TW593297B - LFA-1 antagonists - Google Patents

Abstract

The present invention relates to compounds which bind to all or parts of the active binding ""south pole pocket"" of the LFA-1 I-domain and their uses as LFA-1 antagonists. A pharmaceutical composition for use as LFA-1 antagonist, comprising a compound which binds in whole or in part to the south pole pocket of LFA-1 I-domain defined by the amino acids Val 130, Leu 132, Phe 134, Phe 153, Val 157, Leu 161, Tyr 166, Thr 231, Val 233, Ile 235, Ile 255, Tyr 257, Ile 259, Lys 287, Lew 298, Glu 301, Leu 302, Lys 305, in free form or in pharmaceutically acceptable salt form, in association with a pharmaceutically acceptable diluent or carrier therefor.

Description

593297 V. Description of the invention (ι) The present invention relates to compounds that can bind all or part of the "South pole pocket" of the active binding site of the LFA-1 I-region with * and its use as an LFA-1 antagonist use. '' The LFA-1 antigen, which is related to lymphocyte function, belongs to the / 3 2-integrin and plays an important role in T-cell activation and extravasation. LF A-1 interacts with endothelial cells or antigen-expressing cells such as ICA M-1 or ICA M-3. It is an important process for the adsorption and migration of lymphocytes and endothelial cells. Adsorption and migration cause disorders or diseases such as autoimmune diseases, inflammation, ischemia / reperfusion injury and rejection after organ transplantation. The so-called I-region (embedded field) on LFA-1 contains a module of about 190 amino acids (Takada et al., Matrix! Biology, 16, 143-1151, 197 7). X-ray crystallographic analysis | Measured that the I-zone folded into a common structural feature includes a central /?-Sheet and h & lice (A. Qu & D. Leahy, Proc. Natl. Acad. Sci. USA, 92, 10277- 10281, 1995). I compactin and mevinolin are metabolites of fungus i and their structural formula is as follows:

C: \ Prografn Files \ Patent \ 54618. Ptci, page 5, 593297 丨 V. Description of the invention (2) | These metabolites are disclosed in, for example, `` Progress in the Chemical Synthesis of Antibiotics and Related Microbial Products " by Chapleur. , Springer Verlag, 19S3, vol. 2, 829-937. Mimicrin and most known analogs such as (Pavastatin) pravastatin, (Mevastatin) mevastatin, (Sivavastatin) swvastatm, etc. It has been found to be very useful, for example, as an inhibitor of 3-methyl-3-pentylglutaridine coenzyme a reductase (HMG coAAR). According to the present invention ', we are now very difficult to find that micronolin and it can be combined with LFA_1. Based on this, the present invention proposes compounds that can be used to ... treat 'prevent autoimmune diseases, inflammation, ischemia / reperfusion (= ,: 10η) injury and organ rejection, and these compounds are substantially better than 1 ^ = 2 The above specific LFA] binding molecule is, for example, specific or substantially specific-inhibitory of the LFA-: 1 or 1CAM-3 interaction. Mei Jia is that these compounds are not LFA_i2 antibodies. Even more: The other thing is that we found that the location of Mevinorin and H is between c-terminal helix α 7 Ang θ〆 (hereinafter referred to as "Antarctic capsule". Take X-shoot ::: V \ (sheet) The complex formed between the side-regions shows rice 4: analysis = Lin and LFA_1 I (π-dependent metal ion adsorption site ,,) Guang Yawei, 5 to ... Shape of I-zone,-micronorlin (100% solution in 0 纟 丨 30) 1 compound. The preparation of the material is rice < 10 "MMgS〇4), and then) :: 彳Protein solution (I2.

C: \ Prograin Files \ Pateat \ 54618. Ptd

593297 I ~-~~~~~~ __ I. V. Description of the invention (3) ^ — Substitution method to unlock (using ap0) LFA — 1 Equivalent of BD (CDordmate), A Qu & D · Uahy, ibid.) And using the meaning of the analysis range of 4-2 · 6 / \-ray amplitude to resolve it to an R factor of 19.4% (Rfree = 2o · 9%). The final model contains 2xΐ82 amino acids (LFA-jα-chain amino acid residues 128 to 309, corresponding to this region), 2 micronolin molecules and all 86 water molecules. Data Collection Status LFA-1 I-Zone / Micronome Temperature Temperature Wavelength Resolution Range Space Group Single Crystal Size Measurement Used Unique Reflection Completeness Multiple Average I / sig (I) R Combine

293K I. 5418A 15.0 into -2.60 into P21212l a = 72.7A, b = 77.7 people, c = 91.8 into 87179 16457: 99.9% (99.8% in the shell 2.69 into -2.60 into) 5.3 (5.2) 13.9 (2.1) II 6% (48.4%) The Antarctic sac is an amino acid between the middle / 5-fold (/ 5 1, 3/3, 4, 5, 5), preferably the amino acid Side chain) and β-helix α 1, a 7 (LF A-1 I-region secondary structure). Preferably, the Antarctic capsule is composed of amino acids Val 130 and Leu in the I-region of LFA-1.

Page 7 C: \ Program Files \ Patent \ 54618 · ptd 593297 Five 'invention description (4) 132, Phe 134, Phe 153, Val 157, Leu 161, Tyr 166, Thr. 23 1, Val 2 3 3, Ue 2 3 5, lie 2 5 5, Tyr 257, Ile 259, Lys 287, Leu 298, Glu 301 vLeu 3 0 2 'Lys 3 0 5, especially Leu 132, Phe 153, Val 157, Val 23.3, lie 235, Spaces defined by Thr 257, lie 259, Lys 287, Leu 2 98, Glu 301, Leu 302, LyS 3 0 5 and more specifically spaces defined by the side chains of these amino acids. 5 人。 In this capsule, the preferred interaction distance of the non-hydrogen atom of miromerin is <58, and the better is 4-4. 5 people. The Antarctic Antarctic Capsule / Micinoline complex is hydrophobic in energy, van der Waals force and / or static electricity. Gravity is bound by == indirect hydrogen bonding. They will accept that each asymmetric unit has two 丨 -zones / miwanorin complexes whose correlation is twice that of an amorphous one. Figure 1 shows that the monomeric part of the I-region of LFA-1 is first connected to its adjacent monomer ^ carboxyl terminal region-located in the knot "asymmetric unit". The ligands are shown in the CPK-model.

i Yang, · Sunrise Miwei Youlin and LFA-1 I 丨-enlarged view of the Antarctic capsule. · '! Its shape shows, as described above, the funeral soil β 軎; The Antarctic Capsule of the Heart and My Mind is not only conducive to combining with Micronorlin but also can be used as a farmer! With his chemical f-body or coordination early 丨Combined. These chemical entities or ligands 4 D a 1 D and LFA-1 inhibitor a ^ LFA-1 / | CAM-1 or CAM-3 interaction inhibitor. ^ ^ The present invention provides a chemical entity or compound that can be partially or fully combined with the aforementioned Ding Sha TP, i τ \ dou, Renru "· Human Yi UA — 1 丨-Antarctic capsule of the region ☆ 2 ^ Dingbei limbs X ligand. The chemical facts

C: \ Program Fi ies \ Patent \ 54618.ptd

593297 V. Description of the invention (5) The working distance of the body or the ligand <5 again, especially 4-4. 5 people. Appropriate examples of such chemical entities include, for example, milinoline derivatives. By elucidating the combined interactions of Mavinorline in the LFA-1 I-Zone Antarctic Capsule provides important information to design new interactions with LFA-1 I-Zone Antarctic Capsule provides important information to design a new A chemical entity or compound that partially or fully binds to the South Pole of the LFA-1 I-region. Therefore, with the present invention, molecular design technology (such as computer model design technology) can be used to identify, screen and design chemical entities or compounds that can be partially or fully combined with the Antarctic capsule. Designing a compound for binding to the Antarctic capsule according to the present invention usually requires consideration of two factors. First, the chemistry: the entity must be physically and structurally bound to the Antarctic capsule in part or in whole. Non-covalent molecular interaction forces that are important in this binding include hydrophobicity, van der Waals force interactions, hydrophobic interactions and / or electrostatic force interactions, and possible hydrogen tritium binding. = Second, the chemical entity must be able to be configured to bind directly to the Antarctic capsule. Although some specific parts of these chemical entities do not directly participate in their interactions, specific parts of their chemical entities still affect the overall configuration of these chemical entities. In the end, the potency of these chemical entities will still be seriously affected. The structural requirements include the relationship between the overall three-dimensional spatial structure and arrangement of the chemical entities and some or all of the Antarctic capsule, or the functional group of any entity on the chemical entity that directly interacts with the Antarctic capsule Spatial location. Will interact with the Antarctic capsule in part or in whole (preferably with Miwei

593297! ^ --- 丨 V. Description of the invention (6) | Norlin's chemical method can be further used J urkat or Hut 7 8 cells described in A) described below to test its inhibition lj a — 1 / ICAM-1 or I CAM-3 interaction capabilities. According to the present invention, the representative compounds that can be combined with the Antarctic capsule are compounds that can inhibit the adsorption of Jurkat or Hut 78 cells to 1CAM-1 by 1 C5G $ 3 0 / ζ M. These compounds are called 1 ^ \-1 antagonists or 1 ^ human-1 / 10 six} ^-1 or 1041 ^ 3 interaction inhibitors. The compounds according to the present invention used in accordance with the present invention are meminolins (hereinafter referred to as “•, mesonorlins of the present invention,”), preferably these compounds have no or only limited legs GC OAR inhibitory activity. Accordingly, the present invention proposes:-a compound that is habitually used to treat and / or x-'inflammatory 'ischemia / reperfusion (a homologous or heterogeneous organ or group of compounds. Systemic immunity 1. Disease, acute or chronic 1 ο η) 傜 &, acute or chronic miscellaneous ^ or infectious diseases θ A ', especially 4-4. 疋 All combined. 1 $ 4 &amp; Sex is used to treat and / or ruler, ischemia / reperfusion (same or heterogeneous organs or groups; micro-norlins. 1. Use the LFA itself to prevent autoimmune diseases , Acute or slow reperfusi ο η) injury, rejection or infection after acute or slow tissue transplantation 1.1 for the treatment and / or prevention of spontaneous inflammation, ischemia / reperfusion (reperfus after allogeneic or xenogeneic organ or tissue transplantation A compound with an action distance as defined above, for example, the Antarctic capsule as described above or 1.2 using its own LFA or preventing autoimmune diseases, acute or reperfusi ο) injury, acute or slow rejection after transplantation or疋 Infectious diseases

C: \ Program Files \ Patent \ 54618. Ptd Page 10 5933297 I. Explanation of the invention (7) '&quot; ~ ---, 1 · 3 are used to treat and / or prevent autoimmune diseases, acute or chronic inflammation , Ischemia / reperfusion injury, acute or chronic rejection after transplantation of allogeneic or xenogeneic organs or tissues, or Zhu Wei, an infectious disease. 5 A, especially 4-4 · 5 A combined with, for example, the Antarctic capsule as defined above or all 1 · 4 according to 1, 2 or 1, 3 for the treatment and / or prevention of autoimmune diseases, acute or chronic Inflammation, ischemia / reperfusion (reperfusión) damage 'acute or chronic allogeneic or xenogeneic organs or tissues after transplantation or infectious diseases of miminoline, such miminoline compounds are described in The following results of the in vitro microsomal (In Vitro Microsomal) HMG C〇AR inhibition assay show that it inhibits the activity of HMG c〇AR IC5Q ^ 1 // Μ 〇2 · — a species that can be combined with the LFA-1 I-region Antarctic capsule Chemical entity or ligand method, the method includes the following steps:! A · 利Computer operations are used to match these chemical entities with the Antarctic capsule. Fitting operations; and! B, analyze the results of the matching operations to quantify the binding force between these chemical entities and the Antarctic capsule. i In vitro microsomal HMG-CoA reductase inhibition assay | Analysis of freshly prepared liver microsomes from male Spa rgu e-Da w 1 ey rats (weight 1 50-2 2 5 g) containing 10 mmol Suspension of thiol-butanediol in buffer A 200 // 1 part (1.08-1. 50 mg / ml) and 1 0 // 1 test compound dissolved in diethylacetamide and grown in accordance with | Ackerman et al., J. Lipid Res. 18, 408-413 (

C: \ Program Files \ Patent \ 54618. Ptd Page 11 593297 I V. Description of the invention (8) 丨 1 7 7) The method described above was used to detect and analyze the activity of MG-COAAR. In this assay, microsomes act as a source of HMG CoA R enzymes to catalyze the reduction of HMG CoA R to mevalonate. In this detection analysis, chloroform extraction was used to separate the [UC] mevalono-lactone product formed from the substrate [UC] HMG CoA via the HMG CoA R reaction. [3H] mivalonide was added as an internal reference. Inhibition of HMG CoA R was calculated by comparing the decrease in the specific activity of [14C cadaver] and Zhuvalonate tested with the control group, and expressed as IC5Q (the concentration of the test compound that inhibits 50% of HMG CoA R activity). The use of a compound of the present invention (for example, the Miminorolins of the present invention) as an inhibitor of LFA-1 / ICAM-1 or ICAM-3 interaction can be exemplified by the following test methods; A. In vitro

Will be obtained from eight (::: 1 ^ 1 ^ 1; or 111 ^ 78 cells supplemented with 10% ί FCS'L -glutamine, non-essential amino acids and 0 · "5ιηΜ 2-sulfur Alcohol-based ethanol was cultured in RPMI- 1640, centrifuged, washed once with PBS, and then resuspended in a binding buffer (1.5% BSA) containing 5 / zg / ml BCECF-AM (Molecular Probes) , 5mM glucose, 2mM MgCl2, 2mM i MnCl2 in TBS, pH 7) to make 0.5 x 106 cells / ml. The cells were incubated at 37 ° C: in the dark for 30-45 minutes. Then the cells were centrifuged and then pipetted to make them丨 Resuspend in the binding buffer and use it immediately for experiments. I Flat-bottomed micro-calibration culture dish (NUNC Maxi sorp) in carbonate buffer (15mM Na2C03, 35mM NaHC03, ρΗ8 · 0) l &quot; g / 丨 in 1 sheep anti-mouse C k was incubated for 2 hours at 3 7 C. The culture jui was emptied

593297 I V. Description of the invention (9) After I, 1.5% BSA and 0.5% Tween-20 were used in carbonate buffer solution to cover it at 37 ° C for 90 minutes. The dish was emptied and rinsed again with TBS containing 15% BSA. Next, the baculovirus-i mouse Ck fusion protein (100 ng / ml in DBS / 1 · 5% BSA) was added to each i-slot chamber. The dish was incubated at 37 ° C for 90 minutes. After washing twice with dibutyl bs / 1.5%, the compound to be tested is diluted in binding buffer (as described above but without glucose and BSA) and added to each tank. Then, 10,000 Jurkat or Hut 78 cells were added to each chamber, and incubated at 37 ° C for 30 minutes for attachment. Rinse 2 ~ 4 times with binding buffer to separate the attached and unattached cells. The attached cells were quantified with a fluorescent EL 丨 SA reader, and the filters were set to emit light at 48 5 nm and 5 3 Onm.

In this assay, the compound of the present invention inhibits the attachment of Jurkat or Hut 78 cells to ICAM-1 at a concentration of C5q $ 3 0 # M (preferably 0.05 to 30 / zM). | B. In the body. i i) Rat thioglycolate-induced peritonitis i I Intraperitoneal injection of thioglycolate into small white gas and immediately | τίί 4 people tested the compound by subcutaneous injection. After 4 hours, the mice were killed and lavaged their peritoneal cavity, and the total number and number of neutrophils in the lavage fluid were measured. In this assay, a compound of the present invention, such as lenoline, can inhibit neutrophil migration induced by thioacetate I via subcutaneous injection at a dose of 0.001-50 mg / kg.丨 i i) Ischemia / reperfusion injury These compounds can be used in a model of ischemia / reperfusion injury (

C: \ Program Files \ Patent \ 54618. Ptd 苐 page 13 593297 5. Description of the invention (ίο)

Circulation of Abdeslam Oubenaissa et al., 94, Suppl · I II, 2 5 4-2 5 8, 1 9 9 6) or test according to the following method:! Reset the mice in 20-2og to different After anesthesia with isoflurane, the right renal tubule was clamped with a microvascular clamp for one minute. After 60 minutes of ischemia, the microvascular clip was removed. Connect the tubing of the left kidney (arteries, veins and ureters) with 4-0 surgical sutures. Remove the left (non-ischemic) kidney and close the abdominal cavity with 3-0 surgical suture. The control group was treated in the same way as the ischemic group but without clamping the right kidney tubing. At 24 hours of reperfusion, the animals were killed by inhaling CO 2 at the 1st and 2nd week. Immediately after the animal was killed, blood samples were collected by heart blood collection in a 3.0 ml Vacutaine r® containing 0.04 ml of 7.5% K3EDTA. Plasma was stored separately at -20 ° C until further analysis. Creatine in plasma and urea nitrogen (BUN) in blood were analyzed by Sigma procedure. After the animals were killed, the kidneys were lavaged with physiological * saline, frozen with liquid nitrogen and stored at -7O ° C until analysis. according to

The method of Bradley et al. (J. Invest. Dermatol., 78, 206-209, 1 982) was used to measure the activity of renal muscle peroxidase (MP0). In this mode, the compound of the present invention, such as meminoline, is particularly administered for 4 days at a dose of 0.0000 to 50 mg / kg before the ischemic treatment. It can reduce the plasma muscle caused by ischemia. Acid and blood urea nitrogen. I 111) Vascularized heterologous heart transplantation. The donor heart is implanted into the recipient's abdominal blood vessels: an end-to-side (e nd 丨 -to-side) vascular anastomosis using 11/0 Ethilcon sutures (Ethicon,

C: \ Program Files \ Patent \ 546l8.ptd Page 14 593297 V. Description of the Invention (11)

Norderstedt, Germany) connected the head and arm veins to the inferior vena cava. After the 6 / 〇 γ i artery and the right lung were moved with two layers of sutures, the animals were kept warm with straight suture sutures (EthiCon), and the ischemic time of the animals was within 40-50 minutes, and the period was up to 25-2. All tube anastomosis operations (10-15 minutes) were performed at 4C with a knife / knife *. After the blood is planted, the two plants remain cold. (Heart palpitations) Come and watch. Beware of heartbeat; Mother: Assess the beating of the graft During the experiment, tissue inspection of finger ΐ3 was not completely rejected. All compounds of the invention, such as minocycline in f / F fields, can significantly improve the function of the graft when administered to animals. A dose of = mg / ml is used to: ΐ 卞 / fat month compounds such as the minororin of the present invention can be = or dysregulated 'such as ischemia / reperfusion injury, for example: wind' intestinal ischemia, renal failure or Hemorrhagic rest is the same as: infarction II. ^ 7 or genital rejection, acute or chronic inflammation, or autologous disease, such as wind arthritis, asthma: conditions, skin diseases, such as psoriasis, Compulsive symptoms, inflammatory bowel disease, x, adult respiratory tightness ^ ^ ^, disease or ocular inflammatory disease, infectious disease 4 diarrhea ^ shock 'traumatic shock. First, from the above two methods of use, of course, the required dose depends on the way of signing 'what you want: the specific condition of the combined therapy and the effect you want to achieve'; the same but 逋 ¥ at a dose of 0.5 to 80 mg / kg of animal body weight can achieve the desired effect. For larger mammals such as humans-the amount is from about 20 邶 to 丨. 5 ~ for example 丨. "…~/day

C: \ Program Files \ Patent \ 54618. Ptd page 15 5933297 I. Description of the invention (12) | and can be administered once a day, or divided into 2-4 times / day, or as a sustained release dosage form . The unit dosage form may suitably include from about 5 mg to 0.750 g of a compound of the invention and a pharmaceutically acceptable diluent or carrier. The juveniles of the present invention can be made in free form or with a pharmaceutically acceptable salt form such as an acid addition salt or an alkali salt such as sodium, potassium, substituted ammonium or unsubstituted Ammonium salts are administered. These salts can be prepared by conventional methods and have the same effect as the free form. It is further proposed according to the present invention; 3. A method for preventing or treating disorders or diseases mediated by the interaction of LEA-1 / ICAM-1 (such as those mentioned above in a recipient in need of such treatment) Disorders or diseases), the method comprises administering to the subject an effective amount of a compound of the invention, such as a mecorinoline of the invention, or a pharmaceutically acceptable salt thereof. 4. A pharmaceutical composition for use in the above 3) method, the composition comprising a compound of the present invention, such as a microninoline in a free form or a pharmaceutically acceptable salt form, and a pharmaceutically acceptable | Accept the diluent or its carrier. 5. A compound of the present invention, such as a micronorline or a pharmaceutically acceptable salt thereof, these compounds are used to prepare a pharmaceutical composition required for the method 3) above. . These pharmaceutical compositions can be manufactured in a conventional manner. The compounds of the present invention, for example

C: \ Program Files \ Patent \ 54618. Ptd Page 16 5'Explanation of the invention (13) — ^ --One ^^ ~ 2 Conventional administration, such as enteral (preferably oral), such as for The drug or capsule is administered by injection, for example, it can be made into a heart solution or suspension, or administered nasally or in the form of suppositories. The meconorinoids of the present invention can be administered in the form of a single active ingredient. They can also be used in the course of immunosuppressive therapy with other drugs or with other anti-inflammatory agents to prevent or treat acute or chronic allogeneic or xenotransplantation. Rejection or inflammation or autoimmune disorders. For example, users that can be combined with it include cyclosporins, rapamycins, or ascomycins, or their immunosuppressive analogs, such as Cyclosporin A 'Ciclosporin G, FK-506, rapamycin, 40-〇_ (2-Alkyl) ethyl-rapamycin, etc .; Corticosteroids; Cyclophosphamide; Atazite Azathioprene; methotrexate; brequinar; FTY 720; leflunomide; mizribine; myco phenolic acid); mycophenolate mofetil; 15-deoxyspergualine; immunosuppressive single-source antibodies, such as single-source antibodies of lymphocyte receptors, examples of lymphocytes The receptors are MHC, CD2, CD3, CD4, CD7, CD25, CD28, B7, CD45, or CD58 or their ligands; or other immunomodulatory compounds, such as CTLA4Ig, or other molecular adhesion inhibitors, such as m Abs or low molecular weight inhibitors, including selectin junction inhibitors and VLA-4 junction inhibitors

Page 丨 7C: \ Program Files \ Patent \ 54618.ptd 593297 V. Description of the invention (14) When the microninoline of the present invention is used in combination with other immunosuppressive / immunomodulatory therapies or other anti-inflammatory therapies When coadministered (for example, to prevent or treat the aforementioned chronic rejection), the doses of immunosuppressive / immunomodulatory or other anti-inflammatory compounds that are jointly used will of course depend on the type of drug used together (for example, A steroid or a cyclosporine), the particular drug used, and the symptoms you want to manage, etc. Based on the foregoing, the present invention further proposes the following viewpoints: 6.-A method as described above, which method comprises the simultaneous use (simultaneously or sequentially) of an effective amount of the free-form or pharmaceutically-acceptable salt form of the microveno of the present invention Lin and a second drug, the second drug is an immunosuppressant, immunomodulatory or anti-inflammatory agent, such as those specified above. 7, a kit or combination for use in any of the methods defined in 3) above, the kit or combination comprising the free micronose or pharmaceutically acceptable salt form of the microninoline of the present invention and at least one comprising A pharmaceutical composition of an immunosuppressant, immunomodulatory or anti-inflammatory drug. Such kits or combinations may include instructions for their administration. The representative miconorins used according to the present invention are those which contain a molecular structure A ^.

C: \ Program Files \ Patent \ 54618.ptd Page 18 593297 I V. Description of the invention (15) -b and α --- / 3 are single or double bonds. Each of a --- b and α --- β may each be a part of a cyclopropyl group. It is known that the literatures of the microminolins describe that the 4, 5, 6 and / or 7 rhyme positions of the molecular structure A may be substituted by one or more substituents, for example, as disclosed in Y. C hap 1 eur ( (See above). A preferred substituent at position 4 is a substituted amidino. The preferred substituent at position 5 is connected to the bicyclic moiety through a "0-C0-"; more preferably, it is connected through "0-c0-r2, where R2 • Ci-Cs alkyl, c3_7 cycloalkyl , Aryl, hetero. Aryl, c3_7 cycloalkyl-Chalkyl, aryl-Chalkyl or heteroaryl-Chalkyl. Preferred LF A-1 antagonists of the present invention are the micronolin compounds of the present invention having a free form or a pharmaceutically acceptable salt form of the following structural formula I

Where R2 is as defined above, h is ... Η,... Ch, or 0Ra;

Ra is 11; (: Bu 6 alkyl; substituted with 0H or Ch alkoxy (: Bu 6 alkyl; C 2-6 alkenyl; or aryl alkyl; R 3 is formula (i), (ii), ( iii) or (iv)

C: \ Prograni Files \ Patent \ 54618. Ptd page 19 593297 V. Description of the invention (16)

Where Xi is (} 1, Η), (Η, 0H) or = 0; X2 and y2 are two 0 or (R, R) where each R is Η, (V3 alkyl, substituted Ci_3 alkyl) Or X2 and Y2 together with the carbon atoms to which they are attached form a 4-, 5-, 6- or 7-membered carbocyclic or heterocyclic residue, R4.0Ra, where 1 is as defined above; or -0-C0Rb, Wherein Rb is a Ci_8 alkyl group optionally substituted with OH, C3_7 cycloalkyl, C3_7 cycloalkyl-C ^ alkyl, aryl, aryl-Ch alkyl, heteroaryl or heteroaryl-Ch alkyl; Or NReRd, in which each R. and Rd is milked as a Cy alkyl group, or forms a heterocyclic residue with the nitrogen to which it is attached, which optionally contains one oxygen or another nitrogen atom; R5 series 11, Cy alkyl, C3_9 alkenyl, C3_9 alkynyl, aryl-Ch alkyl, or C3_7 cycloalkyl-Ch alkyl; R6 is -CHRn-CO-NR12Ri3, where Rii is one of the aforementioned definitions of R5, and each of R12 and R13 is respectively Η, Ci_4 alkyl, or substituted Ci_4 alkyl; R7 is = 0 or (H, 0H); R8 is 〇1; * NReRf, where each Re ARf is Η, 1_6 alkane

C: \ PrograuiFiles \ Patent \ 54618.ptd Page 20 593297 5. Description of the invention (17) group, Ci_6 alkyl group substituted with 0H or (: 4 alkoxy group, or five-membered heterocyclic group; or R7AR8 together form dioxy- (V4 elongation group or -0-QD-0-; R9 is one of the definitions of R5 above; R10 is COORa; CH2ORc, where Rc is Ra or CD; * CONR14R15 or CH2NR14R15, Wherein each of R14 and R15 is (^ _4 alkyl, substituted with (^ _4 alkyl, fluorenylamine-fluorenyl, alkyl) -methylamine fluorenyl-fluorenyl or bis (Cp4 alkyl) -fluorenylamine Fluorenyl-fluorenyl, or one of R14 and R15 is hydrogen and the other is CN6 alkyl, selected via OH and / or fluorenylfluorenyl-fluorenyl, (Ci_4 alkyl) -fluorenamine fluorenyl-fluorenyl, di ( Ch alkyl) -Ci_6 alkyl substituted with a methyl group and a methyl and heteroaryl alkyl group substituted with a Ci_6 alkyl group, 'Cn alkyloxy group, a stilbenzyl group, adamantyl group, methyl group, c3_7 cycloalkyl group, and c ^ Alkyl, aryl-cw alkyl, wherein the aryl group may be substituted and the (V4 alkyl group may be substituted by amidinofluorenyl or Gy alkoxy-carbonyl, or a heteroaryl-c ^ alkyl, Wherein the heteroaryl group may be substituted with a fluorenylamino group or a cv4 alkoxy-carbonyl group to end the Gy The group may be substituted by methylamine glutamate, or R14 and R15 together with the nitrogen atom to which it is attached form a heterocyclic residue, the heterocyclic residue may optionally contain another nitrogen atom and optionally be alkyl, ((^ _4 alkoxy) -carbonyl, amido stilbene, dioxyalkylene, aryl-c ^ alkyl or heteroaryl substituted 'wherein the heteroaryl may be substituted by alkyloxy- several groups; R16 is Η (^ 4 alkyl; aryl-Ch alkyl, where the aryl can be substituted with halogen, 0H, optionally substituted amine, COOH, CF3, Ci_4 alkoxy or cyano; or C3_7 cycloalkane -CiMaryl; | each a --- b and β --- /? Are single or double bonds, respectively.

C: \ Prograni Files \ Patent \ 54618. Ptci 页 p. 21 593297 I. 5 'Description of the invention (18) I Hexyl groups such as Ra, Rb, R5' Rll 'Rl2 or Rl3 or its radical moiety may have Branched or straight chain. When R, R12 or 'is a substituted alkyl group, it is preferred that these substituents are located at the end of the alkyl chain and may be halide, OH, C3_7 cycloalkyl or aryl group, when Re or Rf When it is a substituted Ci-6 alkyl group, these substituents are preferably located at the end of the alkyl chain. The cycloalkyl group is either partially cyclopentyl or cyclohexyl. The aryl or aryl moiety is preferably a phenyl group and is preferably substituted with, for example, halogen, OH, optionally substituted amine, COOH, CF3, (4, 4 alkoxy or cyano, more preferably Substituted by alkoxy group at the position of 1, 2 or 3. The preferred aryl alkyl group is benzyl-C ^ alkyl group, such as benzyl or benzyl. Heteroaryl is preferably derivatized. Selective fusion from a 5- or 6-membered heterocyclic ring to a stupid ring, such as pharynyl, miso, sulfanyl, erythro | And Rd = —when it forms a heterocyclic ring with the adjoining nitrogen atom, it can be a 5- or 6-membered ring, such as 1¾ pyridyl, hexahydropyridyl, hexahydropyridyl, 4- Fluorenyl-hexahydropyridyl. When one of RuSRb is a heteroaryl-Ch alkyl, the heteroaryl moiety is a 5- or 6-membered heterocyclic ring optionally fused to a stupid ring or a Heterocyclic groups such as furyl, morpholinyl, hexahydropyridyl or indolyl. When R14 and R15 together form a heterocyclic ring with the nitrogen atom to which it is attached, it may be, for example, Pyrrolidinyl, N-hexahydropyridine Hexahydropyridyl. When x2 and Y2 together form a carbon- or heterocyclic ring group, it may

C: \ Program Files \ Patent \ 54618. Ptd Page 22 593297 V. Description of the invention (19) Cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, pyrrolidinyl, pyrrolidone . The alkylene portion of the dioxy-Ci_4alkylene can be straight chain, such as -CH2-, -CH2_CH2_, or it can have a branch bond, such as = C (CH3) 2. The compounds of formula I may exist in free form or in the form of salts, for example, as acid addition salts with organic or inorganic acids, such as the hydrochloride, or salts with bases in the presence of COOH, such as the base Metal salts such as sodium or bell salts, or saturated or unsaturated salts. We should all understand that carbon atoms with R4, R5, R6, R7 and R8 in formulas (i), (i i) and (i i i) may be asymmetric. When the molecule of formula I does not specifically indicate its stereochemical properties, we should all understand that the invention encompasses all enantiomers and mixtures thereof. The same considerations apply to related starting compounds with asymmetric carbon atoms as previously described. In compounds of formula I, the following are preferred definitions: 1. I is} 1 or ch3, preferably ch3;. 2. R2 € C4_8 alkyl, preferably -CH (CH3) -CH2-CH3-CH (CH2- ch2-ch3) 2; -ch (ch2ch3) 2-;

/ CH, -CH3 C—CH.-CH,; I ·. Cr: 3 or a CH \; 3. R3 is a group of formula (1); 4, R3 is a group of formula (1i1); 5. R3 is a group of formula (U1); Among them R7 series (1 0H);

C: \ Program Files \ Pateat \ 546l8. Ptd page 23 593297 V. Description of the invention (20) 6. R3 is a group of formula; wherein R? Is 20; I 7. R3 is a group of formula; Where R8 is ⑽; 8. R3 is a group of formula (ii i); wherein R? And I together form a dioxo-Ch-alkylene group or -o-co-o-; 9 R3 is a formula (iii ); Wherein Rs is NReRf; 103 is a group of the formula (un of which 1 is a leg 1 of which 1 is a c 6 alkynyl group, wherein the alkoxy group is selectively passed through η or 〇1-4 Substitution; 1 1 · R3 is a group of formula (iii); wherein R9 is H, CH3, benzyl or propynyl; 1 2, R3 is a group of formula (i ^); where is CDNR &quot; R15 13. R3 is a group of formula (i ii)-; where riq is CONHR15 and D5 is (^ 4 alkyl which is optionally substituted with 0H; 14. R3 is a formula (iH) Group; its center is CONHRIi5 and 1 ^ 5 is a benzyl-Ch alkyl group or a heteroaryl-Ch alkyl group, wherein the benzyl 'heteroaryl group and the CV4 alkyl moiety may have the Substituted. It is preferred that the phenyl group be substituted with Ci_4 alkoxy group, especially 0CH3 at the position of 1, 2 or 3; | 15 · R15 CH2-benzyl or CH (CO-OCH3) -benzyl, in which the phenyl I is substituted at position 1, 2 or 3 with 4 alkoxy groups, especially 0CH3; I 16.1 ^ 5 system ^ 2-furanyl or [; 11 ((: 0_2) — (;) 12-3—indolyl;! 丨 7 · R1G is a CONR14R15 where R14 and Ri5 together with the nitrogen atom to which it is attached form a k • selectively substituted Hexahydrophthyl is, for example, substituted with dioxoalkyl and preferably substituted with dioxoethyl; | 1 8 · R3 is a group of formula (i V). Among the Norrins, the compound of Formula 11 or its a is

C: \ Program Files \ Patent \ 54618.ptd 593297 V. Description of the invention (21) The novel compounds form part of the present invention

Where R! 'R2' R3 and dashed lines a --- b and α-are not defined as described above, the conditions are 1) when K is 11, CH3 or C2H5, R2 is not .. Gy alkyl or aryl- Ch alkyl, and R3 is a group of formula (i), wherein K is ⑽ or OCH3, &amp; or C4 alkyl, and 11 and ¥ 1 are = 0; or 2) when Rs is of formula (iii ) Is not a Gy alkyl group, in which r9 is Η and R1Q is COORa. Particularly preferred are compounds of formula 11 in which 1 · R is H or CH3, preferably ch3; 2 · R? Is a Cos alkyl group, preferably as disclosed above + 3; R3 is formula (iii) Compounds; 4.1 is a group of formula (iii), where Rs.NHRf is a (^ _6 alkyl group, which is optionally substituted with 0H or (^ _4 alkoxy group); 5, R3 is a A group of the formula (iij〇, wherein Riq is (; ^ 015 and 1-15 are Ch alkynyl groups which are optionally substituted by 0H, preferably Ci_4 alkynyl groups substituted by 〇Η;

C: \ Program Files \ Patent \ 54618.ptd Page 25 593297 V. Description of the invention (22) 6. R3 is a group of formula (iii), in which R1G is CONHR15 and R15 is phenyl-Ci_4 alkyl or hetero Elementary aryl-C ^ alkyl, wherein the phenyl, heteroaryl and Ci_4 alkyl moiety may be substituted as mentioned above. Preferably, the benzyl group is substituted with a Ci_4 alkoxy group, particularly 0CH3, at positions 1, 2, or 3. The invention also includes a method for preparing a compound of formula II, which method comprises: a) when preparing a compound of formula II wherein R3 is a group of formula (0 or (ii),

In which a --- b and a --- / 3 are defined as described above, R, is defined as except that it must not be 0 因为, because it must exist in a protected form, and R'3 is formula (i) or ( ii) reacts with a compound of formula IV

r2cooh IV in which R2 is as defined above, or it is a functional group derivative 'or (b) converts micronolin or compressin to a compound of formula I; and, if necessary, removes the protective group The resulting compound of formula II | | is recovered in free form or in the form of a salt. If the oh in the starting product does not participate in the reaction, it must first be protected according to the known method. The 0H protection group is well known in the technical field, such as j tertiary butyl-difluorenyl_shixiyanyl. | Method step a) can be carried out according to known esterification methods. One type

C: \ Program Files \ Patent \ 54618. Ptd page 26 593297 I. Description of the invention (23) The functional group derivative of the IV compound includes, for example, an acid compound, an ester, or an acid anhydride. Step b) of the method may be a substitution reaction at the 5-position or a reduction reaction of a piperane group, such as disclosed in Example 3. The R2-C0-0- group of these micronolins can be reduced to 0H and then esterified to another R2-C0-0- group. In order to produce a compound of formula II in which R3 is formula (iii) and R1G is CON14R15, a compound of formula II in which R3 is a group of formula (i) or (ii), such as meminoline or compressin, Amines (such as alkylamines, HO-alkyl-amines, heterocyclic amines) may undergo ring opening via azide formation. When R7 is (0, 0H), it can be oxidized to = 0 by a known method such as by making sulfur trioxide with a bite ratio compound or by performing a Swaern oxidation method. A compound of formula II in which R7 and Rs in formula (iii) together form a aryl group or a dioxyalkylene group can be produced according to known methods, for example, using a preferred carbonyl diimidazole to generate a carbonyl group and dioxygenation through an acid condensation method Extension burning base. __ Method step b) may also be a compound of formula I I wherein 1 ^ 3 is a group of formula (iii) and cyclized to form a compound of formula I I where R3 is a group of formula (i) or (i v). The cyclization reaction can be advantageously performed in the presence of a base such as H un i g 's base and an activator such as trifluorosulfonate needle. R3 can be used as (i 1 i) a group of compounds of formula II in which R1Q is CONHR15 and 1 is oxidized to = 0 is conveniently made into compounds of formula II in which R3 is a (b) group. The cyclization reaction can be performed by acid treatment, such as trifluoroacetic acid. . As of now, there is no specific description of the preparation method of the starting material.

C: \ Program Files \ Patent \ 54618.ptd Page 27 593297 V. Description of the Invention (24) Substances are known or can be followed by methods well known in the art, such as disclosed in Y.Chapleur, Progress in The Chemical Synthesis of Antibiotics and Related microbial Products'1, Springer Verlag, 1993, voi. 2, 829-93. The present invention further proposes: 8. A compound of formula II or a pharmaceutically acceptable salt thereof as a medicine for preventing or treating the aforementioned disorders or diseases. 9. A pharmaceutical composition comprising a humanoid of formula I 丨, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable diluent or a carrier thereof. The following examples are provided to illustrate the present invention. Conventional Example 1: 2-ethyl-butyric acid 8- [2- (4-carboxy-6-oxy-tetrahydro ~? Sigma-2-yl) -ethyl] -3,7-monomethyl 2,3 , 7,8,8a-Hexahydro · Mu-Yi Diao added 927 mg (4.39 mmol) of 2-ethyl-butyric anhydride to 9 (0.35 0 mmol) of 4- (Third-Butyl) -Difluorenyl-silylamino mg- (8-hydroxy-2, 6-difluorenyl-1,2, 6, 7, 8, 8a-hexahydro-fluorene ~ ^ odorous) 6 [2 radical] tetra Hydropiperan-2-one in a solution of 2 m 1 of pyridine. The mixture was stirred overnight at ^ B temperature. The solution was quenched by the addition of the aqueous solution of sodium bicarbonate of Yuwa; Third-order butyl bond extraction and two organic phases were combined, and then extracted with 10% citric acid solution and then treated with sodium chloride. Water was added. The crude product was dissolved in 5 ml of butyl acetate containing 75 mg (1.3 mmol) of acetic acid. Add 0,3 g (1 mmOl) of tetrabutylammonium fluoride tetrahydrate. After 20 hours at * temperature, terminate with saturated aqueous sodium bicarbonate solution.

C: \ Prograin Files \ Patent \ 54618. Ptcl • page 28 593297 V. Description of the invention (25)

should. After separating the liquid phases, the aqueous phase was extracted with ethyl acetate. Enter the machine phase, extract and wash with brine, and dehydrate with sodium sulfate. The solvent was distilled off, and the product was purified by silica gel, chromatography (methyl tertiary butyl), and the desired product was obtained, followed by diethyl ether / hexane. T mp .120 -122 ° C (diethyl ether / hexane) day by day. MS (ESI) 441 (M + Na), 419 (MH + H) Example 2: 2-methyl-butyric acid 8 — [2 — (5 —benzyl-4 -hydroxyloxy-tetrachloro-piperan-2 -Yl) -ethyl] -1,3,7-difluorenyl-1,2,3,7,8,83 ~ hexa | 1-methyl 1-yl ester octadecene to 0.63 m 1 (1.0 mm) i) A solution of 1.6 M butyllithium in hexane was added to a solution of 101 mg (1.0 mmo 1) diisopropylamine in 5 ml THF under ice-cooling (-77 ° C) In solution. After 15 minutes had elapsed, 202 mg (0.5 Ommo 1) of microminolin was added and the reaction mixture was maintained at -77 C 30 minutes. Then, 17 lmg (1.0 mmo I) of benzyl bromide was added. After 2 hours, the reaction was gradually warmed to room temperature and then poured into 0, 1 n η C 1 aqueous solution. After separating the liquid phases, the aqueous phase was extracted with ethyl acetate for two-person. a The organic phase 'was washed with brine and then dehydrated with sodium sulphate. After distilling off the solvent, the crude product was purified by shijia gel chromatography (diethyl ether / hexane 2/1) to obtain the desired product as a colorless oil. MS (FAB) 495 (M + H)? 393 Example 3: 2-fluorenyl-butanoic acid 8- [2- (4 ~ hydroxy-tetrahydro-piperan-2-yl) _ethyl] -3, 7 -Difluorenyl-1,2,3, 7, 8, 8a-Hexahydro-fluoren-1-yl ester Add 22 mg (1.0 mmol) of lithium borohydride to 65 mg (0.15 mm) of rice Micronolin in a 5 m 1 ethanol solution and stir the mixture at room temperature

C: \ PrograniFiles \ Patent \ 54618.ptd Page 29 593297 f I. Description of the invention (26) I Jing Ye. 0. IN HC1 aqueous solution was added to stop the reaction. After separating the liquid phases, the aqueous phase was extracted twice with ethyl acetate. The organic phases were combined, washed with brine and dehydrated with sodium sulfate. After distilling off the solvent, the crude product was purified by silica gel chromatography (ethyl acetate) to obtain 2-fluorenyl-butanoic acid 3,7-difluorenyl-8- (3,5,7-trimeryl- Heptyl) -1,2,3,7,8,8a-hexahydro-fluoren-1-yl§ is intended to be an oil. MSCESI) 431 (M + iNa), 40 9 (M + H) Add 0.9 g (7, 〇ιηπιοί) of Tiger Niger base and 0.08 ml (0.5 mmol) of trifluoroacetic anhydride to 73 mg (0, 18 mmol) ) Of 2-fluorenyl-butanoic acid 3, 7-difluorenyl-8- (3,5,7-trisyl-heptyl) -1,2,3,7,8,8a -hexahydro-fluorene The -1-yl ester was placed in an ice-cold (-7 7 ° C) solution of 5 m 1 of dichloromethane. After 30 minutes, a saturated aqueous sodium hydrogen carbonate solution was added to terminate the reaction. The phases were separated and the aqueous phase was extracted twice with ethyl acetate. The organic phases were combined, washed with brine and dried over sodium sulfate. After the solvent was distilled off, the amidine product was purified by silica gel chromatography (diethyl ether / hexane 1/4) to obtain the desired product as a colorless oil. MS (FAB) 391 (M + H), 289 Example 4: 2-fluorenyl-butanoic acid 8- (6-benzylphosphoniumamino-3, 5-dihydroxy-hexyl) -3,7 --fluorene 2,3,7,8,8a-Hexahydro-fluoren-1-yl vinegar 0.5 ml (5mniol) of benzylamine was added to a solution of 0. BmgO, 32mmol) Miwei Zhulin in 15ml THF. The stirred reaction mixture was heated to reflux temperature for 7 hours. When it has cooled down to room temperature, add 20m of sulfonyl-tertiary butyl ether and dilute it, and then extract and wash with HC1 and brine in sequence. The organic phase is then dehydrated with sodium sulfate and the solvent is evaporated off. Layered by Shixi gelatin

C: \ ProgramFiles \ Patent \ 54618.ptd 页 Page 30 593297 V. Description of the invention (27) | Analytical method (ethyl acetate) The crude product can be purified to obtain the desired product as an oil. MS (ESI) 5 34 (M + Na), 512 (M + H) ', Example 5: Preparation of compound 3 3 in Table 3 200 mg meminoline in 5 ml THF and 1 ml hydrazine hydrate (25% In water) and stirred at room temperature for 15 hours. After concentration, the reaction mixture was diluted with ethyl acetate, washed with water and brine, dehydrated (Na2S04), and concentrated. With (Et) 20, 2-fluorenyl-butanoic acid 8- (6-trapylcarbonyl-3, 5-dihydroxy-hexyl)-3, 7-difluorenyl-1, 2, 3, 7, 8, 8a -Hexa-Hydroxy- 蓁 -Bugi § purpose recrystallization. Add 1.0 ml of a solution of 5N HC1 in (Et) 20 and 0.36 ml of 10% third butyl nitrite in DMF to a stirred 86 mg ice-cold (-15 ° C) hydrazine solution . After stirring at -15 C for 15 minutes, 0.15 ml of NEt3 and 0.06 ml of 1,4-dioxa-8-azaspiro 5] decane were added. The reaction mixture was stirred for 15 hours at 0 ° C and then evaporated under reduced pressure. It was diluted with ethyl acetate, washed with 0.1N HC1, saline, bicarbonate, extracted with brine, dehydrated (NadO4), and concentrated. . The product of Example 33 can be obtained after diisopropyl junction formation. °! MS (ESI): 548 μΜ Example 6: 2-Amino-butyric acid 8- [3-hydroxy-5-(2-hydroxy-ethylamino) (2-hydroxy-ethylamino) —Hexyl]-3, 7-dimethyl-1,2, [7 8 8a-hexahydro-fluoren-1-yl § intent I will add 20ml of acetic anhydride at room temperature to 4. Og (l 〇mm〇i) Zhu Weinuo | Lin at 20 m 1 swallowed the solution and swallowed the solution. After 1 hour, the reaction was carried out under vacuum.

593297 V. Description of the invention (28) | The mixture should be concentrated. The residue was dissolved in 10 ml of fluorenyl tertiary butyl ether and then extracted with water, 0.1 N H C 1 and brine in order. The organic phase was then dehydrated with sodium sulfate and the solvent was removed in vacuo. The crude product was purified by silica gel chromatography (fluorenyl tertiary butyl ether) to obtain 2-fluorenyl-butyric acid 3,7-difluorenyl-8- [2- (6-oxo-3,6 -Dihydro-2} 1-piperan-2-yl) -ethyl] -1,2,3,7,8,8a -Hexahydro-fluoren-1-yl ester is a white powder. Add 1.5 ml (25 mmol) of ethanolamine at room temperature to a 1.22 (3.0111111〇1) 2-fluorenyl-butyric acid 3,7-diamidino-8- [2 -(6-oxo-3,6-dihydro-piperan-2-yl) -ethyl] -1,2,3,7,8,8a-hexahydro-fluoren-1 -yl ester. After 12 hours, the solvent was distilled off and the residue was dissolved in 10 m of ethyl acetate. The solution was sequentially washed with 5 ml of a saturated sodium hydroxide solution and 5 ml of brine. After the solution was dehydrated with steel sulfate, the solvent was distilled off, and the title compound was obtained as a foam which was susceptible to moisture. MSCESI) 515 (M + Li), 5 0 9 CM + H) By following the steps in the previous example in sequence, the compound of formula Xi can be prepared by using appropriate starting materials.

In which R15 is defined in Table 1 below.

C: \ Prograni Fi les \ Patent \ 54618. Ptcl page 32 593297

C: \ Prograin Files \ Patent \ 54618. Ptd page 33 593297 V. Description of the invention (30) 19 / (: ¾ OH CH \. 0 and 2 ESI 507 MH. 20 CHrCO-N (CH3) 2 FAB 507 MH & quot • 21 c〇och3-ESI 568 MH- 22 CH (CH3) -CONH2 ESI 491 MH · By following the steps in the previous example, using the appropriate. Starting material j co-nh-ck2-ch2-oh can be prepared The compound of formula x2 is ho-ch2-ck2-hn \

R2 is defined in Table 2 below. Table 2

Ex r2 MS 23 ch (ch2-ch2-ch3) 2 FAB 557 M + Li + 24 1 CCH--CH, I 3 FAB 529 M + Li + ch3

C: \ Program Files \ Patent \ 54618.ptd Page 34 593297 I. Description of the invention (32)

31 OH OH CO-N 3-CH.-CH • \ OH '' 2 ESI 538 MIT 32 OH OH C〇-N (CH3) -CHr CO-N (CH3) 2 ESI 521 ΜΗ &quot; 33 OH OH c &quot; CX— ESI 548 MIT 34 OH OH Η_Ν-ς〇CO-N \ ESI 519 MET * 35 = 〇OH CO-NH-C, ~~ <〇ESI 529 NTHCOO · ** 36 OH OH CH2-NH-CH ~ ~ 0〇1} ESI 548 Mir *** 37 〇II / c \ n? 1 CO-NH-CH2—y 0 ESI 528 MH + 38 = 〇OH CO-KH-Oi ~ ^ 〇> FAB 516 M + Lr 39 = 〇OH CO-NHCH3 FAB 440 M + Lf 40 OH OH CC—NK—CH--CSH, \ / FAB 581 ΜίΓ 41 OH OH CK ^ -NH-C? ^-Of ^ ESI 488 MH + 42 OH OH ch 「 nCX- ESI 534 MIT C: \ Prograiii Files \ Patent \ 54618. ptd page 36 593297 V. Description of the invention (33)-43 OH OH-small radical ESI 592 MH · 44 OH OH CO-<.> ESI 540 ΜΗ '* Example of oxidation (EX · 3 5 compound) Slowly treat a solution of 0 · 18ml chloracetin in 10ml CH2C12 with 0.33ml DM SO at -60 ° C. After stirring at -60 ° C for 15 minutes Put fluorenyl-butanoic acid 8-[5- (tertiary-butyl-dimethyl-silyloxy)-3 -Cyclo-6- (4-fluorenyloxy-benzylamine) -hexyl] -3, 7- Difluorenyl-1,2,3, 7,8,8a-hexahydro-fluoren-1-yl § | in 5ml CH2C12 ice-cold (-

7 8 ° C). After 2 hours at -60 ° C, NEt3 of 0.80 m 1 was added and the temperature was slowly raised to room temperature. After 2 hours at room temperature, H20 was added to stop the reaction. After the liquid phase was separated, the organic phase was dehydrated and concentrated. The crude product was purified by silica gel chromatography (CH2Cl2 / MeOH). The resulting compound was dissolved in THF and treated with AcOH and Bu4NF.3H20. After 30 minutes, the mixture was concentrated, diluted with AcOEt and rinsed with H20, saturated NaHC03, brine, and then concentrated with water (Na2S04).

Shrink. The crude product was purified by silica gel chromatography. Pure soluble fractions are combined into cakes! After concentrating, 2--methyl-butyric acid 8- [5-hydroxy-6- (4-methyloxy-benzylamine I fluorenyl) -3 -oxo-hexyl] -3, 7 -difluorenyl-1,2,3,7,8,8a -Hexahydro |! -Fluoren-1 -yl ester is foamed> finished. ί

* * Reduction example (E X. 3 6 · compound) I | Under argon, 2.0 g of 2-fluorenyl-butyric acid 8- {2- [4- (third stage-|

C: \ Program Files \ Patent \ 54618. Ptd Page 37 593297 V. Description of the invention (34) | Butyl-diamidyl-silyloxy) -6-oxo-tetrahydropiperan-2yl] -ethyl 3,7-Difluorenyl-1,2,3,7,8,8a-Hexahydro-fluoren-1-yl §A solution of i2ml anhydrous butane HF at -78 ° C under stirring 8.0ml A solution of 1M diisobutylaluminum hydride in THF was added slowly. After stirring at -78 ° C for 30 minutes, a mixture of 1.5 m 1 M e Η and 3 m 1 T H F was slowly added. After 15 minutes, the reaction mixture was concentrated, diluted with EtOAc and rinsed with 10% citric acid, H20, saturated NaHC03, brine, and then dehydrated (Na2S04) and reduced in vacuo. The obtained galactanol was obtained at a temperature of __ 20 ° C. 300 mg of crude endohydrin was treated with 74 mg of NaCNBH3 and 0.25 ml of 4-methoxybenzylamine in a mixture of 10 ml of DMF and 1 ml of AcOH under argon. After stirring at room temperature for 40 hours, the reaction mixture was diluted with AcOEt and ice-cold IN HC1 and stirred for another 30 minutes. The organic phase was further washed with saturated NaHC03, brine, dehydrated and concentrated. The amidine product was purified by silica gel chromatography (CH2Cl2 / MeOH 95: 5) to obtain 2-fluorenyl-butanoic acid 8- [5- (third-order-butyl-difluorenyl-silica: oxy) ) -3 -hydroxy-7- (4-methoxy-benzylamino) -heptyl] -3, 7-dimethyl-1, 2, 3, 7, 8, 8a-hexahydro-fluorene- 1-based vinegar. To deprotect, the product was treated at room temperature with a mixture of 2 ml of THF containing 20 // 1 AcOH and 77 mg of tetrabutylammonium fluoride trihydrate. After 30 hours of stirring, the mixture was concentrated, diluted with A c OE t and rinsed with H20, saturated NaHC03, brine, then dehydrated (j | Na2S04) and concentrated. 2-Amino-butyric acid 8- [3,5 -dihydroxy- 7- (4-

593297 V. Description of the invention (35)-~ a ~~ * * 氺 Convert Εχ · 17 compound to εχ · 37 compound 100 mg in 2ml CH2Cl2iEx. 17 compound with 73 mg DMAP, and 0.13 ML in 曱 ben Phosgene (2M) treatment inside. After stirring at room temperature overnight, the reaction was terminated with sodium bicarbonate. The reaction mixture was then concentrated, diluted with EtJ, and then rinsed with 1 n HC1, H2O, and NaHC03 successively, then dehydrated and concentrated2. The desired product can be crystallized from Et20 / diisopropyl ether. (Dmap = diamidoaminopyridine). See Example 45: 2-fluorenyl-butanoic acid 8- [2- (1-benzyl-6-oxo-1,6-dihydro_2 1-laz-2-yl-ethyl]-3, 7 -Difluorenyl-1,2,3,7,8,8a-Hexahydro-zirconyl 1-yl vinegar. 2.6 g (5.0mmo: l) of 2-fluorenyl-butyric acid 8-{2-[ 4-(Tertiary monobutyl-monofluorenyl-silyloxy) -6-oxo-tetrahydronanyl] -ethyl 3,7 monofluorenyl-1, 2, 3, 7, 8, 8a -Hexahydro-p--1-yl ester was stirred at 50 mi of benzene) The valley was added at room temperature with 2.0 mmol) of benzylamine and 1 mg of Amberlite IP 120. The reaction was heated at reflux for 8 hours. After cooling to room temperature, the mixture was filtered, rinsed with 10% aqueous citric acid solution and dehydrated with sodium sulfate. After the solvent is distilled off, 2 ~ fluorenyl-butyric acid can be obtained. 8 [6 -Xiafuzhuotai 5- (Secondary-butyl-di-fluorenyl-shixi alkoxy)-3 | yl-hexyl] -3,7-Difluorenyl-1,2, 3, 7,8, 8a-Hexahydro-z--1-yl | Ester is an oil. MS (FAB) m / z 632 ([M + Li] +) | 4 · 4g (28 mmo 1) S03 pyridine complex in 22m 丨 DMS0 solution was added to a 2,9g (4.6 mmol) 2- Fluorenyl-butanoic acid 8- [6-benzylamine fluorenyl i-fluorenyl 5- (tertiary-butyl-difluorenyl-silyloxy) -3 -hydroxy-hexyl]-;

C: \ Program Files \ Patent \ 54618.ptd P.39 5933297 V. Description of the invention (36) 3, 7-difluorenyl-1, 2, 3, 7, 8, 8a-hexahydro-1-1-yl Ester with 5: 2 ml (37 nmol) of triethylamine in a stirred solution of 22 ml DMSO. After 2 hours at room temperature, the mixture was poured onto ice and extracted twice with ethyl acetate. The combined organic phases were dried over sodium sulfate and the solvent was evaporated. Chromatography on silica gel (hexane / ethyl acetate 9/1 to 7/3) can give 2-fluorenyl monobutyric acid 8-[6 -benzylamine methyl alcohol 5- (third grade '• Butyl-difluorenyl-lithium alkoxy) -3-lacto-hexyl] -3,7-difluorenyl-1,2,3,7,8,8a-hexaaza-calyl-ester Oily. MS (FAB) m / z 6 3 0 ([M + Li] +) 0.3 g (2.6 mmol) of trifluoroacetic acid was added to a 200 nig (〇, 32 〇〇1) 2 -fluorenyl-butyric acid 8- [6-benzylaminophenyl 5- (tertiary-butyl-difluorenyl-lithium alkoxy) -3-oxo-hexyl], 3,7-difluorenyl-1, 2, 3 , 7,8,8a-Hexahydro-p--1-yl ester in 5 ml of a stirred solution of dichloroarsine. After 2 hours at room temperature, a saturated aqueous solution of hydrogen carbonate was added to the reaction mixture to terminate the reaction. Water; r Valley liquid phase was extracted twice with ethyl acetate. The combined organic phases were dehydrated with sodium sulfate and the solvent was evaporated. The residue I was chromatographed on silica gel (hexane / ethyl acetate 7/3 to 2/3) to obtain the title compound as a white powdery foam. MS (FAB) in / z 480 ([M + Li] ') Example 46: 2-Amidino-butanoic acid 3, 7-diamidino-8- [2- (6-oxo-1,6-i Dihydro-pharyngeal-2 -yl] -ethyl] -1,2,3,7,8,88 ~ hexahydro-p--1-yl ester | 0.7 g (6.5 mmo 1) trifluoroacetic acid Add to one i.〇g (1.9: -〇1) 2 -fluorenyl-butyric acid 8- [5- (tertiary-butyl-difluorenyl-silyloxy | yl) -6- ¥ amine 3 -oxo-hexyl] -3,7-difluorenyl-1,2,3,7, | 8, 8a-hexahydro-p-1-yl ester in 25 ml of a stirred solution of dichloromethane.丨 After 2 hours at room temperature, add saturated hydrogen carbonate solution to the reaction

C: \ Program Files \ Patent \ 54618.ptd Page 40 593297 V. Description of the invention (37) Mixture to stop the reaction. The aqueous phase was separated and extracted twice with ethyl acetate. The combined organic phases were dried over sodium sulfate and the solvent was evaporated. Chromatography of the residue on silica gel (hexane / acetone 1/1) gave the title compound as a white foam. MS (FAB) ni / z 3 84 ([Μ + ΗΓ) Example 47: 2-Amidino-butanoic acid 3, 7-diamidino-8- [2- (1-Amidino-6-oxo, 6 -Dihydro-¾ bite-2_yl] -ethyl] -1,2,3,7,8,8a-hexahydro- 着 -1 -yl_ O.lg (0.8mmol) carbonate with 0 ·] ^ (0.8mniol) fluorenyl carbon to a 2-fluorenyl-butyric acid 3, 7-difluorenyl-8 [2_ (6-oxo-1,6-dihydro-pharyngeal-2 -yl)- Ethyl] -1,2,3,7,8,8a -Hexahydro-naphthalen-1-yl vinegar in a 5 ml stirred solution of DMF. After 2 hours at room temperature, the reaction mixture was poured into water. Aqueous phase It was separated and extracted twice with ethyl acetate. The combined organic phases were dehydrated with sodium sulfate and then the solvent was distilled off. The residue was chromatographed on stone gum with hexane / acetone 1/1 to obtain the title compound as a white foam. __ MS (FAB) m / z 404 ([M + Li] f) Example 48: 2-ethyl-butyric acid 8- [2- (1-benzyl-6-oxo-1,6-dihydro -Meow bite -2 -yl] -ethyl] -3,7 -dimethyl--1,2,3,7,8,8a -Hexahydro-Cai-1 -Gyron | MSCESI): 487 Μτ Example Compounds of 6, 15 and 3 3 are used to prevent or treat disorders or mediated by LF A-| 1 / ICAM-3 or ICAM-3 interactions. Disease (e | such as ischemia / reperfusion injury, and organ rejection after transplantation). For example the preferred compound words, detection and analysis of the cells disclosed in the foregoing J urkat

C: \ ProgramFiles \ Patent \ 54618.ptd page 41

593297 5. In the description of the invention (38), its I C5Q is 4, 2.2 and 1.2 v Μ, respectively. In the rat model of thioglycolate-induced peritonitis, the compounds of Examples 6 and 33 were administered at doses of 1 and 0.1 mg / kg, respectively, at s.c. after administration. . It is hereby pointed out that when used to treat or prevent the disorder or disease, the compound is administered to a human at a daily dose of from 5 to 750 mg.

C: \ Program Files \ Patent \ 54618. Ptcl Page 42 593297 Attachment HOv 1 〇 I! 1 II 〆 / · = H Compact in CK3? 5 Jf CH, Rrn = Ch: 3 Mevinolin

Current status of Rm data collection LFA-1 I-zone / Micronolin temperature 293K wavelength, 1.5418A resolution range 15.0 into -2.60 into the space group single crystal size a = 72.7 people, b = 77.7 people, c = 91.8 used Measurement 87179 Unique reflection 16457 Completeness 99.9% (99.8% in shell 2.69 in-2.60 in) Multiple 5.3 (5.2) Average I / sig (I) 13.9 (2.1) R combined 11.6% (48.4%) CD a α " AC: \ Program Files \ Patent \ 54618.ptd Eve / Page 593297 Supplement

C: \ Program Files \ Patent \ 54618. Ptd page 593297 supplement

III

C: \ Program Files \ Patent \ 54618.ptd 593297 Appendix Table 1

Ex RlS MS 7 ch2〇ch3 ESI 542 MKT 8 Doxy ESI 606 MH · \ c〇NH2 9 Isobutyl FAB 478 MH + 10 CH2-C0-0-t.butyl FAB 536 MH + 11 FAB 518 MH + / 〇ch3 12 ™ 2- {〇V ~ 〇ch; FAB 572 MH + 13 C (CH2-OH) 3 ESI 524 MH * 14 ch2-ch2- · Merlinyl ESI 535 MH + 15 CH2-^ 〇) ESI 542 MH + x -〇 ch3 16 / CH3 CH — CH | \ ESI 521 MH · CONH2 oh 17 CH.-0 ESI 502 MPT 18 / isobutyl CH \ FAB 535 MHT \ CONH2 19 / CH2〇H ESI 507 MH * r〇NH2 20 CH2- CO-N (CH3) 2 FAB 507 MH &quot; 21 1 ESI 568 MH 'c〇ch3 22 CH (CH3) -CONH2 ESI 491 MH' C: \ Program Fi les \ Patent \ 54618. Ptd Min. Order 593297 Attached page

Table 2

R

Ex r2 MS 23 CH (CH2-CH2-GH3) 2 FAB 557 M + Li + f3 24 1 C-CH ^ -CHj FAB 529 M + Li + 1 ch3 Page C: \ Program Files \ Patent \ 54618.ptd 593297 with Page Table 3

Ex R7 Rs Rio MS 25 OH Hydrogen_Co-OC2H5 FAB 539 M + Li + 26 OH OH c〇-n (ch3) 2 FAB 450 MH + 27 OH OH r ~ \ CO-N N-COOEt ESI 563 MIT 28 OH OH 〒h2-ch3 C〇_N, (ch2) 2-〇h ESI 494 MIT 29 OH OH CO-OCH. ^ Ft c〇—nn ^ i ~ \ j] ESI 562 MH '30 HC, ^ CH3 ff _JT \ CO-NH-CH2- \ 〇ESI 542 MH + 31 OH OH Γ π CO-N CH, -CH \ 2 L_ ok ESI 538 MH + 32 OH OH CO-N (CH3) -CH2- CO-N (CH3) 2 ESI 521 MH + C: \ Program Files \ Patent \ 54618. Ptd Page / ^ 593297 Attachment 33 OH OH c. -nCX— ESI 548 MIT 34 OH OH Η Ν-ςο Vn CO-N \ ESI 519 MH + * 35 = 〇OH CO-NH-CH ^ ~ <〇 ^ ~~ 〇CH3 ESI 529 M'HCOO '** 36 OH OH £ TH2-NH-CH <〇 ^ -OCF ”ESI 548 MH + *** 27 〇II / c \.? 1 _jr \ CO-NK-CH2— 〇ESI 528 MH + 38 = 〇OH CO-NH-CH ^ ~~ 〈〇> FAB 516 M + Li + 39 = 〇OH CO-NHCH3 FAB 440 M + Li + 40 OH OH Λ ~ λ CO— N \ / N— CHj-C ^ FAB 581 MH &quot; 41 OH OH ch2-nk- ch2-zhi) ESI 488 MH + 42 OH OH ESI 534 MH + 43 OH OH ° ^ _Can-indolyl ESI 592 MH_ 44 OH OH CO-NH-CH- (〇> 2 V ^ Z-och3 ESI 540 MH 'C : \ Program Files \ Patent \ 54618.ptd Page 593297

Claims (1)

593297 / Λ- 6. Scope of patent application 87113879 1 · A compound of formula X3 in free form or salt form Wherein (〇1? 7 is meridian, 1 ~ 8 is 2-benzyl-ethylamine, and R1Q is 2-benzyl-ethyl amidinofluorene; C0-] (ii) R7 is a radical '1 to 8 is a radical' and 1 ^. Is 2-methoxy-fluorenylmethylamine hydrazone; or 2. — Please have the patent formula 3. Root control or cure soil) 1-7 is the radical '1 ^ 8 is the radical' and R1Q is the species for LFA- 1 A pharmaceutical composition of an antagonist comprising a compound of formula X3 in a free form or a pharmaceutically acceptable salt form according to item 1 of the scope of application, and a pharmaceutically acceptable diluent or carrier. The pharmaceutical composition according to the scope of patent application No. 2 is used for the pretreatment of chronic transplant rejection. O: \ 54 \ 54618-930129.ptc Page 43