Ojima, "Ca
“Talytic Asymmetry Synthesis”, Verlag
Chemie, 1993, chapter 1, p. 6 and R.I. Noyor
i, "Asymmetric Catalysis in Organic S
synthesis ”, John Wiley, 1994, chapter 2,
p. 16. The use of chiral compounds as metal ligands, as described in US Pat. For example, D- and L
-Synthesis of alpha-amino adipate, pimelate and suberate is described in T.W.
Pham et al. (J. Org. Chem. 59: 3676 (1994).
)).

Further, methods for making optically active alpha-amino acids are described in R. M. Will
iams, "Synthesis of active alpha-amin"
o acids ”Pentagon Press, 1989, and LM O'Don.
nell "alpha-amino acid synthesis",
See Tetrahedral Symposium Bulletin, 44: 5253 (1988). Therefore, chiral amino acids can be synthesized using chiral glycine derivatives in the same manner as described above. After removal or cleavage of this chiral auxiliary, the final chiral amino acid is obtained. This chiral auxiliary gives D or L-amino acids in high enantiomeric excess. The preferred method is U.S.P. The method described by Schoellkopf (Schoellkopf et al., Angew, Chem. Int.
Ed. 18: 863 (1979); Schoellkopf et al., Ange.
w, Chem. Int. Ed. 20: 798 (1981); Sch.
Oellkopf et al., Synthesis, 969 (1981); Schoe
Ilkopf et al., Synthesis, 866 (1982); Schoell
Kopf et al., Synthesis, 861 (1982); Schoellko
pf et al., Synthesis, 37 (1983); Schoellkopf et al., Synthesis, 271 (1984)). M. William
5,6-diphenyl-2,3,5,6-tetrahydro-4H described by s
Method using -1,4-oxazine-2-1 template (Williams et al., J. Am.
Am. Chem. Soc. 113: 9276 (1991); Wil
liams et al., Tetrahedron Letters 29: 6075.
(1988); Solas et al., J. Am. Org. Chem. 61: 1537
(1996); Williams et al., J. Am. Chem. Soc.
110: 1547 (1988); Williams et al., J. Am. Am. Ch
em. Soc. 110: 482 (1988); Williams et al., J.
． Am. Chem. Soc. 108: 1103 (1986)), D
． The method described by Seebach (Fitzi et al., Tetrahed.
ron 44: 5277 (1988), Seebach et al., Liebigs.
Ann. Chem. , 1215 (1989); Schickli et al., Li.
ebigs Ann. Chem. , 1323 (1991); Seebach.
Liebigs Ann. Chem. , 1145 (1992); Mue
Ller et al., Helv. Chim. Acta, 75: 855 (1992).
); Seebach et al., Angew. Chem. 105: 1780 (1
993)); and A. The method described by Myers (Myers et al., J.
． Am. Chem. Soc. 117: 8488 (1995); My
ers et al., J. Am. Chem. Soc. 119: 656 (199
7).

As an example, Fmoc- (6-OH) -Nal-OH or Fmoc- (7-O
The synthesis of (H) Nal-OH is shown in Chemical Formula 22 below.

[0168]

[Chemical formula 22]

Another method of preparation is the electrophoretic amination of chiral enolates followed by hydrolysis of the chiral auxiliary, D. et al. Evans (D. Evans et al., J. Am.
Chem. Soc. 112: 4011 (1990); Evan
s et al., Tetrahedron 44: 5525 (1988)).

Alpha, alpha-disubstituted amino acid derivatives are described in D. Seebach (S
eebach et al., Liebigs Ann. Chem. 217 (1995
); Seebach et al., Tetrahedron Letters, 25: 2
545 (1984)).

Chiral amino acids are described by Sturmer et al. (BASF AG) Ger. Off
en. It is also obtained by decomposition of racemic amino acid esters by enzyme-catalyzed acylation, as described in DE 197 25 517.

Peptidyl such as penta- and tetrapeptidyl intermediates obtained using the above amino acids in a suitably protected form (eg having Boc, Fmoc, Alloc, Cbz) or by the methods 1 to 4 below. The amino acids obtained by the above method may be further modified or converted by performing standard organic reactions such as reductive amination, alkylation, esterification, etherification, Mitsunobu reaction in the environment. The modification or conversion reaction may be carried out in solution or with the amino acid or peptide bound to a suitable solid phase. For example, the solid phase synthesis of Fmoc-N-methyl amino acids is described by Yang et al., Tetrahedron Let.
ters 42: 7307 (1997).

As an example of a modification of the pentapeptide, the reductive amination of a 2-formyl-smp-derivative is shown below in Scheme 23:

[0174]

[Chemical formula 23]

Another example of modification of peptidyl intermediates is the Mitsunobu reaction of Nal-derivatives on the solid phase shown in:

[0176]

[Chemical formula 24]

The novel compounds of formula 1 may be made using known methods of peptide chemistry. Thus, peptidyl derivatives may be constructed sequentially from amino acids or by joining small fragments of suitable peptides. In the case of sequential construction, the peptide chain is extended from the C-terminus by one amino acid each time. When ligating fragments, it is possible to connect multiple fragments of different lengths, and these fragments may be obtained by sequentially constructing amino acids, or they may themselves be obtained by ligating the fragments. May be. In both the case of sequential construction and the case of fragment bond, it is necessary to connect the structural units by forming an amide bond. Enzymatic and chemical methods are suitable for this. Chemical methods for the formation of the amide bond are described in Muller, Methoden der orga.
nichen Chemie Vol. XV / 2, pp 1-364, Zieme Publisher, Stuttgart, 1974; Stewart, Young, Sol.
id Phase Peptide Synthesis, pp 31-34,
71-82, Pierce Chemical Company, Rockford, 1984; B.
odanszky, Klausner, Ondetti, Peptide Sy
nthesis, pp 85-128, John Wiley and Sons, New York, 1976 and other standard peptide chemistry studies. Particularly preferred methods are the azide method, symmetrical mixed anhydride method, in situ generated or preformed active esters, urethane protected N-carboxyanhydrides of amino acids and coupling reagents (activators, especially dicyclohexylcarbodiimide ( DCC), diisopropylcarbodiimide (DIC), 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), 1-ethyl-3.
-(3-Dimethylaminopropyl) carbodiimide hydrochloric acid (EDCI), n-propane-phosphonic anhydride (PPA), N, N-bis (2-oxo-3-oxazolidinyl) imide-phosphoryl chloride (BOP-Cl), bromo -Phosphonium tris-pyrrolidinohexafluorophosphate (PyBrop), diphenyl-phosphoryl azide (DPPA), Castro reagent (BOP, PyBop), O-benzotriazolyl-N, N, N ', N'-tetramethyluro Nium salt (HBTU), diethylphosphoryl cyanide (DEPCN), 2,5-diphenyl-2,3-dihydro-3-oxo-4-hydroxy-thiophene dioxide (Stegrich reagent; HOTDO), 2- (1H-benzotriazole-
1-yl) -1,1,3,3-tetramethyltetrafluoroborate uronium (T
BTU), O- (7-azabenzotriazol-1-yl) -1,1,3,3-
Formation of an amide bond using uronium tetramethylhexafluorophosphate (HATU) and 1,1′-carbonyl-diimidazole (CDI). The binding reagent may be used alone or N, N-dimethyl-4-aminopyridine (D
MAP), N-hydroxy-benzotriazole (HOBt), 1-hydroxy-7-azabenzotriazole (HOAt), N-hydroxybenzotriazine (HOOBt), N-hydroxysuccinimide (HOSu) or 2-hydroxypyridine. You may use it in combination with an agent.

In enzymatic peptide synthesis, it is usually possible to omit the protecting group, but in chemical synthesis both reactives reversibly protect the reactive groups not involved in the amide bond. There is a need to. In chemical peptide synthesis, four conventional protecting group methods: benzyloxycarbonyl (Cbz), t-butoxycarbonyl (Boc).
), Aryloxycarbonyl (Alloc) and 9-fluorenylmethoxycarbonyl (Fmoc) methods are preferred. What is common in each case is the presence of a protecting group on the α-amino group of the chain extension. A detailed description of amino acid protecting groups can be found in Muller, Methoden der organisch.
en Chemie Vol. XV / 1, pp 20-906, Zieme Publishing Company, Stuttgart, 1974. The moieties used for the construction of this peptide chain are either in solution, in suspension, or according to J. Amer. Chem. So
c. 85: 2149 (1963) may be reacted in a similar manner as described by Merrifield. A particularly preferable method is a method of constructing peptides sequentially or by fragment binding, which comprises Cbz, Boc, Allo.
c or Fmoc protecting group method, wherein one of the reactants of the Merrifield method is bound to an insoluble polymer support (hereinafter also referred to as a resin). This typically includes peptides constructed sequentially on the polymer support using the Boc, Alloc or Fmoc protecting group method, the elongating peptide chain being the C-terminal of the insoluble resin particle. Is covalently bound to. This treatment allows the reagents and by-products to be filtered out and therefore
No recrystallization of the intermediate is necessary. The protected amino acid can be attached to any suitable polymer, but the polymer need only be insoluble in the solution used and in a stable physical form that facilitates filtration. . The polymer must contain a functional group to which the first protected amino acid can be strongly covalently attached. For example, cellulose, polyvinyl alcohol,
Polymethacrylate, sulfonated polystyrene, chloromethylated styrene / divinylbenzene copolymer (Merifield resin), 4-methylbenz-hydrylamine resin (MBHA-resin), phenylacetamidomethyl-resin (Pam-
Resin), Rink amide resin, chlorotrityl chloride resin, p-benzyloxy-
Benzyl-alcohol-resin, benzhydryl-amine-resin (BHA-resin), 4- (hydroxymethyl-)-benzoyl-oxymethyl-resin, Brei
pohl and other resins (Tetrahedron Letters 28 (198
7) 565; provided by Bashem), 4- (2,4-dimethoxyphenylaminomethyl) phenoxy-resin (provided by NovaBiochem) or o-chlorotrityl-resin (provided by Biohelas), for this purpose. Suitable for

Suitable for peptide synthesis in solution are all solvents which are inert under the reaction conditions, in particular water, N, N-dimethylformamide (DMF), dimethylsulfoxide (DMSO), Acetonitrile, dichloromethane (DCM), 1
, 4-dioxane, tetrahydrofuran (THF), N-methyl-2-pyrrolidinone (NMP) and mixtures of these solvents are suitable.

Peptide synthesis on polymer supports can be carried out in all inert organic solvents in which the amino acid derivative used can be dissolved; however, suitable solvents have resin swelling properties, eg DMF. , DCM, NMP, acetonitrile and DMSO
And the like, and mixtures of these solvents. After synthesis is complete, the peptide is cleaved from the polymer support. The conditions under which the various types of resins can be separated are disclosed in the literature. The most commonly used cleavage reactions for such cleavage are acid and palladium catalyzed cleavages, especially in liquid anhydrous hydrogen fluoride, in anhydrous trifluoromethanesulfonic acid, diluted or concentrated trifluoro. Cleavage in acetic acid, eg TH in the presence of a weak base such as morpholine
Pd-catalyzed cleavage in F or THF-DCM-mixture or cleavage in acetic acid / dichloromethane / trifluoroethanol mixture. Depending on the protecting group chosen, they may be retained or may be similarly cleaved off under cleavage conditions.

More specifically, the novel compound of the general formula shown in Chemical formula 1 may be produced by the following production method.

A) Preparation Method 1: Synthesis of R ₁ -A1-A2-A3-A4-A5-U

The above-mentioned peptide sequences are arranged in order from the C terminus to the corresponding Fmoc-amino acids (Fm
oc-A5-OH) to a free amino group on the resin, such as Rink amide AM resin, and then the Fmoc-protecting group is removed to provide the amino group for subsequent coupling with, for example, piperidine. Release the group. This binding and deprotection are carried out for each amino acid by using Fmoc-A4-OH, Fmo-A3-OH, Fmoc-.
A2-OH and repeatedly in the order of Fmoc-A1-OH, intermediate _NH 2 -A1-
A2-A3-A4-A5-NH-resin is obtained.

[0184]   Corresponding acid R_ThreeBy combining -COOH with the above resin intermediate, or
Corresponding acid chloride R_Three-COCl, oxyfluoride R_Three-COF or anhydride (R_Three−
CO)_TwoO may be a base such as a tertiary amine or an alcoholate or an inorganic base.
Final capping by reacting with the resin intermediate in the presence
. In the case of urea, the above-mentioned resin intermediate is converted to the corresponding isocyanate R_Four-NCO or R _Four R₅Treated with NCOCl and in the case of sulfonamides the corresponding sulfonyl chloride
R₆-SO_TwoTreated with Cl and in the case of sulfonylurea R in the presence of a base₇R₈ SO_TwoTreat with Cl.

For example, by treating the resin with an acid such as hydrochloric acid or trifluoroacetic acid,
Depending on the strength of the acid used and the reaction time, which may finally cleave the compound from the resin, it is possible to deprotect the side chains at the same time. The resulting compound may be further purified by standard methods such as column chromatography.

B) Preparation Method 2: Synthesis of R ₁ -A1-A2-A3-A4-A5-NR ₃₈ R ₃₉

[0187]   The final compound has an amino side containing an acid residue such as Glu, Asp or Aad.
When having a chain, the corresponding amino acid Fmoc-Xaa (tBu) -OH is
In solution, the corresponding amine HNR₃₈R₃₉And use standard peptide conjugation methods
And combine to form Fmoc-Xaa (tBu) -NR₃₈R₃₉And generate this
Next, deprotection with 95% trifluoroacetic acid was carried out, and the free carboxylic acid component in the side chain was
Having Fmoc-Xaa-NR₃₈R₃₉To generate. For example, this amino acid
For example, by combining with a resin such as chlorine (trityl chloride resin), a resin-bonded ester
To generate. Here, the Fmoc group is deprotected with a base such as piperidine.
To form the resin-bound amine. This binding and deprotection
Regarding amino acids, Fmoc-A4-OH, Fmo-A3-OH, Fmoc-A
2-OH and Fmoc-A1-OH are repeated in this order to give the intermediate NH_Two-A1-A
2-A3-A4-A5 (resin) -NR₃₈R₃₉To generate. Corresponding acid R_Three−
By combining COOH with the above resin intermediate or the corresponding acid chloride R _Three -COCl, oxyfluoride R_Three-COF or anhydride (R_Three-CO)_TwoCompare O
Said resin intermediate in the presence of an acid or an inorganic base such as a tertiary amine or alcoholate
Final capping is performed by reacting with. In the case of urea, the above tree
Isocyanate R corresponding to fat intermediate_Four-NCO or R_FourR₅Treated with NCOCl
In the case of sulfonamide, the corresponding sulfonyl chloride R₆-SO_TwoTreated with Cl
In the case of sulfonylurea, R in the presence of a base₇R₈SO_TwoTreat with Cl.

For example, by treating the resin with an acid such as hydrochloric acid or trifluoroacetic acid,
The compound may be finally separated from the resin. Depending on the strength of the acid used and the reaction time, it is possible to deprotect the side chains at the same time. The resulting compound may be further purified by standard methods such as column chromatography.

C) Manufacturing method 3: R1-A1-A2-A3-A4-OH and R1-A1-A2-
A3-A4-NR ₃₂ R ₃₃

The tetrapeptide acid sequences were sequenced from the C-terminal to the corresponding Fm as described above.
The oc-amino acid A1 was coupled to the chlorotrityl chloride resin and then the Fmoc-
It is constructed by removing the protecting group and freeing the amino group for subsequent coupling. This binding and deprotection is performed for each amino acid by Fmoc-A3-
OH, Fmo-A2-OH, repeated in the order of Fmoc-A1-OH and Fmoc-A1-OH, to generate the intermediate _{NH 2 -A1-A2-A3-} A4- resin.

[0191]   Corresponding acid R_ThreeBy combining -COOH with the above resin intermediate, or
Corresponding acid chloride R_Three-COCl, oxyfluoride R_Three-COF or anhydride (R_Three−
CO)_TwoO may be a base such as a tertiary amine or an alcoholate or an inorganic base.
Final capping by reacting with the resin intermediate in the presence
. In the case of urea, the above-mentioned resin intermediate is converted to the corresponding isocyanate R_Four-NCO or R _Four R₅Treated with NCOCl and in the case of sulfonamides the corresponding sulfonyl chloride
R₆-SO_TwoTreated with Cl and in the case of sulfonylurea R in the presence of a base₇R₈ SO_TwoTreat with Cl.

For example, by treating the resin with an acid such as hydrochloric acid or trifluoroacetic acid,
The compound may be finally separated from the resin. Depending on the strength of the acid used and the reaction time, it is possible to deprotect the side chains at the same time. The final compound R ₁ -A1-A2-A3-A4-OH obtained may be further purified by standard methods such as column chromatography.

[0193]   These compounds are further used as intermediates to provide the corresponding amine NR₃₂R_{Three Three} And in solution to form the final amide R₁-A1-A2-A3-A4-NR₃₂R ₃₃ And purified by standard methods such as column chromatography.
May be.

D) Preparation Method 4: R ₁ -A1-A2-A3-A4-A5-OH and R ₁ -A1-
A2-A3-A4-A5-NR ₃₈ R ₃₉

Using the above-described method of making a tetrapeptide, a resin such as chlorotrityl chloride resin is linked to amino acid A5 to make the corresponding pentapeptide, and similarly Is repeatedly bound and deprotected, and finally capped to separate from the resin to obtain the final compound R ₁ -A1-A.
2-A3-A4-A5-OH may be produced. These compounds were further used as intermediates to couple in solution with the corresponding amine HNR ₃₈ R ₃₉ to form the final amide R ₁ -A1-A2-A3-A4-NR ₃₈ R ₃₉ , which was It may be further purified by standard methods such as column chromatography.

The amino acids used are either commercially available or their synthetic methods are published in the literature. Amino acid A is considered to be pTyr-mimetic. Examples of non-hydrolyzable phosphorus-containing pTyr mimetics include, for example, phosphonomethylphenylalanine (Pmp, I. Marseigne et al., J. Org. Ch.
em. 53:. 3621-3624 (1988)) and phosphonomethyl difluoromethyl phenylalanine _(F 2 Pmp, T.R Burke Jr. other, J.
Org. Chem. 58: 1336-1340 (1993)) and the like are described in the literature.

Examples of phosphorus-free pTyr mimetics include O-malonyl tyrosine (Ty
r (Mal), K .; H. Kole et al., Biochem. Biophys.
Res. Commun. 209: 817-822 (1995);
Ye et al., J. Med. Chem. 38: 4270-4275 (1995
)), Fluoro-O-malonyl-tyrosine (Tyr (Fmal), TRB.
urke Jr. J. Med. Chem. 39: 1021-1027
(1996)), O-carboxymethyl-tyrosine (TR Burke).
Jr. Tetrahedron 54: 9981-9994 (1998).
)), 3-carboxy-4- (O-carboxymethyl) -tyrosine (TR.
Burke Jr. Tetrahedron 54: 9981-9994, et al.
(1998)), 3,4-di (O-carboxymethyl) -tyrosine (TR.
Burke Jr. Tetrahedron 54: 9981-9994, et al.
(1998)), O- (carboxydifluoromethyl) -tyrosine (HF
retz, Tetrahedron 54: 4849-4858 (1998).
)) And hydroxysulfonylmethylphenylalanine (I. Marsei)
gne et al., J. Med. Chem. 32: 445-449 (1989)
)) Is included.

Prodrugs may be used for a variety of different acid functional groups of compounds of the general structure of Formula I to enhance cell permeability. Typical prodrug forms used for carboxylic acid residues are described in R.S. B. Silverman, The Organ
ic Chemistry of Drug Design and Drug
Action, Academic Press, 1992, Chapter VIII.
Suitable prodrugs for the phosphonate function of amino acid A1 include simple and substituted alkyl and aryl esters, J. Am. P. The acyloxyalkyl esters described in the paper by Krise et al. (Advanced Durg Delivery)
Reviews, 19: 287-310 (1996)), X. S-acyl thioethyl ester described by Li et al. (Bioorg. Med. Ch.
em. Lett. 8: 57-62 (1998)) or C.I. J. Sta
pivaloyl methyl ester described by Nkovic et al. (Bioorg.
Med. Chem. Lett. 7: 1909 (1997)).

In one embodiment, the present invention relates to a method of treating a Cdc25-mediated condition in a patient. The method includes administering to the patient a therapeutically effective amount of the Cdc25 inhibitor described above. The patient may be an animal, preferably a mammal, more preferably a human.

A “Cdc25-mediated condition” is a disease or bodily condition in which the catalytic activity of one or more Cdc25 homologs plays a role, eg in the development of that disease or bodily condition. Or it is a physical condition. For example, in one embodiment, the condition is characterized by excessive cell proliferation.

In one embodiment, the Cdc25-mediated condition is cancer, such as tumor. For example, the condition to be treated includes lymphoma such as Hodgkin's disease and non-Hodgkin's lymphoma, and head tumor, neck tumor, breast tumor, lung tumor such as non-small cell lung cancer,
And abdominal tumors.

The Cdc25-mediated condition may be one in which hyperproliferation of non-cancerous cells plays an important role, such as coronary stenosis or re-occlusion following angioplasty, which are Both are due to abnormal proliferation of smooth muscle cells.

In another embodiment, the Cdc25-mediated condition is an inflammatory disease characterized by abnormal cell proliferation such as rheumatoid arthritis, Reiter's disease, systemic lupus.

The term therapeutically effective amount as used herein refers to an amount that partially or completely inhibits the progression or symptoms of disease. Such amount depends on, for example, the size and sex of the patient, the condition to be treated, the degree of symptoms, and the expected result, and can be determined by those skilled in the art.

The compounds of the invention may optionally be administered in combination with one or more additional agents that treat and / or alleviate the symptoms of a condition mediated by Cdc25, for example. The additional agent may be administered at the same time as the compound of the invention or may be administered sequentially. For example, the Cdc25 inhibitor may be administered in combination with another anticancer agent, as is well known in the art.

The present invention provides pharmaceutical compositions containing one or more of the Cdc25 inhibitors described above. Such a composition comprises a therapeutically (or prophylactically) effective amount of the above-mentioned C.
It contains one or more of the dc25 binding inhibitors and a pharmaceutically acceptable carrier or excipient. Suitable pharmaceutically acceptable carriers include saline, buffered saline, dextrose, water, glycerol, ethanol and combinations thereof. The carrier and composition may be sterile. The dosage form should suit the mode of administration.

Suitable pharmaceutically acceptable carriers include water, saline (eg NaCl), alcohol, acacia, vegetable oils, benzyl alcohol, polyethylene glycol,
Carbohydrate such as gelatin, lactose, amylose or starch, cyclodextrin, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil,
Fatty acid esters, hydroxymethyl cellulose, polyvinylpyrrolidone and the like are included, but are not limited thereto. The pharmaceutical product may be sterile and, if desired, lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for adjusting the osmotic pressure, buffers, colorants, seasonings and It may be mixed with additives that do not deleteriously react with the active compounds, such as fragrances.

The composition may, if desired, also contain minor amounts of wetting or emulsifying agents or pH buffering agents. The composition may be a solution, suspension, emulsion, tablet, pill, capsule, depot or powder. The composition may be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulations can include standard carriers such as mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidinone, sodium saccharin, cellulose, magnesium carbonate.

The composition may be formulated according to the usual procedure as a pharmaceutical composition suitable for intraarterial administration to humans. Often, compositions for intraarterial administration are solutions in sterile and isotonic aqueous buffer. The composition, if desired, can also contain a solubilizing agent and a local anesthetic to ease pain at the injection site. Generally, these ingredients are supplied separately or mixed in a single dose form such as a lyophilized powder or anhydrous concentrate which is hermetically sealed in a container such as an ampoule or sachet indicating the quantity of active substance. To be done. Where the composition is administered by infusion, it may be dispensed with an infusion bottle containing sterile pharmaceutical grade pure water, saline or dextrose / water. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

The pharmaceutical composition of the present invention may be a sustained or sustained release composition containing an agent that suppresses the release of the Cdc25 inhibitor compound.

Cdc25 inhibitors may be administered subcutaneously, intravenously, parenterally, intraperitoneally, intradermally, intramuscularly, topically, enterally (eg orally), rectally, nasally, buccally, sublingually or vaginally. Alternatively,
It may be administered by inhalation spray, by a drug pump or via an implanted reservoir in a dosage form containing conventional non-toxic, physiologically acceptable carriers or excipients. The preferred method of administration is oral administration. The form of administration (eg syrup, elixir, capsule, tablet, liquid, foam, emulsion, gel, sol) will depend in part on the route of administration. For example, transmucosal (eg, oral mucosa, rectal or intestinal mucosa, bronchial mucosa) administration, nasal drops, aerosols, inhalants, nebulizers, eye drops or suppositories may be used. The compositions and agents of the present invention include, for example, analgesics, anti-inflammatory agents, anesthetics, and other agents capable of modulating one or more of the symptoms or causes of a Cdc-25-mediated condition. , May be administered with other biologically active agents.

In one particular embodiment, it may be desirable to administer the agents of the invention locally or to a local area in need of treatment; this may be achieved, for example, by local injection during surgery, application, It may be performed by, but not limited to, a transdermal patch, an injection, a catheter means, a suppository means or an implant means consisting of a perforated, non-perforated or gelatine material including, for example, a silastic membrane or a membrane such as a fiber. There is no such thing. For example,
The drug may be injected intra-articularly.

Example 1 Synthesis of Peptidic Cdc25 Inhibitor General Raw Materials and Methods Here, the following abbreviations for amino acids are used, and the natural amino acids have a three letter code: Asp = aspartic acid, Asn = asparagine, Pro = Proline, Ala = Alanine, Val = Valine, Lys = Lysine, Gly = Glycine, Arg = Arginine, Ile
= Isoleucine, Ser = Serine, Thr = Threonine, Leu = Leucine, Trp =
Tryptophan, Cys = Cysteine, Tyr = Tyrosine, Met = Methionine, Gln
= Glutamine, Glu = Glutamic acid, Phe = Phenylalanine, His = Histidine, other amino acids are Nal = 2-Amino-3- (naphth-1-yl) -propanoic acid
(Or naphthylalanine), Bta = 2-amino-3-benzo [b] thiophen-3-yl-
Propanoic acid (or 3-benzothienylalanine), Aad = a-aminoadipic acid, Smp = Phe (4-CH ₂ -SO ₃ H), Pmp = Phe (4-CH ₂ -PO (OH) ₂ ), F ₂ Pmp = Phe (4-CF _2-
PO (OH) ₂ ), Asu = a-aminosuberic acid, Hyp = 4-hydroxyproline, Nle =
Norleucine, Nva = norvaline, hLeu = homoleucine, Pip = pipecolic acid, 3-MePro = 3-methyl-proline, 2-MePro = 2-methyl-proline, Isc =
1-isoindolinecarboxylic acid, Oic = octahydroindolyl-2-carboxylic acid, Ac6 = 1-amino-1-cyclohexanecarboxylic acid, Ac5 = 1-amino-1-cyclopentanecarboxylic acid, Ac3 = 1-amino- 1-cyclopropanecarboxylic acid, Thiopro =
L-thiazolidine-4-carboxylic acid, Iva = 2-amino-2-methylbutanoic acid, Tyr (m
al) = Tyr (O- (CH (COOH) ₂ ), Tyr (Fmal) = Tyr (O- (CF (COOH) ₂ ), 2-Oxn = (8-hydroxy-quinolin-2-yl) methylglycine, DehydrPro = 3,4-dehydroproline, dehydroVal = dehydrovaline, Aib = a-aminoisobutyric acid, Pra = glycine propargylate, Phg = phenylglycine, SmPhg = Phg (4-CH ₂ -SO ₃ H).

In addition, Xaa = amino acid, hXaa = homoamino acid, (NMe) Xaa = amino acid methylated amino acid, MeXaa = a carbon or amino acid methylated at the indicated position, Xaa (R) = R group Amino acids with functionalized side chains, and D, L-Xaa = D
Represents a mixture of-and L-isomers (50/50 or proportions shown in parentheses below).

During the synthetic process, the amino group of amino acids is usually protected by the Fmoc group, but rarely Al
It may be protected with a loc or Boc group. The carboxylic acid moiety of the amino acid side chain is protected as a tert-butyl ester if that carboxylic acid moiety is required for the final product. The tert-butyl ester is hydrolyzed under the specific conditions used to cleave the peptidyl derivative from the polymer or resin.

Natural amino acids and their corresponding D-isomers were purchased from Nova Biochem or Bachem.

Other amino acids are Fmoc-Nal (1) -OH (Synthetec), Fmoc-Bta-OH (Peptec), Fmoc-Aad (tBu) -OH (Bachem), Fmoc-Smp-OH. (RSP Amino Acid Analogs), Fmoc- (Pmp (Et) ₂ ) -OH (Neosystem), Fmoc-Asu (tBu) -OH (Peninsula Lab), Fmoc-Hyp (tBu) -OH (Nova Biochem , Fmoc-Nle-OH (Nova Biochem), Fmoc-Nva-OH (Nova Biochem), Fmoc-hLeu-OH (Neosystem), Fmoc-Pip-OH (Bachem), Fmoc- (2-Me ) Pro-OH (Bachem), Fmoc-Isc-OH
(Neosystem), Fmoc-Oic-OH (Bachem), Fmoc-Ac6 -OH (Neosystem)
, Fmoc-Ac5-OH (Neosystem), Fmoc-Ac3-OH (Advanced Chemtech),
Fmoc-Thiapro-OH (Neosystem), Fmoc-Iva-OH (Across), Fmoc (Tyr (mal
(tBu) ₂ ) -OH (Bachem), Fmoc-DehydrPro-OH (Bachem), Fmoc-DehydroVal-OH
(Advanced Chem Tech), Fmoc-Aib-OH (Sen Chemicals), Fmoc-Pra-OH (Advanced Chemtech), Fmoc-Phg-OH (Nova Biochem), SmPhg (RSP Amino Acid Analogs), Fmoc -(3-Cl- Phe) -OH (Peptec), Fmoc- (2-Cl-
Phe) -OH (Peptec), Fmoc- (4-Cl- Phe) -OH (Bachem), Fmoc- (4-CONH _2-
Phe) -OH (RSP Amino Acid Analogs), Fmoc- (4-NH ₂ -Phe) -OH (Bachem), Fmoc- (3-NH ₂ -Phe) -OH (RSP Amino Acids Analogs) ,), Fmoc- (4-
NHAc-Phe) -OH (RSP Amino Acid Analogs), Fmoc- (4-COOtBu-Phe) -OH (
Bachem), Fmoc- (4-CF ₃ -Phe) -OH (Apollo), Fmoc- (3-NO ₂ -Phe) -OH (Peptec), Fmoc- (4-Ph-Phe) -OH ( Bachem), Fmoc-3-NH-pyridinone-1-yl-CH _2-
COOH (Neosystem), Fmoc-3-NH-caprolactam-1-CH ₂ -COOH (Neosystem), Fmoc-6-NH-5-oxo-perhydropyrido [2,1-b]-(1,3 ) -Thiazole-3-COOH
(Neosystem), Fmoc-azetidine-carboxylic acid (Neosystem) were purchased.

The following amino acids were prepared according to the following references: Fmoc- (F ₂ Pmp (Et) ₂ ) -OH
Synthesis of TR Burke Jr. et al., J. Org. Chem. 1993, 58, 1336 -1340, synthesis of Fmoc- (Tyr (Fmal (tBu) ₂ ) -OH) is TR Burke Jr. et al. ., J. Med. Chem.
1996, 39, 1021-1027, see TR for synthesis of Fmoc- (O-carboxymethyl) -Tyr-OH.
See Burke Jr. et al., Tetrahedron 1998, 54,9981-9994, Fmoc- (2-Oxn) -O.
For the synthesis of H, see GK Walkup et al., J. Org. Chem. 1998, 63, 6727-6731.
Further, here, the following abbreviations, EDCI = 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, HATU = O- (7-azabenzotriazol-1-yl) -1,1,3,3
Tetramethyluronium hexafluorophosphate, HBTU = 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate, HOAt = 1-hydroxy-7-azabenzotriazole , TBTU = 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium tetrafluoroborate, Fmoc = fluorenylmethoxycarbonyl, Rink amide AM = 4- (2 ', 4'-Dimethoxyphenyl-Fmoc-aminomethyl) -phenoxyacetamide-norleucylaminomethyl is used. Rink amide AM is commercially available from Nova Biochem. 2-Chlorotrityl chloride chloride was purchased from Nova Biochem.

Conditions for HPLC analysis Method AE column: Vydac 300 Å, C18, flow rate: 0.8 mL / min (l = 214 nm) Solvent: solvent A = 0.1% trifluoroacetic acid / water, solvent B = 0.1% trifluoroacetic acid / Acetonitrile gradient: Method A: Solvent B from 30 to 90% for 30 minutes Method B: Solvent B from 5 to 45% for 40 minutes Method C: Solvent B from 5 to 65% for 60 minutes Method D: Solvent B from 15 60% for 30 minutes Method E: Solvent B 40 to 90% for 25 minutes

Method FQ column: C18 (4 mm x 300 mm), flow rate: 0.5 mL / min for CH3CN / H2O (0.1% TFA) Solvent: solvent A = trifluoroacetic acid / water, solvent B = 0.1% trifluoroacetic acid / acetonitrile. Gradient: Method F: Solvent B at 10% for 5 minutes, 10% to 90% for 20 minutes Method G: Solvent B at 50% for 5 minutes, Solvent B at 50% to 90% for 20 minutes Method H: Solvent B at 10% for 5 minutes, solvent B at 10% to 100% for 12.5 minutes Method I: solvent B at 20% for 5 minutes, solvent B at 20% to 70% for 20 minutes Method J: solvent B at 25% 5 minutes, solvent B at 25% to 65% for 13 minutes, 65% to 100% for 4 minutes Method K: Solvent B at 15% for 5 minutes, Solvent B at 15% to 80% for 20 minutes Method L: Solvent B 40% to 100% for 20 minutes Method M: Solvent B 10% to 5 minutes, Solvent B 10% to 90% for 25 minutes Method N: Solvent B 30% to 5 minutes, solvent 30% From 70% to 12.5 minutes, 70% to 100% for 5 minutes Method O: Solvent B at 50% to 90% for 20 minutes Method P: Solvent B at 25% for 7 minutes, Solvent B at 25% to 100% 2 0 minutes Method Q: Solvent B 25% for 5 minutes, Solvent B 25% to 100% for 15 minutes

Method R: Column: Vydac (2.1 mm x 150 mm) 300 Å C18, flow rate: 0
.2 mL / min (l = 214 nm) solvent: solvent A = 0.02% trifluoroacetic acid / 0.08% formic acid / water, solvent B = 0.02% trifluoroacetic acid / 0.08% formic acid / acetonitrile Method R: solvent B from 5% to 98 % For 10 minutes

Synthesis of 2-CH ₃ O-naphthyl-1-CO-Smp-Glu-Glu-Nal-Glu-NH ₂ , cdc1249

[0223]

[Chemical 10]

Construction of peptide sequences is C-terminally linked to the corresponding Fmoc amino acid to a free amino group on the resin, then the Fmoc protecting group is removed to release the amino group for subsequent conjugation. By doing so, it is sequentially performed. The final capping is 2-methoxy-
Performed with naphthyl-carboxylic acid.

Rink amide AM resin (223 mg, 0.1 mmol) in dimethylformamide (2 x 25 mL)
After washing with, the Fmoc protecting group was removed with 20% piperidine in dimethylformamide (2 x 25 mL). All amino acids (0.15 mmol, 1.5 eq) were coupled in dimethylformamide solution using the coupling reagent TBTU (48 mg, 0.15 mmol, 1.5 eq) and the base N, N-diisopropylethylamine (38 mg, 0.3 mmol, 3 eq). FMOc-Phe (4-CH ₂ SO ₃ H) -OH and 2-methoxy-naphth-1-ylcarboxylic acid were added to HATU (57 mg, 0.15 mmol, 1.5 equivalents and 76 mg, 0.2 mmol, 2.0 equivalents, respectively). ) And N,
N-diisopropylethylamine (39 mg, 0.3 mmol, 3.0 eq, and 65 mg, 0.5
mmol, 5.0 eq) in dimethylformamide (25 mL) solution.
Fmoc deprotection was performed with 20% piperidine in dimethylformamide (2 x 25 mL). Completion of binding and deprotection reactions was assessed by Kaiser test (ninhydrin). After each step, add the resin to dimethylformamide (2 x 25 mL), dichloromethane.
(3 x 25 mL) and methanol (2 x 25 mL), and dried under reduced pressure. The final peptide product was dissociated from the resin with trifluoroacetic acid / water (95: 5) for 2 hours at room temperature with side chain deprotection at the same time. Trifluoroacetic acid was removed under reduced pressure, then the residue was dispersed with diethyl ether (30 mL). The solid obtained was dried under reduced pressure.

HPLC purification was carried out on Waters Deltapack C ₁₈ reverse phase silica gel 40 mm x 200 mm.
Performed using a x 300 Angstrom column.

[0227] HPLC conditions: Flow rate: 10 mL / min             Gradient: 57 minutes at 10% -30% B, then 200 minutes at 30% -90% B             A = 0.1% trifluoroacetic acid / water             B = 0.1% trifluoroacetic acid / acetonitrile

[0228] Yield: 25 mg (0.024 mmol) 2 -CH 3 O-1- naphthyl -1-CO-Smp-Glu- Glu-Nal-Glu-NH 2
Analytical data R _t 32.1 min (Method B) MS (ESI): MH ⁺ 1027 H ¹ NMR (d ₆ -DMSO, 400 MHz): d 4.83 (m, 1H), 4.69 (m, 1H), 4.43 (m, 1H ),
4.26 (m, 1H), 4.19 (m, 1H) (specific a-proton)

Synthesis of 2-EtO-1-naphthyl-1-CO-Smp-Glu-Glu-Bta-Glu-NH ₂ , cdc 1659

[0230]

[Chemical 18]

Rink amide AM resin (379 mg, 0.25 mmol) was added to dimethylformamide (2 x 25 mL).
Washed with Fmoc, then 20% piperidine in dimethylformamide (2 x 25 mL).
Deprotected. The 4th amino acid from the C terminus (1.0 mmol) was added to 0.45M HBTU dimethylformamide solution (2 g, 0.9 mmol) and 2M N, N-diisopropylethylamine 1-.
Methyl-2-pyrrolidinone (1.0 mL) solution was used to bind in 1-methyl-2-pyrrolidinone. Then Fmoc-Phe (4-CH ₂ SO ₃ H) -OH (180 mg, 0.375 mmol) was added to TBTU (120 mg, 0.375 mmol) and N, N-diisopropylethylamine (113 mg, in a dimethylformamide (25 mL) solution. 0.875 mmol) was used for coupling. At this point the resin was split and 0.1 mmol was added to HATU (76 mg, 0.2 mmol) and N, N-diisopropylethylamine (5
2-ethoxy-1-naphthoic acid (8 mg, 0.45 mmol) in dimethylformamide solution
43 mg, 0.2 mmol). Fmoc deprotection of each amino acid was performed with 20% piperidine in dimethylformamide (2 x 25 mL). Completion of binding and deprotection reactions was assessed by Kaiser test (ninhydrin). Add the resin to dimethylformamide (2 x
25 mL), dichloromethane (3 x 25 mL), and methanol (2 x 25 mL), and dried under reduced pressure. Trifluoroacetic acid / water (95: 5) at room temperature for about 2 hours,
The peptide was cleaved from the resin and the side chain was deprotected. Trifluoroacetic acid was removed under reduced pressure, then the residue was dispersed with Et ₂ O (30 mL). The solid obtained was dried under reduced pressure. HPLC purification is 40 mm on Waters Deltapack C ₁₈ reverse phase silica gel.
Performed using a x 200 mm x 300 angstrom column.

Yield: 22 mg (0.021 mmol) 2-EtO-1-naphthyl-1-CO-Smp-Glu-Glu-Bta-Glu-NH ₂
Analytical data R _t 34.3 min (Method B) MS (ESI): MH ⁺ 1047 H ¹ NMR (d ₆ -DMSO, 400MHz): d 4.87 (m, 1H), 4.70 (m, 1H), 4.41 (m, 1H ),
4.28 (m, 1H), 4.19 (m, 1H), (specific a-proton), 4.11 (q, 2H, CH ₂ ), 1.19 (t
, 3H, CH ₃ )

2-CH ₃ O-1-naphthyl-CO-Smp-Glu-Glu-Bta-Aad-NHtBu, cdc1671 synthesis

[0234]

[Chemical 11]

A. Mounting 2-Chlorotrityl Chloride Resin Fmoc-Aad (tBu) -OH (377 mg, 0.847 mmol) was dissolved in dichloromethane under a nitrogen atmosphere. 2-Chlorotrityl resin (1.5 g, 1.43 mmol) was added to this solution, followed by N, N-diisopropylethylamine (442 mg, 3.43 mmol). The suspension was stirred under nitrogen at room temperature for 6 hours. The completion of the reaction was confirmed by monitoring the amino acid consumption by thin layer chromatography. Filter the resin to dichloromethane / methanol / N, N-diisopropylethylamine (17: 2: 1, 3 x 25 mL), dichloromethane
It was washed with a mixture of (3 x 25 mL) and dimethylformamide (2 x 25 mL). The Fmoc protecting group was removed with 20% piperidine dimethylformamide solution (2 x 25 mL, monitored by Kaiser test). Add the resin to dimethylformamide (2 x 25 mL), dichloromethane (3
x 25 mL), and methanol (2 x 25 mL), and then dried under reduced pressure. Wearing: About 0.6 mmol / g.

B. Amino Acid Binding Trityl resin (417 mg, 0.25 mmol) equipped with Aad (tBu) was suspended in 1-methyl-2-pyrrolidinone (4 mL). Amino acids Fmoc-Bta-OH, Fmoc-Glu (tBu) -OH, and
Fmoc-Glu (tBu) -OH (1.0 mmol each) was added to 0.45 M HBTU dimethylformamide (2 g, 0
1.9 mmol) solution and 2M N, N-diisopropylethylamine in 1-methyl-2-pyrrolidinone (1.0 mL) solution for coupling. At this point the resin is split and the resin 176mg
(0.083 mmol), TBTU (40 mg, 0.125 mmol) and N, N-diisopropylethylamine (43 mg, 0.332 mmol) as a coupling reagent Fmoc-Phe (4-CH ₂ SO ₃ H) -OH (60 mg
, 0.125 mmol) was used for binding. The final capping of the peptide is HATU
(63 mg, 0.166 mmol) and N, N-diisopropylethylamine (48 mg, 0.374 mmol) with 2-methoxy-1-naphthoic acid (34 mg, 0.166 mmol). After each join step
Fmoc deprotection was performed with 20% piperidine dimethylformamide solution (2 x 25 mL).
Completion of binding and deprotection reactions was assessed by Kaiser test (ninhydrin). The resin was washed with dimethylformamide (2 x 25 mL), dichloromethane (3 x 25 mL), and methanol (2 x 25 mL). Dichloromethane / trifluoroacetic acid / acetic acid (8: 1: 1
, 10 mL) at room temperature for 1 hour to release the peptide from the resin. The side chain protected pentapeptide acid was collected as a white solid (52 mg).

C. C-Terminal Amide Synthesis Side chain protected pentapeptide acid 2-EtO-1-naphthoyl-Phe (4-CH ₂ SO ₃ H) -Glu (tB
u) -Glu (tBu) -Bta-Glu-OH (52 mg, 0.042 mmol) was dissolved in dichloromethane (3 mL), and then tert-butylamine (6.2 mg, 0.084 mmol), HOAt (5.7 mg, 0.042 mmol).
), EDCI (16 mg, 0.084 mmol), and N, N-diisopropylethylamine (22 mg
, 0.168 mmol) was added. The reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was diluted with dichloromethane (150 mL) and washed with 1: 1 water / brine (75 mL). The organic layer was dried over sodium sulfate, concentrated under reduced pressure and the residual solid dried under reduced pressure. The tert-butyl ester was deprotected with trifluoroacetic acid / water (95: 5) at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure and the residue was dispersed with diethyl ether (30 mL). The solid obtained was dried under reduced pressure. HPLC purification was performed on Waters Deltapack C ₁₈ reverse phase silica gel using a 40 mm x 200 mm x 300 Angstrom column.

[0238] HPLC conditions: Flow rate: 10 mL / min             Gradient: 20% -40% B 57 minutes, then 40% -100% B 257 minutes                   A = 0.1% trifluoroacetic acid / water                   B = 0.1% trifluoroacetic acid / acetonitrile

Yield: 13 mg (0.012 mmol) 2-CH ₃ O-1-naphthyl-CO-Smp-Glu-Glu-Bta-Aad-NHtBu Analytical data R _t 39 min (Method B) MS (ESI): MH ⁺ 1103 H ¹ NMR (d ₆ -DMSO, 400 MHz): d 4.82 (m, 1H), 4.71 (m, 1H), 4.45 (m, 1H),
4.30 (m, 1H), 4.17 (m, 1H) (specific a-proton), 1.24 (s, 9H, tBu)

[0240] Synthesis of 2-EtO-1-naphthyl-CO-Smp-Nva- (3-Me) Pro-Bta-Aad-NHtBu, cdc 1747

[0241]

[Chemical 19]

A) Synthesis of 6- (tert-butylamino) -5-(((9H-9-fluorenylmethoxy) carbonyl) amino) -6-oxohexanoate Fmoc-La-aminoadipic acid-d -tert.-Butyl ester (2 g, 4.55 mmol) was dissolved in dichloromethane (80 mL), then tert-butylamine (0.665 g, 9.1 mmol), 1-hydroxy-7-azabenzotriazole (0.619 g, 4.55 mmol). , 1- (3-Dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (1.74 g, 9.1 mmol), and diisopropylethylamine (2.05 g, 15.92 mmol) were added. This yellow solution was stirred at room temperature for 2
Stir for 1 hour. The reaction mixture was diluted with dichloromethane then washed with saturated aqueous sodium dicarboxylate solution, 5% aqueous citric acid solution and 1: 1 water / brine. The organic layer is dried over sodium sulfate and concentrated under reduced pressure to give tert-butyl 6- (tert-
2.07 g (4.19 mmol) of butylamino) -5-{[9H-9-fluorenylmethoxy) carbonyl] amino} -6-oxohexanoate was obtained.

MS (ESI): MH ⁺ = 495 R _t = 23.5 (Method E)

Tert-Butyl 6- (tert-butylamino) -5-{[9H-9-fluorenylmethoxy) carbonyl] amino} -6-oxohexanoate (2.07 g, 4.19 mmol) crude product Dissolved in the minimum amount of dichloromethane then diluted with 95% trifluoroacetic acid / water (30 mL). The reaction mixture was stirred at room temperature for 1.5 hours and then concentrated under reduced pressure. The residual oil was dispersed with diethyl ether and filtered. The diethyl ether filtrate was concentrated under reduced pressure to give 6- (tert-butylamino) -5-(((9H-9-fluorenylmethoxy) carbonyl).
2.48 g of amino) -6-oxohexanoate was obtained.

MS (ESI): MH ⁺ = 439 R _t = 14.6 (Method E)

B) Attachment of 5-amino-6- (tert-butylamino) -6-oxohexanoic acid to chlorotrityl resin chloride 6- (tert-butylamino) -5-(((9H-9-fur Olenylmethoxy) carbonyl) amino
) -6-Oxohexanoate (2.48 g, 5.66 mmol) was dissolved in dichloromethane (80 ml) under a nitrogen atmosphere, then chlorotrityl resin chloride (4.85 g, 7.23 mmol) and diisopropylethylamine (2.92 g, 22.64 mmol). ) Was added. The dark purple reaction mixture was stirred under a nitrogen atmosphere for 5 hours. The suspension was filtered and the resin was then washed 3 times with a mixture of dichloromethane / methanol / diisopropylethylamine (17: 2: 1), 3 times with dichloromethane and 2 times with dimethylformamide. The resin was suspended in dimethylformamide (25 mL), then treated with 20% piperidine in dimethylformamide for the first 5 minutes, the second 20 minutes, and then washed 5 times with dimethylformamide. Finally, the resin was washed 3 times with dichloromethane and 2 times with methanol, and dried under reduced pressure to obtain 5.21 g of mounted resin.

C) 2-EtO-1-naphthyl-CO-Smp-Nva- (3-Me) Pro-Bta-Aad-NHtBu Peptide sequence was constructed by starting the C-terminus with the corresponding Fmoc amino acid on the resin. Sequentially by conjugating to the free amino group of ## STR1 ## and then freeing the amino group by removing the Fmoc protecting group for subsequent conjugation. The final capping is 2-methoxy-
Performed with naphthyl-carboxylic acid.

Preloaded trityl resin (resin 187 mg, 0.15 mmol) was suspended in dimethylformamide (25 ml). 4 kinds of amino acids (Fmoc-Bta-OH, Fmoc
-(3-Me) Pro-OH, Fmoc-Nva-OH, Fmoc-Smp-OH) (0.225 mmol, 1.5 equiv.), Coupling reagent TBTU (72 mg, 0.225 mmol, 1.5 equiv.) And base N, N -Diisopropylethylamine (77 mg, 0.6 mmol, 4 eq) was coupled using a dimethylformamide solution, and the final 2-ethoxy-naphth-1-yl-carboxylic acid bond was HATU (11
4 mg, 0.3 mmol, 2 eq) and N, N-diisopropylethylamine (87 mg, 0.675
mmol, 4.5 equivalents) in dimethylformamide (25 mL). Fmoc deprotection was performed with 20% piperidine in dimethylformamide (2 x 25 mL). Completion of binding and deprotection reactions was assessed by Kaiser test (ninhydrin). After each step, the resin was added to dimethylformamide (2 x 25 mL), dichloromethane (3 x 2 mL).
5 mL) and methanol (2 x 25 mL), and dried under reduced pressure. The final peptide product was cleaved from the resin with dichloromethane / trifluoroethanol / acetic acid (8: 1: 1, 10 mL) for 45 minutes at room temperature. The suspension was filtered and washed with dichloromethane. The filtrate was concentrated under reduced pressure and the residue was dispersed with diethyl ether. The obtained solid was dried under reduced pressure to give 2-EtO-naphth-1-yl-as a pale yellow solid.
CO-Smp-Nva- (3-Me) Pro-Bta-Aad-NHtBu was obtained (105 mg, 66%).

MS (ESI): MH ⁺ = 1069 R _t = 15.8 (Method A)

The compounds shown in Table 1-6 were obtained in a manner similar to the above four pentapeptides. The Cdc number column shows the reference number of the compound. Columns R ₁ , A1, A2, A3, A4, A5, U, R ₂ are the residues of the peptidyl compound as defined in Formula 1 above. The table shows mass spectral data (MH +) and analytical data for compounds (retention time and HPLC used).
Method) is also described.

[0251]

[Table 1]

[0252]

[Table 2]

[0253]

[Table 3]

[0254]

[Table 4]

[0255]

[Table 5]

[0256]

[Table 6]

[0257]

[Table 7]

Synthesis of 2-EtO-1-naphthyl-CO-Smp-Nva-Pro-Bta-NH-CH _2- (3-HOOC-CH ₂ ) -Ph, cdc2276

[0259]

[Chemical 20]

The tetrapeptide acid sequence was constructed as described above (see Example 3), with the corresponding Fmoc amino acid (Fmoc-Bta-OH) linked to the chlorotrityl resin chloride, with the C-terminus first, and then the next linkage. Was done sequentially by removing the Fmoc protecting group and liberating the amino group. Final capping is done with 2-ethoxy-naphthyl-carboxylic acid.
The tetrapeptide is cleaved from the resin in a manner similar to that described in Example 3.

A) 2-EtO-1-naphthyl-CO-Smp-Nva-Pro-Bta-NH-CH _2- (3-MeOOC-CH ₂ ) -Ph Tetrapeptide acid (50 mg, 0.058 mmol) in dichloromethane (5 mL), then methyl 2- [3- (aminomethyl) phenyl] acetic acid hydrochloride (23 mg, 0.116 mmol), 1-
Hydroxy-7-azabenzotriazole (8 mg, 0.058 mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (22 mg, 0.116 mmol), and diisopropylethylamine (49 mg, 0.377 mmol) added. The reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was diluted with dichloromethane and washed with 5% aqueous citric acid solution (1x). The organic layer was dried over sodium sulfate and concentrated under reduced pressure. The residue was redissolved in dichloromethane. Heptane was then added until precipitation occurred. The mixture was concentrated under reduced pressure and the residual solid was dried under reduced pressure to give the white solid product 2-EtO-1-naphthyl-CO-Smp-Nva-Pro-Bta-NH-CH _2- (3- MeOOC-CH ₂ ) -Ph)
60 mg was obtained.

MS (ESI): MH ⁺ = 1018.0 R _t = 16.49 (Method A)

A) Crude product of 2-EtO-1-naphthyl-CO-Smp-Nva-Pro-Bta-NH-CH _2- (3-HOOC-CH ₂ ) -Ph tetrapeptide ester (60 mg, 0.06 mmol ) To tetrahydrofuran (5
mL) and water (1 mL), and then 1N aqueous lithium hydroxide solution (150 ml, 0.15 mmol) was added. The reaction mixture was stirred at room temperature for 2 days. The reaction was incomplete as determined by HPLC, so more 1N aqueous lithium hydroxide solution (60 ml, 0.06 mmol) was added at room temperature and the reaction mixture was heated to 40 ° C. for 3 hours. The reaction mixture was cooled to 0 ° C. and quenched with 1N hydrochloric acid (240 ml, 4 eq). The mixture was concentrated under reduced pressure. The residue was redissolved in the minimum amount of acetonitrile / water and freeze dried to give 2-EtO-1-naphthyl-CO-Smp-Nva-Pro-Bta-NH-CH _2- (3-HOOC-CH ₂ ) -Ph 50 mg was obtained as a mixture of diastereomers (ratio 85: 6).

MS (ESI): MH ⁺ = 1004 R _t = 14.51, 14.98 (Method A)

[0265] The following tetrapeptide was prepared by the method described above.

[0266]

[Table 8]

Example 2 Protein Purification GST-Tagged Proteins (DN1A, DN1B, DN1C) Expression cloning of an open reading frame for tagging and expressing GST in the catalytic domains of Cdc25A, Cdc25B, and Cdc25C was performed using EcoRI and HinDIII.
The site was used to clone the appropriate fragment into PGX-2T or PGEX-KT (Pharmacia) (see Table 9). The obtained plasmid is BL-2
1 (DE3) was transformed, and the protein was expressed in a large amount by inducing mid-log cells with 0.5 mM IPTG at 25 ° C. for 3 hours.

Purification of Cdc25 catalytic domain from GST-Cdc25 Cell pellets obtained from approximately 4 liters of E. coli concentrated fermentations were thawed. The cells were mixed with 600 ml phosphate buffered saline (PBS), 1/1000 pepstatin A (10 mg / ml, final concentration 10 u).
g / ml) and resuspended in 10 mM dithiothreitol and protease inhibitor cocktail (complete tablet Boehringer Mannheim Catalog No. 1697489).
The cells were lysed at 15,000 psi through a microfluidizer via two routes. The obtained suspension was 1 at 12,000 rpm with a GSA rotor (Beckman).
Centrifuge for hours.

The supernatant was batched and shaken gently at 4 ° C. for 1 hour while GSH Sepharose 4-
B (Amersham Pharmacia Biotech) 250 ml was bound. The supernatant was gently removed, resuspended with GSH Sepharose 4-B and loaded onto an xk50 column at a rate of 20 ml / min. Load the column with 5-10 volumes of 50 mM Tris pH8.0, 0.500 m
It was washed with M NaCl, 1 mM EDTA, 1 mM DTT. The column was then washed with 5-10 volumes of column 50 mM tris, pH 8.0, 1 mM EDTA, 1 mM DTT at a rate of 5 ml / min.
The column was then loaded with 50 mM tris pH 8.0, 25 mM reduced GSH, 1 mM EDTA, 1 mM DTT.
At a rate of 1.5 ml / min. The eluent was collected in 4 mL fractions.

The fraction was then subjected to size exclusion column chromatography using a S300 Sephacryl xk50 / 100 column, eluting at 4 ml / min, 50 mM tris pH 8.0, 1
Equilibration was carried out with 50 mM NaCl, 1 mM EDTA, 5 mM DTT. The eluate was collected as 10 mL fractions. Both aggregate and dimer peaks in the exclusion volume of the column were eluted from GST-CDC25. The fraction corresponding to the dimer peak was saved.

The dimeric GST-CDC25 was bound to fresh GSH Sepharose beads at a rate of 4 mg fusion protein per ml GSH beads. Load the beads with 10 volumes of 25 mM tris on the column.
The cells were washed with pH 8.0, 150 mM NaCl, 2.5 mM CaCl2, 1 mM DTT, 100 uM EDTA.
The beads were resuspended in 2 volumes of buffer and then 5 units of thrombin per mg of fusion protein (Calbiochem Catalog No. 604980 specific activity 1900 units / mg).
), And allowed to digest at room temperature for 90 minutes with gentle rocking.

The beads were filtered using a 0.45 mm cellulose acetate bottle top filtration system (Corning Co.) to remove the supernatant, and the beads were washed with 1 volume of buffer.
The wash solution was added to the stock solution. Thrombin was removed using ATIII agarose beads (Sigma Catalog No. A-8293) at a ratio of 1 mL of beads to 100 ug of added thrombin. The solution was incubated at 4 ° C. for 1 hour with gentle rocking, then the beads were removed using a 0.45 mm cellulose acetate bottle top filtration system (Corning). Next, 10 mM EDTA and 0.5 mM AEBSF (Calbiochem catalog number 101500) were added to inactivate all remaining thrombin. Next, the solution was sent to Centriprep 10,000 Dalton molecular weight cut-off device (Millipore).
Concentrated to 6 mL with and filtered through a 0.22 mm filter.

The concentrated and filtered solution was treated with 50 mM NaPi pH 6.75, 100 mM NaCl, at a rate of 2 ml / min.
It was injected onto a Superdex 75 xk26 / 100 column equilibrated with 1 mM DTT, 1 mM EDTA, and the eluate was collected as a 2.5 mL fraction. Fractions containing the Cdc25 catalytic domain were pooled and concentrated to 20 mg / ml with BSA. EDTA at a final concentration of 5
Similarly, DTT was added to 10 mM, AEBSF was added to 0.5 mM, and sodium azide was added to 0.02%.

Native protein (DN5A, DN8A, DN8A-c17, DN5B, DN8B, DN8B-c17, DN8B-c18, D
N9C) Expression cloning of an open reading frame for the native expression of the catalytic domains of Cdc25A, Cdc25B, and Cdc25C, by incorporating the NcoI at the start codon and the HinDIII site following the stop codon into the pET-3d vector (Novagen).
Was cloned with the appropriate fragment (see Table 9). The obtained plasmid was transformed into BL-21 (DE3), and its protein was digested with 0.5 mM IPTG at 25 ° C.
It was made to express in large quantities by induction of mid-log cells for 3 hours. All steps in the purification were performed at 4 ° C. and the assay was performed using pNPP as substrate after activation of phosphatase. In a typical preparation, 33 g of frozen cell pellet is added to 150 mL of buffer A (
3 mM potassium phosphate (pH 7.4), 75 mM NaCl, 1 mM EDTA, 1 mM DTT, and a protease inhibitor cocktail (0.001 mg / ml aprotinin, 0.001 mg / ml leupeptin, 0.01 mg / ml soybean trypsin inhibitor, 0.01 mg / ml L-1-chloro-3- (4-tosylamide) -7-amino-2-heptanone hydrochloride and 0.01 mg / ml L-1-chloro-3- (4-tosylamide) -4-phenyl -2-butanone)). After centrifugation at 18K for 30 minutes, the clear lysate was bound to 15 mL SP-Sepharose equilibrated with buffer A. Cdc25 protein was eluted with buffer A containing 150 mM NaCl, or with a gradient of NaCl concentration up to 250 mM in buffer A, and phosphatase reaction buffer (50 mM Tris-HCl (pH 8.0), 50 mM NaCl, 1 mM EDTA, 1 mM DTT) S-200
Purified by chromatography. Protein yield is 1 per liter of cell culture
It was within the range of -25 mg.

[0275]

[Table 9]

Example 3 Crystallization of Polypeptide and Determination of Crystal Structure Crystallization of Cdc25A (DN1A) Frozen Cdc25A (25 mM Tris.HCl, pH 7.5, 100 mM NaCl, 10 mM DTT, 5 mM ED
Thaw the DN1A preparation (25 mg / ml) in TA, 0.5 mM AEBSF 25 mL, and add 1 mL DTT (100 m
M), 1 mL Na ₂ WO ₄ (100 mM), and 23 mL H ₂ O. This protein solution (1 mL)
15% (w / v) polyethylene glycol (PEG) 4000, 100 mM sodium citrate pH 5.
It was mixed with 1 mL of a reservoir solution containing 6 and attached to the underside of a silicone treated cover glass at 4 ° C. and suspended above the reservoir solution. Elongated pyramidal crystals formed in one day. Crystals also grew under conditions such as varying the amount of PEG4000, the presence of 0-400 mM ammonium acetate, and varying pH values from 4.8 to 5.6.

Cryoprotection of Cdc25A (DN1A) Crystals Cdc25A (DN1A) crystals (Crystal 1) grown as described above were transformed into 21-24% (w / v) PEG 4
000, 100 mM sodium citrate, pH 5.6, 2 mM Na ₂ WO ₄ , and 0, ₅ , 10, 15
, And 20% (v / v) glycerol sequentially in a cryoprotective buffer. The crystals were first soaked in 0% glycerol buffer for 2 minutes and then continuously soaked in 5, 10, 15 and 20% glycerol buffer for 5 minutes each. The crystals were lifted with a ring of fibers, placed in liquid nitrogen and quenched. The crystals were stored in a liquid nitrogen freezer.

Collection of X-Ray Diffraction Data for Cdc25A (DN1A) Crystals Grown from PEG X-ray diffraction data for Crystal 1 was taken from a Siemens SRA anode rotary generator (50 kV with a MAR Research image plate detector). , 108 mA, 40% bias, Cu K _a
The lines were collected by the spinning method using graphite (monochromized). Cdc25A (DN1A)
The crystals were maintained at 100K with an Oxford Cryosystems Cliostream chiller during data collection. For each data frame (225 frames in total),
The crystals were rotated by 0.4 ° C. After that, the crystal was repositioned (about 60
(° C rotation) and an additional 225 data frames were collected. This data is used in the CCP4 program suite (Collaborative Computational Project No.4,
1994). REFIX (Kabsch, 1993) and IDXREF (joint calculation program No.4
, 1994) to determine the crystallographic orientation, and then accumulated the data with MOSFLM (Leslie, 1992) (space group P4 ₁ or P4 ₃ , a = 43.79 Å, c = 117.37 Å) and scaled with SCALA (Evans, 1997). Then, merging was performed, and the amplitude of the structure factor was converted by TRUNCATE (French & Wilson, 1978) after placing on absolute coordinates. free
To calculate the R factor (R _free ), 5% of the eigenreflections were randomly assigned to the free set and the working set for calculating the R factor (R) was constructed with the remaining 95% of the reflections. This data is summarized in Table 10.

Structural Comparison of Cdc25A (DN1A) in Crystals Grown from Ammonium Sulfate or PEG Diffraction data for Crystal 1 described above was displayed in the space group P4 ₁ (or P4 ₃ ) of the tetragonal unit cell. In the case of space groups P4 ₁ and P4 ₃ , the direction of the polar c-axis cannot be determined without reference to the molecular model of (a part of) the contents of the unit cell. Therefore, first
The data displayed as "UP" is converted into h´ = -k, k´ = -h, l´ = -l and
Displayed again in "DOWN". Calculation of structure factors in space group P4 ₁ (Joint calculation program (Collaborative Computational Project) No.4, 1994 ) , the grown from ammonium sulfate as described below, to form a tetragonal unit cell of similar size Cdc2
Partially refined structure coordinates determined from the 5A (DN1A) crystal (FIGS. 16A to 16I) were used. The structure factor calculated in this way was scaled to the data displayed in UP and the data displayed in DOWM. The scaling of the data displayed in DOWN was significantly better than the data displayed in UP (R _free was 39.1 vs. 52.4%, respectively), confirming that DOWN is the correct representation of the data. As a result of examining 2F _o -F _c and F _o -F _c electron density maps in which Sigma A is weighted (Joint calculation program No. 4, 1994), the Cdc25A (DN1A) molecule in the crystal grown from ammonium sulfate was examined. In partially refined structural coordinates,
It was revealed that most of the Cdc25A (DN1A) structures in crystals grown from polyethylene glycol (PEG) could be explained.

Growth of Cdc25A (N1A) Crystals from Ammonium Sulfate Cdc25A (N1A) is 1.9-2.3 M (NH ₄ ) ₂ SO ₄ , 50 mM sodium phosphate at 4 ° C.
Crystallized from 2 mM sodium tungstate, pH 6.5-7.0. Over a period of two weeks, a long square pyramidal crystal, which is often slightly tapered in the longitudinal direction and often has sharp apexes, is grown. The unit cell size of the crystal is a = b = 44.17 angstrom, c = 118.65 angstrom, and belongs to the tetragonal space group P4 (1). One molecule of protein was a crystallographic asymmetric unit. Three kinds of heavy atom derivatives were prepared by contacting (1) KAu (CN) ₂ , (2) K ₂ PtCl ₄ , and (3) thiomersal with these crystals, and electron density that can be interpreted using them. The phase required to obtain the figure was obtained. The phase of heavy atoms was improved by solvent smoothing. Diffraction data up to a resolution of 2.1 Å (X-Plor) was used to refine the atomic structure. The R factor was 26% and R (free) was 31%. The atomic coordinates obtained are shown in FIGS. 16A to 16I.

Pre-Refinement of PEG Cdc25A (DN1A) Crystal Structure (Crystal 1) Partially refined structural coordinates derived from Cdc25A (DN1A) crystal grown from ammonium sulphate, relative to the crystal data shown in DOWN above. X-PLOR program (Bru
nger, 1992). Rigid body refinement, Powell minimization, slow cool simulated annealing molecular dynamics, and temperature factor refinement results for all / F /> 2.0s _F reflections in the 20 to 1.8 Angstrom resolution range. , R was 31.7% (R _free 36.3%). 2F _o -F _c with weighting Sigma A and
The electron density map of F _o -F _c revealed that the active site loop (residues 431-434) was severely impaired, and no ligand (ie tungstate) was bound to the active site. . Residues 493-523 at the C-terminal of the protein were not identified in the electron density map and were not included in this structural coordinate. This data is summarized in Table 10.

Immersion of Cdc25A (DN1A) crystals in cdc1316 Cdc25A (DN1A) crystals grown as described above were treated with 24% PEG 4000, 5 m at 4 ° C.
Transferred to 100 mL of a solution of M sodium citrate, pH 5.6. After soaking for 2 minutes, the crystals were transferred to a fresh solution additionally containing 2 mM cdc1316. After 23 hours, the crystals were crystallized from 24% (w / v) PEG 4000, 10 mM sodium citrate, pH 5.6, 2 mM cdc1316.
, And 5, 10, and 20% (v / v) glycerol (5 minutes each) were sequentially transferred to a cryoprotection buffer. After soaking in 20% glycerol buffer for an additional 5 minutes, the crystals were lifted with a ring of fibers and placed in liquid nitrogen to quench.

Collection of X-Ray Diffraction Data from cdc1316 Soaked Cdc25A (DN1A) Crystals A total of 235 data frames (0.4 ° each) were collected from the cdc1316 soaked Cdc25A (DN1A) crystals (Crystal 2). The crystal was maintained at 100K during data collection. Diffraction data were processed and space group P4 ₁ , a = 4 as described above for space group crystal 1.
3.69 Angstrom, c = 117.27 Converted to the structure factor amplitude at Angstrom. Eigenreflections were assigned to the same "free" and "working" sets as used for crystal 1. This data is summarized in Table 10. further,
Additional data for this crystal was collected on a US Synchrotron Light Source (NSLS, beamline X25, l = 1.100 Å, Brandeis B4 CCD detector). The crystals were maintained at 100K with an Oxford Cryosystems cryostream cooler during data collection. Diffraction data was processed and converted to structure factor amplitudes as described above. Only a small portion of the unique data was due to instrument limitations. However, the data show that the X-ray diffraction resolution of this crystal is up to 1.35 Å.
This additional data is also summarized in Table 10.

Improved Refinement of PEG Cdc25A (DN1A) Crystal Structure The partially refined structural coordinates of Crystal 1 were further refined against the diffraction data collected from Cdc25A (DN1A) Crystal 2 using X-PLOR. . The refinement was alternated with the manual reconstruction of structural coordinates (“model”) using the molecular graphics program o (Jones et al., 1991). Rigid body, Powell minimization, slow cool simulated annealing molecular dynamics, and refinement of individual temperature factors resulted in / F /> 1.5s _F reflections in the 20 to 1.8 Å resolution range for all reflections. R is 29.9% (
R _free 32.6%). The reconstruction of this model was done while checking the simulated annealing omit map. After several more rebuilds and refinements, R was 27.0% (R _free 28.9%; / F />
1.5s _F , 20-1.80 Angstrom). Furthermore, REFMAC (Murshudov et al., 1997)
As a result of refinement with, R became 23.0% (R _free 24.3%; / F /> 0.0 s _F , 20
-1.80 Angstrom). This model (Refmac1) contains Cdc25A residues 335-413, 419-
It contains 431 and 435-492 and 74 water molecules. Active site loop (residues 432-434
There was no uninterpretable electron density for the rest of the) or for ligands such as tungstate or cdc1316. Also, residues 493-523 at the C-terminal of the protein were not identified in the electron density map and were not included in the structural coordinates. For residues 414-418 there was a faint but easily interpretable density. This data is summarized in Table 10.

Crystallization of Cdc25A (DN8A) Frozen Cdc25A (50 mM Tris.HCl, pH 7.5, 50 mM NaCl, 1 mM DTT, 1 mM EDTA 150
Thaw DN8A product (17 mg / ml) in mL, DTT (1 M) 1.5 mL, NaN ₃ (1.5 M) 0.
It was mixed with 3 mL and 1.5 mL of Na ₂ WO ₄ (100 mM). 20% of this protein solution (1 mL)
(w / v) PEG 3000, 600 mM Li ₂ SO ₄ , 100 mM sodium citrate, mixed with 1 mL of reservoir solution containing pH 5.6, attached to the lower surface of the cover glass treated with silicone at 4 ° C, and put on the reservoir solution. I hung it up. The pyramidal crystals were formed in about two weeks. Crystals change the amount of PEG 3000 or Li ₂ SO ₄ , other salts (instead of Li ₂ SO ₄ (
It was also grown under conditions such as the presence of ammonium acetate or ammonium sulphate), complete absence of PEG 3000, and varying pH values between 5.6 and 5.8.

Cryoprotection of Cdc25A (DN8A) Crystals Frozen Cdc25A (DN8A) crystals (Crystal 3) grown as described above were 20% (w / v) PEG 30.
Cryoprotection buffer containing 00, 100 mM sodium citrate, pH 5.6, and 0 and 5% (v / v) glycerol, and 10% glycerol and 25% or 30%
Sequential transfer to the same buffer containing PEG 3000. The crystals were mixed with 0% glycerol, 5% glycerol, 25% PEG 3000/10% glycerol, and 30% PEG 3000/1.
It was continuously immersed in 0% glycerol buffer for 5 minutes each. The crystals were lifted with a ring of fibers, placed in liquid nitrogen and quenched. The crystals were stored in a liquid nitrogen freezer.

Collection of X-Ray Diffraction Data for Cdc25A (DN8A) Crystals A total of 225 data frames (0.4 ° each) were collected for Crystal 3 described above. The crystal was maintained at 100K during data collection. Processing the diffraction data, space group P4 ₁ as described above, a = 43.94 Å, was converted to the structure factor amplitude at c = 117.39 angstroms. Eigenreflections were assigned to the same "free" and "working" sets as used for crystal 1.

Annealing of Cdc25A (DN8A) Crystals Crystal 3 was 30% (w / v) PEG 3000, at 4 ° C. after collecting X-ray data.
Thaw in a cryoprotective buffer containing 100 mM sodium citrate, pH 5.6, and 10% (v / v) glycerol. After 15 minutes, the crystals were lifted with a ring of fibers, placed in liquid nitrogen and quenched.

Collection of X-Ray Diffraction Data for Annealed Cdc25A (DN8A) Crystals A total of 180 data frames (0.5 ° each) were collected for Crystal 3 using the method described above. The crystal was maintained at 100K during data collection. Processing the diffraction data, space group P4 ₁ as described above, a = 43.92 Å, was converted to the structure factor amplitude at c = 117.38 angstroms. The resolution of the data after annealing was higher than that of the data before annealing (2.15 vs 2.40 angstroms). Crystals after annealing also had low mosaic spread (0.3 ° vs. 0.6 °)
. The intrinsic reflections were assigned to the same "free" and "working" sets used for Crystal 1. The data for this final crystal 3 are summarized in Table 1.

Refinement of Cdc25A (DN8A) Crystal Structure Refinement of Cdc25A (DN8A) crystal structure (Crystal 3) was carried out by the above-mentioned Cdc25A (D
N1A) starting from the intermediate "Powell 8" structural coordinates. Rigidity, Powell minimization, and refinement of global and individual temperature factors resulted in R of 27.1% (R _{fre e for} all reflections of / F /> 2.0s _F at resolutions in the range 20 to 2.15 Angstroms. 29.2%). From the 2F _o -F _c and F _o -F _c electron density maps with weights added to Sigma A,
The active site loop (residues 431-434) was partially ordered and distinct from the conformations found in other phosphatases. When further refined with REFMAC, R became 22.8% (R _free 27.1%, / F /> 0.0s _F , 20-2.15 angstrom).
This model (Refmac1) contains Cdc25A residues 335-413 and 419-492, and 67 water molecules. There were no uninterpretable electron densities for the tungstate ligand. For residues 414-418 there was a weak but easily interpretable electron density. Active site loop residues 431-433 constructed alanine. Tungstate, 335
Earlier residues, and residues 493-523, were not identified in the electron density and were not included in this structural coordinate. This data is summarized in Table 10.

Immersion of Cdc25A (DN8A) in Na ₂ WO ₄ and Collection of X-Ray Diffraction Data Crystal 3 was placed in a buffer supplemented with 10 mM Na ₂ WO ₄ and annealed for 3 days at 4 ° C. The crystals were placed in liquid nitrogen and quenched. A total of 40 data frames (1.0 ° each) were collected as described above for that crystal (Crystal 4). The crystal was maintained at 100K during data collection. The diffraction data is processed and the space group P4 ₁ , a =
43.98 angstroms, c = 117.72 angstroms converted to structure factor amplitudes. The data after immersion was much weaker than that of crystal 3 (3.00 vs. 2.
15 Angstrom). The intrinsic reflections were assigned to the same "free" and "working" sets used for Crystal 1. The data for this final crystal 3 is summarized in Table 10.

Refinement of Cdc25A (DN8A) Crystal Structure Soaked in Na ₂ WO ₄ Refinement of Cdc25A (DN8A) Crystal Structure Soaked in Na ₂ WO ₄ (Crystal 4) Start with coordinates "Powell 8". As a result of rigid body, Powell minimization, and temperature factor refinement, / F /> 2 in the 20 to 300 angstrom range.
R was 22.7% (Rfree 25.7%) for all 0s _F reflections. The 2F0-Fc and F0-Fc electron density maps with weighting on Sigma A revealed the absence of tungstate at the active site. This model was not refined any further. This data is summarized in Table 10.

Crystallization of Cdc25B (DN1B) .cdc1249 inhibitor complex Frozen Cdc25B (10 mM sodium phosphate, pH 6.7, 50 mM NaCl, 10 mM DTT, 5 mM
Thaw 17.5 mg / ml DN1B preparation in 212 mL EDTA and 0.4 mL NaN ₃ (1.5 M)
And 7.4 mL cdc1249 (25 mM HEPES sodium, 30 mM in pH 7.5).
This protein solution was aged at 1 ° C for 4 days. The slightly formed precipitate was removed by centrifugation. The supernatant (1 mL) was mixed with 1 mL of a reservoir solution containing 4.4 M NaCl, 50 mM sodium HEPES, pH 7.0, attached to the underside of a silicone treated cover glass at 4 ° C. and suspended above the reservoir solution. Block-like crystals formed within 2-7 days. Crystals also grew under conditions such as varying the amount of NaCl and varying pH values from 5.75 to 8.0.

Crystallization of Cdc25B (DN8B) .cdc1249 inhibitor complex Frozen Cdc25B (50 mM Tris.HCl, pH 7.5, 50 mM NaCl, 1 mM DTT, 1 mM EDTA 2
27.5 mg / ml of DN8B product was thawed in 5 mLn, 0.25 mL of NaN ₃ (0.3 M), DTT (1 M
) 0.2 mL and cdc1249 0.85 mL (30 mM in 25 mM HEPES sodium, pH 7.5). This protein solution (2 mL) was mixed with 2 mL of a reservoir solution containing 82.5% saturated NaCl (~ 4.5 M), 50 mM MES sodium, pH 6.5, and placed on the underside of a silicone-coated cover glass at 4 ° C. I hung it up. Block-shaped crystals were formed in 1 day.

Cdc25B (DN8B-c17) .cdc1249, Cdc25B (DN8B-c18) .cdc1249, Cdc25B (DN1B) .cdc16
71, Cdc25B (DN1B) .cdc1885, Cdc25B (DN8B) .cdc1659, and Cdc25B (DN8B) .cdc1671
The complex was also crystallized using this general procedure.

Cryoprotection of Cdc25B (DN1B), Cdc25B (DN8B), Cdc25B (DN8B-c17), and Cdc25B (DN8B-c18) inhibitor complex crystals Cdc25B inhibitor complex crystals grown as described above were 4.5 M NaCl, 50 mM M
Sodium ES, pH 6.5, or 50 mM HEPES Sodium, pH 7.0, and 0, 5, 1
Sequential transfer to 0, and 16.5% (v / v) glycerol. The crystals were soaked in 0 and 5% glycerol buffer for 5 minutes consecutively and then in 10 and 16.5% glycerol buffer (also containing 0.5-2.0 mM of the appropriate inhibitor, respectively).
The crystals were picked up with a ring of fibers, placed in liquid nitrogen and quenched. The crystals were stored in a liquid nitrogen freezer.

Collection of X-Ray Diffraction Data for Cdc25B (DN1B) .cdc1249 Inhibitor Complex Crystals A total of 359 data frames (each 0.25 °) in the Cdc25B (DN1B) .cdc1249 inhibitor complex crystals (Crystal 5) were described above. It was collected by using the above device. The crystal was maintained at 100K during data collection. Diffraction data processed, space group P4 ₁ 2 ₁ 2 as described above
Or P4 ₃ 2 ₁ 2, a = 70.15 angstrom, c = 130.35 angstrom was converted to the structure factor amplitude. The free R-factor (Rfree) was calculated by randomly assigning 5% of the eigenreflection to the freeset, and the R-factor (R) was calculated with the remaining 95% of the reflex constituting the working set. This data is summarized in Table 10.

Molecular Substitution Solution of Cdc25B (DN1B) .cdc1249 Inhibitor Complex Crystal Structure For the above Cdc25B (DN1B) .cdc1249 inhibitor complex crystal, the cross-rotation fnction was calculated using the AMORE program. did. The search model was partially refined structural coordinates of Cdc25A (DN1A) (crystal 1). The cross-rotation function had one obvious solution at Eulerian angles [27.66, 63.02, 94.53] that was 11.3 standard deviations larger than the mean value of the cross-rotation function, and the next highest peak was 6.9 standard deviations larger than the mean. The translation function was calculated in the space group P4 ₁ 22, P4 ₃ 22, P4 ₁ 2 ₁ 2, and P4 ₃ 2 ₁ 2 (AMORE). One solution in the space group P4 ₃ 2 ₁ 2 was clear with an R factor of 49.3% and a correlation coefficient of 31.4% (resolution 15-3.0 Å).

Collection of Synchrotron X-Ray Diffraction Data for Cdc25B (DN1B) .cdc1249 Inhibitor Complex Crystals Crystal 5 at US Synchrotron Light Source (NSLS, Beamline X25, l = 1.100 Å, Brandy's B4 CCD Detector) A total of 35 data frames (1.0 ° each) were collected for. The crystal was maintained at 100K in an Oxford Cryosystems cryostream cooler during data collection. Diffraction data processed, space group P4 ₃ 2 ₁ 2, a = 70.15 Å, c = 13 as described above.
Converted to structure factor amplitude at 0.35 Å. Eigen reflections were assigned to the same "free" and "working" sets used for the crystal data for crystal 5. This data is summarized in Table 10.

Cdc25B (DN1B) .cdc1249 Refinement of Inhibitor Complex Crystal Structure Partially refined structure coordinates of Cdc25A (DN1A) (crystal 1) were converted to Cdc25B (DN1B) using X-PLOR.
B) .cdc1249 Inhibitor complex crystal data refined. Alternate refinement and manual model reconstruction using the molecular graphics program o (Jones et al., 1991). The molecular substitution model was modified by substituting a large number of amino acid side chains that differ between Cdc25A and Cdc25B with amino acids of Cdc25B. This side chain was localized in a clear electron density. The ambiguous amino acid was truncated as alanine. Bulk solvent modification (volume ratio of solvent in this crystal ~ 60%), Powell minimization,
Overall temperature factor refinement and then group temperature factor refinement and manual reconstruction resulted in / F /> 1.5 within a resolution range of 20 to 1.8 angstroms.
For all reflections of s _F , R was reduced to 37.7% (R _free 43.9%). Twist angle molecular dynamics allows us to construct two invisible loops (residues 456-464 and 474-477, active site loop) to a very clear density, improving the electron density map I was able to. Cdc1 bound to the active site at this stage of refinement
The electron density was also clear for most of 249. By continuing refinement (including individual temperature factors) and reassembly, residues 533-548 (C-terminal a-helix), cd
Of the total 6 residues of c1249, the 5th residue from the front and several water molecules could be added (R 27.0%, R _free 31.2%). Further refinement, including the dynamics of the slow-cool simulated annealing molecule, revealed that another Cdc25B (DN1B) molecule, which binds to the non-active site of cdc1249 and is related by crystallographic symmetry, is present. The existence of the molecule was revealed. After further refinement by adding this second molecule of cdc1249, several water molecules, several Na ⁺ ions, and some Cl ⁻ ions, R
Was reduced to 22.8% (R _free 26.1%). This model was inspected and reconstructed from a simulated annealing omission map. As a result of repeating refinement several times, R
Was 20.3% (R _free 22.9%, / F /> 0.5s _F , 20-2.30 angstrom). C
This structural coordinate (Powell 14) of the dc25B (DN1B) .cdc1249 inhibitor complex is Cdc2
It consists of 5B residues 377-548, 2 molecules of cdc1249, 129 water molecules, 1 Na ⁺ ion, and 5 Cl ⁻ ions. At this point, the synchrotron data described above is available. Further refinement with REFMAC, including the addition of several side chains of alternative conformations, resulted in R of 21.8% (R _free 24.4%; / F /> 0.0s _F , 20-1.95 angstroms). This final structural coordinate (Refmac1) of the Cdc25B (DN1B) .cdc1249 inhibitor complex is derived from Cdc25B residues 377-548, two molecules of cdc1249, 158 water molecules, 1 Na ⁺ ion, and 5 Cl ^- ions. Become. This data is summarized in Table 10.

X-Ray Diffraction Data Collection of Cdc25B (DN1B) .cdc1249 Inhibitor Complex Crystals Soaked in cdc1316 Collection of Cdc25B (DN1B) .cdc1249 inhibitor complex crystals prepared as described above at 4.5 MN
Buffer 5 containing aCl, 50 mM HEPES sodium, pH 7.0, and 2 mM cdc1316
Transferred to 0 mL. After 70 minutes, the crystals were cryoprotected and quenched (2 mM cdc1316 containing buffer). A total of 180 data frames (0.5 ° each) for this immersion crystal (crystal 6)
Collected. The crystal was maintained at 100K during data collection. Diffraction data processed, space group P4 ₃ 2 ₁ 2, a = 70.21 Å, c = 130.09 as described above.
It was converted to the structure factor amplitude in Angstrom. The intrinsic reflections were assigned to the same "free" and "working" sets used for crystal 5. This data is summarized in Table 10.

Refinement of Cdc25B (DN1B) .cdc1249 Inhibitor Complex Crystal Structure Immersed in cdc1316 Refinement of the data collected on Crystal 6 was performed by eliminating both molecules of cdc1249 from C.
Starting with an intermediate model of the dc25B (DN1B) .cdc1249 inhibitor complex (Powell 11). Slow-cool simulated annealing As a result of molecular dynamics, Powell minimization, and refinement of individual temperature factors, R was 27.4% (R _free 32.9%; / F />
1.0s _F , 20-2.50 angstrom). When the electron density map was examined, a strong and clear electron density of cdc1249 was observed in the active site. Active site cdc1316
There was no evidence of replacement with cdc1249. However, the positive F _o -F _c electron density was not strong at the crystal symmetry site, suggesting that partial substitution of cdc1249 and cdc1316 may have occurred at this site. This data is summarized in Table 11.

Collection of X-Ray Diffraction Data for Cdc25B (DN8B) .cdc1249 Inhibitor Complex Crystals A total of 201 data frames (0.5 ° each) for two Cdc25B (DN8B) .cdc1249 inhibitor complex crystals (crystals 7 and 8). Collected. The crystal was maintained at 100K during data collection. Diffraction data were processed and converted to structure factor amplitudes in the space group P4 ₃ 2 ₁ 2, a = 70.28 Å, c = 130.97 Å as described above. The intrinsic reflection is the same "free" and "working" as that used for crystal 5.
Assigned to the set. This data is summarized in Table 10.

Cdc25B (DN8B) .cdc1249 Inhibitor Complex Crystal Structure Refinement Cdc25B (DN8B) .cdc1249 Inhibitor Complex Crystal Data Refinement is Cdc25B (DN1B).
Starting with the structural coordinates "Powell 14" of the cdc1249 inhibitor complex. Rigid body,
As a result of Powell minimization and refinement of individual temperature factors, R was 23.7% (R _free 2
5.5%; / F /> 2.0s _F , 20-2.00 angstrom). From the electron density map, it was revealed that the N-terminal amino acid residue was added as expected in the Cdc25B (DN8B) construct as compared with the Cdc25B (DN1B) construct. Both molecules of cdc1249 were in the expected positions of the active site and the crystallographic symmetry site. After further refinement and reconstruction, R decreased to 22.6% (R _free 24.3%; / F /> 1.5s _F , 20-2.00 angstrom). These structural coordinates (Powell 12) of Cdc25B (DN8B) .cdc1249 inhibitor complex show Cdc25B residues 370-548, cdc12492 molecule, water molecule 128.
, One Na ⁺ ion, and five Cl ⁻ ions. This data is summarized in Table 11.

Immersion of Cdc25B (DN8B) .cdc1249 inhibitor complex crystals in low molecular weight organic compounds Eight crystals of Cdc25B (DN8B) .cdc1249 inhibitor complex were added to 4.5 M NaCl at 4 ° C.
, 50 mM HEPES sodium, pH 7.0, 0.5-1.5 mM cdc1249, and increased concentration (0, 50, 100, 250, and 1000 mM) t-BuNH ₂ , imidazole, (R) -3-hydroxypyrrolidine, Alternatively, transfer continuously to 2-methyl-1-propanol (up to ~ 500 mM, saturated solution). Soak the crystals in each solution for 10-15 minutes, then from 3 to the final solution.
I left it for 23 hours. The crystals were cryoprotected by the addition of glycerol (7.5% (v / v), 5 minutes, 17.5% (v / v), 5 minutes) and then placed in liquid nitrogen to quench. From the test X-ray diffraction images of all eight crystals, it was revealed that the order of each crystal was not affected by the immersion treatment at all, and the maximum resolution of the crystal X-ray diffraction was 2.2 to 2.6 angstroms. The crystals were stored in a liquid nitrogen freezer.

[0306] t-BuNH₂Cdc25B (DN8) dipped in imidazole, imidazole, or 2-methyl-1-propanol
B) .cdc1249 Inhibitor complex crystal X-ray diffraction data collection   Cdc soaked in ~ 0.5 M 2-methyl-1-propanol for 3 hours using the above equipment
25B (DN8B) .cdc1249 Total 100 data frames for inhibitor complex crystal (crystal 9).
The cells (0.5 ° each) were collected. The crystal was maintained at 100K during data collection. Times
Fold data is processed and space group P4₃2₁2, a = 70.01 angstrom, c = 129.92
It was converted to the structure factor amplitude in Angstrom. Similarly, 1 M t-BuNH₂To 2
Cdc25B (DN8B) .cdc1249 inhibitor complex crystal (crystal 10) soaked for 3 hours
A total of 225 data frames (0.4 ° each) were collected. This crystal was collected during data collection
, Maintained at 100K. Diffraction data processed, space group P4₃2₁2, a = 70.38 angs
The strom, c = 131.15 Angstrom was transformed into the structure factor amplitude. same
Similarly, the Cdc25B (DN8B) .cdc1249 inhibitor compound was immersed in 1 M imidazole for 23 hours.
A total of 112 data frames (0.5 ° each) were collected for the coalesced crystals (Crystal 11).
The crystal was maintained at 100K during data collection. Diffraction data processed, space group P4 ₃ 2₁2, a = 70.46 angstrom, c = 131.36 angstrom structure
Converted to factor amplitude. Eigenreflections of all three data sets were used for crystal 5.
Assigned to the same "free" and "working" sets as These 3 sets
Data are summarized in Table 10.

[0307] t-BuNH₂ Or (R) -3-hydroxypyrrolidine soaked in Cdc25B (DN8B) .cdc1249
Collection of X-ray diffraction data of inhibitor complex crystals   X-ray diffraction data was collected on NSLS at 100K as described above. 1 M t-BuNH₂To 3
Cdc25B (DN8B) .cdc1249 inhibitor complex crystal (crystal 12) soaked for 2 hours
A total of 60 data frames (1.0 ° each) were collected. This diffraction data is processed and
Group P4₃2₁2, a = 70.24 Å, c = 131.57 Å
It was converted into the amplitude of the structure factor. Similarly, at 1 M (R) -3-hydroxypyrrolidine at 3 o'clock
Cdc25B (DN8B) .cdc1249 inhibitor complex crystal (Crystal 13) immersed in water
A total of 67 data frames (1.0 ° each) were collected. This diffraction data is processed into space
Group P4₃2₁2, a = 70.04 angstroms, c = 130.34 angstroms
Converted to structure factor amplitude. Also, soak in 1 M (R) -3-hydroxypyrrolidine for 23 hours.
Total of 9 dipped Cdc25B (DN8B) .cdc1249 inhibitor complex crystals (crystal 14)
Nine data frames (0.5 ° each) were collected. This diffraction data is processed and the space group P4₃2 ₁ 2, a = 70.46 angstroms, c = 131.00 angstroms
Converted to child amplitude. The intrinsic reflections of these three data sets were used for crystal 5.
They were assigned to the same "free" and "working" sets. These three types
The data set is summarized in Table 10.

Refinement of structure of dc25B (DN8B) .cdc1249 inhibitor complex crystal immersed in low molecular weight organic compound Refinement of dc25B (DN8B) .cdc1249 inhibitor complex crystal data immersed in cdc1
Dc25B (DN8B) from which water molecules and Cl ^- ions in the "swimming pool" region of the active site, adjacent to the Nal residue of 249, have been removed, starting with structure coordinate "Powell 12" of the cdc1249 inhibitor complex . As a result of performing rigid body, Powell minimization, refinement of individual temperature factors, and kinetics of slow-cool simulated annealing molecule on the crystal 12, R was 24.2% (R _free 27.7%, / F /> 1.0s _F , 20-1.60 angstroms). From the electron density map, it was found that t-BuNH ₂ did not exist in the “swimming pool”. As a result of similarly refining the model of the crystal 13, R is 24.
It was 7% (R _free 27.8%, / F /> 1.0s _F , 20-1.60 angstrom). From the electron density map, it was found that (R) -3-hydroxypyrrolidine did not exist in the “swimming pool”. As a result of refining the model of the crystal 9 as described above except for the slow cool simulated annealing molecular dynamics, R was 21.3% (R _free 25.1%, /
F /> 2.0s _F, 30-2.45 Angstrom). From the electron density map, it was found that 2-methyl-1-propanol did not exist in the "swimming pool". As a result of similarly refining the model of the crystal 10, R was 22.3% (R _free 25.6%, / F /> 2.0s _F , 20-2.15 angstrom). From the electron density map, it was found that t-BuNH ₂ did not exist in the “swimming pool”. As a result of refining the model of the crystal 14 in the same manner as the crystal 10, the R was 23.2% (R _free 25.2%, / F /> 2.0s _F , 30-2.30 angstrom). From the electron density map, it was found that there was no imidazole in the "swimming pool". Finally, as a result of refining the model of the crystal 14 in the same manner as the crystals 12 and 13 (adding Powell minimization and refinement of individual temperature factors one more time), R is 24.6% (R _free 27.9%, / F /> 0.0s _F , 20-1.60 angstrom). From the electron density map, (R) -3-
It was found that hydroxypyrrolidine was absent. This data is summarized in Table 11.

Collection of X-Ray Diffraction Data for Cdc25B (DN1B) .cdc1671, Cdc25B (DN8B) .cdc1659, and Cdc25B (DN8B) .cdc1671 Inhibitor Complex Cdc25B (DN1B) .cdc1671 Inhibitor Complex Crystals (Crystal 15) A total of 195 data frames (0.4 ° each) were collected for. The crystal was maintained at 100K during data collection. Processing this diffraction data, the space group P4 ₃ 2 ₁ 2, a = 70.03 Angstrom,
c = 130.07 angstrom converted to structure factor amplitude. Similarly, Cdc25B
A total of 120 data frames (0.5 ° each) were collected for the (DN8B) .cdc1659 inhibitor complex crystals (Crystal 16). This diffraction data is processed and the space group P4 ₃ 2 ₁ 2, a = 70.1
Converted to structure factor amplitudes at 0 Å, c = 129.47 Å. Cdc25B (DN8B) .cdc1671 inhibitor complex crystal (crystal 17) space group P4 ₃ 2 ₁ 2, a
= 70.10 angstroms, c = 130.13 angstroms), the test data was collected, but the diffraction was weaker than the Cdc25B (DN1B) .cdc1671 inhibitor complex crystal (maximum resolution 3.00 angstroms), and complete data collection is performed. I decided it wasn't worth it. The intrinsic reflections of crystals 15 and 16 were assigned to the same "free" and "working" sets used for crystal 5. The two types of data sets are summarized in Table 10.

[0310] Collection of X-ray diffraction data for Cdc25B (DN1B) .cdc1885 inhibitor complex crystals   Cdc25B (DN1B) .cdc1885 Inhibitor complex crystal (Crystal 18) for a total of 100 days
Taframes (0.5 ° each) were collected. In addition, data (32
Frames, 1.0 ° each, were collected. This crystal was maintained at 100K during data collection.
It was Diffraction data collected in the laboratory and NSLS were processed and merged into space group P4₃2 ₁ 2, structure at a = 70.10 angstroms, c = 130.13 angstroms
Converted to factor amplitude. The intrinsic reflection is the same "free" as that used for crystal 5.
And assigned to the "working" set. This data set is summarized in Table 10.
.

Refinement of Cdc25B (DN1B) .cdc1671 Inhibitor Complex Crystal Structure Refinement of Cdc25B (DN1B) .cdc1671 Inhibitor Complex Crystalline Data (Crystal 15)
Starting with the structural coordinates "Powell 15" of the c25B (DN1B) .cdc1249 inhibitor complex. The cdc1249 molecule, water molecule and ions were removed from the model, followed by rigid body, Powell minimization, and individual temperature factor refinement. Electron density maps showed that both molecules of cdc1671 were in the expected (equivalent to cdc1249) position in the active and crystallographic symmetry sites. This inhibitor was added to the model. As a result of alternating refinement and reconstruction, R decreased to 20.2% (R _free 22.8%, / F /> 1.0s _F , 20-3.00 angstrom). This structural coordinate of the Cdc25B (DN1B) .cdc1671 inhibitor complex (Powell 16) consists of Cdc25B residues 377-548, cdc16712 molecules, 32 water molecules, 1 Na ⁺ ion, and 5 Cl ⁻ ions. This data is summarized in Table 11.

Refinement of Cdc25B (DN8B) .cdc1659 Inhibitor Complex Crystal Structure Refinement of Cdc25B (DN8B) .cdc1659 Inhibitor Complex Crystalline Data (Crystal 16)
Starting with the structural coordinates "Powell 12" of c25B (DN8B) .cdc1249. The cdc1249 molecule was removed from the model and subjected to rigid body, Powell minimization, and individual temperature factor refinement. Electron density maps showed that both molecules of cdc1659 were at the predicted (equivalent to cdc1249) position in the active and crystallographic symmetry sites. This inhibitor was added to the model. As a result of further alternating refinement and reconstruction, R is 2
It decreased to 2.2% (R _free 25.3%, / F /> 1.0s _F , 20-2.20 angstrom). Cd
This structural coordinate (powell 13) of the c25B (DN8B) .cdc1659 inhibitor complex is Cdc25B
It consists of residues 370-548, 2 molecules of cdc1659, 128 water molecules, 1 Na ⁺ ion, and 5 Cl ⁻ ions. This data is summarized in Table 11.

Refinement of Cdc25B (DN1B) .cdc1885 Inhibitor Complex Crystal Structure Refinement of Cdc25B (DN1B) .cdc1885 Inhibitor Complex Crystal Data (Crystal 18)
Starting with the structural coordinates "Powell 15" of the c25B (DN8B) .cdc1249 inhibitor complex. The cdc1249 molecule was removed from the model and subjected to rigid body, Powell minimization, and individual temperature factor refinement. Electron density maps showed that both molecules of cdc1885 were at the predicted (equivalent to cdc1249) position in the active and crystallographic symmetry sites. This inhibitor was added to the model. As a result of further alternating refinement and reconstruction, R was reduced to 22.8% (R _free 25.1%, / F /> 1.0s _F , 30-1.70 angstrom). This structural coordinate of the Cdc25B (DN1B) .cdc1659 inhibitor complex (Powell 12)
Is Cdc25B residues 377-548, 2 molecules of cdc1885, 128 water molecules, 1 Na ⁺ ion, and
Consists of 5 Cl ^- ions. This data is summarized in Table 11.

Collection of Synchrotron X-Ray Diffraction Data for Cdc25B (DN8B) .cdc1249 Inhibitor Complex Crystals Grown in the Presence of cdc1900 Cdc25B (DN8B) .cdc1249 Inhibitor Complex Crystals Grown in the Presence of cdc1900 A total of 121 data frames (0.5 ° each) for (Crystal 19) were collected in NSLS as described above. The crystal was maintained at 100K during data collection. The diffraction data were processed and converted to structure factor amplitudes in the space groups P4 ₃ 2 ₁ 2, a = 70.29 Å, c = 130.59 Å. The intrinsic reflections were assigned to the same "free" and "working" sets used for crystal 5. This data set is summarized in Table 10.

High resolution refinement of Cdc25B (DN8B) .cdc1249 inhibitor complex crystal structure Cdc25B (DN8B) .cdc for synchrotron data collected on crystal 19.
Refinement of the 1249 inhibitor complex crystal structure was initiated using the structural coordinates of the Cdc25B (DN8B) .cdc1249 inhibitor complex, "Powell 12. X-PLOR for rigid bodies, Powell minimization, and individual temperature factors. As a result of refining and then refining with REFMAC by adding several side chains to the alternative conformation, R was 22.4% (R _free 24.1%, / F /> 0.0s _F
, 15-1.45 angstroms). As a result of further refinement of individual anisotropic temperature factors, R is 21.1% (R _free 23.1%, / F /> 0.0s _F , 15-1.45 angstrom)
Met. This final structural coordinate of the Cdc25B (DN8B) .cdc1249 inhibitor complex (Ref
mac3) is cdc25B residues 369-548, cdc12492 molecule, 235 water molecules, 1 Na ⁺ ion,
And 6 Cl ⁻ ions. The inhibitor cdc1900 was included in the crystallization mixture but was not localized. This data is summarized in Table 11.

Collection of X-Ray Diffraction Data for Cdc25B (DN8B-c17) .cdc1249 Inhibitor Complex Crystals Cdc25B (DN8B-c17) .cdc1249 inhibitor complex crystals (crystal 2
A total of 113 data frames (0.5 ° each) were collected for 0). The crystal was maintained at 100K during data collection. Processes the diffraction data, as described in 3, space group _{P4 3 2 1 2, a =} 70.05 Å, was converted to the structure factor amplitude at c = 131.44 angstroms. The intrinsic reflection is the same as that used for the crystal 5.
Assigned to the "Free" and "Working" sets. This data set is summarized in Table 10.

Refinement of the Cdc25B (DN8B-c17) .cdc1249 Inhibitor Complex Crystal Structure A refinement of the Cdc25B (DN8B-c17) .cdc1249 inhibitor complex crystal structure to the data collected on Crystal 20 was performed using Cdc25B (DN8B ) .cdc1249 Inhibitor complex structure starting with "Powell 12". The cdc1249 molecule was removed from the model and rigid body, Powell minimization, and refinement of individual temperature factors were performed with X-PLOR. Electron density maps showed that both molecules of cdc1249 were at the predicted positions in the active and crystallographic symmetry sites. This inhibitor was added to the model. As a result of further alternating refinement and reconstruction, R decreased to 22.6% (R _free 27.4%, / F /> 1.0s _F , 30-2.70 angstrom). The final structural coordinates of Cdc25B (DN8B-c17) .cdc1249 inhibitor complex are from Cdc25B residues 370-548, 2 molecules of cdc1249, 128 water molecules, 1 Na ⁺ ion, and 5 Cl ⁻ ions. Become. This structure is very similar to that of the Cdc25B (DN8B) .cdc1249 complex. This data is summarized in Table 11.

Collection of X-ray diffraction data for Cdc25B (DN8B) .cdc1249 inhibitor complex crystals grown in the presence of cdc1973. For Cdc25B (DN8B) .cdc1249 inhibitor complex crystals grown in the presence of cdc1973, A total of 140 data frames (0.5 ° each) were collected. The crystal was maintained at 100K during data collection. This diffraction data is processed and the space group P4 ₃ 2 ₁ 2, a = 70.22
Angstrom, c = 131.29 Angstrom converted to structure factor amplitude. The intrinsic reflections were assigned to the same "free" and "working" sets used for crystal 5. This data set is summarized in Table 10.

Refinement of the Cdc25B (DN8B) .cdc1249 inhibitor complex crystal structure grown in the presence of cdc1973 Refinement of the Cdc25B (DN8B) .cdc1249 inhibitor complex crystal structure on the data collected from crystal 21 Starting with the structural coordinates "Powell 12" of the dCdc25B (DN8B) .cdc1249 inhibitor complex. The cdc1249 molecule was removed from the model and rigid body, Powell minimization and refinement of individual temperature factors were performed with X-PLOR. From the electron density map, cdc124
Both 9 molecules were shown to be at predicted positions in the active and crystallographic symmetry sites. This inhibitor was added to the model. As a result of further alternating refinement and reconstruction, R decreased to 20.5% (R _free 24.4%, / F /> 1.0s _F , 30-2.52 angstrom). Cdc25B (DN8B) .cdc1249 inhibitor complex final structure coordinates (
Powell 12) consists of two molecules of Cdc25B residues 369-548, cdc1249, 128 water molecules, 1 Na ⁺ ion, and 5 Cl ⁻ ions. Cdc1249 is cdc1 at both sites
There was no evidence of substitution with 973. This data is summarized in Table 11.

Immersion of Cdc25B (DN8B) .cdc1249 Inhibitor Complex Crystals in Other Inhibitors Four crystals of Cdc25B (DN8B) .cdc1249 inhibitor complex prepared as described above were added to 4.5M.
It was immersed at 4 ° C. in a buffer containing NaCl, 50 mM HEPES sodium, pH 7.0.
This buffer also contained 2 mM cdc1659, cdc1671, cdc1748, or cdc1973. One day later, the crystals dipped in cdc1748 were dissolved, but the other crystals were safe. Similar immersion fluids using cdc1659, cdc1671, or cdc1973 were set at room temperature. After an additional 4 days, the crystals were cryoprotected as described above. Additional crystals were similarly soaked (1 mM inhibitor) for 1 day and then cryoprotected as described above.

Collection of X-Ray Diffraction Data for Cdc25B (DN8B) .cdc1249 Inhibitor Complex Crystals Soaked in the Presence of cdc1659, cdc1671, or cdc1973 The X-ray diffraction properties of the crystals soaked as described above were verified. The crystal was maintained at 100K during data collection. Only the crystals dipped in 2mM cdc1973 at 4 ℃ had X
The lines were diffracted. A total of 88 data frames (0.5 ° each) for this crystal and crystal 22
Collected. Diffraction data were processed and converted to structure factor amplitudes in the space group P4 ₃ 2 ₁ 2, a = 70.03 Å, c = 131.30 Å. Eigen reflection is
Assigned to the same "free" and "working" sets used for crystal 5. This data set is summarized in Table 10.

Refinement of Cdc25B (DN8B) .cdc1249 Inhibitor Complex Crystal Structure Soaked in the Presence of cdc1973 Cdc25B (DN8B) .cdc1249 Inhibitor Complex Crystals for the data collected from Crystal 22, Cdc25B (DN8B). Starting with the structural coordinates "Powell 12" of the cdc1249 inhibitor complex. The cdc1249 and water molecules were removed from the model and rigid body, Powell minimization and refinement of individual temperature factors were performed with X-PLOR. The electron density map showed that both molecules of cdc1249 were at the predicted positions in the active site and the crystallographic symmetry site. This inhibitor was added to the model. As a result of further alternation of refinement and reconstruction, R decreased to 21.6% (R _free 25.3%, / F /> 2.0s _F , 30-3.50 angstrom). The final structural coordinates (Powell 13) of Cdc25B (DN8B) .cdc1249 inhibitor complex (soaked in cdc1973) are Cdc25B residues 370-548, two molecules of cdc1249, Na ⁺
It consists of one ion and five Cl ^- ions. In the F ₀ -F _C electron density map with weighted sigma A, no interpretable density of differences of cdc1249 molecules was found in the active site.
However, a strong negative peak (> 3s) was found in the central part of the sulfonate moiety of cdc1249 in the crystallographic symmetry lattice site, suggesting that the substitution of cdc1973 with cdc1249 occurred only at this site. This data is summarized in Table 11.

Cdc25B (DN8B) .cdc1249 soaked at room temperature in the presence of cdc1659 or cdc1671.
Collection of X-Ray Diffraction Data of Inhibitor Complex Crystals The X-ray diffraction characteristics of the crystals soaked as described above (immersion time at room temperature for 1 day) were verified using the apparatus described above. The crystal was maintained at 100K during data collection. 2 mM
Only crystals immersed in cdc1659 diffracted X-rays. A total of 59 data frames (0.75 ° each) were collected for this crystal, crystal 23. Diffraction data were processed and converted to structure factor amplitudes in the space group P4 ₃ 2 ₁ 2, a = 70.05 Å, c = 129.95 Å. The intrinsic reflections were assigned to the same "free" and "working" sets used for crystal 5. This data set is summarized in Table 10.

Refinement of Cdc25B (DN8B) .cdc1249 inhibitor complex crystal structure soaked at room temperature in the presence of cdc1659 Refinement of Cdc25B (DN8B) .cdc1249 inhibitor complex crystal structure for data collected from crystal 24 Was initiated using the structural coordinates "Powell 12" of the Cdc25B (DN8B) .cdc1249 inhibitor complex. cdc1249 and water molecules were removed from the model, rigid body and Powell minimization, and global temperature factor refinement were performed with X-PLOR. Electron density maps showed that both molecules of cdc1249 were at the predicted positions in the active and crystallographic symmetry sites. As a result of further alternation of refinement and reconstruction, R is 24.
3% (R _free 26.3%, / F /> 1.0s _F , 30-2.70 angstrom). Cdc
The final structural coordinates (Powell 12) of 25B (DN8B) .cdc1249 inhibitor complex (soaked in cdc1659) are Cdc25B residues 370-548, two molecules of cdc1249, Na ⁺ ion 1
And 5 Cl ⁻ ions. In the F ₀ -F _C electron density map with weighted sigma A, no interpretable density of differences for cdc1249 molecules was found at the active site, and cdc1659 and cdc124
It was suggested that the substitution of 9 did not occur. This data is summarized in Table 10.

[0325]

[Table 10]

[0326]

[Table 11]

References Brunger, AT (1992) X-PLOR Version 3.1, A System for Crystallography a
nd NMR (Yale University Press, New Haven, CT). Collaborative Computational Project, Number 4. (1994). Acta Cryst. D50,
760-763. Evans, PR (1997). Joint CCP4 and ESF-EACBM Newsletter 33, 22-24. French, S. & Wilson, K. (1978) .Acta Cryst. A34, 517-525. Jones, TA, Zou, JY, Cowan, SW & Kjelgaard, M. (1991). Acta Cryst.
A47, 110-119. Kabsch, W. (1993). J. Appl. Cryst. 24,795-800. Leslie, AGW (1992). CCP4 and ESF-EACMB Newsletter on Protein Crystall
ography No. 26. Murshudov, GN, Vagin, AA, and Dodson, EJ (1997) Acta Crystallogr. D53, 240 255. Navaza, J. (1994). Acta Cryst. A50, 157-163.

While the preferred embodiment of the invention has been described, the form and details of the invention can be modified in various ways without departing from the scope of the invention, which is encompassed by the above claims. It will be appreciated by those skilled in the art that there is none.

[Brief description of drawings]

FIG. 1 is an amino acid sequence of human Cdc25A (SEQ ID NO:
1) is shown.

FIG. 2 is an amino acid sequence of human Cdc25B (SEQ ID NO:
2) is shown.

FIG. 3 is an amino acid sequence of human Cdc25C (SEQ ID NO:
3) is shown.

FIG. 4 shows the amino acid sequence of the polypeptide Cdc25A ΔN1A (S
EQ ID NO: 4).

FIG. 5 shows the amino acid sequence of the polypeptide Cdc25B ΔN1B (S
EQ ID NO: 5).

FIG. 6 shows the amino acid sequence of the polypeptide Cdc25A ΔN5A (S
EQ ID NO: 6).

FIG. 7 shows the amino acid sequence of the polypeptide Cdc25C ΔN1C (S
EQ ID NO: 7).

FIG. 8 shows the amino acid sequence of the polypeptide Cdc25A ΔN8A (S
EQ ID NO: 8).

FIG. 9 shows the amino acid sequence of the polypeptide Cdc25BΔN8A-c17 (SEQ ID NO: 9).

FIG. 10 shows the amino acid sequence (SEQ ID NO: 10) of the polypeptide Cdc25B ΔN5B.

FIG. 11 shows the amino acid sequence of the polypeptide Cdc25B ΔN8B (SEQ ID NO: 11).

FIG. 12 shows the amino acid sequence (SEQ ID NO: 12) of the polypeptide Cdc25BΔN8B-c17.

FIG. 13 shows the amino acid sequence of polypeptide Cdc25BΔN8B-c18 (SEQ ID NO: 13).

FIG. 14 shows the amino acid sequence of the polypeptide Cdc25BΔN9C (SEQ ID NO: 14).

15A to 15PPP show Cdc25B (ΔN8B) / c.
9 shows atomic coordinates of a dc1249 complex (crystal 19).

16A to 16I show atomic coordinates of Cdc25A (ΔN1A).

17A to 17EE are Cdc25B (ΔN1B) / cd.
6 shows atomic coordinates of the c1249 complex (crystal 5).

18A-18C show Cdc25A (ΔN8A) (Crystal 3).
The atomic coordinates of are shown.

19A to 19I show Cdc25B (ΔN1B) / cdc.
16 shows the atomic coordinates of the 1671 composite (crystal 15).

FIG. 20 shows the complex of the pentapeptide cdc1249 with the catalytic domain of Cdc25B, with the ligand bound at the catalytic loop (thick bond line) and the ligand bound at the terminal site (thin bond line). It is illustrated in the form of protein secondary structure.

FIG. 21 shows the catalytic domain of Cdc25B and the pentapeptide cdc.
A complex with 1249 is shown with two symmetrical protein molecules interacting with a ligand bound to the catalytic site; water molecules and ions are not shown.

FIG. 22 shows the catalytic domain of Cdc25B and the pentapeptide cdc.
FIG. 12 is a top view of the molecular surface around the ligand binding region of the complex with 1249.

FIG. 23 shows the catalytic domain of Cdc25B and the pentapeptide cdc.
FIG. 12 is a side view of a complex with 1249.

FIG. 24 shows the catalytic domain of Cdc25B and the pentapeptide cdc.
FIG. 7 is a top view of a complex with 1249 in which protein residues are labeled.

FIG. 25 shows the catalytic domain of Cdc25B and the pentapeptide cdc.
FIG. 12 is a side view of a complex with 1249.

FIG. 26 shows the catalytic domain of Cdc25B and the pentapeptide cdc.
FIG. 9 is a top view of the surface of the molecule around the ligand binding region of the complex with 1249, with each subsite labeled.

FIG. 27 shows the catalytic domain of Cdc25B and the pentapeptide cdc.
FIG. 9 is a top view of the complex with 1249, labeled at subsites 1-6.

FIG. 28 is a side view of a potential strong binding inhibitor bound to the catalytic domain of Cdc25B.

[Sequence list]

                                SEQUENCE LISTING <110> BASF Aktiengesellschaft       GPC Biotech, Inc.       Taylor, Neil R.       Borhani, David       Epstein, David       Rudolph, Johannes       Ritter, Kurt       Fujimori, Taro       Robinson, Simon       Eckstein, Jens       Haupt, Andreas       Walker, Nigel       Dixon, Richard W.       Choquette, Deborah       Blanchard, Jill       Kluge, Arthur       Pal, Kollol       Bockovich, Nicholas       Come, Jon       Hediger, Mark <120> Method of Identifying Inhibitors of   CDC25 <130> 2079.1038-007 <150> 60 / 172,215 <151> 1999-08-31 <160> 14 <170> FastSEQ for Windows Version 4.0 <210> 1 <211> 523 <212> PRT <213> Homo Sapien <400> 1 Met Glu Leu Gly Pro Ser Pro Ala Pro Arg Arg Leu Leu Phe Ala Cys  1 5 10 15 Ser Pro Pro Pro Ala Ser Gln Pro Val Val Lys Ala Leu Phe Gly Ala             20 25 30 Ser Ala Ala Gly Gly Leu Ser Pro Val Thr Asn Leu Thr Val Thr Met         35 40 45 Asp Gln Leu Gln Gly Leu Gly Ser Asp Tyr Glu Gln Pro Leu Glu Val     50 55 60 Lys Asn Asn Ser Asn Leu Gln Arg Met Gly Ser Ser Glu Ser Thr Asp 65 70 75 80 Ser Gly Phe Cys Leu Asp Ser Pro Gly Pro Leu Asp Ser Lys Glu Asn                 85 90 95 Leu Glu Asn Pro Met Arg Arg Ile His Ser Leu Pro Gln Lys Leu Leu             100 105 110 Gly Cys Ser Pro Ala Leu Lys Arg Ser His Ser Asp Ser Leu Asp His         115 120 125 Asp Ile Phe Gln Leu Ile Asp Pro Asp Glu Asn Lys Glu Asn Glu Ala     130 135 140 Phe Glu Phe Lys Lys Pro Val Arg Pro Val Ser Arg Gly Cys Leu His 145 150 155 160 Ser His Gly Leu Gln Glu Gly Lys Asp Leu Phe Thr Gln Arg Gln Asn                 165 170 175 Ser Ala Gln Leu Gly Met Leu Ser Ser Asn Glu Arg Asp Ser Ser Glu             180 185 190 Pro Gly Asn Phe Ile Pro Leu Phe Thr Pro Gln Ser Pro Val Thr Ala         195 200 205 Thr Leu Ser Asp Glu Asp Asp Gly Phe Val Asp Leu Leu Asp Gly Glu     210 215 220 Asn Leu Lys Asn Glu Glu Glu Thr Pro Ser Cys Met Ala Ser Leu Trp 225 230 235 240 Thr Ala Pro Leu Val Met Arg Thr Thr Asn Leu Asp Asn Arg Cys Lys                 245 250 255 Leu Phe Asp Ser Pro Ser Leu Cys Ser Ser Ser Thr Arg Ser Val Leu             260 265 270 Lys Arg Pro Glu Arg Ser Gln Glu Glu Ser Pro Pro Gly Ser Thr Lys         275 280 285 Arg Arg Lys Ser Met Ser Gly Ala Ser Pro Lys Glu Ser Thr Asn Pro     290 295 300 Glu Lys Ala His Glu Thr Leu His Gln Ser Leu Ser Leu Ala Ser Ser 305 310 315 320 Pro Lys Gly Thr Ile Glu Asn Ile Leu Asp Asn Asp Pro Arg Asp Leu                 325 330 335 Ile Gly Asp Phe Ser Lys Gly Tyr Leu Phe His Thr Val Ala Gly Lys             340 345 350 His Gln Asp Leu Lys Tyr Ile Ser Pro Glu Ile Met Ala Ser Val Leu         355 360 365 Asn Gly Lys Phe Ala Asn Leu Ile Lys Glu Phe Val Ile Ile Asp Cys     370 375 380 Arg Tyr Pro Tyr Glu Tyr Glu Gly Gly His Ile Lys Gly Ala Val Asn 385 390 395 400 Leu His Met Glu Glu Glu Val Glu Asp Phe Leu Leu Lys Lys Pro Ile                 405 410 415 Val Pro Thr Asp Gly Lys Arg Val Ile Val Val Phe His Cys Glu Phe             420 425 430 Ser Ser Glu Arg Gly Pro Arg Met Cys Arg Tyr Val Arg Glu Arg Asp         435 440 445 Arg Leu Gly Asn Glu Tyr Pro Lys Leu His Tyr Pro Glu Leu Tyr Val     450 455 460 Leu Lys Gly Gly Tyr Lys Glu Phe Phe Met Lys Cys Gln Ser Tyr Cys 465 470 475 480 Glu Pro Pro Ser Tyr Arg Pro Met His His Glu Asp Phe Lys Glu Asp                 485 490 495 Leu Lys Lys Phe Arg Thr Lys Ser Arg Thr Trp Ala Gly Glu Lys Ser             500 505 510 Lys Arg Glu Met Tyr Ser Arg Leu Lys Lys Leu         515 520 <210> 2 <211> 566 <212> PRT <213> Homo Sapien <400> 2 Met Glu Val Pro Gln Pro Glu Pro Ala Pro Gly Ser Ala Leu Ser Pro  1 5 10 15 Ala Gly Val Cys Gly Gly Ala Gln Arg Pro Gly His Leu Pro Gly Leu             20 25 30 Leu Leu Gly Ser His Gly Leu Leu Gly Ser Pro Val Arg Ala Ala Ala         35 40 45 Ser Ser Pro Val Thr Thr Leu Thr Gln Thr Met His Asp Leu Ala Gly     50 55 60 Leu Gly Ser Arg Ser Arg Leu Thr His Leu Ser Leu Ser Arg Arg Ala 65 70 75 80 Ser Glu Ser Ser Leu Ser Ser Glu Ser Ser Glu Ser Ser Asp Ala Gly                 85 90 95 Leu Cys Met Asp Ser Pro Ser Pro Met Asp Pro His Met Ala Glu Gln             100 105 110 Thr Phe Glu Gln Ala Ile Gln Ala Ala Ser Arg Ile Ile Arg Asn Glu         115 120 125 Gln Phe Ala Ile Arg Arg Phe Gln Ser Met Pro Val Arg Leu Leu Gly     130 135 140 His Ser Pro Val Leu Arg Asn Ile Thr Asn Ser Gln Ala Pro Asp Gly 145 150 155 160 Arg Arg Lys Ser Glu Ala Gly Ser Gly Ala Ala Ser Ser Ser Gly Glu                 165 170 175 Asp Lys Glu Asn Asp Gly Phe Val Phe Lys Met Pro Trp Lys Pro Thr             180 185 190 His Pro Ser Ser Thr His Ala Leu Ala Glu Trp Ala Ser Arg Arg Glu         195 200 205 Ala Phe Ala Gln Arg Pro Ser Ser Ala Pro Asp Leu Met Cys Leu Ser     210 215 220 Pro Asp Arg Lys Met Glu Val Glu Glu Leu Ser Pro Leu Ala Leu Gly 225 230 235 240 Arg Phe Ser Leu Thr Pro Ala Glu Gly Asp Thr Glu Glu Asp Asp Gly                 245 250 255 Phe Val Asp Ile Leu Glu Ser Asp Leu Lys Asp Asp Asp Ala Val Pro             260 265 270 Pro Gly Met Glu Ser Leu Ile Ser Ala Pro Leu Val Lys Thr Leu Glu         275 280 285 Lys Glu Glu Glu Lys Asp Leu Val Met Tyr Ser Lys Cys Gln Arg Leu     290 295 300 Phe Arg Ser Pro Ser Met Pro Cys Ser Val Ile Arg Pro Ile Leu Lys 305 310 315 320 Arg Leu Glu Arg Pro Gln Asp Arg Asp Thr Pro Val Gln Asn Lys Arg             325 330 335 Arg Arg Ser Val Thr Pro Pro Glu Glu Gln Gln Glu Ala Glu Glu Pro             340 345 350 Lys Ala Arg Val Leu Arg Ser Lys Ser Leu Cys His Asp Glu Ile Glu         355 360 365 Asn Leu Leu Asp Ser Asp His Arg Glu Leu Ile Gly Asp Tyr Ser Lys     370 375 380 Ala Phe Leu Leu Gln Thr Val Asp Gly Lys His Gln Asp Leu Lys Tyr 385 390 395 400 Ile Ser Pro Glu Thr Met Val Ala Leu Leu Thr Gly Lys Phe Ser Asn                 405 410 415 Ile Val Asp Lys Phe Val Ile Val Asp Cys Arg Tyr Pro Tyr Glu Tyr             420 425 430 Glu Gly Gly His Ile Lys Thr Ala Val Asn Leu Pro Leu Glu Arg Asp         435 440 445 Ala Glu Ser Phe Leu Leu Lys Ser Pro Ile Ala Pro Cys Ser Leu Asp     450 455 460 Lys Arg Val Ile Leu Ile Phe His Cys Glu Phe Ser Ser Glu Arg Gly 465 470 475 480 Pro Arg Met Cys Arg Phe Ile Arg Glu Arg Asp Arg Ala Val Asn Asp                 485 490 495 Tyr Pro Ser Leu Tyr Tyr Pro Glu Met Tyr Ile Leu Lys Gly Gly Tyr             500 505 510 Lys Glu Phe Phe Pro Gln His Pro Asn Phe Cys Glu Pro Gln Asp Tyr         515 520 525 Arg Pro Met Asn His Glu Ala Phe Lys Asp Glu Leu Lys Thr Phe Arg     530 535 540 Leu Lys Thr Arg Ser Trp Ala Gly Glu Arg Ser Arg Arg Glu Leu Cys 545 550 555 560 Ser Arg Leu Gln Asp Gln                 565 <210> 3 <211> 473 <212> PRT <213> Homo Sapien <400> 3 Met Ser Thr Glu Leu Phe Ser Ser Thr Arg Glu Glu Gly Ser Ser Gly  1 5 10 15 Ser Gly Pro Ser Phe Arg Ser Asn Gln Arg Lys Met Leu Asn Leu Leu             20 25 30 Leu Glu Arg Asp Thr Ser Phe Thr Val Cys Pro Asp Val Pro Arg Thr         35 40 45 Pro Val Gly Lys Phe Leu Gly Asp Ser Ala Asn Leu Ser Ile Leu Ser     50 55 60 Gly Gly Thr Pro Lys Cys Cys Leu Asp Leu Ser Asn Leu Ser Ser Gly 65 70 75 80 Glu Ile Thr Ala Thr Gln Leu Thr Thr Ser Ala Asp Leu Asp Glu Thr                 85 90 95 Gly His Leu Asp Ser Ser Gly Leu Gln Glu Val His Leu Ala Gly Met             100 105 110 Asn His Asp Gln His Leu Met Lys Cys Ser Pro Ala Gln Leu Leu Cys         115 120 125 Ser Thr Pro Asn Gly Leu Asp Arg Gly His Arg Lys Arg Asp Ala Met     130 135 140 Cys Ser Ser Ser Ala Asn Lys Glu Asn Asp Asn Gly Asn Leu Val Asp 145 150 155 160 Ser Glu Met Lys Tyr Leu Gly Ser Pro Ile Thr Thr Val Pro Lys Leu                 165 170 175 Asp Lys Asn Pro Asn Leu Gly Glu Asp Gln Ala Glu Glu Ile Ser Asp             180 185 190 Glu Leu Met Glu Phe Ser Leu Lys Asp Gln Glu Ala Lys Val Ser Arg         195 200 205 Ser Gly Leu Tyr Arg Ser Pro Ser Met Pro Glu Asn Leu Asn Arg Pro     210 215 220 Arg Leu Lys Gln Val Glu Lys Phe Lys Asp Asn Thr Ile Pro Asp Lys 225 230 235 240 Val Lys Lys Lys Tyr Phe Ser Gly Gln Gly Lys Leu Arg Lys Gly Leu                 245 250 255 Cys Leu Lys Lys Thr Val Ser Leu Cys Asp Ile Thr Ile Thr Gln Met             260 265 270 Leu Glu Glu Asp Ser Asn Gln Gly His Leu Ile Gly Asp Phe Ser Lys         275 280 285 Val Cys Ala Leu Pro Thr Val Ser Gly Lys His Gln Asp Leu Lys Tyr     290 295 300 Val Asn Pro Glu Thr Val Ala Ala Leu Leu Ser Gly Lys Phe Gln Gly 305 310 315 320 Leu Ile Glu Lys Phe Tyr Val Ile Asp Cys Arg Tyr Pro Tyr Glu Tyr                 325 330 335 Leu Gly Gly His Ile Gln Gly Ala Leu Asn Leu Tyr Ser Gln Glu Glu             340 345 350 Leu Phe Asn Phe Phe Leu Lys Lys Pro Ile Val Pro Leu Asp Thr Gln         355 360 365 Lys Arg Ile Ile Ile Val Phe His Cys Glu Phe Ser Ser Glu Arg Gly     370 375 380 Pro Arg Met Cys Arg Cys Leu Arg Glu Glu Asp Arg Ser Leu Asn Gln 385 390 395 400 Tyr Pro Ala Leu Tyr Tyr Pro Glu Leu Tyr Ile Leu Lys Gly Gly Tyr                 405 410 415 Arg Asp Phe Phe Pro Glu Tyr Met Glu Leu Cys Glu Pro Gln Ser Tyr             420 425 430 Cys Pro Met His His Gln Asp His Lys Thr Glu Leu Leu Arg Cys Arg         435 440 445 Ser Gln Ser Lys Val Gln Glu Gly Glu Arg Gln Leu Arg Glu Gln Ile     450 455 460 Ala Leu Leu Val Lys Asp Met Ser Pro 465 470 <210> 4 <211> 190 <212> PRT <213> Artificial Sequence <220> <223> Polypeptide with deletion mutation <400> 4 Gly Ser Leu Ile Gly Asp Phe Ser Lys Gly Tyr Leu Phe His Thr Val  1 5 10 15 Ala Gly Lys His Gln Asp Leu Lys Tyr Ile Ser Pro Glu Ile Met Ala             20 25 30 Ser Val Leu Asn Gly Lys Phe Ala Asn Leu Ile Lys Glu Phe Val Ile         35 40 45 Ile Asp Cys Arg Tyr Pro Tyr Glu Tyr Glu Gly Gly His Ile Lys Gly     50 55 60 Ala Val Asn Leu His Met Glu Glu Glu Val Glu Asp Phe Leu Leu Lys 65 70 75 80 Lys Pro Ile Val Pro Thr Asp Gly Lys Arg Val Ile Val Val Phe His                 85 90 95 Cys Glu Phe Ser Ser Glu Arg Gly Pro Arg Met Cys Arg Tyr Val Arg             100 105 110 Glu Arg Asp Arg Leu Gly Asn Glu Tyr Pro Lys Leu His Tyr Pro Glu         115 120 125 Leu Tyr Val Leu Lys Gly Gly Tyr Lys Glu Phe Phe Met Lys Cys Gln     130 135 140 Ser Tyr Cys Glu Pro Pro Ser Tyr Arg Pro Met His His Glu Asp Phe 145 150 155 160 Lys Glu Asp Leu Lys Lys Phe Arg Thr Lys Ser Arg Thr Trp Ala Gly                 165 170 175 Glu Lys Ser Lys Arg Glu Met Tyr Ser Arg Leu Lys Lys Leu             180 185 190 <210> 5 <211> 191 <212> PRT <213> Artificial Sequence <220> <223> Polypeptide with deletion mutation <400> 5 Gly Ser Leu Ile Gly Asp Tyr Ser Lys Ala Phe Leu Leu Gln Thr Val  1 5 10 15 Asp Gly Lys His Gln Asp Leu Lys Tyr Ile Ser Pro Glu Thr Met Val             20 25 30 Ala Leu Leu Thr Gly Lys Phe Ser Asn Ile Val Asp Lys Phe Val Ile         35 40 45 Val Asp Cys Arg Tyr Pro Tyr Glu Tyr Glu Gly Gly His Ile Lys Thr     50 55 60 Ala Val Asn Leu Pro Leu Glu Arg Asp Ala Glu Ser Phe Leu Leu Lys 65 70 75 80 Ser Pro Ile Ala Pro Cys Ser Leu Asp Lys Arg Val Ile Leu Ile Phe                 85 90 95 His Cys Glu Phe Ser Ser Glu Arg Gly Pro Arg Met Cys Arg Phe Ile             100 105 110 Arg Glu Arg Asp Arg Ala Val Asn Asp Tyr Pro Ser Leu Tyr Tyr Pro         115 120 125 Glu Met Tyr Ile Leu Lys Gly Gly Tyr Lys Glu Phe Phe Pro Gln His     130 135 140 Pro Asn Phe Cys Glu Pro Gln Asp Tyr Arg Pro Met Asn His Glu Ala 145 150 155 160 Phe Lys Asp Glu Leu Lys Thr Phe Arg Leu Lys Thr Arg Ser Trp Ala                 165 170 175 Gly Glu Arg Ser Arg Arg Glu Leu Cys Ser Arg Leu Gln Asp Gln             180 185 190 <210> 6 <211> 201 <212> PRT <213> Artificial Sequence <220> <223> Polypeptide with deletion mutation <400> 6 Gly Thr Ile Glu Asn Ile Leu Asp Asn Asp Pro Arg Asp Leu Ile Gly  1 5 10 15 Asp Phe Ser Lys Gly Tyr Leu Phe His Thr Val Ala Gly Lys His Gln             20 25 30 Asp Leu Lys Tyr Ile Ser Pro Glu Ile Met Ala Ser Val Leu Asn Gly         35 40 45 Lys Phe Ala Asn Leu Ile Lys Glu Phe Val Ile Ile Asp Cys Arg Tyr     50 55 60 Pro Tyr Glu Tyr Glu Gly Gly His Ile Lys Gly Ala Val Asn Leu His 65 70 75 80 Met Glu Glu Glu Val Glu Asp Phe Leu Leu Lys Lys Pro Ile Val Pro                 85 90 95 Thr Asp Gly Lys Arg Val Ile Val Val Phe His Cys Glu Phe Ser Ser             100 105 110 Glu Arg Gly Pro Arg Met Cys Arg Tyr Val Arg Glu Arg Asp Arg Leu         115 120 125 Gly Asn Glu Tyr Pro Lys Leu His Tyr Pro Glu Leu Tyr Val Leu Lys     130 135 140 Gly Gly Tyr Lys Glu Phe Phe Met Lys Cys Gln Ser Tyr Cys Glu Pro 145 150 155 160 Pro Ser Tyr Arg Pro Met His His Glu Asp Phe Lys Glu Asp Leu Lys                 165 170 175 Lys Phe Arg Thr Lys Ser Arg Thr Trp Ala Gly Glu Lys Ser Lys Arg             180 185 190 Glu Met Tyr Ser Arg Leu Lys Lys Leu         195 200 <210> 7 <211> 206 <212> PRT <213> Artificial Sequence <220> <223> Polypeptide with deletion mutation <400> 7 Gly Ser Gly Gly Gly Gly Gly Arg Leu Val Pro Arg Gly Ser Leu Ile  1 5 10 15 Gly Asp Phe Ser Lys Val Cys Ala Leu Pro Thr Val Ser Gly Lys His             20 25 30 Gln Asp Leu Lys Tyr Val Asn Pro Glu Thr Val Ala Ala Leu Leu Ser         35 40 45 Gly Lys Phe Gln Gly Leu Ile Glu Lys Phe Tyr Val Ile Asp Cys Arg     50 55 60 Tyr Pro Tyr Glu Tyr Leu Gly Gly His Ile Gln Gly Ala Leu Asn Leu 65 70 75 80 Tyr Ser Gln Glu Glu Leu Phe Asn Phe Phe Leu Lys Lys Pro Ile Val                 85 90 95 Pro Leu Asp Thr Gln Lys Arg Ile Ile Ile Val Phe His Cys Glu Phe             100 105 110 Ser Ser Glu Arg Gly Pro Arg Met Cys Arg Cys Leu Arg Glu Glu Asp         115 120 125 Arg Ser Leu Asn Gln Tyr Pro Ala Leu Tyr Tyr Pro Glu Leu Tyr Ile     130 135 140 Leu Lys Gly Gly Tyr Arg Asp Phe Phe Pro Glu Tyr Met Glu Leu Cys 145 150 155 160 Glu Pro Gln Ser Tyr Cys Pro Met His His Gln Asp His Lys Thr Glu                 165 170 175 Leu Leu Arg Cys Arg Ser Gln Ser Lys Val Gln Glu Gly Glu Arg Gln             180 185 190 Leu Arg Glu Gln Ile Ala Leu Leu Val Lys Asp Met Ser Pro         195 200 205 <210> 8 <211> 194 <212> PRT <213> Artificial Sequence <220> <223> Polypeptide with deletion mutation <400> 8 Glu Asn Ile Leu Asp Asn Asp Pro Arg Asp Leu Ile Gly Asp Phe Ser  1 5 10 15 Lys Gly Tyr Leu Phe His Thr Val Ala Gly Lys His Gln Asp Leu Lys             20 25 30 Tyr Ile Ser Pro Glu Ile Met Ala Ser Val Leu Asn Gly Lys Phe Ala         35 40 45 Asn Leu Ile Lys Glu Phe Val Ile Ile Asp Cys Arg Tyr Pro Tyr Glu     50 55 60 Tyr Glu Gly Gly His Ile Lys Gly Ala Val Asn Leu His Met Glu Glu 65 70 75 80 Glu Val Glu Asp Phe Leu Leu Lys Lys Pro Ile Val Pro Thr Asp Gly                 85 90 95 Lys Arg Val Ile Val Val Phe His Cys Glu Phe Ser Ser Glu Arg Gly             100 105 110 Pro Arg Met Cys Arg Tyr Val Arg Glu Arg Asp Arg Leu Gly Asn Glu         115 120 125 Tyr Pro Lys Leu His Tyr Pro Glu Leu Tyr Val Leu Lys Gly Gly Tyr     130 135 140 Lys Glu Phe Phe Met Lys Cys Gln Ser Tyr Cys Glu Pro Pro Ser Tyr 145 150 155 160 Arg Pro Met His His Glu Asp Phe Lys Glu Asp Leu Lys Lys Phe Arg                 165 170 175 Thr Lys Ser Arg Thr Trp Ala Gly Glu Lys Ser Lys Arg Glu Met Tyr             180 185 190 Ser Arg <210> 9 <211> 181 <212> PRT <213> Artificial Sequence <220> <223> Polypeptide with deletion mutation <400> 9 Glu Asn Ile Leu Asp Asn Asp Pro Arg Asp Leu Ile Gly Asp Phe Ser  1 5 10 15 Lys Gly Tyr Leu Phe His Thr Val Ala Gly Lys His Gln Asp Leu Lys             20 25 30 Tyr Ile Ser Pro Glu Ile Met Ala Ser Val Leu Asn Gly Lys Phe Ala         35 40 45 Asn Leu Ile Lys Glu Phe Val Ile Ile Asp Cys Arg Tyr Pro Tyr Glu     50 55 60 Tyr Glu Gly Gly His Ile Lys Gly Ala Val Asn Leu His Met Glu Glu 65 70 75 80 Glu Val Glu Asp Phe Leu Leu Lys Lys Pro Ile Val Pro Thr Asp Gly                 85 90 95 Lys Arg Val Ile Val Val Phe His Cys Glu Phe Ser Ser Glu Arg Gly             100 105 110 Pro Arg Met Cys Arg Tyr Val Arg Glu Arg Asp Arg Leu Gly Asn Glu         115 120 125 Tyr Pro Lys Leu His Tyr Pro Glu Leu Tyr Val Leu Lys Gly Gly Tyr     130 135 140 Lys Glu Phe Phe Met Lys Cys Gln Ser Tyr Cys Glu Pro Pro Ser Tyr 145 150 155 160 Arg Pro Met His His Glu Asp Phe Lys Glu Asp Leu Lys Lys Phe Arg                 165 170 175 Thr Lys Ser Arg Thr             180 <210> 10 <211> 203 <212> PRT <213> Artificial Sequence <220> <223> Polypeptide with deletion mutation <400> 10 Met Asp Glu Ile Glu Asn Leu Leu Asp Ser Asp His Arg Glu Leu Ile  1 5 10 15 Gly Asp Tyr Ser Lys Ala Phe Leu Leu Gln Thr Val Asp Gly Lys His             20 25 30 Gln Asp Leu Lys Tyr Ile Ser Pro Glu Thr Met Val Ala Leu Leu Thr         35 40 45 Gly Lys Phe Ser Asn Ile Val Asp Lys Phe Val Ile Val Asp Cys Arg     50 55 60 Tyr Pro Tyr Glu Tyr Glu Gly Gly His Ile Lys Thr Ala Val Asn Leu 65 70 75 80 Pro Leu Glu Arg Asp Ala Glu Ser Phe Leu Leu Lys Ser Pro Ile Ala                 85 90 95 Pro Cys Ser Leu Asp Lys Arg Val Ile Leu Ile Phe His Cys Glu Phe             100 105 110 Ser Ser Glu Arg Gly Pro Arg Met Cys Arg Phe Ile Arg Glu Arg Asp         115 120 125 Arg Ala Val Asn Asp Tyr Pro Ser Leu Tyr Tyr Pro Glu Met Tyr Ile     130 135 140 Leu Lys Gly Gly Tyr Lys Glu Phe Phe Pro Gln His Pro Asn Phe Cys 145 150 155 160 Glu Pro Gln Asp Tyr Arg Pro Met Asn His Glu Ala Phe Lys Asp Glu                 165 170 175 Leu Lys Thr Phe Arg Leu Lys Thr Arg Ser Trp Ala Gly Glu Arg Ser             180 185 190 Arg Arg Glu Leu Cys Ser Arg Leu Gln Asp Gln         195 200 <210> 11 <211> 196 <212> PRT <213> Artificial Sequence <220> <223> Polypeptide with deletion mutation <400> 11 Met Glu Asn Leu Leu Asp Ser Asp His Arg Glu Leu Ile Gly Asp Tyr 1 5 10 15 Ser Lys Ala Phe Leu Leu Gln Thr Val Asp Gly Lys His Gln Asp Leu             20 25 30 Lys Tyr Ile Ser Pro Glu Thr Met Val Ala Leu Leu Thr Gly Lys Phe         35 40 45 Ser Asn Ile Val Asp Lys Phe Val Ile Val Asp Cys Arg Tyr Pro Tyr     50 55 60 Glu Tyr Glu Gly Gly His Ile Lys Thr Ala Val Asn Leu Pro Leu Glu 65 70 75 80 Arg Asp Ala Glu Ser Phe Leu Leu Lys Ser Pro Ile Ala Pro Cys Ser                 85 90 95 Leu Asp Lys Arg Val Ile Leu Ile Phe His Cys Glu Phe Ser Ser Glu             100 105 110 Arg Gly Pro Arg Met Cys Arg Phe Ile Arg Glu Arg Asp Arg Ala Val         115 120 125 Asn Asp Tyr Pro Ser Leu Tyr Tyr Pro Glu Met Tyr Ile Leu Lys Gly     130 135 140 Gly Tyr Lys Glu Phe Phe Pro Gln His Pro Asn Phe Cys Glu Pro Gln 145 150 155 160 Asp Tyr Arg Pro Met Asn His Glu Ala Phe Lys Asp Glu Leu Lys Thr                 165 170 175 Phe Arg Leu Lys Thr Arg Ser Trp Ala Gly Glu Arg Ser Arg Arg Glu             180 185 190 Leu Cys Ser Arg         195 <210> 12 <211> 183 <212> PRT <213> Artificial Sequence <220> <223> Polypeptide with deletion mutation <400> 12 Met Glu Asn Leu Leu Asp Ser Asp His Arg Glu Leu Ile Gly Asp Tyr  1 5 10 15 Ser Lys Ala Phe Leu Leu Gln Thr Val Asp Gly Lys His Gln Asp Leu             20 25 30 Lys Tyr Ile Ser Pro Glu Thr Met Val Ala Leu Leu Thr Gly Lys Phe         35 40 45 Ser Asn Ile Val Asp Lys Phe Val Ile Val Asp Cys Arg Tyr Pro Tyr     50 55 60 Glu Tyr Glu Gly Gly His Ile Lys Thr Ala Val Asn Leu Pro Leu Glu 65 70 75 80 Arg Asp Ala Glu Ser Phe Leu Leu Lys Ser Pro Ile Ala Pro Cys Ser                 85 90 95 Leu Asp Lys Arg Val Ile Leu Ile Phe His Cys Glu Phe Ser Ser Glu             100 105 110 Arg Gly Pro Arg Met Cys Arg Phe Ile Arg Glu Arg Asp Arg Ala Val         115 120 125 Asn Asp Tyr Pro Ser Leu Tyr Tyr Pro Glu Met Tyr Ile Leu Lys Gly     130 135 140 Gly Tyr Lys Glu Phe Phe Pro Gln His Pro Asn Phe Cys Glu Pro Gln 145 150 155 160 Asp Tyr Arg Pro Met Asn His Glu Ala Phe Lys Asp Glu Leu Lys Thr                 165 170 175 Phe Arg Leu Lys Thr Arg Ser             180 <210> 13 <211> 182 <212> PRT <213> Artificial Sequence <220> <223> Polypeptide with deletion mutation <400> 13 Met Glu Asn Leu Leu Asp Ser Asp His Arg Glu Leu Ile Gly Asp Tyr  1 5 10 15 Ser Lys Ala Phe Leu Leu Gln Thr Val Asp Gly Lys His Gln Asp Leu             20 25 30 Lys Tyr Ile Ser Pro Glu Thr Met Val Ala Leu Leu Thr Gly Lys Phe         35 40 45 Ser Asn Ile Val Asp Lys Phe Val Ile Val Asp Cys Arg Tyr Pro Tyr     50 55 60 Glu Tyr Glu Gly Gly His Ile Lys Thr Ala Val Asn Leu Pro Leu Glu 65 70 75 80 Arg Asp Ala Glu Ser Phe Leu Leu Lys Ser Pro Ile Ala Pro Cys Ser                 85 90 95 Leu Asp Lys Arg Val Ile Leu Ile Phe His Cys Glu Phe Ser Ser Glu             100 105 110 Arg Gly Pro Arg Met Cys Arg Phe Ile Arg Glu Arg Asp Arg Ala Val         115 120 125 Asn Asp Tyr Pro Ser Leu Tyr Tyr Pro Glu Met Tyr Ile Leu Lys Gly     130 135 140 Gly Tyr Lys Glu Phe Phe Pro Gln His Pro Asn Phe Cys Glu Pro Gln 145 150 155 160 Asp Tyr Arg Pro Met Asn His Glu Ala Phe Lys Asp Glu Leu Lys Thr                 165 170 175 Phe Arg Leu Lys Thr Arg             180 <210> 14 <211> 196 <212> PRT <213> Artificial Sequence <220> <223> Polypeptide with deletion mutation <400> 14 Met Glu Glu Asp Ser Asn Gln Gly His Leu Ile Gly Asp Phe Ser Lys  1 5 10 15 Val Cys Ala Leu Pro Thr Val Ser Gly Lys His Gln Asp Leu Lys Tyr             20 25 30 Val Asn Pro Glu Thr Val Ala Ala Leu Leu Ser Gly Lys Phe Gln Gly         35 40 45 Leu Ile Glu Lys Phe Tyr Val Ile Asp Cys Arg Tyr Pro Tyr Glu Tyr     50 55 60 Leu Gly Gly His Ile Gln Gly Ala Leu Asn Leu Tyr Ser Gln Glu Glu 65 70 75 80 Leu Phe Asn Phe Phe Leu Lys Lys Pro Ile Val Pro Leu Asp Thr Gln                 85 90 95 Lys Arg Ile Ile Ile Val Phe His Cys Glu Phe Ser Ser Glu Arg Gly             100 105 110 Pro Arg Met Cys Arg Cys Leu Arg Glu Glu Asp Arg Ser Leu Asn Gln         115 120 125 Tyr Pro Ala Leu Tyr Tyr Pro Glu Leu Tyr Ile Leu Lys Gly Gly Tyr     130 135 140 Arg Asp Phe Phe Pro Glu Tyr Met Glu Leu Cys Glu Pro Gln Ser Tyr 145 150 155 160 Cys Pro Met His His Gln Asp His Lys Thr Glu Leu Leu Arg Cys Arg                 165 170 175 Ser Gln Ser Lys Val Gln Glu Gly Glu Arg Gln Leu Arg Glu Gln Ile             180 185 190 Ala Leu Leu Val         195

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 35/00 C12N 9/16 B 43/00 111 9/99 C12N 9/16 15/00 ZNAA 9/99 A61K 37/02 (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD, SL , SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID , IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Bolhany David United States Massachusetts 01609 Worcester Willowbrook Lane 122 (72) Inventor Epstein David United States Massachusetts 02478 Belmont Pine Street 56 (72) Inventor Dolph Johannes USA North Carolina 27705 Durham Stony Brook Drive 3423 (72) Inventor Littercart USA Massachusetts 02492 Newton Ashmont Avenue 30 (72) Inventor Fujimori Taro USA Massachusetts 01545 Shrewsbury Lantern Lane 10 (72) Inventor Robinson Simon United States Pennsylvania 19366 West Chester Brandy Ridge Drive 6 (72) Inventor Eckstein Jen United States Massachusetts 02476 Arlington Gracetreat 303 (72) Inventor Haopt Andreas Schwezengen Day-68723 Scherzichvek 52 (72) Inventor Walker Nigel California, USA 94010-6033 Burlingham Howland Hill Lane 20 (72) Inventor Dixon Richard W. United States Massachusetts 01536 North Grafton Samuel Drive 6 (72) Inventor Chocket Deborah United States Massachusetts 01543 Ratland 1 Central Tree Road 38 (72) Inventor Blanchard Jill United States Massachusetts State 02476-4109 Arlington Massachusetts Avenue 1265-A (72) Inventor Cluj Arthur United States Massachusetts 01773 Lincoln Old Country Road 111 (72) Inventor Pal Koror United States Massachusetts 02492 Newton Tudor Road 205 (72) Inventor Bokovich Nicholas 02148 Malden 2 McCormack, Massachusetts, USA Claude 83 (72) Inventor Kam John 02139 Cambridge Witney Avenue, USA Massachusetts 12 (72) Inventor Hediger Mark 01752 Marlborough Stearns Road 418 F Term (Reference) 4B024 AA01 BA11 CA04 DA06 EA04 GA11 4B050 CC01 CC03 DD11 LL01 4C084 AA02 AA07 AA17 BA01 BA10 BA32 BA44 CA59 NA14 ZA392 ZB112 ZB262

Claims (147)

[Claims] 1. A crystalline polypeptide comprising the Cdc25B protein or the catalytic domain of the Cdc25C protein. 2. The crystalline polypeptide of claim 1, wherein the polypeptide comprises the catalytic domain of human Cdc25B. 3. A crystalline polypeptide-ligand complex, wherein the polypeptide comprises the catalytic domain of the Cdc25 protein. 4. The crystalline polypeptide / ligand complex of claim 3, wherein the polypeptide comprises the catalytic domain of mammalian Cdc25. 5. The crystalline polypeptide / ligand complex of claim 4, wherein the mammalian Cdc25 protein is Cdc25A, Cdc25B or Cdc25C. 6. The mammalian Cdc25 protein is human Cdc25A, human Cdc25B or human C.
The crystalline polypeptide / ligand complex of claim 5, which is dc25C. 7. The crystalline polypeptide / ligand complex of claim 6, wherein the polypeptide comprises amino acids 336-523 of SEQ ID NO: 1. 8. The polypeptide comprises amino acids 351-540 of SEQ ID NO: 2,
The crystalline polypeptide according to claim 2. 9. The polypeptide comprises amino acids 351-540 of SEQ ID NO: 2,
The crystalline polypeptide / ligand complex of claim 4. 10. The ligand has the formula: 10. The crystalline polypeptide / ligand complex of claim 9, which is 11. The unit cell parameters a and b are about 70 Å,
11. The crystalline polypeptide / ligand complex of claim 10, wherein c is about 130 Å and α = β = γ = 90 °. 12. A method for determining the three-dimensional structure of a first polypeptide containing the catalytic domain of Cdc25 protein, comprising the steps of: (a) obtaining a crystal of the first polypeptide containing the catalytic domain of Cdc25; ) Obtaining x-ray diffraction data of the crystal, (c) Resolving the crystal structure of the crystal using atomic coordinates of a second polypeptide and the x-ray diffraction data, The second polypeptide is Cdc25B
Comprising a catalytic domain of a protein. 13. The method of claim 12, wherein the crystal of the first polypeptide comprises the first polypeptide complexed with a ligand. 14. The method of claim 12, wherein the first polypeptide comprises the catalytic domain of the mammalian Cdc25 protein. 15. The method of claim 14, wherein the first and second polypeptides individually comprise the catalytic domain of human Cdc25 protein. 16. The method of claim 15, wherein the first polypeptide comprises the catalytic domain of human Cdc25A, Cdc25B or Cdc25C and the second polypeptide comprises the catalytic domain of human Cdc25B. 17. The method of claim 16, wherein the first polypeptide comprises the catalytic domain of human Cdc25A. 18. The method of claim 16, wherein the first polypeptide comprises the catalytic domain of human Cdc25B. 19. The method of claim 16, wherein the first polypeptide comprises the catalytic domain of human Cdc25C. 20. A method for identifying a compound that is an inhibitor of Cdc25 protein, comprising the steps of: (a) obtaining a crystal containing a polypeptide containing the catalytic domain of cdc25 protein; and (b) atomic coordinates of the polypeptide. And (c) using the atomic coordinates to define a catalytic domain of Cdc25, (d) identifying a compound that matches the catalytic domain, wherein The compound is an inhibitor of the Cdc25 protein, and the steps. 21. The method of claim 20, further comprising assessing the ability of the compound identified in step (d) to inhibit Cdc25. 22. The Cdc25 protein is a mammalian protein.
The method described in. 23. The method of claim 22, wherein the Cdc25 protein is a human protein. 24. The Cdc25 protein is human Cdc25A, human Cdc25B or human Cdc25.
24. The method of claim 23, which is C. 25. The method of claim 20, wherein the crystal further comprises a ligand bound to the catalytic domain. 26. The method of claim 23, wherein the polypeptide comprises amino acids 351-540 of SEQ ID NO: 2. 27. The ligand has the formula: 25. The method of claim 24, which is 28. The method of claim 27, wherein the crystal has unit cell parameters a = b = 70.15 Å, c = 130.35 Å, and α = β = γ = 90 °. 29. A method for identifying a compound which is a potential inhibitor of Cdc25 protein, wherein the compound which will interact with one or more subsites in the catalytic domain of Cdc25 protein is A method comprising designing, based on crystal structure coordinates of a polypeptide comprising, wherein said compound is identified as a potential inhibitor of Cdc25 protein. 30. The Cdc25 protein is a mammalian Cdc25 protein.
9. The method according to 9. 31. The Cdc25 protein is a human Cdc25 protein.
The method described in. 32. The Cdc25 protein is human Cdc25A, human Cdc25B or human Cdc25.
32. The method of claim 31, which is C. 33. The method of claim 32, wherein the polypeptide comprises amino acids 336-540 of SEQ ID NO: 2. 34. The method of claim 33, wherein the polypeptide has the amino acid sequence set forth in SEQ ID NO: 5. 35. The method of claim 34, wherein the polypeptide has the amino acid sequence set forth in SEQ ID NO: 11. 36. The method of claim 35, wherein the crystal structure coordinates are as set forth in Figures 15A-15PPP. 37. The method of claim 32, wherein the crystal structure coordinates are those set forth in Figures 18A-18X. 38. The method of claim 32, wherein the crystal structure coordinates are as set forth in Figures 17A-17EE. 39. The method of claim 32, wherein the crystal structure coordinates are as set forth in Figures 19A-19I. 40. The method of claim 31, wherein the compound interacts with one or more of subsites 1-16. 41. The method of claim 40, wherein the compound interacts with two or more of subsites 1-16. 42. The method of claim 41, wherein the compound interacts with three or more of subsites 1-16. 43. The method of claim 41, wherein the compound interacts with a set of subsites including subsite 1 and subsite 2. 44. The method of claim 42, wherein the compound interacts with a set of subsites including subsite 1, subsite 2 and subsite 3. 45. The method of claim 41, wherein the compound interacts with a set of subsites, including subsite 1 and subsite 5. 46. The method of claim 41, wherein the compound interacts with a set of subsites including subsite 1 and subsite 3. 47. The method of claim 42, wherein the compound interacts with a set of subsites including subsite 1, subsite 4 and subsite 5. 48. The method of claim 42, wherein the compound interacts with a set of subsites including subsite 1, subsite 5, and subsite 6. 49. The method of claim 42, wherein the compound interacts with a set of subsites including subsite 1, subsite 7 and subsite 8. 50. The method of claim 42, wherein the compound interacts with a set of subsites including subsite 1, subsite 2 and subsite 9. 51. The compound is subsite 1, subsite 2, or subsite 4.
43. The method of claim 42, wherein the method interacts with a set of subsites, including and subsite 9. 52. The method of claim 42, wherein the compound interacts with a set of subsites including subsite 1, subsite 3 and subsite 9. 53. The sub-site 1, sub-site 3, sub-site 4
43. The method of claim 42, wherein the method interacts with a set of subsites, including and subsite 9. 54. (a) a negatively charged functional group at a position of interacting with Arg479 of human Cdc25B; (b) one or more of Cys426, Tyr428, Pro444, Glu446 and Thr547 of human Cdc25B; A hydrogen bond donor or a positively charged functional group at a position of interaction; (c) a hydrogen bond acceptor or a negatively charged position at a position of interaction with one or more of Tyr428, Arg482 and Arg544 of human Cdc25B. (D) a hydrophobic moiety at a position that interacts with one or more of Leu445, Glu446, Arg479, Met483, Thr547 and Arg548; (e) one or more of Arg482 and Arg544 of human Cdc25B. A negatively charged functional group at a position that interacts with (f) a hydrophobic portion at a position that interacts with one or more of Glu478, Arg479, Met531 and Arg544 of human Cdc25B; One of Tyr428, Glu478, Arg479, Met531, Leu540, and Arg544 (H) a hydrophobic portion in a position that interacts with the above; (h) a hydrophobic portion in a position that interacts with one or more of human Cdc25B Phe475, Met531, Asn532, and Leu540; (i) human Cdc25B Phe475 and (J) A hydrophobic moiety at a position that interacts with one or more of Ser477; (j) a hydrophobic moiety at a position that interacts with one or more of Glu474, Phe475, Met531, and Asn532 of human Cdc25B; ) A hydrophobic moiety at a position that interacts with one or more of Tyr428, Met531, Lys537, Lys541, Leu540 and Arg544 of human Cdc25B; (l) a hydrogen bond donor at a position that interacts with Ser477 of human Cdc25B. Or a hydrogen bond acceptor; (m) a hydrogen bond donor or a positively charged functional group at a position interacting with Glu478 of human Cdc25B; (n) a negative charge at a position interacting with Lys394 of human Cdc25B Functional group: (o) Interaction with Arg482 of human Cdc25B A negatively charged functional group at a position (p) a negatively charged functional group that interacts with Arg544 of human Cdc25B; and (q) a hydrophobic moiety at a position that interacts with Asn532 of human Cdc25B. And a Cdc25 inhibitor comprising two or more of a hydrogen bond donor or a hydrogen bond acceptor. 55. The Cdc25 inhibitor according to claim 54, which comprises (a) and (e). 56. The Cdc25 inhibitor according to claim 54, comprising (a) and at least one of (b), (c) and (d). 57. The Cdc25 inhibitor according to claim 56, further comprising (e). 58. The Cdc2 of claim 54, which comprises (a), (e) and (f).
5 inhibitors. 59. The Cdc25 inhibitor according to claim 54, which comprises (a) and (g). 60. The Cdc2 of claim 54, which comprises (a), (f) and (g).
5 inhibitors. 61. Cdc2 according to claim 54, which comprises (a), (g) and (h).
5 inhibitors. 62. It includes (a) and at least one of (i) and (j),
The Cdc25 inhibitor according to claim 54. 63. The Cdc25 inhibitor according to claim 54, comprising (a), (k), and at least one of (b), (c) and (d). 64. The Cdc25 inhibitor according to claim 63, further comprising (f). 65. The Cdc25 inhibitor according to claim 55, further comprising (k). 66. The Cdc25 inhibitor according to claim 61, further comprising (f). 67. A method of treating a Cdc25-mediated condition in a patient, comprising administering to the patient a therapeutically effective amount of the Cdc25 inhibitor of claim 54. 68. The method of claim 67, wherein the patient is a human. 69. The method of claim 67, wherein the Cdc25-mediated condition is characterized by excessive cell proliferation. 70. The method of claim 69, wherein the Cdc25-mediated condition is cancer, coronary restenosis, reocclusion, and inflammation. 71. Formula I: Or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable prodrug thereof.
Or a combination thereof, wherein:   R₁Is R₃-CO ； R_FourR_FiveN-CO ； R₆SO₂; R₇R₈NSO₂And   However, in the formula, R₃, R_Four, R_Five, R₆, R₇, R₈Are individually hydrogen, substituted or
Substituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted cycloalkyl
Group, substituted or unsubstituted heterocycloalkyl, E- or Z-aryl-C₂-C_Four-
Alkenyl or aryl-C₂-C_Four-Alkynyl (original: alkinyl); yes
I R_FourAnd R_FiveForms a 4- to 7-membered heterocycle with a nitrogen atom; or
R₇And R₈Forms a 4- to 7-membered heterocycle with a nitrogen atom; or   R₃-CO is the formula R₉-CO-A₆Amino acid residue (in the formula,     R₉Is hydrogen, C_1-6Alkyl, phenyl, benzyl, naphthyl, benzyloxy
Or C_1-6Is alkoxy;     A6 is aspartyl, asparagyl, prolyl, alanyl, valyl, lysyl,
Glycyl, arginyl, isoleucyl, ceryl, threonyl, leucyl, tryp
Tofanyl, cysteinyl, tyrosyl, methionyl, glycyl, glutamyl, fe
Is nilanalanyl or histidyl); A1 is of formula II: Is an amino acid residue of   However, in the formula, R_TenAnd R₁₁Are individually hydrogen or C_1-6Alkyl;   n is 0, 1 or 2;   X is SO₃H, SO₂NR₁₂R₁₃, CH₂-SO₃H, CF₂-SO₃H, CH₂-SO₂NR₁₂R₁₃, CF₂-SO₂NR₁₂ R₁₃(In the formula, R₁₂And R₁₃Are hydrogen and C_1-6Alkyl or substituted
Is an unsubstituted phenyl, benzyl, furanyl, thiophenyl, thiazolyl, isothiphenyl.
Azolyl, pyrazolyl, isoxazolyl or oxazolyl; or R_{1 2} Is hydrogen, and R₁₃Is hydroxy, C_1-6Alkoxy, C_1-6Alkylcarbonyl or
Is substituted or unsubstituted benzolyl); or   X is PO₃H₂, OCH₂PO₃H₂, CH₂PO₃H₂, CF₂PO₃H₂, COOH, CH₂-COOH, CF₂-CO₂H, O
CH₂CO₂H, OCF₂CO₂H, OCH (CO₂H)₂, OCF (CO₂H)₂Or;   X is NH-SO₂-R₁₄(In the formula, R₁₄Is C_1-6Alkyl, benzyl, phenyl
); Or   X is NH-CO-COO-R₁₅(In the formula, R₁₅Is C_1-6Alkyl, benzyl, or phenyl
Is);   Z is hydrogen, C_1-6Alkyl, halogen, hydroxy, C_1-6Alkylamino, di-
C_1-6Alkylamino, C_1-6Alkoxy, C_1-6Alkylthio, C_1-6Alkyl carbo
Nyl, halogen substitution C_1-6Alkylcarbonyl, formyl-, phenylcarbonyl
, Benzylcarbonyl, C_1-6Alkyl-sulfonyl, C_1-6Alkyl-sulfonyl-
Amino, carboxyl, O-C_1-6Alkyl carboxyl, carboxyl alk
Nil, O-C_1-6Alkyl carboxyl alkenyl, C_1-6Alkyl carbamyl,
Is it cyano, nitro, trifluoromethyl, oxytrifluoromethyl;
Ruiha   Z is (CH₂)_m-NR₁₆R₁₇Where m is 0, 1 or 2 and R₁₆ And R ₁₇ Are individually hydrogen, C_1-6Alkyl, C_1-6Alkyl-carbonyl, amino
-C_2-6Alkyl, C_1-6Alkyl-amino-C_2-6Alkyl, di-C_1-6Alkyl-amino-
C_2-6Alkyl, hydroxy-C_1-6Alkyl, C_1-6Alkoxy-C_1-6Alkyl, ant
-C_1-6Alkyl, C_3-8Cycloalkyl-C_1-6Alkyl and heterocyclo
Alklyl-C_1-6Selected from the group consisting of alkyl);   A2 has the formula III: Is an amino acid residue of   However, in the formula, R₁₈ And R₁₉ Are individually hydrogen or C_1-6Alkyl;
Or   R₂₀Is glycine, alanine, valine, leucine, isoleucine, norvaline
, Norleucine, aspartic acid, glutamic acid, lysine, asparagine, glu
Tamine, phenylalanine, histidine, homoleucine, C_1-6Alkyl-gluta
Minic acid, C_1-6Selected from the group consisting of alkyl-aspartic acid and lysine- (Boc)
Is a side chain of an amino acid that is   R₂₀Is? (CH₂)_o-COOR_{twenty one} Where o is from about 3 to about 5 and R_{twenty one}Is
Hydrogen or C_1-6Is alkyl); or   R₁₉ And R₂₀Forms a 3- to 7-membered carbocyclic ring system with the alpha carbon;
Ruiha   R₁₈ And R₂₀Together with the α-position carbon atom and nitrogen atom together with one substituted or unsubstituted
Form a substituted 4- to 7-membered heterocyclic ring system; or   R₁₈ And R₂₀Is an 8- to 12-membered ring compound together with a carbon atom and a nitrogen atom at the α-position.
Forming an elementary double ring system;   A3 has the formula IV: Is an amino acid of   However, in the formula, R_{twenty two} And R_{twenty three} Are individually hydrogen or C_1-6Is it alkyl
; Or   R_{twenty four}Is glycine, alanine, valine, leucine, isoleucine, norvaline
, Norleucine, aspartic acid, glutamic acid, lysine, asparagine, glu
Tamine, phenylalanine, histidine, homoleucine, C_1-6Alkyl-gluta
Minic acid, C_1-6Selected from the group consisting of alkyl-aspartic acid and lysine- (Boc)
The side chain of an amino acid_{twenty four}Is (CH₂)_o-COOR_{twenty one} (In the formula, o
Is about 3 to about 5 and R_{twenty one}Is hydrogen or C_1-6Is alkyl)
;   R_{twenty three} And R_{twenty four} May together form a 3- to 7-membered carbocyclic ring system
Or;   R_{twenty two} And R_{twenty four}Represents, together with the carbon atom and the nitrogen atom in the α-position, a single substitution or
Forming an unsubstituted 4 to 7 membered heterocyclic ring system; or   R_{twenty two} And R_{twenty four}Is an 8- to 12-membered group together with the carbon atom and the nitrogen atom in the α-position.
Forming a heterobicyclic ring system of rings; or   A2 and A3 are both   6-amino-5-oxoperhydropyrido [2,1-b] [1,3] thiazole-3-carboxylic acid;   6-amino-5-oxoperhydro-3-indolizinecarboxylic acid; (S, R) -6-ami
No-5-oxoperhydro-8a-indolizinecarboxylic acid; (R, R) -6-amino-5-oxy
Soperhydro-8a-indolizinecarboxylic acid; (R, S) -6-amino-5-oxoper
Hydro-8a-indolizinecarboxylic acid; (S, S) -6-amino-5-oxoperhydro-8a
-Indolizinecarboxylic acid; 2- (3-amino-2-oxo-1,2-dihydro-1-pyridini
) Acetic acid; 2- (3-amino-2-oxo-6-phenyl-1,2-dihydro-1-pyridinyl) acetic acid
3-Aminobenzoic acid; 4-Aminobenzoic acid; 3-Aminomethylbenzoic acid; (S)-
3- (1-aminoethyl) benzoic acid; (R) -3- (1-aminoethyl) benzoic acid; (S) -3- (1-a
Minopropyl) benzoic acid; (R) -3- (1-aminopropyl) benzoic acid; (S) -3- (1-a
Minobutyl) benzoic acid; (R) -3- (1-aminobutyl) benzoic acid; 2- (3-amino-2-o
Xo-1-azepanyl) acetic acid; 2- [8- (aminomethyl) -3,6-dimethyl-9,10,10-trio
Xo-9,10-dihydro-10⁶-Thioxanthen-1-yl] acetic acid; 2- (2-oxopiperazi
No) acetic acid; 2- [8- (aminomethyl) -2-oxo-5-phenyl-2,3-dihydro-1H-1,4-be
Nazodiazepin-1-yl] acetic acid; 2- [8- (aminomethyl) -2-oxo-5-methyl-2,3-di
Hydro-1H-1,4-benzodiazepin-1-yl] acetic acid; 3-aminopropanoic acid; 4-amino
Butanoic acid; 5-aminopentanoic acid; 2- [2- (2-aminoethoxy) ethoxy] acetic acid; and
2- (3-amino-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepin-1-yl) vinegar
One residue selected from the group consisting of acids;   A4 is the formula V: Is an amino acid of   R_{twenty five}Is hydrogen or C_1-6Alkyl;   R₂₆Is hydrogen or C_1-6Is alkyl; and   R₂₇Is (CH₂)_p-(CH (R₂₈))_q-Aryl, where p is 0, 1 or 2
Q is 0, 1 or 2; and R₂₈Is hydrogen or methyl); and   R₂NR₃₂R₃₃And   Where R₃₂Is hydrogen or C_1-6Alkyl; and   R₃₃Is (CH₂)_w-W- (CH₂)_x-V, where     W is a single bond, and the sum of w and x is 1 to 6; or
Is     W is a substituted or unsubstituted aryl or aryl-T, where T is O
, S or NH; w is 0, 1, 2 or 3 and x is 0, 1, 2 or 3
Yes); or     W is C_3-8Cycloalkyl; w is 0, 1, 2 or 3 and x is
, 0, 1, 2 or 3; and     V is COOR₃₄ And (where R is₃₄Is hydrogen and C_1-6Is alkyl); is
Iha     V is COC_1-6Alkyl, CONH₂, SO₃H or NO₂; Is; or   R₂Is of formula VI:   Is the amino acid A5 of However, in the formula, R₃₅ And R₃₆ Are individually hydrogen or C_1-6Alkyl;   R₃₇Is aspartic acid, asparagine, glutamic acid, glutamine, aspa
Rutile-C_1-6Alkyl ester, glutamyl-C_1-6Is the side chain of an alkyl ester
; Or   R₃₇Is (CH₂)_y-COOR₄₂Where y is 3, 4 or 5 and R₄₂Is
Hydrogen or C_1-6Alkyl); or   R₃₇Is (CH₂)_z-CONR₄₀R₄₁Where z is 1 to 5 and R₄₀Over
And R₄₁ Are individually hydrogen or C₁-₆-Alkyl or R₄₀, R₄₁ as well as
The nitrogen atoms together form a 5- to 8-membered heterocycle); or   R₃₇Is (CH₂)_a-SO₃H (where a is 1, 2, 3, 4 or 5); or (CH₂)_b-Te
Is trazolyl, where b is 1, 2, 3, 4 or 5; or   R₃₇Is (CH₂)_d-Phenyl- (CH₂)_e-COOR₄₃ (Where d is 0 to 2 and
, E is 0 to 2 and R₄₃Is hydrogen or C_1-6Alkyl); yes
Is   R₃₇Is (CH₂)_d-Phenyl- (CH₂)_e-CONR₄₄R₄₅ (Where d is 0 to 2 and
, E is 0 to 2, and R₄₄And R₄₅ Individually represents hydrogen, C_1-6Al
Kill or R₄₄And R₄₅ And nitrogen atoms together form a single 5-8
Form a membered heterocycle);   U is hydroxy, C_1-6Alkoxy or NR₃₈R₃₉(Where R is₃₈ And R₃₉ 
Are each individually hydrogen; substituted or unsubstituted C_1-10-Alkyl, substituted
Is an unsubstituted aryl or a substituted or unsubstituted cycloalkyl or bicycloalkyl
Rukiru); or   R₃₈And R₃₉Forms a single 4- to 7-membered heterocycle with the nitrogen atom
, Compound, or pharmaceutically acceptable salt thereof, pharmaceutically acceptable prodrug thereof
, Or a combination of these. 72. R ₃ , at least one of R ₄ and R ₅ , R ₆ , or R ₇ and
At least one of R _8, but, hydroxy, C _1-6 alkoxy, phenoxy, benzyloxy, halogen, amino, C _1-6 alkylamino, di -C _1-6 alkylamino, C _1-6 alkyl -CO-NH 72. The compound of claim 71, which is an alkyl substituted with one or more substituents individually selected from the group consisting of: substituted and unsubstituted aryl, and substituted and unsubstituted cycloalkyl. 73. R ₃ , at least one of R ₄ and R ₅ , at least one of R ₆ , or at least one of R ₇ and R ₈ is substituted and unsubstituted phenyl, naphthyl, anthracenyl, phenanthrenyl. , Fluorenyl, pyridyl, pyridazinyl, pyridinonyl, furanyl, thienyl, thiazolyl, isothiazolyl, imidazolyl,
Substituted with at least one aryl group selected from the group consisting of triazolyl, pyrrolyl, tetrazolyl, benzimidazolyl, pyrazinyl, pyrimidyl, quinolyl, isoquinolyl, benzofuranyl, benzothienyl, pyrazolyl, indolyl, purinyl, isoxazolyl, oxazolyl, dibenzofuranyl. 73. The compound of claim 72, which is alkyl. 74. The aryl group is C _1-6 alkyl, halogen, hydroxy,
C _1-6 alkylamino, di-C _1-6 alkylamino, C _1-6 alkoxy, C _1-6 alkylthio, C _1-6 alkylcarbonyl, phenylcarbonyl, benzylcarbonyl,
C _1-6 alkyl-sulfonyl, C _1-6 alkyl-sulfonyl-amino, C _1-6 alkyl-carbonyl-amino, carboxyl, O-C _1-6 alkyl carboxyl, carboxyl alkenyl, O-C _1-6 alkyl carboxyl alkenyl, C _1-6 alkyl carbamyl, cyano, nitro, substituted with one or more substituents selected individually from the group consisting of trifluoromethyl and oxy trifluoromethyl a compound according to claim 73. 75. R ₃ , at least one of R ₄ and R ₅ , R ₆ , or R ₇
And at least one of R ₈ is alkyl substituted with at least one cycloalkyl selected from the group consisting of C _3-8 -cycloalkyl, adamantyl and bicyclooct [3.3.0] -yl. 72. The compound according to 72. 76. The cycloalkyl group is C _1-6 alkyl, halogen, hydroxy, C _1-6 alkylamino, di-C _1-6 alkylamino, C _1-6 alkoxy, C _1-6
Alkylthio, and C _1-6 from the group consisting of alkylcarbonyl is substituted with one or more substituents selected individually, the compound according to claim 75. 77. R ₃ , at least one of R ₄ and R ₅ , R ₆ , or R ₇
And at least one of R ₈ is a cycloalkyl group selected from the group consisting of substituted and unsubstituted C _3-8 -cycloalkyl, adamantyl and bicyclooct [3.3.0] -yl. The compound according to. 78. The cycloalkyl group, C _1-6 alkyl, halogen, hydroxy, C _1-6 alkylamino, di -C _1-6 alkylamino, C _1-6 alkoxy, C _1-6 alkylthio, and C _78. The compound of claim 77, which is substituted with one or more substituents individually selected from the group consisting of _1-6 alkylcarbonyl. 79. R ₃ , at least one of R ₄ and R ₅ , R ₆ , or R ₇
And at least one of R ₈ is one aryl-EC _2-4 -alkenyl, aryl-
A ZC _2-4 -alkenyl or aryl-C _2-4 -alkynyl (original term alkinyl) group,
In the formula, the aryl group is a substituted or unsubstituted phenyl, naphthyl, anthracenyl, phenanthrenyl, fluorenyl, pyridyl, pyridazinyl, pyridinonyl, furanyl, thienyl, thiazolyl, isothiazolyl, imidazolyl, triazolyl, pyrrolyl, tetrazolyl, benzimidazolyl, pyrazinyl, 72. The compound of claim 71 selected from the group consisting of pyrimidyl, quinolyl, isoquinolyl, benzofuranyl, benzothienyl, pyrazolyl, indolyl, purinyl, isoxazolyl, oxazolyl, and dibenzofuranyl. 80. The aryl group is C _1-6 alkyl, halogen, hydroxy,
C _1-6 alkylamino, di-C _1-6 alkylamino, C _1-6 alkoxy, C _1-6 alkylthio, C _1-6 alkylcarbonyl, phenylcarbonyl, benzylcarbonyl,
C _1-6 alkyl-sulfonyl, C _1-6 alkyl-sulfonyl-amino, C _1-6 alkyl-carbonyl-amino, carboxyl, OC _1-6 alkyl carboxyl, carboxyl alkenyl, O-C _1-6 alkyl carboxyl alkenyl, One or more individually selected from the group consisting of C _1-6 alkylcarbamyl, cyano, nitro, trifluoromethyl, oxytrifluoromethyl, substituted and unsubstituted cycloalkyl and substituted and unsubstituted heterocycloalkyl 80. The compound of claim 79, which is substituted with a substituent. 81. R ₃ , at least one of R ₄ and R ₅ , R ₆ , or R ₇
And at least one of R ₈ is a substituted or unsubstituted pyrrolidinyl, piperazinyl, tetrahydropyranyl, tetrahydrofuranyl, pyrrolidinonyl, and one substituted or unsubstituted heterocycloalkyl group selected from the group consisting of morpholinyl, Item 72. The compound according to Item 71. 82. R ₃ , at least one of R ₄ and R ₅ , R ₆ , or R ₇
And at least one of R ₈ is a substituted or unsubstituted phenyl, naphthyl, anthracenyl, phenanthrenyl, fluorenyl, pyridyl, pyridazinyl, pyridinonyl, furanyl, thienyl, thiazolyl, isothiazolyl, imidazolyl, triazolyl, pyrrolyl, tetrazolyl, benzimidazolyl, pyrazinyl. 72. The compound of claim 71, which is one aryl group selected from the group consisting of :, pyrimidyl, quinolyl, isoquinolyl, benzofuranyl, benzothienyl, pyrazolyl, indolyl, purinyl, isoxazolyl, oxazolyl, and dibenzofuranyl. 83. The aryl group is C _1-6 alkyl, halogen, hydroxy,
C _1-6 alkylamino, di -C _1-6 alkylamino, C _1-6 alkoxy, C _1-6 alkylthio, C _1-6 alkylcarbonyl, phenylcarbonyl, benzylcarbonyl, C _{1 -6} alkyl - sulfonyl, C _1-6 alkyl-sulfonyl-amino, C _1-6 alkyl-carbonyl-amino, carboxyl, OC _1-6 alkyl carboxyl, carboxyl alkenyl, O-C _1-6 alkyl carboxyl alkenyl, C _1-6 alkyl carbamyl, cyano , Nitro, trifluoromethyl, oxytrifluoromethyl, substituted and unsubstituted cycloalkyl and substituted with one or more substituents individually selected from the group consisting of substituted and unsubstituted heterocycloalkyl. 71. The compound according to 71. 84. W is aryl or aryl-T, wherein the aryl group is substituted or unsubstituted phenyl, naphthyl, pyridyl, furanyl, thienyl.
72. The compound of claim 71 selected from the group consisting of: and pyrimidyl. 85.] R ₂ is of the formula VI, and in U is NR ₃₈ R ₃₉ (wherein, at least one for R ₃₈ and R ₃₉ is hydroxy, halogen, substituted and unsubstituted aryl and substituted and 72. A compound according to claim 71, which is one C _1-6 -alkyl group substituted with one or more substituents individually selected from the group consisting of unsubstituted cycloalkyl). 86. At least one of R ₃₈ and R ₃₉ is substituted or unsubstituted phenyl, naphthyl, anthracenyl, phenanthrenyl, fluorenyl, pyridyl,
Pyridazinyl, pyridinonyl, furanyl, thienyl, thiazolyl, isothiazolyl, imidazolyl, triazolyl, pyrrolyl, tetrazolyl, benzimidazolyl, pyrazinyl, pyrimidyl, quinolyl, isoquinolyl, benzofuranyl, benzothienyl, pyrazolyl, indolyl, purinyl, isoxazolyl, oxazolyl, benzoazolyl, benzoazolyl It becomes one of C to be replaced individually one or more aryl groups selected from the group _1-6 - alkyl group a compound according to claim 85. 87. At least one of R ₃₈ and R ₃₉ is one C _1-6 -alkyl group substituted with one or more aryl groups, and at least one of the aryl groups is C _1-6 alkyl. , Halogen, hydroxy, C _1-6 alkylamino, di-C _1-6 alkylamino, C _1-6 alkoxy, C _1-6 alkylthio, C _1-6 alkylcarbonyl, phenylcarbonyl, benzylcarbonyl, C _1-6 Alkyl-sulfonyl, C _1-6 alkyl-sulfonyl-amino, C _1-6 alkyl-carbonyl-amino, carboxyl, O-
One or more selected from the group consisting of C _1-6 alkyl carboxyl, carboxyl alkenyl, O-C _1-6 alkyl carboxyl alkenyl, C _1-6 alkylcarbamyl, cyano, nitro, trifluoromethyl and oxytrifluoromethyl 87. The compound of claim 86, which is substituted with the substituents of. 88. At least one of R ₃₈ and R ₃₉ is a substituted or unsubstituted C _3-8-.
Cycloalkyl, one of C _1-6 substituted with one or more cycloalkyl groups selected individually from adamantyl and the group consisting bicyclooctyl -. Alkyl group, A compound according to claim 87. 89. are those wherein R ₂ is of formula VI, and in U is NR ₃₈ R ₃₉ (wherein, at least one for R ₃₈ and R ₃₉ are substituted and unsubstituted phenyl, naphthyl, anthracenyl, phenanthrenyl, fluorenyl , Pyridyl, pyridazinyl, pyridinonyl, furanyl, thienyl, thiazolyl, isothiazolyl, imidazolyl, triazolyl, pyrrolyl, tetrazolyl, benzimidazolyl, pyrazinyl, pyrimidyl, quinolyl, isoquinolyl, benzofuranyl, benzothienyl, pyrazolyl, indolyl, purinyl, isoxazolyl, oxazolyl, oxazolyl, oxazolyl, oxazolyl 72. A compound according to claim 71 which is one aryl group selected from the group consisting of furanyl). 90. At least one of R ₃₈ and R ₃₉ is C _1-6 alkyl, halogen, hydroxy, C _1-6 alkylamino, di-C _1-6 alkylamino, C _1-6 alkoxy, C _{1- 6} alkylthio, C _1-6 alkylcarbonyl, phenylcarbonyl, benzylcarbonyl, C _1-6 alkyl - sulfonyl, C _1-6 alkyl - sulfonyl - amino, C _1-6 alkyl - carbonyl - amino, carboxyl, O-C _{1 -6} alkyl carboxyl,
Substituted with one or more substituents individually selected from the group consisting of carboxylalkenyl, O-C _1-6 alkylcarboxylalkenyl, C _1-6 alkylcarbamyl, cyano, nitro, trifluoromethyl and oxytrifluoromethyl. 90. The compound of claim 89, which is one aryl group represented by: 91.] are those wherein R ₂ is of formula VI, and in U is NR ₃₈ R ₃₉ (wherein, R ₃₈ and at least one of substituted and unsubstituted C of R _{39 3-8} - cycloalkyl, adamantyl and 72. A compound according to claim 71 which is one cycloalkyl group selected from the group consisting of bicyclooctyl). 92. The cycloalkyl group is C _1-6 alkyl, halogen, hydroxy, C _1-6 alkylamino, di-C _1-6 alkylamino, C _1-6 alkoxy, C _1-6 alkylthio or C 1. _92. The compound of claim 91, which is substituted with one or more substituents individually selected from the group consisting of _1-6 alkylcarbonyl. 93. At least one of R ₁₆ and R ₁₇ is one aryl-C _1-6 alkyl group, wherein the aryl group is a substituted or unsubstituted phenyl, naphthyl, anthracenyl, phenanthrenyl, fluorenyl group. , Pyridyl, pyridazinyl, pyridinonyl, furanyl, thienyl, thiazolyl, isothiazolyl, imidazolyl, triazolyl, pyrrolyl, tetrazolyl, benzimidazolyl, pyrazinyl, pyrimidyl, quinolyl, isoquinolyl, benzofuranyl, benzothienyl, pyrazolyl, indolyl, purinyl, isoxazolyl, oxazolyl, oxazolyl, oxazolyl, oxazolyl 72. The compound of claim 71 selected from the group consisting of furanyl. 94. At least one of R ₁₆ and R ₁₇ is one aryl-C _1-6 alkyl group, wherein the aryl group is C _1-6 alkyl, halogen, hydroxy, C _1-6. Alkylamino, di-C _1-6 alkylamino, C _1-6 alkoxy, C _1-6 alkylthio, C _1-6 alkylcarbonyl, phenylcarbonyl, benzylcarbonyl, C _1-6 alkyl-sulfonyl, C _1-6 alkyl -Sulfonyl-amino, C _1-6 alkyl-carbonyl-amino, carboxyl, O-C _1-6 alkylcarboxyl, carboxylalkenyl, O-C _1-6 alkylcarboxylalkenyl, C _1-6 alkylcarbamyl, cyano, nitro 10. Substituted with one or more substituents individually selected from the group consisting of, trifluoromethyl and oxytrifluoromethyl.
The compound according to 3. 95. At least one of R ₁₆ and R ₁₇ is one heterocycloalkyl-C _1-6 alkyl group, wherein the heterocycloalkyl group is a substituted or unsubstituted pyrrolidinyl, piperazinyl, tetrahydro group. 72. The compound of claim 71 selected from the group consisting of pyranyl, tetrahydrofuranyl, pyrrolidinonyl and morpholinyl. 96. R ₂₇ is — (CH ₂ ) _p — (CH (R ₂₈ )) _q -aryl, wherein the aryl group is substituted and unsubstituted phenyl, naphthyl, anthracenyl, phenanthrenyl, Fluorenyl, pyridyl, pyridazinyl, pyridinonyl, furanyl,
Thienyl, thiazolyl, isothiazolyl, imidazolyl, triazolyl, pyrrolyl, tetrazolyl, benzimidazolyl, pyrazinyl, pyrimidyl, quinolyl,
72. A compound according to claim 71 selected from the group consisting of isoquinolyl, benzofuranyl, benzothienyl, pyrazolyl, indolyl, purinyl, isoxazolyl, oxazolyl, tetrahydronaphthyl, benzodihydrofuranyl, quinazoline and dibenzofuranyl. 97. The aryl group is C_1-6Alkyl, halogen, hydroxy,
C_1-6Alkylamino, di-C_1-6Alkylamino, C_1-6Alkoxy, C_1-6Alkyl
Thio, C_1-6Alkylcarbonyl, halogen substitution C_1-6Alkyl carbonyl,
Mill, phenylcarbonyl, benzylcarbonyl, C_1-6Alkyl-sulfonyl, C _1-6 Alkyl-sulfonylamino, carboxyl, O-C_1-6Alkyl carboxyl,
Carboxylalkenyl, O-C_1-6Alkylcarboxyalkenyl, C_1-6Alkyl
Carbamyl, cyano, nitro, trifluoromethyl, oxytrifluoromethyl
, Aryl; Y- (CH₂)_s-C_3-8-Cycloalkyl and Y- (CH₂)_s-Aryl (wherein Y
Is O, S or NH, and s is 0, 1, 2 or 3); Y- (CH₂)_t-F
Telocycloalkyl (wherein t is 1 to 6); Y- (CH₂)_u-R₂₉ (Where Y is
, O, NH, or S, u is 2 to 6, and R₂₉Is OH, CH₂-OH, NH₂ or
Is NH (C = NH) NH₂); Y- (CH₂)_v-R₃₀ (In the formula, Y is O, NH, or S, and v is
, 1 to 6 and R₃₀Is the COC_1-6Alkyl, COOH or CONH₂); And
And Y-(CH = CH) -R₃₁(In the formula, R₃₁Is the COC_1-6Alkyl, COOH, CONH₂ Or pheny
Is substituted with one or more substituents individually selected from the group consisting of
The compound according to claim 96. 98. The aryl groups in the aryl substituent are each independently hydroxy, amino, carboxyl, carboxamide, halogen, hydroxy,
Substituted or unsubstituted, which can be substituted with one or more of C _1-6 alkylamino, di-C _1-6 alkylamino, C _1-6 alkoxy, C _1-6 alkylthio, and C _1-6 alkylcarbonyl. Phenyl, pyridyl, furanyl, thienyl, thiazolyl, isothiazolyl, imidazolyl, pyrazinyl, pyrimidyl, pyrazolyl, isoxazolyl and
98. The compound of claim 97 selected from the group consisting of oxazolyl. 99. The aryl group is substituted with Y- (CH ₂ ) _t -heterocycloalkyl, wherein the heterocycloalkyl group is morpholinyl, pyrrolidinyl, piperazinyl, N-substituted piperazinyl, piperidinyl, tetrahydropyraniyl. 10. Tetrahydrofuranyl and pyrrolidinonyl selected from the group consisting of:
7. The compound according to 7. 100. The substituent X of formula II is in the 3- or 4-position of the phenyl ring,
72. The compound of claim 71. 101. The compound of claim 71, wherein A2 is aspartyl or an ester thereof; glutamyl or an ester thereof; alpha-aminoadipic acid or an ester thereof; valyl, norvalyl or leucyl. 102. A3 is aspartyl or its ester glutamyl or its ester; alpha-aminoadipic acid or its ester; valyl, norvalyl, leucyl, prolyl, or thiaprolyl; or R ₂₃ and R ₂₄ together are 3 or. 7-membered ring to form; or R ₂₂ and R ₂₄ form a heterocyclic ring substituted or unsubstituted with the nitrogen compound of claim 71. 103. The compound of paragraph 71, wherein R ₂₇ is substituted or unsubstituted phenyl, naphthyl or benzothienyl. 104. R ₂ is (CH ₂ ) _w— W— (CH ₂ ) _x —COOR ₃₄ , wherein W is one single bond, phenyl or C ₆ -cycloalkyl. Item 72. The compound according to Item 71. 105. R ₃₇ is a side chain of aspartic acid or glutamic acid; (CH ₂ ) _y —COOR ₄₂ , wherein y is 3 to 5, and R ₄₂ is hydrogen or C _1-6 alkyl. Or -phenyl- (CH ₂ ) _e- COOR ₄₃ , wherein e is 0, 1 or 2 and R ₄₃ is hydrogen or C _1-6 alkyl. Item 72. The compound according to Item 71. 106. U is OH or NHR _38, wherein R ₃₈ is t-butyl, isopropyl, 2,4-dimethylpent-3-yl, cyclopentyl, cyclohexyl, or bicyclooct [3.3.0] yl. Or R ₃₈ and R ₃₉ , together with the nitrogen atom,
106. forming a single pyrrolidinyl or piperazinyl ring). 107. The compound of claim 100, wherein A2 is aspartyl or an ester thereof; glutamyl or an ester thereof; alpha-aminoadipic acid or an ester thereof; valyl, norvalyl or leucyl. 108. A3 is aspartyl or an ester thereof; glutamyl or an ester thereof; alpha-aminoadipic acid or an ester thereof; valyl, norvalyl, leucyl, prolyl, or thiaprolyl; or R ₂₃ and R ₂₄ together, 3 to form a 7-membered ring; or together R ₂₂ and R ₂₄ with the nitrogen to form a substituted or unsubstituted heterocyclic compound of claim 100. 109. The compound of paragraph 100, wherein R ₂₇ is substituted or unsubstituted phenyl, naphthyl or benzothienyl. Is wherein 110] _{R 2, (CH 2) w} -W- (CH 2) a _x -COOR _34, wherein, W is one of a single bond, phenyl, or C ₆ - cycloalkyl, claim The compound according to 100. 111. R ₃₇ is a side chain of aspartic acid or glutamic acid; (CH ₂ ) _y —COOR ₄₂ , wherein y is 3 to 5, and R ₄₂ is hydrogen or C _1-6 alkyl. Or -phenyl- (CH ₂ ) _e- COOR ₄₃ , wherein e is 0, 1 or 2 and R ₄₃ is hydrogen or C _1-6 alkyl. Item 100. A compound according to Item 100. 112. U is OH or NHR ₃₈ (wherein R ₃₈ is t-butyl, isopropyl, 2,4-dimethylpent-3-yl, cyclopentyl, cyclohexyl, or bicyclooct [3.3.0] yl. Or R ₃₈ and R ₃₉ together with the nitrogen atom form one pyrrolidinyl or piperazinyl ring).
11. The compound according to 11. 113. A3 is aspartyl or an ester thereof; glutamyl or an ester thereof; alpha-aminoadipic acid or an ester thereof; valyl, norvalyl, leucyl, prolyl, or thiaprolyl; or R ₂₃ and R ₂₄ together, to form a single 3 to 7-membered ring; or R ₂₂ and R _24, together with the nitrogen, form a heterocyclic ring of a substituted or unsubstituted, a compound according to claim 107. 114. The compound of paragraph 107, wherein R ₂₇ is substituted or unsubstituted phenyl, naphthyl or benzothienyl. Wherein 115] R _{2 is, (CH 2) w -W- (} CH 2) a _x -COOR _34, wherein, W is one of a single bond, phenyl, or C ₆ - cycloalkyl, wherein Item 107. A compound according to Item 107. 116. R ₃₇ is a side chain of aspartic acid or glutamic acid; (CH ₂ ) _y —COOR ₄₂ , wherein y is 3 to 5, and R ₄₂ is hydrogen or C _1-6 alkyl. Or -phenyl- (CH ₂ ) _e- COOR ₄₃ , wherein e is 0, 1 or 2 and R ₄₃ is hydrogen or C _1-6 alkyl. The compound according to. 117. U is OH or NHR ₃₈ (wherein R ₃₈ is t-butyl, isopropyl, 2,4-dimethylpent-3-yl, cyclopentyl, cyclohexyl, or bicyclooct [3.3.0] yl. Or R ₃₈ and R ₃₉ together with the nitrogen atom are
108. forming a single pyrrolidinyl or piperazinyl ring). 118. The compound of claim 113, wherein R ₂₇ is substituted or unsubstituted phenyl, naphthyl or benzothienyl. Wherein 119] R _{2 is, (CH 2) w -W- (} CH 2) x -COOR 34 ( wherein, W is one of a single bond, phenyl, or C ₆ - cycloalkyl) a, wherein Item 113. A compound according to item 113. 120. R ₃₇ is a side chain of aspartic acid or glutamic acid; (CH ₂ ) _y —COOR ₄₂ , wherein y is 3 to 5, and R ₄₂ is hydrogen or C _1-6 alkyl. Or -phenyl- (CH ₂ ) _e- COOR ₄₃ , where e is 0, 1 or 2 and R ₄₃ is hydrogen or C _1-6 alkyl, The compound of claim 113. 121. U is OH or NHR ₃₈ (wherein R ₃₈ is t-butyl, isopropyl, 2,4-dimethylpent-3-yl, cyclopentyl, cyclohexyl, or bicyclooct [3.3.0] yl. Or R ₃₈ and R ₃₉ together with the nitrogen atom form one pyrrolidinyl or piperazinyl ring).
The compound according to. Wherein 122] R _{2 is, (CH 2) w -W- (} CH 2) x -COOR 34 ( wherein, W is one of a single bond, phenyl, or C ₆ - cycloalkyl) a, wherein Item 118. A compound according to Item 118. 123. R ₃₇ is a side chain of aspartic acid or glutamic acid; (CH ₂ ) _y —COOR ₄₂ , wherein y is 3 to 5, and R ₄₂ is hydrogen or C _1-6 alkyl. Or -phenyl- (CH ₂ ) _e- COOR ₄₃ , wherein e is 0, 1 or 2 and R ₄₃ is hydrogen or C _1-6 alkyl. Item 118. A compound according to Item 118. 124. U is OH or NHR ₃₈ (wherein R ₃₈ is t-butyl, isopropyl, 2,4-dimethylpent-3-yl, cyclopentyl, cyclohexyl, or bicyclooct [3.3.0] yl. Or R ₃₈ and R ₃₉ together with the nitrogen atom are
124) forming one pyrrolidinyl or piperazinyl ring). 125. R ₂ is (CH ₂ ) _w- W- (CH ₂ ) _x- COOR ₃₄ , wherein W is one single bond, phenyl or C ₆ -cycloalkyl. The compound of claim 114. 126. In the formula, R ₃₇ is a side chain of aspartic acid or glutamic acid; (CH ₂ ) _y —COOR ₄₂ (wherein y is 3 to 5, and R ₄₂ is hydrogen or C _{1- 6} alkyl); or -phenyl- (CH ₂ ) _e- COOR ₄₃ , where e is 0, 1 or 2 and R ₄₃ is hydrogen or C _1-6 alkyl. The compound of claim 114. 127. In the formula, U is OH or NHR ₃₈ (wherein R ₃₈ is t-butyl, isopropyl, 2,4-dimethylpent-3-yl, cyclopentyl, cyclohexyl, or bicycloocto [3.3.0]. Or R ₃₈ and R ₃₉ together with the nitrogen atom form one pyrrolidinyl or piperazinyl ring). 128. In the formula, R ₂ is (CH ₂ ) _w- W- (CH ₂ ) _x- COOR ₃₄ , wherein W is one single bond, phenyl or C ₆ -cycloalkyl. 110. The compound according to claim 109. 129 wherein R ₃₇ is a side chain of aspartic acid or glutamic acid; (CH ₂ ) _y —COOR ₄₂ (wherein y is 3 to 5, and R ₄₂ is hydrogen or C _1-6. Or -phenyl- (CH ₂ ) _e- COOR ₄₃ , where e is 0, 1 or 2 and R ₄₃ is hydrogen or C _1-6 alkyl. 110. The compound according to claim 109. 130. In the formula, U is OH or NHR ₃₈ (wherein R ₃₈ is t-butyl, isopropyl, 2,4-dimethylpent-3-yl, cyclopentyl, cyclohexyl, or bicyclooct [3.3.0]. Or R ₃₈ and R ₃₉ together with the nitrogen atom form one pyrrolidinyl or piperazinyl ring).
The compound according to. 131. In the formula, A2 and A3 are both 6-amino-5-oxoperhydropyrido [2,1-b] [1,3] thiazole-3-carboxylic acid; 6-amino-5-oxo Perhydro
-3-Indolizinecarboxylic acid; (S, R) -6-amino-5-oxoperhydro-8a-indolizinecarboxylic acid; (R, R) -6-amino-5-oxoperhydro-8a-indo Lysine carboxylic acid; (R, S) -6-amino-5-oxoperhydro-8a-indolizine carboxylic acid;
(S, S) -6-Amino-5-oxoperhydro-8a-indolizinecarboxylic acid; 2- (3-amino-2-oxo-1,2-dihydro-1-pyridinyl) acetic acid; 2- (3 -Amino-2-oxo-6-phenyl-1,2-dihydro-1-pyridinyl) acetic acid; 3-aminobenzoic acid; 4-aminobenzoic acid; 3-aminomethylbenzoic acid; (S) -3- (1 -Aminoethyl) benzoic acid; (R) -3- (
1-aminoethyl) benzoic acid; (S) -3- (1-aminopropyl) benzoic acid; (R) -3- (1-aminopropyl) benzoic acid; (S) -3- (1-aminobutyl) Benzoic acid; (R) -3- (1-aminobutyl) benzoic acid; 2- (3-amino-2-oxo-1-azepanyl) acetic acid; 2- [8- (aminomethyl) -3,6-dimethyl -9,10,10-Trioxo-9,10-dihydro-10 ^6- thioxanthen-1-yl] acetic acid; 2- (2-oxopiperazino) acetic acid; 2- [8- (aminomethyl) -2-oxo- 5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-1-yl] acetic acid; 2- [8- (
Aminomethyl) -2-oxo-5-methyl-2,3-dihydro-1H-1,4-benzodiazepine-1-
Il] acetic acid; 3-aminopropanoic acid; 4-aminobutanoic acid; 5-aminopentanoic acid; 2- [
2- (2-aminoethoxy) ethoxy] acetic acid; and 2- (3-amino-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepin-1-yl) acetic acid 132. The compound of claim 131, which is one residue that is 132. The compound of claim 131, wherein the substituent X is in the 3- or 4-position of the phenyl ring in formula II. 133. The compound of claim 131, wherein R ₂₇ is substituted or unsubstituted phenyl, naphthyl or benzothienyl. 134. wherein R ₂ is (CH ₂ ) _w —W— (CH ₂ ) _x —COOR ₃₄ , where W is one single bond, phenyl or C ₆ -cycloalkyl. 132. The compound of claim 131, wherein 135. In the formula, R ₃₇ is a side chain of aspartic acid or glutamic acid; (CH ₂ ) _y —COOR ₄₂ (wherein y is 3 to 5, and R ₄₂ is hydrogen or C _{1- 6} alkyl); or -phenyl- (CH ₂ ) _e- COOR ₄₃ , where e is 0, 1 or 2 and R ₄₃ is hydrogen or C _1-6 alkyl. 132. The compound of claim 131. 136. In the formula, U is OH or NHR ₃₈ (wherein R ₃₈ is t-butyl, isopropyl, 2,4-dimethylpent-3-yl, cyclopentyl, cyclohexyl, or bicycloocto [3.3.0. ] Or R ₃₈ and R ₃₉ together with the nitrogen atom form one pyrrolidinyl or piperazinyl ring).
35. The compound according to 35. 137. The compound of claim 132, wherein R ₂₇ is phenyl, naphthyl or benzothienyl. 138. In the formula, R ₂ is (CH ₂ ) _w- W- (CH ₂ ) _x- COOR ₃₄ (wherein W is one single bond, phenyl or C ₆ -cycloalkyl). 132. The compound of claim 132, wherein 139 wherein R ₃₇ is a side chain of aspartic acid or glutamic acid; (CH ₂ ) _y —COOR ₄₂ (wherein y is 3 to 5 and R ₄₂ is hydrogen or C _{1 - 6} alkyl); or - phenyl - (CH _{2) e} -COOR ₄₃ (wherein e is 0, 1 or 2 and R ₄₃ is a a a) hydrogen or C _1-6 alkyl 133. The compound of claim 132. 140. In the formula, U is OH or NHR ₃₈ (wherein R ₃₈ is t-butyl, isopropyl, 2,4-dimethylpent-3-yl, cyclopentyl, cyclohexyl, or bicyclooct [3.3.0]. ] Or R ₃₈ and R ₃₉ together with the nitrogen atom form one pyrrolidinyl or piperazinyl ring).
39. The compound according to 39. 141. In the formula, R ₂ is (CH ₂ ) _w- W- (CH ₂ ) _x- COOR ₃₄ (W is one single bond, phenyl or C ₆ -cycloalkyl), 138. The compound of claim 137. 142. In the formula, R ₃₇ is a side chain of aspartic acid or glutamic acid; (CH ₂ ) _y —COOR ₄₂ (wherein y is 3 to 5, and R ₄₂ is hydrogen or C _{1 - 6} alkyl); or - phenyl - (CH _{2) e} -COOR ₄₃ (wherein e is 0, 1 or 2 and R ₄₃ is a a a) hydrogen or C _1-6 alkyl 138. The compound of claim 137. 143. In the formula, U is OH or NHR ₃₈ (wherein R ₃₈ is t-butyl, isopropyl, 2,4-dimethylpent-3-yl, cyclopentyl, cyclohexyl, or bicycloocto [3.3.0]. ]; Or R ₃₈ and R ₃₉ together with the nitrogen atom form one pyrrolidinyl or piperazinyl ring). 144. A method of treating a Cdc25-mediated condition in a patient, comprising the step of administering to the patient a therapeutically effective amount of the Cdc25 inhibitor of claim 71. 145. The method of claim 144, wherein the patient is a human. 146. The method of claim 145, wherein said Cdc25-mediated condition is characterized by excessive cell proliferation. 147. The Cdc25-mediated condition is cancer, coronary restenosis,
146. The method of claim 146, which is occlusion and inflammation.