High throughput method for constructing and screening compound library and reaction device

[0001] This application is a Section 371 of International Application No. PCT/CN2020/099034 filed Jun. 29, 2020, which was published in the Chinese language Jan. 14, 2021, under International Publication No. WO 2021/004326 A1, which claims priority to Chinese Patent Application No. 201910605212.5 filed Jul. 5, 2019, the disclosures of which are incorporated herein by reference in their entireties.

[0002] The present invention relates to the chemical and biological fields, in particular to a high-throughput method for constructing and screening compound library and a reaction device.

[0003] Triazole is an important five-membered nitrogen heterocyclic compound, which involves a wide range of industrial applications, such as agricultural chemicals, corrosion inhibitors, dyes, fluorescent brighteners, and bioactive agents, etc. Azide functional groups are very important and widely used in organic synthesis. However, this kind of compounds have great potential safety hazards in the process of synthesis, storage and transportation due to their high energy, which leads to difficulty in synthesis and use. At present, the synthetic methods of azide compounds are very limited, among which alkyl, acyl and sulfonyl azide are mostly obtained by nucleophilic substitution reactions with NaN₃ being used to replace the leaving functional groups in polar solvents, and the method need further purification, and the syn thesis efficiency is not high, resulting tedious post-treatment when used for further activity screening.

[0004] In the development of pharmaceuticals and agrochemicals, commercial compound libraries and compound libraries produced by combinatorial synthesis have been screened to find biologically active substances (such as leading compounds). However, in the synthesis of compounds used as drugs, separation and purification take a lot of time, and it is difficult to find leading compounds that can be used as drugs. The compound library produced by combinatorial chemistry is a mixture of many compounds, and the structure of products is difficult to control. In addition, in order to establish a compound library, it is necessary to identify the structure of compounds during the synthesis of compounds and their derivatives, so as to determine the suitable methods for modifying compounds and explore the reaction conditions for synthesizing derivatives. The resulting adverse result is that it takes a lot of time to synthesize compounds.

[0005] Therefore, it is necessary to develop a simple, efficient and high-throughput method for synthesizing compound libraries and screening their activities.

[0006] The invention provides a simple, efficient and high-throughput method for synthesizing a compound library and screening their activities.

[0007] In a first aspect of the invention, provided is a method for constructing and screening a high-throughput compound library comprising:

• • [0008] (a) providing a reactor comprising n reaction chambers, wherein the reaction chambers are each independent and addressable, and the n reaction chambers constitute an addressable array of reaction chambers;
  • [0009] (b) performing m each independent synthesis reactions in t e n reaction chambers, and obtaining respective synthetic products in each of reaction chambers in which the synthesis reactions are carried out, thereby constructing and obtaining a compound library;
  • [0010] wherein, the synthesis reaction comprises the following steps:
  • [0011] (b1) in a reaction chamber, in an inert solvent, carrying out a 1, 3-dipolar cycloaddition reaction with a 1, 3-dipolar cyclization reagent and a reaction substrate containing terminal unsaturated bond, thereby forming a reaction product containing 1, 3-dipolar ring;
  • [0012] and (c) optionally, adding activity test reagent into the reaction chamber where the synthesis reaction is carried out, respectively to carry out activity test, thereby carrying out activity screening on each synthetic product;
  • [0013] wherein, n is a positive integer ≥10, and m is a positive integer ≤n and m≥10.

[0014] In another preferred embodiment, in step (c), after adding activity test reagent, performing incubation so that the activity test reagent reacts or contacts with each synthesis reaction.

[0015] In another preferred embodiment, the reactor comprises an array reactor.

[0016] In another preferred embodiment, n is 20-50000, preferably 36-10000, more preferably 48-5000, most preferably 96-2500.

[0017] In another preferred embodiment, the 1, 3-dipolar cyclization reagent is R—N₃, where R is the molecular moiety or fragment attached to the azide group.

[0018] In another preferred embodiment, in two or more reaction chambers, performing the same synthesis reactions.

[0019] In another preferred embodiment, different synthesis reactions are carried out in each reaction chamber.

[0020] In another preferred embodiment, in the synthesis reaction, m kinds of 1, 3-dipolar cyclization reagents located in m reaction chambers respectively, are used to react with the same kind of reaction substrates containing terminal unsaturated bond.

[0021] In another preferred embodiment, the “terminal unsaturated bond” means that there is a double bond or triple bond between the terminal atom (except hydrogen atom) located at the terminal and its adjacent subterminal atom (except hydrogen atom).

[0022] In another preferred embodiment, the terminal atom and t e adjacent subterminal atom are each independently selected from the group consisting of C, N, and combinations thereof.

[0023] In another preferred embodiment, the “terminal unsaturated bond” is selected from the group consisting of C═C, C═C, C═N, and combinations thereof.

[0024] In another preferred embodiment, the reaction chamber is a micro or small reaction chamber.

[0025] In another preferred embodiment, the volume of each of the reaction chambers is independently 5 μL-5000 μL.

[0026] In another preferred embodiment, the volume of the reaction chamber is 10-2000 μL, more preferably from 20 μL-1500 μL.

[0027] In another preferred embodiment, in a single reaction chamber, the volume of the reaction system in which the synthesis reaction is carried out is 52 ml, preferably ≤1 ml, more preferably 50.6 ml.

[0028] In another preferred embodiment, in a single reaction chamber, the volume of the reaction system in which the synthesis reaction is carried out is 5-2000 μL, preferably 10-1000 μL, more preferably 20-600 μL.

[0029] In another preferred embodiment, the activity test reagent is selected from the group consisting of small molecule compounds, proteins, nucleic acids, cells and combinations thereof.

[0030] In another preferred embodiment, the activity test reagent comprises an enzyme activity test reagent composition, or a cell activity test reagent composition.

[0031] In another preferred embodiment, the activity test reagent is a compound having physiological activity, preferably selected from the group consisting of enzyme inhibitor, ligand/receptor binding inhibitor, angiogenesis inhibitor, cell adhesion inhibitor, gene expression inhibitor and growth factor-like active substance.

[0032] In another preferred embodiment, the enzyme inhibitor is elected from the group consisting of tyrosinase inhibitor, cyclooxygenase inhibitor telomerase inhibitor, matrix metalloprotein inhibitor, prostaglandin D synthesis inhibitor, phosphodiesterase inhibitor, cholinesterase inhibitor, viral protease inhibitor, reverse transcriptase inhibitor, and a combination thereof.

[0033] In another preferred embodiment, the protein comprises an antibody, a ligand, an antigen, or a combination thereof.

[0034] In another preferred embodiment, the step (c) comprises a step of detecting each of reaction chamber thereby obtaining activity test results in each chamber, respectively.

[0035] In another preferred embodiment, a step of separating or purifying the reaction product is not included between the steps (b) and (c).

[0036] In another preferred embodiment, after the end of step (b) (e.g. step (b1)), it is directly applied in step (c) for subsequent activity testing without separation.

[0037] In another preferred embodiment, in the step (b), the reaction formula for the 1, 3-dipolar cycloaddition reaction is selected from the group consisting of

[0038]

• • [0039] wherein, R, R′, R″ and R″ are each independently R¹ or

[0040]

• • [0041]  and R¹ and R² are each independently a substrate fragment.

[0042] In another preferred embodiment, in the step (b), the reaction formula for the 1, 3-dipolar cycloaddition reaction is as follows:

[0043]

• • [0044] wherein, R and R″ are each independently R¹ or

[0045]

• • [0046]  R¹ and R² are each independently a substrate fragment.

[0047] In another preferred embodiment, R¹ and R² are the remaining molecular moiety of the reaction substrate other than molecular moiety of the terminal unsaturated bond.

[0048] In another preferred embodiment, R¹ and R² each are independently drug active fragments.

[0049] In another preferred embodiment, R¹ and R² are each independently substituted or unsubstituted groups selected from the group consisting of alkyl, cycloalkyl, heteroalkyl, heterocyclyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl, sulfonyl, or a combination thereof.

[0050] In another preferred embodiment, R¹ and R² are each independently selected from the group consisting of substituted or unsubstituted C₁-C₁₂ alkyl, substituted or unsubstituted 3-12 heteroalkyl, substituted or unsubstituted 5-16 heteroaryl, substituted or unsubstituted C₆-C₁₈ aryl, substituted or unsubstituted 3-20 heterocyclyl, substituted or unsubstituted C₃-C₁₂ cycloalkyl; wherein the “substituted” means that one or more hydrogen atoms in the groups are substituted by substituent Ra, and Ra is selected from the group consisting of halogen, substituted or unsubstituted C₁-C₆ alkyl, substituted or unsubstituted C₂-C₆ alkenyl, substituted or unsubstituted C₂-C₆ alkynyl, substituted or unsubstituted C₃-C₁₂ cycloalkyl, oxo (i.e. ═O), Rf—SO₂—, ═NRf—CN, hydroxyl, —ORf, NRdRe, substituted or unsubstituted C₁-C₆ amino, substituted or unsubstituted —(C₁-C₆ alkylene)-NH—(C₁-C₆ alkylene), carboxyl, substituted or unsubstituted C₆-C₁₀ aryl, substituted or unsubstituted 5-12-membered heteroaryl having 1-3 heteroatoms, substituted or unsubstituted 5-12-membered heterocyclyl having 1-4 heteroatoms, wherein the substituent is selected from group consisting of halogen, hydroxyl, carboxyl, cyano, C1-C6 alkoxy, and C1-C6 alkylamino;

[0051] Rd, Re, Rf are each independently selected from the group consisting of H, substituted or unsubstituted C₁-C₆ alkyl, substituted or unsubstituted C₃-C₁₀ cycloalkyl, substituted or unsubstituted C₆-C₁₀ aryl, substituted or unsubstituted 5-12-membered heteroaryl having 1-3 heteroatoms, substituted or unsubstituted 5-12-membered heterocyclyl having 1-4 heteroatoms;

• • [0052] the “substituted” in Ra, Rd, Re and Rf means that being substituted by one or more (e.g. 2, 3, 4, etc.) substituents selected from the group consisting of halogen, —CH₂Cl, —CHCl₂, —CCl₃, —CH₂F, —CHF₂, —CF₃, —O(CH₂)_XO(CH₂)_YCH₃, oxo —CN, hydroxy, amino, substituted amino, carboxyl, —NHAc, a group selected from the group consisting of, which is unsubstituted or substituted by one or more substituents selected from the group consisting of, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₆-C₁₀ aryl, C₃-C₈ cycloalkyl, halogenated C₆-C₁₀ aryl, 5-10-membered heteroaryl having 1-3 heteroatoms selected from N, S and O, 5-10-membered heterocyclyl having 1-3 heteroatoms selected from N, S and 0;
  • [0053] x is 1, 2, 3, 4, 5 or 6;
  • [0054] y is 0, 1, 2, 3, 4, 5 or 6;
  • [0055] in each of the above-mentioned formula, any one of the heteroatoms is independently selected from the group consisting of N, S and O.

[0056] In another preferred embodiment, any one of the reaction substrates and the reaction products, or the groups or atoms thereof, conforms to the relevant rules of the valence state of the compound (for example, the highest valence state of C is 4, and the highest valence state of N is 5).

[0057] In another preferred embodiment, the reaction substrates and reaction products are organic compounds.

[0058] In another preferred embodiment, the R—NH₂ is unprotected free amine or a salt of acid-protected amine.

[0059] In another preferred embodiment, the 1, 3-dipolar cycl addition reaction is a modular synthesis using the click chemical reaction principle.

[0060] In another preferred embodiment, the 1, 3-dipolar cyclization reagent is prepared in situ or added to the reaction chamber.

[0061] In another preferred embodiment, in the step (b), prior to step (b1), further comprises:

• • [0062] (b0) in an inert solvent, in the presence of a base, react ng an amine fragment (preferably R—NH₂) with FSO₂N₃ to obtain a 1,3-dipolar cyclization reagent;

[0063]

• • [0064] wherein, R is the molecular moiety or fragment attached to the azide group.

[0065] In another preferred embodiment, the step (b0) is performed in or out of the reaction chamber.

[0066] In another preferred embodiment, in the step (b0), reacting m different amines respectively located in m reaction chambers with FSO₂N₃ respectively to obtain different R—N₃.

[0067] In another preferred embodiment, the molar ratio of R—NH₂ and FSO₂N₃ is preferably about 1:2 to 2:1, such as about 1:1.

[0068] In another preferred embodiment, the molar ratio of the base and FSO₂N₃ is about 10:1 to 2:1, preferably about 4:1.

[0069] In another preferred embodiment, in the step (b0), the reaction for preparation of azide compound can be carried out at 0° C.-60%° C., preferably 25° C.-30° C.

[0070] In another preferred embodiment, the azide R—N₃ prepared in the step (b0) can be directly used in the subsequent 1, 3-dipolar cycloaddition reaction without separation.

[0071] In another preferred embodiment, in the step (b) (comprising steps (b0) and/or (b1)), the inert solvent is selected from the group consisting of water, nitrile solvent, alcohol solvent, aromatic solvent, haloalkane solvent, sulfur dioxide, alkane solvent, ester solvent, ketone solvent, ether solvent, sulfoxide solvents amide solvent and N-methylpyrrolidone, or a combination thereof.

[0072] In another preferred embodiment, the nitrile solvent is acetonitrile.

[0073] In another preferred embodiment, the alcohol solvent is selected from the group consisting of methanol, ethanol, tert-butyl alcohol, or a combination thereof.

[0074] In another preferred embodiment, the aromatic solvent is selected from the group consisting of benzene, toluene, trifluorotoluene, fluorobenzene, or a combination thereof.

[0075] In another preferred embodiment, the haloalkane solvent is elected from the group consisting of dichloromethane, chloroform, 1, 2-dichloroethane, or a combination thereof.

[0076] In another preferred embodiment, the alkane solvent is selected from the group consisting of petroleum ether 30-60, petroleum ether 60-90, n-hexane, or a combination thereof.

[0077] In another preferred embodiment, the ester solvent is ethyl acetate.

[0078] In another preferred embodiment, the ketone solvent is acetone.

[0079] In another preferred embodiment, the ether solvent is selected from the group consisting of methyl tert-butyl ether, 1, 4-dioxane, diethyl ether, tetrahydrofuran, or a combination thereof.

[0080] In another preferred embodiment, the sulfoxide solvent is dimethyl sulfoxide.

[0081] In another preferred embodiment, the amide solvent is N, N-dimethylformamide.

[0082] In another preferred embodiment, the solvent is a mixed solvent of an ether solvent, a sulfoxide solvent and water, or a mixed solvent of an ether solvent, a nitrile solvent and water.

[0083] In another preferred embodiment, the solvent is a mixture of methyl tert-butyl ether, dimethyl sulfoxide and water, or a mixture of methyl tert-butyl ether, acetonitrile and water.

[0084] In another preferred embodiment, when the solvent is a mixed solvent of ether solvent, sulfoxide solvent and water, the volume ratio of the ether solvent, sulfoxide solvent and water is 2-7: 10-20:0.8-1.2; preferably 5:15:1.

[0085] In another preferred embodiment, when the solvent is a mixed solvent of ether solvent, nitrile solvent and water, the volume ratio of the mixed solvent of ether solvent, nitrile solvent and water is 15-25:0.8-1.2:15-25; preferably 20:1:20.

[0086] In another preferred embodiment, in the step (b) (comprising steps (b0) and/or (b1)), the base is an inorganic base and/or an organic base.

[0087] In another preferred embodiment, when the base is an inorganic base, the inorganic base is one or more of sodium carbonate, potassium carbonate, cesium carbonate, sodium hydroxide, potassium hydroxide, sodium bicarbonate, potassium bicarbonate and potassium phosphate.

[0088] In another preferred embodiment, when the base is an organic base, the organic base is one or more of triethylamine, N, N-diisopropyl ethylamine, pyrrole, pyridine, 4-dimethylaminopyridine, sodium acetate, potassium acetate, sodium methoxide, sodium ethoxide, potassium tert-butoxide, 1, 8-diazabicycloundecan-7-ene and tetramethylethylenediamine.

[0089] In another preferred embodiment, the method further comprises: correlating the reaction substrate, reaction product, and activity test data based on the addressable address of the reaction chambers to obtain an addressable data set.

[0090] In a second aspect of the invention, provided is a high-throughput addressable reaction device, and the device comprising:

• • [0091] (i) a reaction module for carrying out a 1, 3-dipolar cycloaddition reaction; and the reaction module comprises at least one reactor comprising n reaction chambers, wherein the reaction chambers are each independent and addressable, and the n reaction chambers constitute an addressable array of reaction chambers, the reaction chamber is used for carrying out the 1, 3-dipolar cycloaddition reaction, resulting in the formation of a synthetic product in the reaction chamber.
  • [0092] wherein, n is a positive integer ≥10, and m is a positive integer ≤n and m≥10;
  • [0093] and (ii) optionally, an activity test module for carrying out an in-situ activity test on a synthetic product formed in the reaction chamber, thereby obtaining activity test data.

[0094] In another preferred embodiment, the reactor comprises m reaction reagents respectively located in m reaction chambers; wherein, the reaction reagent is a 1, 3-dipolar cyclization reagent or an amine precursor (i.e. an amine fragment) thereof.

[0095] In another preferred embodiment, the device further comprises (iii) an automatic pipetting module for adding a reaction reagent and/or an activity test reagent to predetermined each of addressable reaction chambers.

[0096] In another preferred embodiment, the device further comprises (iv) a processor for controlling one or more of the reaction module, the activity test module, and the automatic pipetting module.

[0097] In another preferred embodiment, the processor is also used for correlating the reaction substrate, the reaction product, and the activity test data based on the addressable address of the reaction chamber to obtain an addressable data set, and sending or storing the data set.

[0098] In another preferred embodiment, the reactor is selected from the group consisting of a microplate, an array of PE tubes, an array of test tubes, an array of reaction flasks, or a combination thereof.

[0099] In another preferred embodiment, the reactor is an array reactor.

[0100] In a third aspect of the invention, provided is a method for constructing a compound library, comprising the steps of:

• • [0101] (1) using the high-throughput addressable reaction device escribed in the second aspect to construct the compound library.

[0102] In another preferred embodiment, the step (1) comprises the steps of:

• • [0103] (1a) when the reaction reagent is an amine fragment, reacting FSO₂N₃ with amine fragment compounds in each reaction chamber to prepare 1, 3-dipolar cyclization reagent, thereby obtaining a first compound library composed of 1, 3-dipolar cyclization reagent; and
  • [0104] (1b) optionally, in the reaction chamber, reating the reaction substrates containing terminal unsaturated bonds with the 1, 3-dipolar cyclization r agents respectively to form 1, 3-dipolar ring compounds, thereby obtaining a second compound library composed of the 1, 3-dipolar ring compounds.

[0105] In another preferred embodiment, the method further comprises:

• • [0106] (2) carrying out activity test in situ in the reaction chamber on the 1, 3-dipolar ring compound in the second compound library.

[0107] A fourth aspect of the invention provides a use of a high-throughput addressable reaction device according to the second aspect for one or more uses selected from the group consisting of

• • [0108] a) synthesizing different compounds simultaneously;
  • [0109] b) high throughput (HTS) random screening of physiologically active compounds;
  • [0110] c) searching for drugs or agricultural chemicals; and
  • [0111] d) searching for leading compounds for drugs or agrochemicals.

[0112] It should be understood that in the present invention, any of the technical features specifically described above and below (such as in the Example can be combined with each other, thereby constituting new or preferred technical solutions. Limited by space, it will not be repeated here.

[0119] Through extensive and in-depth research, the present inventors have developed a high throughput method for constructing and screening a compound library and a device for the method for the first time. In the method of the invention, n addressable reaction chamber array formed by a plurality of independent and addressable reaction chambers is adopted for the first time, and specific synthetic reaction are independently carried out in each reaction chamber, thus a large number of different reaction products with well-defined structures could be prepared in high throughput and a high throughput compound library could be obtained. Unexpectedly, the experiment shows that subsequent compound activity determination in situ can be directly carried out in the addressable reaction chamber array (or corresponding compound library) containing the reaction product without separation and purification, thus greatly improving the construction and screening efficiency of compound library. On this basis, the present invention was completed.

[0120] In a preferred embodiment, the present inventor employs reaction chamber array to synthesize thousands of different 1, 3-dipolar ring reagents in high throughput (as a library of azides, or a first compound library), and then thousands of different 1, 3-dipolar ring reaction products (as triazole derivative library, or a second compound library) were synthesized in high throughput. Through in-situ activity screening of the compound library of the invention, the discovery of leading compound of drug is greatly accelerated, the screening speed is improved, and the development and research cycle of new inhibitors are shortened (which can be shortened from several years to several weeks or several days).

[0121] As used herein, the “reaction chamber” is any kind of devices capable of carrying out chemical reactions, including but not limited to micropore of microplate, PE tube, test tube, reaction flask, etc.

[0122] Definitions of standardized terms can be found in references including Carey and Sundberg “ADVANCED ORGANIC CHEMISTRY 4TH ED.” Vols. A (2000) and B (2001), Plenum Press, New York. Unless otherwise indicated, conventional methods within the scope of the art, such as mass spectrometry, NMR, IR and UV/VIS spectroscopy and pharmacological methods are employed. Unless otherwise indicated, terms used herein in the description of analytical chemistry, organic synthetic chemistry and pharmaceutical and pharmaceutical chemistry are known in the art. For example, the reaction and purification may be carried out using the manufacturer's instructions for the use of the kit or in a manner well known in the art or in accordance with the instructions of the present invention. The above-described techniques and methods may generally be practiced in accordance with conventional metho s well known in the art in accordance with the description in a plurality of summary and more specific documents cited and discussed in this specification. In this specification, groups and their substituents may be selected by those skilled in the art to provide stable structural moieties and compounds.

[0123] When a substituent is described by a conventional chemical formula written from left to right, the substituent also includes a chemically equivalent substituent obtained by writing a structural formula from right to left. For example, —CH₂O— is equivalent to —OCH₂—.

[0124] The chapter headings used in this article are for organizational purposes only and should not be construed as limiting the subject matter. All documents or portions of documents cited in the present application including but not limited to patents, patent applications, articles, books, manuals and papers are incorporated herein by reference in their entirety.

[0125] Certain chemical groups defined herein are preceded by simplified symbols to indicate the total number of carbon atoms present in that group. For example, C1-C6 alkyl is an alkyl having a total of 1 to 6 carbon atoms as defined below. The total number of carbon atoms in the simplified symbol does not include carbon that may be present in the substituent of the group.

[0126] In addition to the foregoing, when used in the specification and claims of present application, the following terms have the following meanings unless otherwise specifically indicated.

[0127] As used herein, the term “halogen” refers to fluorine, chlorine, bromine or iodine.

• • [0128] “Hydroxyl” refers to —OH.
  • [0129] “Carbonyl” refers to —C(═O)—.
  • [0130] “Cyano” refers to —CN.
  • [0131] “Amino” refers to —NH₂.

[0132] “Substituted amino” refers to an amino substituted with one or two alkyl, alkylcarbonyl, aralkyl, heteroaralkyl as defined below, for example, monoalkylamino, dialkylamino, alkylamido, aralkylamino, heteroaralkylamino.

[0133] “Carboxyl” refers to —COOH.

[0134] As used herein, as a group or part of other group (for example, in halogen-substituted alkyl), the term “alkyl” refers to a fully saturated straight or branched hydrocarbon chain group consisting only of carbon and hydrogen atoms, having for example 1 to 12 (preferably 1 to 8, preferably 1 to 6) carbon atoms, and connected to the rest of the molecule by a single bond. Examples include but not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-amyl, 2-methyl-butyl, 2, 2-dimethyl-propyl, n-hexyl, heptyl, 2-methyl-hexyl, 3-methyl-hexyl, octyl, nonyl and decyl, etc. Examples of cycloalkyl include monocycloalkyl or polycycloalkyl such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl and cyclodecyl.

[0135] “Alkylene” is used alone or in combination words such as optionally substituted alkylene to denote the same group as “alkyl” as defined above, except that another hydrogen is removed to form a divalent group. It should be understood that the optional substituent may be attached to or form part of the alkylene chain.

[0136] “Alkenyl” used alone or in combination words such as “optionally substituted alkenyl” denotes a group formed from a straight-chain, branched or cyclic olefin, including an ethylene mono-, di- or polyunsaturated alkyl or cycloalkyl, preferably a C2-6 alkenyl, as defined above. Examples of the alkenyl include vinyl, allyl, 1-methylvinyl, butenyl, isobutenyl, 3-methyl-2-butenyl, 1-pentenyl, cyclopentenyl, 1-methylcyclopentenyl, 1-hexenyl, 3-hexenyl, cyclohexenyl, 1-heptenyl, 3-heptenyl, 1-octenyl, cyclooctenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 1-decenyl, 3-decenyl, 1, 3-butadienyl, 1, 4-pentadienyl, 1, 3-cyclopentadienyl, 1, 3-hexadienyl, 1, 4-hexadienyl, 1, 3-cyclohexadienyl, 1, 4-cyclohexadienyl, 1, 3-cycloheptadienyl, 1,3, 5-cycloheptrienyl and 1, 3, 5, 7-cyclooctatetraenyl.

[0137] “Alkynyl” used alone or in combination words such as “optionally substituted alkynyl” denotes a group formed from a straight, branched or mono-, di- or polycyclic alkyne, preferably a C2-6 alkynyl. Examples of alkynyl include ethynyl, 1-propynyl, 1- and 2-butyynyl, 2-methyl-2-propynyl, 2-pentynyl, 3-pentynyl, -pentynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl, 10-undecynyl, 4-ethyl-1-octy 1-3-yl, 7-dodecynyl, 9-dodecynyl, 10-dodecynyl, 3-methyl-1-dodecyn-3-yl, 2-tridecynyl, 11-tridecynyl, 3-tetradecynyl, 7-hexadecynyl, 3-octadecynyl, and the like.

[0138] As used herein, the term “heteroalkyl” as a group or part of other group means a fully saturated straight or branched hydrocarbon chain group consisting of 2 to 14 carbon atoms and 1 to 6 heteroatoms selected from the group consisting of nitrogen, phosphorus, oxygen and sulfur. The nitrogen, carbon or sulfur atoms in the heteroalkyl can be optionally oxidized; the nitrogen atoms can optionally be quaternized.

[0139] As used herein, the term “heterocyclyl” as a group or part of other means a stable 3 to 20 nonaromatic cyclic group consisting of 2 to 14 carbon atoms and 1 to 6 heteroatoms selected from the group consisting of nitrogen, phosphorus, oxygen and sulfur. Unless otherwise specifically specified in this specification, the heterocyclyl may be a monocyclic, bicyclic, tricyclic, or more ring system, which may include a fused ring system, a bridge ring system, or a spiro ring system. The nitrogen, carbon or sulfur atoms in the heterocyclyl are optionally oxidized. The nitrogen atom can optionally be quaternized. And the heterocyclyl can be partially or completely saturated.

[0140] Heterocyclyl can be connected to the rest of the molecule via carbon atoms or heteroatoms and through single bond. Among the heterocyclyl containing fused rings, one or more of the rings may be aryl or heteroaryl as defined below, provided that the connection point with the rest of the molecule is a non-aromatic ring atom. For purposes of the present invention, the heterocyclyl is preferably a stable 4-11 membered non-aromatic monocyclic, bicyclic, bridged or spiro group containing 1 to 3 heteroatoms selected from nitrogen, oxygen and sulfur, and more preferably a stable 4-8 non-aromatic monocyclic, bicyclic, bridged or spiro group containing 1 to 3 heteroatoms selected from nitrogen, oxygen and sulfur. Examples of heterocyclic groups include, but are not limited to pyrrolidinyl, morpholinyl, piperazinyl, homopiperazinyl, piperidinyl, thiomorpholinyl, 2, 7-diaza-spiro [3.5]nonan-7-yl, 2-oxa-6-aza-spiro [3.3]heptan-6-yl, 2,5-diaza-bicyclo[2.2.1]heptan-2-yl, a etidinyl, pyranyl, tetrahydropyranyl, thiopyranyl, tetrahydrofuranyl, oxazinyl, dioxycyclopentyl, tetrahydroisoquinolinyl, decahydroisoquinolinyl, imidazoinyl, imidazolidinyl, quinazinyl, thiazolidinyl, isothiazolidinyl, isoxazolidinyl, indolnyl, octahydroindolinyl, octahydroisoindolinyl, pyrrolidinyl, pyrazolidinyl, phthalimido and the like.

[0141] As used herein, the term “aryl” as a group or a part of other group means a conjugated hydrocarbon ring system group having 6 to 18 carbon atoms (preferably 6 to 10 carbon atoms). For the purposes of the invention, the aryl may be a ring system of monocyclic, bicyclic, tricyclic or more rings and may also be fused with a cycloalkyl or heterocyclyl as defined above, provided that the aryl is connected to the rest of the molecule by a single bond via an atom on the aromatic ring. Examples of the aryl include, but are not limited to phenyl, naphthyl, anthryl, phenanthryl, fuorenyl, 2, 3-dihydro-1H-isoindolyl, 2-benzoxazolinone, 2H-1, 4-benzoxazin-3 (4H)-keto-7-yl, and the like.

[0142] As used herein, the term “heteroaryl” as a group or part of other group means a 5- to 16-membered conjugated ring group having 1 to 15 carbon atoms (preferably 1 to 10 carbon atoms) and 1 to 6 heteroatoms selected from nitrogen, oxygen and sulfur in the ring. Unless otherwise specifically specified in this specification, the heteroaryl may be a ring system of monocyclic, bicyclic, tricyclic or more rings and may also be fused with a cycloalkyl or heterocyclyl as defined above, provided that the heteroaryl is connected to the rest of the molecule by a single bond via an atom on the aromatic ring.

[0143] The nitrogen, carbon or sulfur atoms in the heteroaryl can be optionally oxidized; the nitrogen atoms can optionally be quaternized. For purposes of the present invention, the heteroaryl is preferably a stable 5- to 12-membered aromatic group comprising 1 to 5 heteroatoms selected from nitrogen, oxygen and sulfur, and more preferably a stable 5- to 10-membered aromatic group comprising 1 to 4 heteroatoms selected from nitrogen, oxygen and sulfur or a 5- to 6-membered aromatic group comprising 1 to 3 heteroatoms selected from nitrogen, oxygen and sulfur. Examples of heteroaryl include but are not limited to thienyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl, xadiazolyl, isoxazolyl, pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, benzimidazolyl, b nzopyrazolyl, indolyl, furyl, pyrrolyl, triazolyl, tetrazolyl, triazinyl, indazinyl, isoindolyl, indazolyl, isoindazolyl, purinyl, quinolinyl, isoquinolinyl, diazanaphthyl, naphthyridyl, quinoxolinyl, pterridinyl, carbazolyl, carbolinyl, phenanthridinyl, phenanthrolinyl, acridinyl, phenazinyl, isothiazolyl, benzothiazolyl, benzothiophenyl, oxtriazolyl, cinolinyl, quinazolinyl, phenylthio, indolizinyl, o-diazophenyl, isoxazolyl, phenoxazinyl, phenothiazinyl, 4, 5, 6, 7-tetrahydrobenzo [b] thienyl, naphthopyridyl, [1, 2, 4]triazolo[4, 3-b]pyridazinyl, [1, 2, 4]triazolo[4, 3-b]pyrzinyl, [1, 2, 4]triazolo [4, 3-c]pyrimidinyl, [1, 2, 4]triazolo[4, 3-a]pyridinyl, imidazolo [1, 2-a]pyridinyl, imidazolo [1,2-b]pyridinyl, imidazolo [1,2-b]pyridazin 1, imidazolo [1, 2-a]pyrazinyl, etc.

[0144] As used herein, “optionally” or “substituted or unsubstituted” means that a subsequently described event or condition may or may not occur, and that the description includes both the occurrence and non-occurrence of the event or condition.

[0145] For example, “optionally substituted aryl” means substituted or unsubstituted aryl, and the description includes both substituted and unsubstituted aryl. The “optional” substituent as described in the claims and specification sections of the present invention is selected from the group consisting of alkyl, alkenyl, alkynyl, halogen, haloalkyl, haloalkenyl, haloalkynyl, cyano, nitro, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cyclic hydrocarbon, optionally substituted heterocyclic hydrocarbon.

[0146] As used herein, the term “substrate fragment” refers to the molecular portion of a reaction substrate other than a functional group (e. g., an unsaturated bond) involved in a particular reaction.

[0147] As used herein, the terms “moiety”, “structural moiety”, “chemical moiety,” “group” and “chemical group” refer to a particular fragment or functional group in a molecule. Chemical moieties are usually regarded as chemical entities embedded or attached to molecules.

Construction and Application of Compound Library

[0148] In a preferred embodiment of the present invention, a synthesis reaction based on CuAAC is employed to realize chemical connection between two molecules, thereby constructing a compound library in high throughput.

[0149] Because of the simplicity and high efficiency of the method of the invention, the diazotransfer can have certain industrial practicability even in a small laboratory. For example, in the case of obtaining 1000 primary amines, a person can construct a library of 1000 primary amine compounds with a concentration of 0.1 mol/L within 2 weeks, and the 1000 primary amine compounds can be azidated in one day, thus constructing an azide compound library containing 1000 azide compounds. The 1000 azide compounds can react with terminal alkynyl compounds, for example, 100 terminal alkynyl compounds to obtain 100,000 triazole compounds which can be directly used for subsequent activity screening. Compared to conventional compound libraries produced by combinatorial chemistry, the compound libraries constructed by the present invention contain mainly one compound in each reactor, rather than a mixture of many compounds, and are easy to control.

[0150] The structures of the compounds in the library can be analyzed by any known structural analyse methods, such as mass spectrometry, tandem mass spectrometry, ultraviolet/visible absorption spectroscopy, proton nuclear magnetic resonance spectroscopy, C¹³ nuclear magnetic resonance spectrum, infrared absorption spectrum and X-ray diffraction crystal spectrum, or their combination.

[0151] The invention provides a method for constructing and screening a high-throughput compound library, which can simultaneously synthesize different compounds in large quantities at one time, and the constructed compound library can be used for high-throughput (HTS) random screening, searching for drugs or agricultural chemicals, searching for leading compounds of drugs or agricultural chemicals, etc.

[0152] For example, in order to obtain compounds with physiological activity, the compound library can be used for various screening. Example of compounds having physiological activity include, but are not limited to enzyme inhibitor, ligand/receptor binding inhibitor, angiogenesis inhibitor, cell adhesion inhibitor, gene expression inhibitor and growth factor-like active substance. Examples of enzyme inhibitors include tyrosinase inhibitor, cyclooxygenase inhibitor, telomerase inhibitor, matrix metalloprotein inhibitor, prostaglandin D synthesis inhibitor, phosphodiesterase inhibitor, cholinesterase inhibitor, viral protease inhibitor and reverse transcriptase inhibitor. Examples of receptors include adrenergic receptor, histamine receptor, leukotriene receptor and opioid receptor.

1, 3-Dipolar Ring Reagent

[0153] In the present invention, a 1, 3-dipolar cyclization reaction is carried out on a substrate fragment having an unsaturated bond at the terminal with a 1, 3-dipolar ring reagent to obtain a compound containing a 1, 3-dipolar ring structure, such as a triazole compound. Exemplary reaction routes are as follows:

[0154]

• • [0155] wherein, R, R′, R″ and R″′ are each independently R¹ or

[0156]

• • [0157]  and R¹ and R² are each independently a substrate fragment.

[0158] In the present invention, R¹ and R² are the remaining molecular moiety of the reaction substrate other than the molecular moiety of the terminal unsaturated bond.

[0159] Preferably, R¹ and R² each are independently drug active fragments from drug molecules.

[0160] In the present invention, there is no particular limitations for R, R′, R″ and R″′ or corresponding R¹ and R², they can be a variety of suitable organic groups. Typically, the representative Rand R² include organic groups formed of elements such as C, H, O, N, S, F, Cl, etc., such as a molecular moiety consisting of substituted or unsubstituted groups selected from the group consisting of alkyl, cycloalkyl, heteroalkyl, heterocyclyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl, sulfonyl, or a combination thereof.

[0161] Although exemplary R, R′, R″ and R″′ are enumerated in the present invention, it should be understood that any compound having an azide structure can be used as a 1, 3-dipolar cyclization agent of the present invention for reacting with a substrate having an unsaturated group (e.g. alkenyl, alkynyl, cycloalkenyl, cyano etc.) at the terminal to obtain a compound containing nitrogen heterocycle.

[0162] A particularly preferred type of substrate is a drug molecule (e.g. erlotinib molecule) known in the art or a known drug molecule modified y an unsaturated group.

[0163] In preferred embodiments, the modification may be obtained by means of methods known in the art, using inverse synthetic analysis, for example, modification of unsaturated groups may be performed using coupling reactions known in the art (e.g. Suzuki reaction, etc.).

1, 3-Dipolar Cyclization Reagent (Azide Compound)

[0164] In the present invention, an azide compound used as a 1, 3-dipolar cyclization reagent can also be prepared by reacting an amine fragment with a fluorosulfonyl azide.

[0165] Exemplary reactions are as follows:

[0166]

• • [0167] wherein the substituent R may be all substituted or unsubstituted alkyl, cycloalkyl, heteroalkyl, heterocyclyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl, sulfonyl, or a combination thereof. See the definitions of R, R′, R″ and R″′ above for the exemplary definitions of R.

[0168] Although the present invention enumerates an exemplary R group, it should be understood that any substrate with a primary amine structure can be used as an amine fragment of the present invention for constructing an azide compound as a 1, 3-dipolar cyclization agent under the process of the present invention. It should be understood that due to the abundance of primary amine compounds in nature, the method can be carried out using any existing primary amine fragment or any subsequently developed primary amine fragment.

[0169] The main advantages of the present invention include:

• • [0170] 1 The reaction is simple and efficient, and hundreds or thousands of structure well-defined compounds can be rapidly synthesized on a large scale, thus obtaining an addressable library of compounds.
  • [0171] 2. The library of 1, 3-dipolar cyclization reagent and 1, 3-dipolar ring reagent can be constructed quickly.
  • [0172] 3. Compared to conventional compound libraries produced by combinatorial chemistry, the compound libraries constructed by the present invention contain mainly one compound in each reactor, rather than a mixture of many compounds, and are easy to control.
  • [0173] 4. The high-throughput method for constructing and screen ng a compound library of the present invention can be applied to fragment-based drug discovery because the stoichiometric reagents are almost quantitative.
  • [0174] 5. The high-throughput method for constructing and screening a compound library of the present invention can be directly applied to activity research or even phenotype screening, which cannot be realized by previous combinatorial chemistry.

[0175] The present invention will be further illustrated below with reference to the specific examples. It should be understood that these examples re only to illustrate the invention but not to limit the scope of the invention. The experimental methods without specific conditions in the following examples usually follow conventional conditions, or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentage and parts are calculated by weight.

[0176]

[0177] 1-(fluorosulfonyl)-2, 3-dimethyl-1H-imidazole trifluoromethanesulfonate in acetonitrile (6 mmol, 1 ml MeCN) was added to a mixed system of sodium azide aqueous solution (0.25 M, 20 ml; containing 5 mmol NaN₃) and methyl tert-butyl ether (20 ml) under ice bath. The reaction system was stirred under ice bath for 10 minutes, followed by letting the reaction solution stand at room temperature (25° C.) for 5 minutes. The water phase in the reaction system was removed to obtain the organic phase which was fluorosulfonyl azide (FSO)₂N₃) solution with a yield of 92% (through ¹⁹F NMR determination, relative to the number of moles of sodium azide used; in methyl tert-butyl ether (MTBE), the chemical shift of the product is +61.5 ppm, and

[0178]
with known quantity can be used as internal standard (δ+36.7 ppm) for quantification, and the total amount of products in the reaction system was calculated by the integral ratio of the signal of the product and the internal standard in fluorine spectrum, so as to calculate the reaction yield). GC-MS (tR): 1.69 min; EI-MS (m/z): 125 [M]⁺ (GC-MS (EI) spectra were determined by Agilent 7890A GC system and Agilent 5975C Inert MSD system. Methods: T₀=40° C., 1=10 min, ramp=20° C./min; T₁=200° C., t=10 min). Dimethyl sulfoxide (DMSO, bout 20 ml) was added to the solution of fluorosulfonyl azide (FSO₂N₃), and the resulting solution can be directly used for the diazotransfer reaction of primary amine compounds (see EXAMPLE 3).

[0179] 1128 different primary amine compounds (including alkyl primary amine, aryl primary amine and heteroaryl primary amine) were respectively prepared into a solution with a concentration of about 100 mM, the solvent was dimethyl sulfoxide, the volume was about 1 ml, and the solution was stored in a 96-well microplate with a size of 1.2 ml.

[0180] When the primary amine compound is an aryl primary amine or heteroaryl primary amine, and the compound contains n primary amine functional groups that can be converted to azide, 0.10/n mmol primary amine was dissolved in 1.0 ml of dimethyl sulfoxide in a 1.5 ml centrifuge tube, and then transferred to the wells in the corresponding locations of its corresponding microplate.

[0181] When the primary amine compound is an ammonium salt obtained by neutralizing alkyl primary amine with hydrochloric acid, methanesulfonic acid, tartaric acid, p-toluenesulfonic acid, or hydrobromic acid, and the compound contains n primary amine functional groups that can be converted to azide, 0.10/n mmol primary amine was dissolved in 1.0 ml of dimethyl sulfoxide in a 1.5 ml centrifuge tube, and then transferred to the wells in the corresponding locations of its corresponding microplate.

[0182] When the primary amine compound is a free alkyl primary amine, and the compound contains n primary amine functional groups that can be converted to azide, 0.10/n mmol primary amine was dissolved in 1.0 ml 100/n mM methanesulfonic acid solution (solvent is dimethyl sulfoxide) in a 1.5 ml centrifuge tube. If the compound contains two or more free basic functional groups (primary amine, secondary amine, tertiary amine, guanidine, etc.), increase the concentration of methanesulfonic acid solution to 200/n mM. The prepared primary amine solution was then transferred to wells at in the corresponding locations of its corresponding microplate.

[0183] Primary amines with poor solubility in dimethyl sulfoxide could also be prepared into azide compounds by diazotransfer, and most of these azide products can be dissolved in dimethyl sulfoxide and classified into azide compound library in solution form. However, these primary amines with poor solubility could not be stored in the primary amine compound library in solution form. In addition to the above mentioned 1128 primary amine compound libraries stored in solution form in microplates (distributed in 12 96-well microplates), another 96 primary amines (some of which have poor solubility) were selected as substrates for diazotransfer. Thus, a library contained 1224 primary amines with different structures was constructed, which can be used for the subsequent preparation of azide compound library (FIG. 1).

[0184]

[0185] The fluorosulfonyl azide solution prepared by the method of EXAMPLE 1 (the solvent was methyl tert-butyl ether, the concentration was about 400 mM through ¹⁹F NMR determination, about 40 ml) diluted with dimethyl sulfoxide (40 ml) to obtain a fluorosulfonyl azide solution (the concentration was about 20 C mM through ¹⁹F NMR measurement, 80 ml, the solvent was dimethyl sulfoxide/methyl tert-butyl ether 1:1).

[0186] Using a 96-well pipette to transfer the primary amine solution (100 mM in the solvent dimethyl sulfoxide; took out 200 μl, containing 20 μmol of primary amine) in each well of a 96-well plate in the primary amine compound library prepared in Example 2 to an empty 1.2 ml 96-well microplate. Aqueous potassium bicarbonate solution (3.0 M, 26.7 μl, containing 80 μmol of potassium bicarbonate) and the above diluted fluorosulfonyl azide solution (200 mM, 100 μl, containing 20 μmol of fluorosulfonyl azide) were added to each well. Additional dimethyl sulfoxide (73 μl) was added to bring the total volume about 400 μl. The microplate was sealed and placed in a shaker, shaken for 1 hour at 800 rpm under 30° C. After shaking, a 50 mM solution of the corresponding azide compound (the solvent system was about 3:1 of dimethyl sulfoxide/methyl tert-butyl ether) was obtained with a yield close to quantitative in each well in the microplate. The reaction solution in each well in the microplate was detected by UPLC-MS. Exemplary UPLC chromatography were shown in FIGS. 2a-f.

[0187] From the compound library of 1128 primary amines in Example 2, 1128 corresponding azides were obtained by this method. The 1128 azide solutions were distributed in 12 96-well microplates (serial number of the microplates were 1-7 and 10-14). The 96 separately stored primary amines mentioned in EXAMPLE 2 (some of which have poor solubility) were reacted by the same method in this example to yield 96 soluble azide solutions and transferred to a new 96-well microplate (see plate 8 of azide library for details). Thereby, a compound library containing 1224 azide solutions with different structures was constructed.

[0188] The compound library containing 1224 azide solutions with different structures were distributed in 13 96-well microplates. The microplate of the azide compound library could be sealed and stored in a refrigerator at a refrigeration temperature (4° C.), stable for at least 6 months. The compound library could be directly used for reaction without separation.

[0189] The structures in azide compound library and their locations in the microplates were as follows, and the corresponding locations of primary amine library (except the eighth plate) and triazole library remained unchanged.

[0190]

[0191] Sodium ascorbate (2.48 g, 12.5 mmol), disodium hydrogen phosphate (7.0 g, 49.3 mmol) and citric acid (4.87 g, 25.4 mmol) were mixed and dissolved in distilled water to 100 ml aqueous solution to obtain a sodium buffer solution of ascorbate/disodium hydrogen phosphate/citric acid with a pH value of about 5.

[0192] From the eighth plate of the azide library obtained in EXAMPLE 2, the solution (100 μl, the concentration of about 50 mM, containing about 5 μl mol of azide) was taken from each well and transferred to a new 96-well plate with a size of 350 μl. The above buffer solution of ascorbic acid/disodium hydrogen phosphate/citric acid (40 g 1) was successively added to each well, and after sealing film, shaken at 800 rpm for 15 minutes at 30° C. Tearing off the sealing film, 3-acetamidophenylacetylene solution (compound 4a, 100 mM, the solvent was dimethyl sulfoxide; 47.5 μl, containing 4.75 μmol of 3-acetaminophenylacetylene) and copper sulfate/THPTA aqueous solution (the concentrations of both copper sulfate and THPTA were 20 mM respectively, 12.5 g 1, 0.25 μmol) were added to each well. The microplate was sealed and shaken for 6 hour at 800 rpm under 40° C. After the reaction was completed, the in-plate mixture was transferred one-to-one to an empty 1.2 ml 96-well plate, diluted by adding methanol (400 μl) and acetonitrile (400 μl) to each well, and the resulting 1, 2, 3-triazole products were detected by UPLC-MS.

[0193] Exemplary UPLC chromatograms of 1, 2, 3-triazole products were shown in FIGS. 3a-f.

[0194]

Plate 1 A1
Plate 1 A2
Plate 1 A3
Plate 1 A4
Plate 1 B3
Plate 1 C1
Plate 1 C2
Plate 1 C3
Plate 1 C4
Plate 1 D1
Plate 1 D2
Plate 1 D3
Plate 1 D4
Plate 1 E1
Plate 1 E2
Plate 1 E3
Plate 1 E4
Plate 1 F1
Plate 1 F2
Plate 1 F3
Plate 1 F4
Plate 1 G1
Plate 1 G2
Plate 1 G3
Plate 1 G4
Plate 1 H1
Plate 1 H2
Plate 1 H3
Plate 1 H4
Plate 1 A5
Plate 1 A6
Plate 1 A7
Plate 1 A8
Plate 1 C5
Plate 1 C6
Plate 1 C7
Plate 1 C8
Plate 1 D5
Plate 1 D6
Plate 1 D7
Plate 1 D8
Plate 1 E5
Plate 1 E6
Plate 1 E7
Plate 1 E8
Plate 1 F5
Plate 1 F6
Plate 1 F7
Plate 1 F8
Plate 1 G5
Plate 1 G6
Plate 1 G7
Plate 1 G8
Plate 1 H5
Plate 1 H6
Plate 1 H7
Plate 1 H8
Plate 1 A9
Plate 1 C9
Plate 1 C10
Plate 1 C11
Plate 1 C12
Plate 1 D9
Plate 1 D10
Plate 1 D11
Plate 1 D12
Plate 1 E9
Plate 1 E10
Plate 1 E11
Plate 1 E12
Plate 1 F9
Plate 1 F10
Plate 1 F11
Plate 1 F12
Plate 1 G9
Plate 1 G10
Plate 1 G11
Plate 1 G12
Plate 1 H9
Plate 1 H10
Plate 1 H11
Plate 1 H12

[0195]

Plate 2
	1	2	3	4
A
B
C
D
E
F
G
H
	5	6	7	8
A
B
C
D
E
F
G
H
	9	10	11	12
A
B
C
D
E
F
G
H

[0196]

Plate 3
	1	2	3	4
A
B
C
D
E
F
G
H
	5	6	7	8
A
B
C
D
E
F
G
H
	9	10	11	12
A
B
C
D
E
F
G
H

[0197]

Plate 4
	1	2	3	4
A
B
C
D
E
F
G
H
	5	6	7	8
A
B
C
D
E
F
G
H
	9	10	11	12
A
B
C
D
E
F
G
H

[0198]

Plate 5
	1	2	3	4
A
B
C
D
E
F
G
H
	5	6	7	8
A
B
C
D
E
F
G
H
	9	10	11	12
A
B
C
D
E
F
G
H

[0199]

Plate 6
	1	2	3	4
A
B
C
D
E
F
G
H
	5	6	7	8
A
B
C
D
E
F
G
H
	9	10	11	12
A
B
C
D
E
F
G
H

[0200]

Plate 7 A1
Plate 7 A2
Plate 7 A3
Plate 7 A4
Plate 7 B1
Plate 7 B2
Plate 7 B3
Plate 7 B4
Plate 7 C1
Plate 7 C2
Plate 7 C3
Plate 7 C4
Plate 7 D1
Plate 7 D2
Plate 7 D3
Plate 7 D4
Plate 7 E1
Plate 7 E2
Plate 7 A6
Plate 7 A7
Plate 7 A8
Plate 7 B5
Plate 7 B6
Plate 7 B7
Plate 7 B8
Plate 7 C5
Plate 7 C6
Plate 7 C7
Plate 7 C8
Plate 7 D5
Plate 7 D6
Plate 7 D7
Plate 7 D8
Plate 7 E5
Plate 7 E6
Plate 7 E7
Plate 7 E8
Plate 7 F5
Plate 7 F6
Plate 7 F7
Plate 7 F8
Plate 7 G5
Plate 7 G6
Plate 7 G7
Plate 7 G8
Plate 7 H5
Plate 7 H6
Plate 7 H7
Plate 7 H8
Plate 7 A9
Plate 7 A10
Plate 7 A11
Plate 7 A12
Plate 7 B9
Plate 7 B10
Plate 7 B11
Plate 7 B12
Plate 7 C9
Plate 7 C10
Plate 7 C11
Plate 7 C12
Plate 7 D9
Plate 7 D10
Plate 7 D11
Plate 7 D12
Plate 7 E9
Plate 7 E10
Plate 7 E11
Plate 7 E12
Plate 7 F9
Plate 7 F10
Plate 7 F11
Plate 7 F12
Plate 7 G9
Plate 7 G10
Plate 7 G11
Plate 7 G12
Plate 7 H9

[0201]

Plate 8 G4
Plate 8 H1
Plate 8 H2
Plate 8 H3
Plate 8 H4
Plate 8 A5
Plate 8 A6
Plate 8 A7
Plate 8 A8
Plate 8 B5
Plate 8 B6
Plate 8 B7
Plate 8 B8
Plate 8 C5
Plate 8 C6
Plate 8 C7
Plate 8 C8
Plate 8 D5
Plate 8 D6
Plate 8 D7
Plate 8 D8
Plate 8 E5
Plate 8 E6
Plate 8 E7
Plate 8 E8
Plate 8 F5
Plate 8 F6
Plate 8 F7
Plate 8 F8
Plate 8 G5
Plate 8 G6
Plate 8 G7
Plate 8 G8
Plate 8 H5
Plate 8 H6
Plate 8 H7
Plate 8 H8
Plate 8 A9
Plate 8 A10
Plate 8 A11
Plate 8 A12
Plate 8 B9
Plate 8 B10
Plate 8 B11
Plate 8 B12
Plate 8 F12
Plate 8 G9
Plate 8 G10
Plate 8 G11
Plate 8 G12
Plate 8 H9
Plate 8 H10
Plate 8 H11
Plate 8 H12

[0202]

Plate 10 A1
Plate 10 A2
Plate 10 A3
Plate 10 A4
Plate 10 B1
Plate 10 B2
Plate 10 B3
Plate 10 B4
Plate 10 C1
Plate 10 C2
Plate 10 C3
Plate 10 C4
Plate 10 D1
Plate 10 D2
Plate 10 D3
Plate 10 D4
Plate 10 E1
Plate 10 E2
Plate 10 E3
Plate 10 E4
Plate 10 F1
Plate 10 B5
Plate 10 B6
Plate 10 B7
Plate 10 B8
Plate 10 C5
Plate 10 C6
Plate 10 C7
Plate 10 C8
Plate 10 D5
Plate 10 D6
Plate 10 D7
Plate 10 D8
Plate 10 E5
Plate 10 E6
Plate 10 E7
Plate 10 E8
Plate 10 F5
Plate 10 F6
Plate 10 F7
Plate 10 F8
Plate 10 G5
Plate 10 G6
Plate 10 G7
Plate 10 G8
Plate 10 H5
Plate 10 H6
Plate 10 H7
Plate 10 H8
Plate 10 A9
Plate 10 A10

[0203]

Plate 11 D4
Plate 11 E1
Plate 11 E2
Plate 11 E3
Plate 11 E4
Plate 11 F1
Plate 11 F2
Plate 11 F3
Plate 11 F4
Plate 11 G1
Plate 11 G2
Plate 11 G3
Plate 11 G4
Plate 11 H1
Plate 11 H2
Plate 11 D6
Plate 11 D7
Plate 11 D8
Plate 11 E5
Plate 11 E6
Plate 11 E7
Plate 11 E8
Plate 11 F5
Plate 11 F6
Plate 11 F7
Plate 11 F8
Plate 11 G5
Plate 11 G6
Plate 11 G7
Plate 11 G8
Plate 11 H5
Plate 11 H6
Plate 11 H7
Plate 11 H8
Plate 11 A9
Plate 11 A10
Plate 11 A11
Plate 11 A12
Plate 11 B9
Plate 11 B10
Plate 11 B11
Plate 11 B12
Plate 11 C9
Plate 11 C10
Plate 11 C11
Plate 11 C12
Plate 11 D9
Plate 11 D10
Plate 11 D11
Plate 11 D12
Plate 11 E9
Plate 11 E10
Plate 11 E11
Plate 11 E12
Plate 11 F9
Plate 11 F10
Plate 11 F11
Plate 11 F12
Plate 11 G9
Plate 11 G10
Plate 11 G11
Plate 11 G12
Plate 11 H9
Plate 11 H10
Plate 11 H11
Plate 11 H12

[0204]

Plate 12 A1
Plate 12 A2
Plate 12 A3
Plate 12 A4
Plate 12 B1
Plate 12 B2
Plate 12 B3
Plate 12 B4
Plate 12 C1
Plate 12 C2
Plate 12 C3
Plate 12 C4
Plate 12 D1
Plate 12 D2
Plate 12 D3
Plate 12 D4
Plate 12 E1
Plate 12 E2
Plate 12 E3
Plate 12 E4
Plate 12 F1
Plate 12 F2
Plate 12 F3
Plate 12 F4
Plate 12 G1
Plate 12 G2
Plate 12 G3
Plate 12 G4
Plate 12 H1
Plate 12 H2
Plate 12 H3
Plate 12 H4
Plate 12 A5
Plate 12 A6
Plate 12 A7
Plate 12 A8
Plate 12 B5
Plate 12 B6
Plate 12 B7
Plate 12 B8
Plate 12 C5
Plate 12 C6
Plate 12 C7
Plate 12 C8
Plate 12 D5
Plate 12 D6
Plate 12 D7
Plate 12 D8
Plate 12 E5
Plate 12 E6
Plate 12 E7
Plate 12 E8
Plate 12 F5
Plate 12 F6
Plate 12 F7
Plate 12 F8
Plate 12 G5
Plate 12 G6
Plate 12 G7
Plate 12 G8
Plate 12 H5
Plate 12 H6
Plate 12 H7
Plate 12 H8
Plate 12 A9
Plate 12 A10
Plate 12 A11
Plate 12 A12
Plate 12 B9
Plate 12 B10
Plate 12 B11
Plate 12 B12
Plate 12 C9
Plate 12 C10
Plate 12 C11
Plate 12 C12
Plate 12 D9
Plate 12 D10
Plate 12 D11
Plate 12 D12
Plate 12 E9
Plate 12 E10
Plate 12 E11
Plate 12 E12
Plate 12 F9
Plate 12 F10
Plate 12 F11
Plate 12 F12
Plate 12 G9
Plate 12 G10
Plate 12 G11
Plate 12 G12
Plate 12 H9
Plate 12 H10
Plate 12 H11
Plate 12 H12