US 20140046022A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2014/0046022 A1 Takahashi (43) Pub. Date: Feb. 13, 2014

(54) COMPOUND (30) Foreign Application Priority Data (71) Applicant: Ajinomoto Co., Inc., Tokyo (JP) Mar. 12, 2009 (JP) ...... 2009-06O291 (72) Inventor: Daisuke Takahashi, Mie (JP) Publication Classification (73) Assignee: Ajinomoto Co., Inc., Tokyo (JP) (51) Int. Cl. C07K L/06 (2006.01) (21) Appl. No.: 14/027,961 CD7C 43/21 (2006.01) (52) U.S. Cl. (22) Filed: Sep. 16, 2013 CPC ...... C07K I/062 (2013.01); C07C 43/21 (2013.01) Related U.S. Application Data USPC ...... 530/335:568/634 (62) Division of application No. 12/723,027, filed on Mar. (57) ABSTRACT 12, 2010, now Pat. No. 8,569,453. Particular compounds having a fluorene skeleton are Superior (60) Provisional application No. 61/159,998, filed on Mar. in broad utility and stability, as a protecting reagent for liquid 13, 2009. phase synthesis of amino acids and/or peptides. US 2014/0046022 A1 Feb. 13, 2014

FLUORENE COMPOUND carrier, an isolation target compound can be selectively pre cipitated from a homogeneous solution state, in other words, CROSS REFERENCES TO RELATED a particular compound can be isolated after a liquid phase APPLICATIONS reaction when other soluble components still remain in a Solution, thus obviating the need to consider extraction and 0001. This application is a division of U.S. Ser. No. precipitation conditions for each compound. 12/723,027, filed Mar. 12, 2010, and claims priority to U.S. 0010. However, when a polymer is used as a carrier mol Provisional Patent Applications No. 61/159,998, filed on ecule, the reaction becomes non-homogeneous due to the Mar. 13, 2009, and Japanese Patent Application No. 2009 molecular weight distribution, as in the Solid phase method, 060291, filed on Mar. 12, 2009, both of which are incorpo where tracking of reaction and analysis of the reaction prod rated herein by reference in their entireties. uct on a carrier are difficult to perform, since the compound is bound to a carrier. BACKGROUND OF THE INVENTION 0011 Thus, a method using a protecting group (anchor) 0002 1. Field of the Invention capable of irreversible change from a dissolved state to an 0003. The present invention relates to fluorene com insolubilized state (precipitated State) of a particular compo pounds which are useful as protecting reagents for organic nent according to the varying solvent composition has been synthesis reactions. The present invention also relates to developed. For example, JP-A-2000-44493 and Bull. Chem. organic synthesis reactions which use Such a compound and Soc. Jpn., 74, 733–738 (2001) disclose methods including the like. More particularly, the present invention relates to developing an anchor by introducing a long chain aliphatic fluorene compounds which are usable as protecting reagents group into a benzyl alcohol type compound (see the following for a C-terminal and/or a side chain of amino acid or peptide structure), dissolving and reacting the anchor in a haloge in peptide synthesis, particularly liquid phase synthesis of a nated solvent, and precipitating a reacted product with metha peptide, and methods of peptide synthesis and organic Syn nol or acetonitrile to allow peptide chain elongation. thesis using such a compound. 0004 2. Discussion of the Background 0005 Methods for the organic synthesis of compounds are OC18H37 generally divided largely into Solid phase methods and a HO liquid phase methods. The Solid phase method is advanta geous in that isolation and purification after the reaction can OC18H37 be performed by filtration and washing alone. However, the solid phase method is problematic in that it essentially OC18H37 includes a non-homogeneous phase reaction, reaction agents and reagents need to be used in excess amounts to compensate 0012 However, when the anchor is used for sequential for the low reactivity, and tracking of reaction and analysis of reactions of peptide synthesis, deprotection of a second resi the reaction product on a carrier are difficult. due results in the production of the by-product diketopipera 0006. In an attempt to perform reactions in a homoge Zine, which causes a sequence showing markedly decreased neous liquid phase while utilizing the advantages of the Solid yield and peptide quality, thus posing problems in broad phase method in that isolation and purification after the reac utility (particularly sequence containing proline). With Such a tion can be performed by filtration and washing alone, a benzyl type anchor, moreover, deprotection under strong method of isolating a particular component dissolved in a acidic conditions is necessary, which prevents dissociation of liquid as a solid has been used. This is because precipitation a desired protecting group. of a particular component alone facilitates isolation and puri (0013 WO2007/122847 discloses a trityl type protecting fication after reaction. group (see the following structure). 0007. A particular component dissolved in a solution can

be precipitated only when predetermined conditions, such as chemical properties, property and relationship with solvents of the compound, are satisfied. 0008. However, determination of precipitation conditions requires trial and error and experimental searching in most cases. In liquid phase synthesis, moreover, some compounds to be synthesized are insoluble in organic solvents used for extraction or show low therein, which necessitates confirmation of the property of each compound to search for isolation and purification methods therefor. Particularly, when sequential and multistep synthesis reactions are required as in peptide synthesis and the like, since isolation and purification conditions such as precipitation, extraction and the like need to be determined based on the properties unique to the compound synthesized in each step, along time wherein m and n are each independently 0 or 1, Za is a and high cost are required. chlorine atom or a bromine atom, and Zb is a hydroxyl group, 0009. To solve such problems, a method using a carrier a chlorine atom or a bromine atom. molecule wherein a dissolved state and an insolubilized state 0014 With a protecting group of this trityl type, the for (precipitated State) irreversibly change according to the vary mation of diketopiperazine as a by-product can be suppressed ing solvent composition has been developed. Using Such a and selective deprotection can be performed. However, its US 2014/0046022 A1 Feb. 13, 2014 property as a protecting group is too low, namely, the com group and a mercapto group; at least one of rings A, B and C pound-anchor bondiseasily cutanda decomposition reaction has an organic group having an aliphatic hydrocarbon group; proceeds in which the anchor is dissociated even in methanol and rings A, B and C each independently optionally have an and the like. Therefore, the method is not satisfactory from electron-withdrawing group, are connected via an organic the aspects of yield and quality. group having an aliphatic hydrocarbon group, which is con tained in the unit structure. SUMMARY OF THE INVENTION 0023 (3) A fluorene compound represented by formula 0015. Accordingly, it is one object of the present invention (I'): to provide novel compounds, which are Superior in broad utility and Stability, which are useful as a protecting group (anchor) for amino acid and/or peptide in liquid phase Syn (I) thesis and the like of peptides. Ra 0016. It is another object of the present invention to pro Y vide novel organic synthesis reaction methods (particularly peptide liquid phase synthesis method) which employ such a s compound. 0017. It is another object of the present invention to pro {y\ Z (Y-NJ.Rc vided novel kits for liquid phase synthesis of peptides, which contain Such a compound. 0018. It is another object of the present invention to pro wherein ring A is an aromatic ring;Y is a group reactive with vided novel intermediates which are useful for producing one or more kinds selected from an amino group, a carboxyl Such a compound. group and a mercapto group; Ra, Rb and Rc are each inde 0019. These and other objects, which will become appar pendently an organic group having an aliphatic hydrocarbon ent during the following detailed description, have been group, a hydrogenatom or an electron-withdrawing group, at achieved by the inventors’ discovery that a particular com least one of Ra, Rb and Rc is an organic group having an pound having a fluorene skeleton can solve the above-men aliphatic hydrocarbon group; and rings A, B and C each tioned problems. independently optionally have an electron-withdrawing 0020. Accordingly, the present invention provides: is as group. described below. 0024 (4) A fluorene compound represented by formula 0021 (1) A fluorene compound represented by formula (II): (I):

(I)

wherein ring A is an aromatic ring;Y is a group reactive with wherein ring A is an aromatic ring;Y is a group reactive with one or more kinds selected from an amino group, a carboxyl one or more kinds selected from an amino group, a carboxyl group and a mercapto group; at least one of rings A, B and C group and a mercapto group; n is an integer of 1 to 19; Ric' is has an organic group having an aliphatic hydrocarbon group; a divalent organic group having an aliphatic hydrocarbon and rings A, B and C each independently optionally have an group; rings A, B and C each independently optionally have electron-withdrawing group. one or more kinds selected from an organic group having an 0022 (2) A fluorene compound wherein 2 to 20 divalent aliphatic hydrocarbon group and an electron-withdrawing unit structures derived from a compound represented by for group; when a plurality of ring A is present, respective rings mula (I): A may be the same or different; when a plurality of Y is present, respective Y may be the same or different; and when a plurality of Rc' is present, respective Rc' may be the same or (I) different. 0025 (5) The fluorene compound of any one of the above mentioned (1)–(4), wherein the organic group having an ali phatic hydrocarbon group and/or the divalent organic group is having an aliphatic hydrocarbon group is bonded on the ring via —O— —S NHCO— or —CONH present in the organic group, or directly bonded to form a carbon-carbon bond. 0026 (6) The fluorene compound of any one of the above wherein ring A is an aromatic ring;Y is a group reactive with mentioned (1)–(5), wherein the organic group having an ali one or more kinds selected from an amino group, a carboxyl phatic hydrocarbon group and/or the divalent organic group US 2014/0046022 A1 Feb. 13, 2014

having an aliphatic hydrocarbon group has a structure com wherein * shows the position of a bond; X, X', X" and X" prising 2 to 10 connections of an aliphatic hydrocarbon group are each independently absent or —O— —S —NHCO— bonded via —O——S——NHCO— or —CONH , oran or—CONH-; R and Rare each independently a hydrogen aliphatic hydrocarbon group directly bonded to form a car atom, an aliphatic hydrocarbon group having a carbon num bon-carbon bond. ber of not less than 5 or a methyl group, R is an organic group 0027 (7) The fluorene compound of the above-mentioned having an aliphatic hydrocarbon group having a carbon num 4, wherein the divalent organic group having an aliphatic ber of not less than 5: n, n., n and n are each independently hydrocarbon group for Ric' is a group represented by formula an integer of 0 to 2: m is an integer of 1 or 2; when a plurality (i): of n, n., n and n are each present, each respective n, n., n and n may be the same or different; when a plurality of X, X" and X" are each present, each respective X', X" and (i) X" may be the same or different; and when a plurality of R --Xa-Rd.--Xa and Ra are each present, each respective RandR may be the same or different; and wherein Xa is absent or —O— —S —NHCO— or 0032 a group represented by the formula (e): —CONH-, Rd is an aliphatic hydrocarbon group having a carbon number of not less than 5; k is an integer of 1-10; when a plurality of Rd is present, respective Rd may be the (e) same or different; and when a plurality of Xa is present, respective Xa may be the same or different. 0028 (8) The fluorene compound of the above-mentioned 4, wherein the divalent organic group having an aliphatic hydrocarbon group for Ric' is a group represented by formula wherein * shows the position of a bond; X is absent or (ii): O— —S —NHCO— or —CONH-; m is an integer of 0 to 15; ns is an integer of 0 to 11; n is an integer of 0 to 5: X, is absent or —O— —S , —COO ... —OCONH . (ii) —NHCO— or CONH : R is a hydrogenatom, a methyl --O-Re--O- group or an aliphatic hydrocarbon group having a carbon number of not less than 5; when a plurality of X, is present, respective X, may be the same or different; and when a wherein Reis analiphatic hydrocarbon group having a carbon plurality of R is present, respective R may be the same or number of 5 to 60; k is an integer of 1 to 3; and when a different. plurality of Re is present, respective Re may be the same or 0033 (10) The fluorene compound of the above-men different. tioned (9), wherein, 0029 (9) The fluorene compound of any one of the above 0034 in the formula (a), X is —O—; R is an aliphatic mentioned (1)–(8), wherein the organic group having an ali hydrocarbon group having a carbon number of 5 to 60; m is phatic hydrocarbon group is one or more kinds of group 1; selected from 0035 in the formula (b), X is - O - or- CONH-;X', 0030 a group represented by formula (a): X" and X" are each independently absent, or—O—; R and Ra are each is independently an aliphatic hydrocarbon group having a carbon number of 5 to 60 or a methyl group; R is an organic group having an aliphatic hydrocarbon group having a carbon number 5 to 60; n n n- and n are the same or different and each is an integer of 0 or 1; m is 1, and wherein * shows the position of a bond; X is absent or 0036 in the formula (e), Xs is —O—; m is 2 or 3; ns is 1: O——S——NHCO— or—CONH : R is an aliphatic n is 2 or 3: X, is —O—; and R in the number of mare each hydrocarbon group having a carbon number of not less than 5: independently an alkyl group having a carbon number of 8 to m is an integer of 1 to 10; when a plurality of X is present, 60. respective X may be the same or different; and when a 0037 (11) The fluorene compound of any one of the plurality of R is present, respective R may be the same or above-mentioned (1)–(6), wherein the aliphatic hydrocarbon different; group of the organic group having an aliphatic hydrocarbon a group represented by the formula (b): group and/or the divalent organic group having an aliphatic 0.031 hydrocarbon group is an aliphatic hydrocarbon group having a carbon number of not less than 5. (b) 0038 (12) The fluorene compound of any one of the X'R above-mentioned (1)–(6), wherein the aliphatic hydrocarbon ( 12 group of the organic group having an aliphatic hydrocarbon group and/or the divalent organic group having an aliphatic pil hydrocarbon group is an aliphatic hydrocarbon group having : X in 3 X." R3 ( in 2 a carbon number of 5 to 60. 1. 0039 (13) The fluorene compound of any one of the X"R4 above-mentioned (1)–(12), wherein the total carbon number derived from an aliphatic hydrocarbon group in one molecule is not less than 20. US 2014/0046022 A1 Feb. 13, 2014

0040 (14) The fluorene compound of any one of the independently an aliphatic hydrocarbon group having a car above-mentioned (1)–(12), wherein the total carbon number bon number of 5 to 60, and k is an integer of 1 to 3. derived from an aliphatic hydrocarbon group in one molecule 0055 (26) The fluorene compound of the above-men is 20 to 200. tioned (4), wherein ring A is a ring;Y is a hydroxyl 0041 (15) The fluorene compound of any one of the group, a bromo group or a chloro group; n is 1; ring A has a above-mentioned (1)-(14), wherein Y is a hydroxyl group, a halogen atom as the electron-withdrawing group; Ric' is a bromo is group, a chloro group, an iodo group, a thiol group group represented by the formula (i) wherein Xa is —O—; Rd or an amino group. is an aliphatic hydrocarbon group having a carbon number of 0042 (16) The fluorene compound of any one of the 5 to 60, and k is an integer of 1 to 3. above-mentioned (1)-(14), wherein Y is a hydroxyl group, a 0056 (27) The fluorene compound of the above-men bromo group or a chloro group. tioned (3) or (4), which is selected from the group consisting 0043 (17) The fluorene compound of any one of the of above-mentioned (1)-(16), wherein the aromatic ring of ring 0057 2-docosyloxy-9-(4-chlorophenyl)-9-; A is a benzene ring. 0.058 2-docosyloxy-9-(4-chlorophenyl)-9-bromofluo 0044) (18) The fluorene compound of the above-men rene; tioned (3), wherein Ra is a halogen atom. 0059 2,7-didocosyloxy-9-(4-chlorophenyl)-9-bromof 0045 (19) The fluorene compound of the above-men luorene; tioned (3), wherein the organic group having an aliphatic 0060 2-(12-docosyloxy-dodecanoxy)-9-(3-fluorophe hydrocarbon group is present at the 2-position and/or the nyl)-9-bromofluorene: 7-position of the fluorene compound. 0061 1,12-bis-12-(2'-O-9-(4-chlorophenyl)-9-fluore 0046 (20) The fluorene compound of the above-men nol)-dodecyloxy-dodecane; tioned (3), wherein Rb and/or Rc are/is 0062 1,12-bis-12-(2'-O-9-(4-chlorophenyl)-9-bromof 0047 a group represented by the formula (a) wherein m is luorene)-dodecyloxy-dodecane; 1: X is —O—, and R is an aliphatic hydrocarbon group 0063. 2-(3-octadecyloxy-2.2-bis-octadecyloxymethyl having a carbon number of 5 to 60, propoxy)-9-(4-chlorophenyl)-9-fluorenol; 0048 a group represented by the formula (b) wherein X. 0064. 2-(3-octadecyloxy-2.2-bis-octadecyloxymethyl X, X" and X" are each —O—; R and R are each inde propoxy)-9-(4-chlorophenyl)-9-bromofluorene: pendently an aliphatic hydrocarbon group having a carbon 0065 9-(4-chlorophenyl)-2-(3,4,5-tris(octadecyloxy)-cy number of 5 to 60; R is an organic group having an aliphatic clohexylmethoxy)-9-fluorenol; and hydrocarbon group having a carbon number of 5 to 60; n, n, 0.066 9-(4-chlorophenyl)-2-(3,4,5-tris(octadecyloxy)-cy n- and n are each 1; and m2 is 1, or clohexylmethoxy)-9-bromofluorene. 0049 a group represented by the formula (e) wherein X is 0067 (28) A protecting reagent for organic synthesis reac —O—; m is 2 or 3; ns is 1: n is 3: X, is —O—; and R in tion, comprising the fluorene compound of any one of the the number is of ms are each independently an alkyl group above-mentioned (1)-(27). having a carbon number of 14 to 30. 0068 (29) A protecting reagent for a carboxyl group of 0050 (21) The fluorene compound of the above-men amino acid or peptide, comprising the fluorene compound of tioned (3), wherein ring A is a benzene ring;Y is a hydroxyl any one of the above-mentioned (1)-(27). group, a bromo group or a chloro group; Rais a halogenatom; 0069 (30) A protecting reagent for the C-terminal of and an organic group having an aliphatic hydrocarbon group amino acid or peptide, comprising the fluorene compound of is a group represented by the formula (a) wherein m is 1: X any one of the above-mentioned (1)-(27). is —O—; and R is an aliphatic hydrocarbon group having a 0070 (31) A method of producing a peptide by a liquid carbon number of 5 to 60, a group represented by the formula phase synthesis process comprising the following steps; (b) wherein X, X, X" and X" are each —O—; R and R. 0071 (a) a step of binding a fluorene compound of any one are each independently an aliphatic hydrocarbon group hav of the above-mentioned (1)-(27) to an amino acid or peptide ing a carbon number of 5 to 60; R is an organic group having (binding step), and an aliphatic hydrocarbon group having a carbon number 5 to 0072 (b) a step of precipitating the bonded product of the 60; n, n2 ns and n are each 1; and m2 is 1, or a group compound and the amino acid or peptide obtained in the represented by the formula (e) wherein Xs is —O—; m is 2 above-mentioned step (precipitation step). or 3; ns is 1; no is 3: X7 is —O—; and R2 in the number of m 0073 (32) A method of producing a peptide by a liquid are each independently an alkyl group having a carbon num phase synthesis process comprising the following steps; ber of 14 to 30, each of which formulas is present at the 0074 (a) a step of obtaining C-fluorene compound-pro 2-position and/or the 7-position of the fluorene compound. tected amino acid or C-fluorene compound-protected peptide 0051 (22) The fluorene compound of the above-men by condensing a fluorene compound of any one of the above tioned (4), wherein n is 1. mentioned (1)-(27) with the C-terminal of an N-protected 0052 (23) The fluorene compound of the above-men amino acid or N-protected peptide (C-terminal fluorene com tioned (4), wherein ring A has an electron-withdrawing pound protection step), group. 0075 (b) a step of removing the protecting group of the 0053 (24) The fluorene compound of the above-men N-terminal of the amino acid or peptide obtained in the tioned (23), wherein the electron-withdrawing group is a above-mentioned step (N-terminal deprotection step), halogen atom. 0076 (c) a step of condensing the N-terminal of the amino 0054 (25) The fluorene compound of the above-men acid or peptide obtained in the above-mentioned step with tioned (4), wherein Rc' is a group represented by the formula N-protected amino acid or N-protected peptide (peptide chain (i) wherein Xa is —O—; Rd in the number of k are each elongation step), and US 2014/0046022 A1 Feb. 13, 2014

0077 (d) a step of precipitating the peptide obtained in the synthesis is performed using a compound of the present above-mentioned step (precipitation step). invention as an anchor, a sequence that easily forms dike 0078 (33) The method of the above-mentioned (32), fur topiperazine can be synthesized in a high yield, and further, ther is comprising one or more repeats of the following steps the anchor can be selectively removed. Thus, a protected (e)-(h): peptide can be directly obtained without removing other pro 0079 (e) a step of deprotecting the N-terminal of the pep tecting groups (peptideside chain protecting group etc.). tide obtained in the precipitation step, I0089. The particular compounds of the present invention 0080 (f) a step of condensing the N-terminal of peptide having a fluorene skeleton function as a Superior protecting obtained in the above-mentioned step with N-protected group and anchor, and the anchor can be removed under weak amino acid or N-protected peptide, and acidic conditions, and selectively removed even when other 0081 (h) a step of precipitating the peptide obtained in the protecting groups (peptide side chain protecting group etc.) above-mentioned step. still remain in the compound obtained by organic synthesis 0082 (34) A method of producing a peptide further com reactions. Namely, the compound is a Substance that enables prising, after the final precipitation step of the above-men easy precipitation in methanol and the like, since it dissolves tioned (32) or (33), a step of deprotecting the C-terminal of only in halogen solvents, THF and the like and scarcely peptide wherein the C-terminal is protected with a fluorene dissolves in polar organic solvents. In the process of elonga compound. tion of peptide chain length wherein the Substance is used as 0083 (35) A method of producing a peptide compound, a protecting group for C-terminal or side chain in the peptide comprising using a fluorene compound of any one of the synthesis and an operation including reaction in a halogen above-mentioned (1)-(27). solvent, followed by precipitation with methanol and the like 0084 (36) A method of producing an , to remove impurity is repeated, a side reaction giving dike comprising using a fluorene compound of any one of the topiperazine is suppressed and the peptide chain can be elon above-mentioned (1)-(27). gated in a high yield and with high quality. Moreover, the 0085 (37) A compound represented by the following for anchor enables selective removal thereof while maintaining mula (III): the protecting group.

DETAILED DESCRIPTION OF THE PREFERRED (III) X'R EMBODIMENTS ( 12 0090. Unless otherwise specified in the sentences, any technique terms and Scientific terms used in the present speci Hal in in 3 X it |- fication, have the same meaning as those generally under ( in 2 stood by those of ordinary skill in the art in the art the present invention belongs to. Any methods and materials similar or equivalent to those described in the present specification can be used for practicing or testing the present invention, and wherein X, X" and X" are each independently absent or preferable methods and materials are described in the follow O— —S —NHCO— or —CONH ; R and Ra are ing. All publications and patents referred to in the Specifica each independently a hydrogen atom, an aliphatic hydrocar tion are hereby incorporated by reference so as to describe bon group having a carbon number of not less than 5 or a and disclose constructed products and methodology methyl group; R is an organic group having an aliphatic described in, for example, publications usable in relation to hydrocarbon group having a carbon number of not less than 5: the described invention. ni, n., n and n are each independently an integer of 0 to 2: m2 is an integer of 1 or 2; when a plurality of n, n., n- and na 1. The Compounds of the Present Invention are each present, each respective n, n., n and na may be the same or different; when a plurality of X, X" and X" are 0091. The compound of the present invention are useful as each present, each respective X', X" and X" may be the a reagent for organic synthesis. Here, the reagent for organic same or different; and a plurality of when R and Ra are each synthesis refers to any reagent relating to organic synthesis present, each respective R and R may be the same or differ reactions, and is a concept including reagents directly ent; and Hal is a halogen atom. involved in the reaction Such as reaction Substrate, reaction I0086 (38) The compound of the above-mentioned (37), promoter, reagent for introducing protecting group, depro wherein, in the formula (III), X, X" and X" are the same or tecting agent and the like, as well as inert solvent and the like. different and each is absent or —O—; R and Ra are each Specifically, reagents to be used for peptide synthesis reaction independently an aliphatic hydrocarbon group having a car are exemplified. Preferred is a reagent introduced as a pro bon number of 10 to 30 or a methyl group; R is an organic tecting group (anchor) of the C-terminal of amino acid or group having analiphatic hydrocarbon group having a carbon peptide in liquid phase synthesis of peptide, and a reagent number of 10 to 30; n., n., n and n are the same or different introduced as a protecting group as a peptideside chain pro and each is an integer of 0 or 1; and m is 1. tecting group in peptide synthesis reaction, and an appropri I0087 (39) The compound of the above-mentioned (37), ate compound can be selected according to the object. Par which is 1-(3-iodo-2,2-bis-octadecanoxymethyl-propoxy) ticularly preferable compounds of the present invention are octadecane. anchors. 0088. The compounds of the present invention are supe 0092. One embodiment of the compound of the present rior in broad utility and stability in organic synthesis reac invention is a fluorene compound represented by the follow tions, particularly peptide synthesis reactions. When peptide ing formula (I): US 2014/0046022 A1 Feb. 13, 2014

wherein ring A is an aromatic ring;Y is a group reactive with (I) one or more kinds selected from an amino group, a carboxyl group and a mercapto group; at least one of rings A, B and C has an organic group having an aliphatic hydrocarbon group; O and rings A, B and C each independently optionally have an electron-withdrawing group, are connected via an organic group having an aliphatic hydro carbon group, which the unit structure has. CO 0096. As the above-mentioned compound, a compound represented by the following formula (II) is also preferable. wherein ring A is an aromatic ring;Y is a group reactive with one or more kinds selected from an amino group, a carboxyl group and a mercapto group; at least one of rings A, B and C (II) has an organic group having an aliphatic hydrocarbon group; and rings A, B and C each independently optionally have an electron-withdrawing group. 0093. A fluorene compound represented by the following formula (I'), wherein at least one of rings A, B and C has “an organic group having an aliphatic hydrocarbon group' and the number of “an organic group having an aliphatic hydro carbon group' of each ring is 1 to 4, preferably 1 to 3, more preferably 1 or 2, is still more preferably 1 or 0, is more preferable. wherein ring A is an aromatic ring;Y is a group reactive with one or more kinds selected from an amino group, a carboxyl group and a mercapto group; n is an integer of 1 to 19; Ric' is (I) a divalent organic group having an aliphatic hydrocarbon group; rings A, B and C each independently optionally have one or more kinds selected from an organic group having an aliphatic hydrocarbon group and an electron-withdrawing group; when a plurality of ring A is present, respective rings A may be the same or different; when a plurality of Y is present, respective Y may be the same or different; and when plurality of Rc' is present, respective Rc' may be the same or different. wherein ring A is an aromatic ring;Y is a group reactive with 0097. The divalent organic group having an aliphatic one or more kinds selected from an amino group, a carboxyl hydrocarbon group for Rc' is preferably represented by the group and a mercapto group; Ra, Rb and Rc are each inde following formula (i). pendently an organic group having an aliphatic hydrocarbon group, a hydrogen atom or an electron-withdrawing group, and at least one of Ra, Rb and Rc is an organic group having (i) an aliphatic hydrocarbon group; and rings A, B and C each --Xa-Rd.--Xa independently optionally have an electron-withdrawing group. wherein Xa is absent or —O— —S —NHCO—, or 0094. A fluorene compound represented by the formula —CONH-; Rd is an aliphatic hydrocarbon group having a (I) is encompassed in a fluorene compound represented by carbon number of not less than 5; k is an integer of 1 to 10; the formula (I). when a plurality of Rd is present, respective Rd may be the 0095 Moreover, the compound of the present invention same or different; and when a plurality of Xa is present, may be a compound having a structure as shown below, respective Xa may be the same or different. wherein a plurality of divalent unit structures derived from a 0098. In formula (i). Xa is preferably —O—, Rd is pref compound represented by the formula (I) are connected. Pre erably an aliphatic hydrocarbon group having a carbon num ferred is a fluorene compound wherein 2 to 20 of a divalent ber of 5 to 60, and k is preferably an integer of 1 to 3. That is, unit structure derived from a compound represented by the preferably formula (i) is represented by the following formula following formula (I): (ii). (I)

(ii) () --O-Re--O-

wherein Reis analiphatic hydrocarbon group having a carbon CO number of 5 to 60; k is an integer of 1 to 3; and when a plurality of Re is present, respective Re may be the same or different. US 2014/0046022 A1 Feb. 13, 2014

0099. In the present specification, the “aromatic ring 0105. The amino group, carboxyl group and/or mercapto means an 'aromatic hydrocarbon ring or an 'aromatic het group are present in an amino acid or peptide, which is a erocycle'. starting material of the peptide synthesis. That is, the com 0100. As the “aromatic hydrocarbon ring', C. arene pound represented by the formula (I), the formula (I) or the (e.g., benzene, naphthalene and the like) is preferable, Co formula (II) of the present invention is bonded to amino acid arene is more preferable, and benzene is particularly prefer or peptide via Y group which is a 'group reactive with an able. amino group, a carboxyl group and/or a mercapto group'. Preferably, in the liquid phase synthesis of peptide, it can 0101 Examples of the “aromatic heterocycle' include a 5 function as an anchor of the C-terminal (C-terminal protect to 7-membered monocyclic aromatic heterocycle or con ing reagent) of an amino acid or peptide to be the starting densed aromatic heterocycle containing, as a ring-constitut material, and/or as a side chain-protecting group (e.g., a car ing atom besides carbon atom, 1 to 4 hetero atoms selected boxyl-protecting reagent). from an oxygen atom, a Sulfur atom and a nitrogen atom. 0106 Here, the “side chain” is a chained structure other Examples of the condensed aromatic heterocycle include a than the main chain present in an amino acid or peptide to be group wherein such 5- to 7-membered monocyclic aromatic the starting material. For example, an acidic amino acid Such heterocycle and a 6-membered ring containing 1 or 2 nitrogen as aspartic acid and glutamic acid also has a carboxyl group in atoms, benzene ring, or a 5-membered ring containing one the side chain, an amino group in lysine and mercapto group Sulfur atom are condensed and the like. in cysteine. 0102 Preferable examples of the “aromatic heterocycle” 0107. In the present specification, the “electron-withdraw include 5 to 7-membered monocyclic aromatic heterocycle ing group' includes those generally used in the art. Specifi Such as furan, thiophene, pyrrole, oxazole, isoxazole, thiaz cally, a halogen atom (chlorine atom, bromine atom, ole, isothiazole, imidazole, pyrazole, 1.2.3-oxadiazole, 1.2, atom, iodine atom), a nitro group, a perhalogenoalkyl group 4-oxadiazole, 1.3,4-oxadiazole, furazan, 1.2.3-thiadiazole, (e.g., trifluoromethyl group), a cyano group, a carboxyl 1,2,4-thiadiazole, 1,3,4-thiadiazole, 1,2,3-triazole, 1,2,4-tria group, an alkoxycarbonyl group (e.g., methoxycarbonyl Zole, tetrazole, pyridine, pyridazine, pyrimidine, pyrazine, group, ethoxycarbonyl group), an aryloxycarbonyl group triazine and the like; 8- to 16-membered (preferably 8- to (e.g., phenyloxycarbonyl group), an alkylamide group (e.g., 12-membered) condensed aromatic heterocycle (preferably acetamide group), a perhalogenoalkylamide group (e.g., tri heterocycle wherein one or two (preferably one) 5- to 7-mem fluoroacetamido group), an optionally Substituted alkanoyl bered monocyclic aromatic heterocycle mentioned above is group (e.g., acetyl group, ethanoyl group, propanoyl group, condensed with one or two (preferably one) benzene rings, or isopropanoyl group, butanoyl group, isobutanoyl group, trif heterocycle wherein the same or different two or three (pref luoroacetyl group), an optionally substituted aroyl group erably two) 5- to 7-membered monocyclic aromatic hetero (e.g., benzoyl group, naphthylcarbonyl group, p-chloroben cycles mentioned above are condensed) such as benzofuran, Zoyl group), an optionally Substituted alkylsulfonyl group isobenzofuran, benzobthiophene, benzocthiophene, (e.g., methanesulfonyl group, trifluoromethanesulfonyl indole, isoindole, 1H-indazole, benzimidazole, benzoxazole, group), an optionally Substituted arylsulfonyl group (e.g., 1.2-benzisoxazole, benzothiazole, 1,2-benzisothiazole, benzenesulfonyl group, p-toluenesulfonyl group), an option 1H-benzotriazole, quinoline, isoquinoline, cinnoline, ally substituted alkylsulfoneamide group (e.g., methane quinazoline, quinoxaline, phthalazine, naphthyridine, purine, Sulfoneamide group, trifluoromethanesulfoneamide group), pteridinee, carbazole, C-carboline, B-carboline, Y-carboline, an optionally substituted arylsulfonamide group (e.g., benze acridine, phenoxathiine, phenothiazine, phenazine, phenox nesulfoneamide group or p-toluenesulfoneamide group) and athiine, thianthrene, phenanthridine, phenanthrolin, indoliz the like can be mentioned. Preferred are fluorine atom, chlo ine, pyrrolopyridine, pyrrolo 1.2-bipyridazine, pyrazolo 1.5- rine atom, nitro group, trifluoromethyl group, cyano group, apyridine, imidazol-2-alpyridine, imidazo 1.5-alpyridine, trifluoroacetamido group, trifluoroacetyl group, p-chloroben imidazo 1.2-bipyridazine, imidazol-2-alpyrimidine, 1,2,4- Zoyl group, methanesulfonyl group, trifluoromethanesulfo triazolo 4.3-alpyridine, 1,2,4-triazolo 4.3-bipyridazine, and nyl group, benzenesulfonyl group, p-toluenesulfonyl group, the like, and the like. methanesulfoneamide group, trifluoromethanesulfoneamide 0103) When desired, the “aromatic ring optionally fur group, benzenesulfoneamide group, p-toluenesulfoneamide ther has substituent(s) besides the organic group having an and the like, and particularly preferred are halogen atom and aliphatic hydrocarbon group in the formula (I) and the Ra trifluoromethyl group. group in the formula (I'). Examples of the further substituent 0108. In the present specification, the “organic group hav include an electron-withdrawing group and the like. Specific ing an aliphatic hydrocarbon group” means a monovalent examples of the further Substituent include a halogen atom organic group (one bond is bonded to at least one of rings A, (chlorine atom, is bromine atom, fluorine atom, iodine atom), B and C) which has an aliphatic hydrocarbon group in the optionally halogenated alkyl group (trifluoromethyl group molecular structure. etc.), nitro group, cyano group, ester group alkoxycarbonyl 0109 The “aliphatic hydrocarbon group' of the “organic group (methoxycarbonyl group, ethoxycarbonyl group, pro group having an aliphatic hydrocarbon group' is a aliphatic poxycarbonyl group etc.), aryloxycarbonyl group (pheny hydrocarbon group consisting of straight chain or branched, loxycarbonyl group etc.) and the like. saturated or unsaturated carbon and hydrogen, and an ali 0104. In the present specification, examples of the “group phatic hydrocarbon group having a carbon number of not less reactive with an amino group, a carboxyl group and/or a than 5 is preferable, an aliphatic hydrocarbon group having a mercapto group' include a hydroxyl group, a bromo group, a carbon number of 5 to 60 is particularly preferable, an ali chloro group, an iodo group, a thiol group, an amino group phatic hydrocarbon group having a carbon number of 5 to 30 and the like, with preference given to a hydroxyl group, a is more preferable, and an aliphatic hydrocarbon group hav bromo group and a chloro group. ing a carbon number of 10 to 30 is further preferable. US 2014/0046022 A1 Feb. 13, 2014

0110. The site of the “aliphatic hydrocarbon group' in the tively preferably bonded to the 2-position and/or the 7-posi organic group having analiphatic hydrocarbon group of is not tion on the fluorene ring in the compound of the present particularly limited, and it may be present at the terminal invention, since the final deanchoring is easy. (monovalent group), or other site (for example, divalent 0116. The “aliphatic hydrocarbon group' in the com group). pound of the present invention is appropriately selected according to the use of the compound to be synthesized. For 0111 Specific examples of the aliphatic hydrocarbon example, when it is use as a peptideside chain-protecting group include monovalent groups such as methyl group, ethyl group in the peptide synthesis, one having a comparatively group, propyl group, isopropyl group, butyl group, isobutyl short chain length, for example, one having a carbon number group, sec-butyl group, tert-butyl group, pentyl group, hexyl of less than 5 can be employed. When it is used as an anchor group, octyl group, decyl group, lauryl group, tridecyl group. at the C-terminal of an amino acid or peptide, one having a myristyl group, cetyl group, Stearyl group, aralkyl group, comparatively long chain length, namely, one having a car behenyl group, oleyl group, isostearyl group and the like and bon number of not less than 5 is preferably employed. Fur a divalent group derived therefrom. thermore, an aliphatic hydrocarbon group having a carbon 0112 In the “organic group having an aliphatic hydrocar number of 5 to 60 is more preferable, an aliphatic hydrocar bon group', a moiety other than the aliphatic hydrocarbon bon group having a carbon number of 5 to 30 is further group can be set freely. For example, it optionally has a preferable, and an aliphatic hydrocarbon group having a car moiety such as —O— —S —NHCO , —CONH . bon number of 10 to 30 is still more preferable. When it is hydrocarbon group and the like. Examples of the “hydrocar used as an anchor, the total number of carbon derived from the bon group' include a monovalent group Such as aliphatic “aliphatic hydrocarbon group” in one molecule of the com hydrocarbon group, monocyclic Saturated hydrocarbon pound of the present invention (hereinafter to be also referred group and aromatic hydrocarbon group and the like, specific to as the total carbon number derived from aliphatic hydro examples include alkyl group, alkenyl group, alkynyl group, carbon group; when a plurality of “aliphatic hydrocarbon cycloalkyl group, aryl group and the like and a divalent group group' is present, the total thereof) is preferably not less than derived therefrom. Examples of the “alkyl group' include 20, more preferably 20 to 200, further preferably 20 to 100, C. alkyl group and the like and preferable examples include and still more preferably 20 to 60. When the carbon number is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, higher, crystallinity of the compound of the present invention tert-butyl, pentyl, hexyl and the like. Examples of the “alk in a polar organic solvent becomes fine even when the peptide enyl group” include Calkenyl group and the like and pref chain is a long chain. erable examples include vinyl, 1-propenyl, allyl, isopropenyl, 0117 Examples of the “organic group having an aliphatic butenyl, isobutenyland the like. Examples of “alkynyl group' hydrocarbon group' include the groups represented by the include C- alkynyl group and the like and preferable following formulas (a) to (e). examples include ethynyl, propargyl, 1-propynyl and the like. Examples of the “cycloalkyl group” include C. cycloalkyl group and the like and preferable examples include cyclopro (a) pyl, cyclobutyl, cyclopenty1 and cyclohexyl. The “aryl ---X-R-i-H group' is preferably, for example, Caryl group Such as phenyl, 1-naphthyl 2-naphthyl, biphenylyl, 2-anthryland the like, and the like, more preferably Co aryl group and, for In the formula (a), * shows the position of a bond; X is absent example, phenyl group and the like. The hydrocarbon group or —O— —S— —NHCO— or —CONH : R is an ali phatic hydrocarbon group having a carbon number of not less is optionally substituted by a substituent selected from a than 5: m is an integer of 1 to 10; when a plurality of X is halogen atom (chlorine atom, bromine atom, fluorine atom, present, respective X may be the same or different; and when iodine atom), an oxo group and the like. a plurality of R is present, respective R may be the same or 0113. The “organic group having an aliphatic hydrocar different. bon group' may be bonded (substitution) to at least one of 0118. In the formula (a), particularly a group whereinX is rings A, B and C via an aliphatic hydrocarbon group present —O—; R is an aliphatic hydrocarbon group having a carbon in the group, namely, directly bonded to form a carbon-carbon number of 5 to 60; and m is 1, is preferable. bond or via —O— —S —NHCO—, —CONH- and the 0119. As the “aliphatic hydrocarbon group having a car like present in the group. Preferably, it is bonded via —O , bon number of not less than 5” for R, an “aliphatic hydro —S or —CONH in view of the easiness of synthesis of carbon group' of the above-mentioned “organic group having the compound. More preferably, it is bonded via —O—. an aliphatic hydrocarbon group', which has a carbon number 0114. The compound of in the present invention is char of not less than 5 can be mentioned, with preference given to acterized in that ring A, B and/or Care/is substituted by 1 to 4, preferably 1 to 3, more preferably 1 or 2, "organic groups on having a carbon number of 5 to 60. having an aliphatic hydrocarbon group” mentioned above. A same ring may be substituted by all “organic groups having an (b) aliphatic hydrocarbon group', a different ring may be substi X'R tuted thereby. In addition, plural aliphatic hydrocarbon groups may be contained in one “organic group having an ( 12 aliphatic hydrocarbon group' by branching and the like. In the compound of the present invention, when a plurality of X pil "Yx R3 aliphatic hydrocarbon group is present, they may be the same ( in 2 or different. 0115. In the compound of the present invention, an organic group having an aliphatic hydrocarbon group is unlimita US 2014/0046022 A1 Feb. 13, 2014

In the formula (b), * shows the position of a bond; X, X', X." In the formula (d), * shows the position of a bond; o and pare and X" are each independently absent, or —O— —S , each 0 or 1; X, X. X", Xs and X are the same or different —NHCO— or —CONH ; R and Ra are each indepen and each is absent, or —O— —S— —NHCO— or dently a hydrogenatom, an aliphatic hydrocarbon group hav —CONH ; R. R. and Rare the same or different and each ing a carbon number of not less than 5 or a methyl group; R. is analiphatic hydrocarbon group and R'' and R are each an is an organic group having an aliphatic hydrocarbon group alkylene group having a carbon number of 1 to 10. having a carbon number of not less than 5: n, n., n and n are 0.126 Examples of the “alkylene group” having a carbon each independently an integer of 0 to 2; m is an integer of 1 number of 1 to 10 include methylene, ethylene, propylene, or 2; when a plurality of n, n., n and n are present, each butylene, hexylene, octylene, dodecylene and the like. respectiven, n., n and n may be the same or different; when a plurality of X, X" and X" are present, each respective I0127. As the “aliphatic hydrocarbon group” for R7-R. X', X" and X" may be the same or different; and when a those similar to the “aliphatic hydrocarbon group' of the plurality of R and Ra are present, each respective R and R. above-mentioned "organic group having an aliphatic hydro may be the same or different. carbon group' can be mentioned, particularly one having a 0120. As the “aliphatic hydrocarbon group having a car carbon number of not less than 5 can be mentioned, prefer bon number of not less than 5” for R, R or R, from among ably one having a carbon number of 5 to 60. the above-mentioned “organic group having an aliphatic I0128. In the formula (d), a group whereino is 0; p is 1:X hydrocarbon group', an “aliphatic hydrocarbon group” hav X' and X" are each —O—: Xsis —NHCO : X is —O—: ing a carbon number of not less than 5 can be mentioned, with R7-R is an aliphatic hydrocarbon group having a carbon preference given to one having a carbon number of 5 to 60. number of 5 to 60; Ro and R are each an alkylene group I0121. In the formula (b), a group wherein X is —O , having a carbon number of 1 to 10, is particularly preferable. —NHCO— or – CONH , preferably - O - or —CONH-X, X" and X" are eachindependently absent or —O—; R and R are each independently an aliphatic (e) hydrocarbon group having a carbon number of 5 to 60 or a methyl group; R is an organic group having an aliphatic Xs ii.5 X ) hydrocarbon group having a carbon number 5 to 60; n., n., n and n are the same or different and each is an integer of 0 or 1; and m is 1, is particularly preferable. 0122. In the formula (b), a group wherein X, X', X" and wherein * shows the position of a bond; X is absent or X" are each —O—, R and Ra are each independently an O— —S , NHCO— or —CONH-; m is an integer aliphatic hydrocarbon group having a carbon number of 5 to of 0 to 15; ns is an integer of 0 to 11; n is an integer of 0 to 5: 60, R is an organic group having an aliphatic hydrocarbon X, is absent or —O —S , —COO , —OCONH , group having a carbon number 5 to 60, n, n., n and n are —NHCO— or —CONH : R is a hydrogenatom, a methyl each 1, and m is 1, is more preferable. group or an aliphatic hydrocarbon group having a carbon number of not less than 5; when a plurality of X, is present, respective X, may be the same or different; and when a In the formula (c), * shows the position of a bond; X and X plurality of R is present, respective R may be the same or are the same or different and each is absent or —O— —S—, different. —NHCO— or —CONH ; Rs and R are the same or dif ferent and each is an aliphatic hydrocarbon group; and Aris I0129. As the “aliphatic hydrocarbon group having a car an arylene group. bon number of not less than 5” for R, the “aliphatic hydro 0123 Examples of the “arylene group' include phe carbon group' of the above-mentioned “organic group having nylene, naphthylene, biphenylene and the like, with prefer an aliphatic hydrocarbon group” having a carbon number of ence given to phenylene. not less than 5 can be mentioned, preferably one having a 0.124. As the “aliphatic hydrocarbon group' for Rs or R. carbon number of 5 to 80. those similar to the “aliphatic hydrocarbon group' of the I0130. In the formula (e), a group whereinX is —O—: m above-mentioned "organic group having an aliphatic hydro is 2 or 3; ns is 1; no is 2 or 3; X, is —O—; and R2 in the carbon group' can be mentioned, particularly one having a number of mare each independently an alkyl group having a carbon number of not less than 5 preferably 5 to 60, can be carbon number of 8 to 60, is preferable. mentioned. 0131. In the formula (e), a group wherein X is —O—, m 0.125. In the formula (c), a group wherein X and X’ are is 2 or 3, ns is 1., n is 3, X, in the number of m is —O—, and each —O—; Rs and R are each independently an aliphatic R in the number of m are each independently an alkyl hydrocarbon group having a carbon number of 5 to 60; and Ar group having a carbon number of 14 to 30, is particularly is phenylene, is particularly preferable. preferable. (d) 0132 Specific examples of the “organic group having an X6 R10 X4R 1. 7 aliphatic hydrocarbon group' from among aliphatic carbon : (RIX: p Cs chain groups having a carbon number of 18 or 22 include the following. In each group, * shows the position of a bond.

: US 2014/0046022 A1 Feb. 13, 2014 10

-continued above-mentioned "organic group having an aliphatic hydro OC18H37 CH carbon group' can be mentioned. 0.136. A fluorene compound wherein 2 to 20 divalent unit O O structures derived from a compound represented by the for mula (I) are connected via the "divalent organic group having 1 -- 1 -- an aliphatic hydrocarbon group' is also within the range of OC18H37 OC18H37 the present invention. OC18H37 0.137 Examples of such divalent organic group include those represented by the following formula (i): O CH3 : O 1 C18H37 Y OC18H37 (i) O --Xa-Rdi-Xa OC18H37 OC18H37 CH3 wherein Xa is absent, or —O— —S , —NHCO— or : —CONH-; Rd is an aliphatic hydrocarbon group having a OC18H37 carbon number of not less than 5; k is an integer of 1 to 10; O when a plurality of Rd is present, respective Rd may be the OC18H37 same or different; and when a plurality of Xa is present, H CH3 respective Xa may be the same or different, and represented : Y N OC18H37 "n O~k C18H37 by the following formula (ii): O C18H37 OC18H37 : --O-Re--O- (ii)

N C18H37 wherein Reis analiphatic hydrocarbon group having a carbon OC18H37 number of 5 to 60; k is an integer of 1 to 3; and when Re is : 1. present in plurality, respective Re may be the same or differ ent, are more preferable. 0.138. As the “aliphatic hydrocarbon group having a car OC18H37 bon number of not less than 5” for Rd, the “aliphatic hydro OC18H37 carbon group' of the above-mentioned “organic group having O an aliphatic hydrocarbon group” having a carbon number of not less than 5 can be mentioned. 1'N-1 ns OC18H37 0.139. As the “aliphatic hydrocarbon group having a car H bon number of 5 to 60' for Re, the “aliphatic hydrocarbon group of the above-mentioned "organic group having an OC18H37 aliphatic hydrocarbon group” having a carbon number of 5 to OC18H37 60 can be mentioned. OC18H37 0140. As a fluorene compound represented by the formula O (I) of the present invention, a compound represented by the formula (I') is preferable, more preferably a compound rep OC18H37 resented by the formula (I), 0141 wherein ring A is a benzene ring; Y is a hydroxyl OC18H37 group, a bromo group or a chloro group; Raisahalogenatom; an organic group having an aliphatic hydrocarbon group is a group represented by the formula (a) wherein m is 1; X is 0133) In addition, the following group is also used. —O—; R is is an aliphatic hydrocarbon group having a carbon number of 5 to 60, a group represented by the formula (b) wherein X, X', X" and X" are each —O : R and Ra are each independently an aliphatic hydrocarbon group hav ing a carbon number of 5 to 60; R is an organic group having -o-call-()-o-cal an aliphatic hydrocarbon group having a carbon number 5 to 60; n, n2 ns and n are each 1; and m2 is 1, or a group represented by the formula (e) wherein Xs is —O—; m is 2 0134. In the present specification, “the divalent organic or 3; ns is 1; n is 3; X, is —O—; R2 in the number of ms are group having an aliphatic hydrocarbon group' is a divalent each independently an alkyl group having a carbon number of organic group having an aliphatic hydrocarbon group in a 14 to 30, each of which formulas is present at the 2-position molecule structure thereof. and/or the 7-position of the fluorene compound. 0135. As the “aliphatic hydrocarbon group' of the "diva 0142. A fluorene compound represented by the formula lent organic group having an aliphatic hydrocarbon group'. (II) of the present invention is preferably a compound repre those similar to the “aliphatic hydrocarbon group' of the sented by the formula (II), US 2014/0046022 A1 Feb. 13, 2014

0143 wherein ring A is a benzene ring; Y is a hydroxyl alkyl group (e.g., methyl, ethyl, propyl), a (C-C)alkoxy group, a bromo group or a chloro group; n is 1; ring A has a group (e.g., methoxy, ethoxy, propoxy), a nitro group and the halogen atom as the electron-withdrawing group; Ric' is a like. group represented by the formula (i) wherein Xa is —O—; Rd 0160 Examples of the amino-protecting group include is an aliphatic hydrocarbon group having a carbon number of formyl group, (C-C)alkyl-carbonyl group (e.g., acetyl, pro 5 to 60, and k is an integer of 1 to 3. pionyl), (C-C)alkoxy-carbonyl group (e.g., methoxycarbo 0144 Preferable examples of the fluorene compound of nyl, ethoxycarbonyl, tert-butoxycarbonyl), benzoyl group, the present invention include the following fluorene com (C7-Co)aralkyl-carbonyl group (e.g., benzylcarbonyl), (C7 pounds. Ca)aralkyloxy-carbonyl group (e.g., benzyloxycarbonyl, 0145 2-docosyloxy-9-(4-chlorophenyl)-9-fluorenol; 9-fluorenylmethoxycarbonyl), trityl group, phthaloyl group, 0146 2-docosyloxy-9-(4-chlorophenyl)-9-bromofluo N,N-dimethylaminomethylene group, silyl group (e.g., trim rene; ethylsilyl, triethylsilyl, dimethylphenylsilyl, tert-butyldim 0147 2,7-didocosyloxy-9-(4-chlorophenyl)-9-bromof ethylsilyl, tert-butyldiethylsilyl), (C-C)alkenyl group (e.g., luorene; 1-allyl) and the like. These groups are optionally substituted 0148 2-(12-docosyloxy-dodecanoxy)-9-(3-fluorophe by 1 to 3 Substituents selected from a halogen atom (e.g., nyl)-9-bromofluorene; fluorine, chlorine, bromine, iodine), a (C-C)alkoxy group 0149 1,12-bis-12-(2'-O-9-(4-chlorophenyl)-9-fluore (e.g., methoxy, ethoxy, propoxy), a nitro group and the like. nol)-dodecyloxy-dodecane; 0.161 Examples of the carboxy-protecting group include 0150 1,12-bis-12-(2'-O-9-(4-chlorophenyl)-9-bromof (C-C)alkyl group (e.g., methyl, ethyl, propyl, isopropyl. luorene)-dodecyloxy-dodecane; butyl, tert-butyl), (C7-Co.)aralkyl group (e.g., benzyl), phe 0151. 2-(3-octadecyloxy-2,2-bis-octadecyloxymethyl nyl group, trityl group, silyl group (e.g., trimethylsilyl, trieth propoxy)-9-(4-chlorophenyl)-9-fluorenol; ylsilyl, dimethylphenylsilyl, tert-butyldimethylsilyl, tert-bu 0152 2-(3-octadecyloxy-2,2-bis-octadecyloxymethyl tyldiethylsilyl, tert-butyldiphenylsilyl), (C-C)alkenyl propoxy)-9-(4-chlorophenyl)-9-bromofluorene; group (e.g., 1-allyl) and the like can be mentioned. These 0153 9-(4-chlorophenyl)-2-(3,4,5-tris(octadecyloxy)-cy groups are optionally substituted by 1 to 3 substituents clohexylmethoxy)-9-fluorenol; and selected from a halogen atom (e.g., fluorine, chlorine, bro 0154 9-(4-chlorophenyl)-2-(3,4,5-tris(octadecyloxy)-cy mine, iodine), a (C-C)alkoxy group (e.g., methoxy, ethoxy, clohexylmethoxy)-9-bromofluorene. propoxy), a nitro group and the like. 0162 Examples of the carbonyl-protecting group include 2. Production Method of the Compound of the Present cyclic acetal (e.g., 1,3-dioxane), acyclic acetal (e.g., di-(C- Invention C.)alkylacetal) and the like. 0155 While the production method of the compounds of 0163 These protecting groups can be removed by a the present invention is not particularly limited, for example, method known per se, for example, the method described in it can be synthesized by the following reactions. Protective Groups in Organic Synthesis, John Wiley and Sons 0156 Unless particularly specified, the starting material (1980) and the like. For example, a method using acid, base, compounds may be a commercially available product, or can ultraviolet rays, hydrazine, phenylhydrazine, sodium N-me be produced according to a method known perse or a method thyldithiocarbamate, tetrabutylammonium fluoride, palla analogous thereto. dium acetate, trialkylsilylhalide (e.g., trimethylsilyliodide, (O157. While the yield of the compound obtained by each trimethylsilylbromide and the like) and the like, a reduction of the following methods may vary depending on the reaction method and the like are used. conditions employed, these resultant products can be isolated and purified by a general method (recrystallization, column Production Method chromatography and the like), and then precipitated by a 0164. An example wherein at least any one of ring A, ring method of changing the solution temperature, a method of B and ring C (hereinafter also to be referred simply as a ring) changing the Solution composition and the like. is Substituted by an organic group having an aliphatic hydro 0158. In each reaction, when the starting material com carbon group via —O— is shown in the following. pound has a hydroxy group, an amino group, a carboxy group or a carbonyl group, a protecting group generally used in the 0.165. In this method, as the “aliphatic hydrocarbon peptide chemistry and the like may be introduced into these group', those similar to the “aliphatic hydrocarbon group of groups, and the object compound can be obtained by remov the above-mentioned “organic group having an aliphatic ing the protecting group as necessary after the reaction. hydrocarbon group' can be mentioned. 0159. Examples of the hydroxyl-protecting group include (C-C)alkyl group (e.g., methyl, ethyl, propyl, isopropyl. Step 1. butyl, tert-butyl), phenyl group, trityl group, (C7-Co)aralkyl group (e.g., benzyl), formyl group, (C-C)alkyl-carbonyl (0166 group (e.g., acetyl, propionyl), benzoyl group, (C7-Co) aralkyl-carbonyl group (e.g., benzylcarbonyl), 2-tetrahydro H-ALK-OH + Hal-ALK-Hal -> pyranyl group, 2-tetrahydrofuranyl group, silyl group (e.g., trimethylsilyl, triethylsilyl, dimethylphenylsilyl, tert-bu (1) (2) tyldimethylsilyl, tert-butyldiethylsilyl), (C-C)alkenyl H-ALK-O-ALK-Hal group (e.g., 1-allyl) and the like. These groups are optionally (3) substituted by 1 to 3 substituents selected from a halogen atom (e.g., fluorine, chlorine, bromine, iodine), a (C-C) US 2014/0046022 A1 Feb. 13, 2014

wherein Hal is a halogenatom (chlorine atom, bromine atom, preferably tetrahydrofuran. The amount of the solvent to be iodine atom, fluorine atom; preferably bromine atom), and used is generally 2- to 50-fold volume relative to compound ALK" and ALK are the same or different and each is an (2). aliphatic hydrocarbon group. 0167 Compound (1) is reacted with 0.2 to 5 molar equiva (0177. The reaction temperature is generally 20° C. to 150° lents of compound (2) to give compound (3). Generally, the C., preferably 50° C. to 100° C. The reaction time is generally reaction is performed in the presence of a base in a solvent that 1 to 30 hours. does not adversely influence the reaction. 0168 Examples of the base include an alkali metal salt Step 3-1. Such as potassium hydroxide, Sodium hydroxide, sodium hydrogen carbonate, potassium carbonate and the like; amine 0178 such as pyridine, triethylamine, N,N-dimethylaniline, 1.8- diazabicyclo5.4.0]undec-7-en and the like; metal hydride Such as potassium hydride, Sodium hydride and the like; O alkali metal alkoxide Such as sodium methoxide, sodium ethoxide, potassium tert-butoxide and the like, and the like. 0169. The amount of the base to be used is preferably / \ H-ALK-Hal about 1 to 5 molar equivalents relative to compound (1). (7) 0170 As such solvent, halogenated hydrocarbon such as chloroform, dichloromethane and the like; and nonpolar organic solvent such as 1,4-dioxane, tetrahydrofuran and the like can be mentioned. These solvents may be used in a mixture of two or more kinds at an appropriate ratio. It is preferably tetrahydrofuran. The amount of the solvent to be used is generally 2- to 50-fold volume relative to compound Y \ (1). H-ALK-O-S (0171 The reaction temperature is generally 20°C. to 150° C., preferably 50° C. to 100° C. The reaction time is generally (8) 1 to 30 hours. Step 2. wherein Hal is a halogenatom (chlorine atom, bromine atom, iodine atom, fluorine atom; preferably bromine atom) and 0172 ALK is an aliphatic hydrocarbon group. 0179 Compound (6) is reacted with 0.3 to 5 molar equiva Hal-ALK?-Hal + HO-ALK-OH -e- lents of compound (7) to give compound (8). Generally, the reaction is performed in the presence of a base in a solvent that (2) (4) Hal-ALK-O-ALK-O-ALK?-Hal does not adversely influence the reaction. 0180 Examples of the base include an alkali metal salt (5) Such as potassium hydroxide, Sodium hydroxide, Sodium hydrogen carbonate, potassium carbonate and the like; amine wherein Hal is a halogenatom (chlorine atom, bromine atom, such as pyridine, triethylamine, N,N-dimethylaniline, 1.8- iodine atom, fluorine atom; preferably bromine atom), and diazabicyclo5.4.0]undec-7-en and the like; metal hydride ALK and ALK are the same or different and each is an Such as potassium hydride, Sodium hydride and the like; aliphatic hydrocarbon group. alkali metal alkoxide Such as sodium methoxide, Sodium 0173 Compound (4) is reacted with about 0.3 to 5 molar ethoxide, potassium tert-butoxide and the like, and the like. equivalents of compound (2) to give compound (5). Gener 0181. The amount of the base to be used is preferably ally, the reaction is performed in the presence of a base in a about 1 to 5 molar equivalents relative to compound (6). solvent that does not adversely influence the reaction. 0.174 Examples of the base include an alkali metal salt 0182 Examples of the solvent include aromatic hydrocar Such as potassium hydroxide, Sodium hydroxide, sodium bon Such as benzene, , Xylene and the like: nitrile Such hydrogen carbonate, potassium carbonate and the like; amine as acetonitrile, propionitrile and the like; ether Such as tet such as pyridine, triethylamine, N,N-dimethylaniline, 1.8- rahydrofuran, 1.4-dioxane, and the like: ketone diazabicyclo5.4.0]undec-7-en and the like; metal hydride Such as , 2-butanone and the like; halogenated hydro Such as potassium hydride, Sodium hydride and the like; carbon such as chloroform, dichloromethane and the like; alkali metal alkoxide Such as sodium methoxide, sodium amide such as N,N-dimethylformamide and the like: sulfox ethoxide, potassium tert-butoxide and the like, and the like. ide such as dimethylsulfoxide and the like, and the like. These (0175. The amount of the base to be used is preferably Solvents may be used in a mixture of two or more kinds at an about 1 to 10 molar equivalents relative to compound (4). appropriate ratio. It is preferably dimethylformamide. The 0176). As such solvent, halogenated hydrocarbon such as amount of the solvent to be used is generally 2- to 50-fold chloroform, dichloromethane and the like; and nonpolar Volume relative to compound (6). organic solvent such as 1,4-dioxane, tetrahydrofuran and the 0183. The reaction temperature is generally 30° C. to 150° like can be mentioned. These solvents may be used in a C., preferably 50° C. to 100° C. The reaction time is generally mixture of two or more kinds at an appropriate ratio. It is 1 to 30 hours. US 2014/0046022 A1 Feb. 13, 2014 13

Step 3-2. 0186 These reactions are generally performed in the pres ence of a base in a solvent that does not adversely influence 0184 the reaction. 0187. Examples of the base include an alkali metal salt Such as potassium hydroxide, Sodium hydroxide, Sodium hydrogen carbonate, potassium carbonate and the like; amine 1) H-ALK-Hal such as pyridine, triethylamine, N,N-dimethylaniline, 1.8- (7) diazabicyclo5.4.0]undec-7-en and the like; metal hydride 2) H-ALK-Hal Such as potassium hydride, Sodium hydride and the like; (7) alkali metal alkoxide Such as sodium methoxide, Sodium HO WS- y (Yled OH ethoxide, potassium tert-butoxide and the like, and the like. (6') 0188 The amount of the base to be used is preferably O about 1 to 10 molar equivalents relative to compound (6'). 0189 Examples of the solvent include aromatic hydrocar bon Such as benzene, toluene, Xylene and the like: nitrile Such as acetonitrile, propionitrile and the like; ether Such as tet rahydrofuran, 1.4-dioxane, diethyl ether and the like: ketone H-ALK-O-CS- -o-ALK"-H Such as acetone, 2-butanone and the like; halogenated hydro carbon such as chloroform, dichloromethane and the like; (8) amide such as N,N-dimethylformamide and the like: sulfox ide such as dimethylsulfoxide and the like, and the like. These wherein Hal is a halogenatom (chlorine atom, bromine atom, Solvents may be used in a mixture of two or more kinds at an iodine atom, fluorine atom; preferably bromine atom) and appropriate ratio. It is preferably dimethylformamide. The ALK and ALK" are the same or different and each is an amount of the solvent to be used is generally 2- to 50-fold aliphatic hydrocarbon group. volume relative to compound (6'). (0190. The reaction temperature is generally 30° C. to 150° 0185. Compound (6') is reacted with halide to give com C., preferably 50° C. to 100° C. The reaction time is generally pound (8). When compound (7) and compound (7") are the 1 to 70 hours. same, the above-mentioned reaction can be performed in one step. When compound (7) and compound (7") are different Step 3-3. compounds, compound (6') is reacted with 0.3 to 5 molar (0191)

O

Her H-ALK-O-ALK-O-ALK?-Hal (7)

-e O-ALK-O-ALK-O-ALK-O S. (9) equivalents of compound (7) and then reacted with 0.3 to 5 wherein Hal is a halogenatom (chlorine atom, bromine atom, molar equivalents of compound (7"). During reaction with iodine atom, fluorine atom; preferably bromine atom) and compound (7), the moiety to be reacted with compound (7") is ALK and ALK are the same or different and each is an preferably protected with a protecting group in advance. As aliphatic hydrocarbon group. the protecting group, hydroxyl-protecting group can be men 0.192 Compound (6) reacted with about 0.5 to 5 molar tioned and, for example, (C-C)alkyl group (e.g., methyl, equivalents of compound (5) to give compound (9). Gener ethyl, propyl, isopropyl, butyl, tert-butyl), phenyl group, trityl ally, the reaction is performed in the presence of a base in a group. (C7-Co) aralkyl group (e.g., benzyl), formyl group. solvent that does not adversely influence the reaction. 0193 Examples of the base include an alkali metal salt (C-C)alkyl-carbonyl group (e.g., acetyl, propionyl), ben Such as potassium hydroxide, Sodium hydroxide, Sodium Zoyl group, (C7-Co)aralkyl-carbonyl group (e.g., benzylcar hydrogen carbonate, potassium carbonate and the like; amine bonyl), 2-tetrahydropyranyl group, 2-tetrahydrofuranyl such as is pyridine, triethylamine, N,N-dimethylaniline, 1.8- group, silyl group (e.g., trimethylsilyl, triethylsilyl, dimeth diazabicyclo5.4.0]undec-7-en and the like; metal hydride ylphenylsilyl, tert-butyldimethylsilyl, tert-butyldiethylsilyl), Such as potassium hydride, Sodium hydride and the like; (C-C)alkenyl group (e.g., 1-allyl) and the like can be men alkali metal alkoxide Such as sodium methoxide, Sodium tioned. ethoxide, potassium tert-butoxide and the like, and the like. US 2014/0046022 A1 Feb. 13, 2014

0194 The amount of the base to be used is preferably (0202) The reaction temperature is generally 10°C. to 100° about 1 to 10 molar equivalents relative to compound (6). C., preferably 30°C. to 70°C. The reaction time is generally 0.195 Examples of the solvent include aromatic hydrocar 1 to 30 hours. bon Such as benzene, toluene, Xylene and the like: nitrile Such as acetonitrile, propionitrile and the like; ether Such as tet rahydrofuran, 1,4-dioxane, diethyl ether and the like: ketone Step 5. Such as acetone, 2-butanone and the like; halogenated hydro carbon such as chloroform, dichloromethane and the like; 0203 amide such as N,N-dimethylformamide and the like: sulfox ide such as dimethylsulfoxide and the like, and the like. These Solvents may be used in a mixture of two or more kinds at an appropriate ratio. It is preferably dimethylformamide. The amount of the solvent to be used is generally 2- to 50-fold Volume relative to compound (6). (0196. The reaction temperature is generally 30° C. to 150° C., preferably 50° C. to 100° C. The reaction time is generally 1 to 70 hours. Step 4. 0197) -o- SOC) (8) (I-2) Hal HAI; Br or Cl

... O wherein Hall is a bromine atom or a chlorine atom, Hal is a halogen atom (chlorine atom, bromine atom, iodine atom, fluorine atom; preferably chlorine atom) and ALK is an aliphatic hydrocarbon group. H-ALK-O1 CS\S O 0204 Compound (1-1) is reacted with a halogenating (I-1) agent generally in a solvent that does not adversely influence the reaction to give compound (I-2). wherein Hal is a halogenatom (chlorine atom, bromine atom, 0205 As the halogenating agent to be used in this step, iodine atom, fluorine atom; preferably chlorine atom), and ALK is an aliphatic hydrocarbon group. acetyl bromide, acetylchloride, thionyl chloride, thionyl bro 0198 Compound (8) is reacted with a Grignard reagent mide, PBr, HBr, NBS and the like can be mentioned. generally in a solvent that does not adversely influence the 0206. The amount of halogenated acyl to be used is pref reaction to give compound (I-1). erably about 1 to 30 molar equivalents relative to compound 0199 As the Grignard reagent used in this step, 4-chlo (I-1). rophenylmagnesium bromide, 3-chlorophenylmagnesium bromide, 3-trifluoromethylphenylmagnesium bromide, 0207 As such solvent, halogenated hydrocarbon such as 3-fluorophenylmagnesium bromide, 3.5-difluorophenylmag chloroform, dichloromethane and the like; and nonpolar nesium bromide and the like can be mentioned. organic solvent such as 1,4-dioxane, tetrahydrofuran and the 0200. The amount of the Grignard reagent to be used is like can be mentioned. These solvents may be used in a preferably about 1 to 10 molar equivalents relative to com mixture of two or more kinds at an appropriate ratio. It is pound (8). preferably chloroform. The amount of the solvent to be used 0201 As such solvent, halogenated hydrocarbon such as is generally 2- to 50-fold volume relative to compound (I-1). chloroform, dichloromethane and the like; and nonpolar (0208. The reaction temperature is generally 10°C. to 100° organic solvent such as 1,4-dioxane, tetrahydrofuran and the C., preferably 50° C. to 80°C. The reaction time is generally like can be mentioned. These solvents may be used in a 1 to 70 hours. mixture of two or more kinds at an appropriate ratio. It is preferably tetrahydrofuran. The amount of the solvent to be 0209 Compound (1-3) can be obtained by successively used is generally 2- to 50-fold volume relative to compound performing the reactions in step 4 and step 5 using compound (8). (3) obtained in step 1 instead of compound (7) in step 3-1. US 2014/0046022 A1 Feb. 13, 2014 15

0214) Examples of the solvent include aromatic hydrocar (I-3) bon Such as benzene, toluene, Xylene and the like: nitrile Such as acetonitrile, propionitrile and the like; ether Such as tet Hal rahydrofuran, 1.4-dioxane, diethyl ether and the like: ketone Such as acetone, 2-butanone and the like; halogenated hydro carbon such as chloroform, dichloromethane and the like; Hall C amide such as N,N-dimethylformamide and the like: sulfox ide such as dimethylsulfoxide and the like, and the like. These Solvents may be used in a mixture of two or more kinds at an / appropriate ratio. It is preferably dimethylformamide. The H-ALK-O-ALK-O1(S amount of the solvent to be used is generally 2- to 50-fold volume relative to compound (7). HAll: Br or Cl 0215. The reaction temperature is generally 20° C. to 150° C., preferably 50° C. to 100° C. The reaction time is generally wherein Hall is a bromine atom or a chlorine atom, Hal is a 1 to 30 hours. halogen atom (chlorine atom, bromine atom, iodine atom, fluorine atom; preferably fluorine atom), and ALK" and Step 7. ALK are the same or different and each is an aliphatic hydro 0216 carbon group.

Step 6. OALK 0210 ( n2 HO pal p3 x -oik -e- iii. OH ( )n4 ( n2 OALK H-ALK-Hal + HOV p3 x -oil -> (11) iii. OALK (7) (l),4 ( #2 OH Hal pal p3 x -oik (10) in2 OALK ( )n4 ( n2 OALK (11) HO pal p3 x in2-oik ( )n4 OALK wherein Hal is a halogenatom (chlorine atom, bromine atom, iodine atom, fluorine atom; preferably iodine atom), ALK is (11) an aliphatic hydrocarbon group, X" in the number of mare each independently absent, or —O— —S —NHCO— or wherein Hal is a halogenatom (chlorine atom, bromine atom, —CONH-, n, n., n and n are each independently an iodine atom, fluorine atom; preferably fluorine atom), ALK" integer of 0 to 2, and m is an integer of 1 or 2, when a plurality is an aliphatic hydrocarbon group, X" in the number of m of n, n., n and n are each present, each respective n, n., n are each independently absent, or —O— —S , and n may be the same or different, and when a plurality of —NHCO— or —CONH-, n, n., n and n are each inde ALK is present, respective ALK may be the same or differ pendently an integer of 0 to 2, and m is an integer of 1 or 2. ent. when a plurality of n, n2 ns and n are each present, each 0217. A hydroxyl group of compound (11) is substituted respective n, n., n and n may be the same or different, and by halogen to give compound (11). when a plurality of ALK is present, respective ALK may be 0218 Triphenylphosphine and a halogen source are the same or different. reacted in a solvent that does not adversely influence the 0211 Compound (7) is reacted with 0.2 to 5 molar equiva reaction. Generally, the reaction is preferably performed in lents of compound (10) to give compound (11). Generally, the the presence of imidazole. As the halogen source, carbon reaction is performed in the presence of a base in a solvent that tetrachloride, hexachloroacetone and triphosgene (chlorine does not adversely influence the reaction. Source), carbon tetrabromide (bromine source), iodomethane 0212. Examples of the base include an alkali metal salt and iodine (iodine source) and the like can be mentioned. The Such as potassium hydroxide, Sodium hydroxide, sodium amount of triphenylphosphine to be used is preferably about hydrogen carbonate, potassium carbonate and the like; amine 0.1-5 molar equivalents relative to compound (11), and the such as pyridine, triethylamine, N,N-dimethylaniline, 1.8- amount of the halogen source to be used is about 1 to 5 molar diazabicyclo5.4.0]undec-7-en and the like; metal hydride equivalents relative to compound (11). When imidazole is Such as potassium hydride, Sodium hydride and the like; used, the amount thereof to be used is about 0.1 to 5 molar alkali metal alkoxide Such as sodium methoxide, sodium equivalents relative to compound (11). ethoxide, potassium tert-butoxide and the like, and the like. 0219. Examples of the solvent include aromatic hydrocar 0213. The amount of the base to be used is preferably bon Such as benzene, toluene, Xylene and the like: nitrile Such about 1 to 5 molar equivalents relative to compound (7). as acetonitrile, propionitrile and the like; ether Such as tet US 2014/0046022 A1 Feb. 13, 2014 16 rahydrofuran, 1,4-dioxane, diethyl ether and the like: ketone 0222. Using compounds (11) and (11') obtained in step 6 Such as acetone, 2-butanone and the like; halogenated hydro and step 7, an intermediate useful for producing the com carbon such as chloroform, dichloromethane and the like; pound of the present invention can be obtained by performing amide such as N,N-dimethylformamide and the like: sulfox the reactions of the above-mentioned step 3-1, step 3-2 and/or ide such as dimethylsulfoxide and the like, and the like. These step 3-3. One example is shown in the following. In the Solvents may be used in a mixture of two or more kinds at an scheme, the carbon number of aliphatic hydrocarbon group, appropriate ratio. It is preferably toluene. The amount of the the kind of halogen atom, reaction reagent and the like are solvent to be used is generally 3-50-fold volume relative to shown for convenience, and can be appropriately changed compound (11). within the range of the above-mentioned definitions. 0220. The reaction temperature is generally 30° C. to 150° C., preferably 40°C. to 120°C. The reaction time is generally Ts: tosyl group, 0.5 to 24 hours. Ph: phenyl group, 0221 Compound (11) and compound (11) produced via Py: pyridine, step 6 and step 7 can be a useful intermediate to produce the compound of the present invention. Et: ethyl group

OC18H37 O C18H37

-->OC18H37 -- co->C18H37 -- -->N O -->O co->O / OH

O

OH Br/PPh3 1. Imidazole CO

O

OH OTs O

TsC/Py C18H37 >. -> (C)() O N-11 "- On-O CHCI OS-O K2CO3/DMF O US 2014/0046022 A1 Feb. 13, 2014

-continued

HN O OC18H37 O OC18H37 O

() -e-OC18H37 () OC18H37

OC18H37 OH

HN OH 4. 18H37/NaH

OH O

O OC18H37 H () OH + BrY- N OC18H37 He O OC18H37 O OC18H37

OC18H37 O O OH

Br -- Hs

(C) O C18H37 C18H37 C18H37 wherein X is a halogen atom (chlorine atom, bromine atom, mentioned step 3-1, step 3-2 and/or step 3-3. One example is iodine atom, fluorine atom). shown in the following. In the scheme, the carbon number of 0223. In addition, even when “an organic group having an aliphatic hydrocarbon group, the kind of halogenatom, reac aliphatic hydrocarbon group' is the following formula (d): tion reagent and the like are shown for convenience, and can be appropriately changed within the range of the above-men tioned definitions. (d) X6 R10 X4R, : 1. t R xt Cs

OC 18H37 Br HO N-1 n NH2 --

OC18H37 wherein each symbol is as defined above, an intermediate useful for producing the compound of the present invention can be obtained by performing the reactions of the above US 2014/0046022 A1 Feb. 13, 2014

-continued -continued C O OC18H37 OH O () HO OC18H37 Brn-1\NH OC18H37 KC O OC18H37 O OC18H37 OC18H37 O O 0225. When a compound wherein an organic group having an aliphatic hydrocarbon group Substitutes a ring via a group (JN-N--N-a-orH other than —O— is to be produced, the compound of the present invention can be produced according to the above OC18H37 mentioned method, or by appropriately changing the above OC18H37 mentioned method. Such change can be made by performing a reaction generally performed in the art. For example, an organic group having an aliphatic hydrocarbon group Substi 0224. In addition, even when “an organic group having an tutes a ring via —NH can be obtained by dehydrative aliphatic hydrocarbon group' is the following formula (e): condensation of a compound having a carboxyl group on end and a compound having an amino group on end. 3. Organic Synthesis Reaction Method (e) (XR2) in 0226. The compound of the present invention can be used as a protecting reagent for various organic synthesis reac tions. For example, the following steps are performed. 0227 (i) a step of binding the compound of the present invention to amino acid or peptide (binding step), and 0228 (ii) a step of precipitating the bonded product of the wherein each symbol is as defined above, an intermediate compound and the amino acid or peptide obtained in the useful for producing the compound of the present invention above-mentioned step (precipitation step). can be obtained by performing the reactions of the above 0229. First, the compound of the present invention is dis mentioned step 3-1, step 3-2 and/or step 3-3. One example is solved in a soluble solvent, then a reaction substrate to be shown in the following. In the scheme, the carbon number of reacted (amino acid or peptide to be a material for peptide aliphatic hydrocarbon group, the kind of halogenatom, reac synthesis here) is added to a reagent dissolved in a soluble tion reagent and the like are shown for convenience, and can solvent to bind the both (binding step). As a solvent to be used be appropriately changed within the range of the above-men for the reaction system, a general organic solvent can be used. tioned definitions. Since superior reactivity can be expected when the solubility in the solvent is higher, a solvent in which the compound of the present invention shows high solubility is preferably O selected. Specifically, halogenated hydrocarbon Such as chlo roform, dichloromethane and the like; and a nonpolar organic Solvent such as 1,4-dioxane, tetrahydrofuran and the like can be mentioned. These solvents may be used in a mixture of two or more kinds at an appropriate ratio. In addition, the above C O OH + mentioned halogenated hydrocarbons and nonpolar organic OC18H37 Solvent may be mixed with aromatic hydrocarbon Such as TSO benzene, toluene, Xylene and the like; nitrile Such as acetoni -e- trile, propionitrile and the like, ketone such as acetone, 2-bu tanone and the like; amide such as N,N-dimethylformamide OC18H37 and the like: sulfoxide such as dimethylsulfoxide and the like OC18H37 at an appropriate ratio and used as long as the compound of MgBr the present invention is dissolved. 0230. For confirmation of the progress of the reaction, a OC18H37 method similar to general liquid phase organic synthesis reac tion can be applied. That is, thin layer silica gel chromatog O OC18H37 C raphy, high performance liquid chromatography and the like can be used to track the reaction. O OC18H37 Step i (Binding Step) 0231. In this step, the compound of the present invention dissolved in a soluble solvent is bonded to a reactive sub Strate. US 2014/0046022 A1 Feb. 13, 2014 19

0238. The reaction temperature is generally 10°C. to 100° C., preferably 20°C. to 70°C. The reaction time is generally 1 to 70 hours. 0239 When a compound of the present invention wherein Y is a is hydroxyl group is used, the reaction Substrate is a P-AA-OH compound having an amino group, and when a compound of the present invention wherein Y is an amino group is used, the reaction Substrate is a compound having a carboxyl group. Step ii (Precipitation Step) 0240. In this step, the solvent used to dissolve the bonded product obtained in the above-mentioned step is changed to cause precipitation to isolate the bonded product. 0241. After the reaction, the solvent used to dissolve the compound is changed (e.g., change of solvent composition, change of Solvent kind) to conveniently isolate the obtained bonded product by precipitation. That is, the bonded product is precipitated and impurity is eliminated by performing a reaction under conditions where the compound is dissolved, Hall; Br or Cl and changing the solvent after the reaction. Examples of the change of solvent include use of a halogen solvent, THF and the like for dissolution, and a polar organic solvent such as wherein Hall is a bromine atom or a chlorine atom, Hal is a methanol, acetonitrile and the like for precipitation. halogen atom (chlorine atom, bromine atom, iodine atom, 0242. The bonded product isolated by precipitation is sub fluorine atom; preferably fluorine atom), P is an amino-pro jected to a desired reaction, and protecting reagent derived tecting group, AA is a group derived from amino acid, and from the compound of the present invention is finally ALK is an aliphatic hydrocarbon group. removed (deprotection step). 0243 For removal of the protecting reagent, various meth 0232. The compound of the present invention (I-2) is ods generally used in the art, particularly in the peptide syn reacted with a protected amino acid (amino acid bound with thesis, are employed. Generally, a method including addition a protecting group: P-AA-OH) generally in a solvent that ofan acid and the like is employed. As the acid, trifluoroacetic does not adversely influence the reaction to give a compound acid (TFA), hydrochloric acid, Sulfuric acid, mesylic acid, (bonded product) wherein compound (I-2) is bound with tosylic acid, trifluoroethanol, hexafluoroisopropanol and the protected amino acid. The amino acid of the 'group derived like are used. Among these, TFA is preferable. from amino acid' is not particularly limited as long as it is 0244. The amount of the acid to be used is appropriately intended to be bonded to compound (1-2), and may be natural determined according to the kind of the acid, and an amount amino acid or non-natural amino acid. Suitable for removing the protecting reagent is used. The 0233. The compound of the present invention such as reaction temperature is generally 0°C. to 80°C., preferably compound (I-2) and the like is bonded to amino acid or 10°C. to 50°C. The reaction time is generally 0.5 to 24 hours. peptide to be a starting material and functions as an anchor in 0245 Peptide can be produced by liquid phase synthesis a peptide synthesis reaction. utilizing the above-mentioned steps. Specifically, the synthe 0234. While the protecting group of protected amino acid sis includes the following steps. (corresponding to P in the above-mentioned scheme) varies, 0246 (1) a step of condensing the fluorene compound of for example, by protection target amino acid, those generally the present invention with the C-terminal of N-protected used, specifically those similar to the above-mentioned amino acid or N-protected peptide to give C-fluorene com groups can be recited as examples. When amino acid is ala pound-protected amino acid or C-fluorene compound-pro nine, P is preferably a benzyloxycarbonyl (Z) group or a tected peptide (C-terminal fluorene compound protection 9-fluorenylmethoxycarbonyl (Fmoc) group. Step), 0247 (2) a step of removing the protecting group from the 0235. The protected amino acid used in this step is of the N-terminal of the amino acid or peptide obtained in the same kind as the amino acid to be bonded to compound (I-2). above-mentioned step (N-terminal deprotection step), For example, when alanine is to be bonded to (I-2), the pro 0248 (3) a step of condensing the N-terminal of the amino tected amino acid is P-alanine. acid or peptide obtained in the above-mentioned step with 0236. The amount of protected amino acid to be used in N-protected amino acid or N-protected peptide (peptide chain this step is preferably about 1 to 10 molar equivalents relative elongation step), and to compound (I-2). 0249 (4) a step of precipitating the peptide obtained in the 0237 As such solvent, halogenated hydrocarbon such as above-mentioned step (precipitation step). chloroform, dichloromethane and the like; and nonpolar organic solvent such as 1,4-dioxane, tetrahydrofuran and the Step 1 (C-Terminal Fluorene Compound Protection Step) like can be mentioned. These solvents may be used in a 0250 In this step, the fluorene compound of the present mixture of two or more kinds at an appropriate ratio. It is invention is condensed with the C-terminal of N-protected preferably chloroform. The amount of the solvent to be used amino acid or N-protected peptide to give C-fluorene com is generally 2- to 50-fold volume relative to compound (I-2). pound-protected amino acid or C-fluorene compound-pro US 2014/0046022 A1 Feb. 13, 2014 20 tected peptide. For example, the step can be performed Step 4 (Precipitation Step) according to the above-mentioned binding step. 0251. In the present invention, the “N-protected amino 0260 This step is performed in the same manner as in the acid and “N-protected peptide' mean amino acid and pep precipitation step in the above-mentioned step ii. tide wherein an amino group is protected and a carboxyl 0261. In the method of producing a peptide of the present group is not protected, which can be indicated as “P-AA-OH' invention, N-protected amino acid or protected peptide (P is an amino-protecting group). obtained in step 4 can be subjected to a desired number of 0252. The condensation reaction of the fluorene com repeats of steps (5)-(7). pound of the present invention with N-protected amino acid 0262 (5) a step of deprotecting the N-terminal of the pep or N-protected peptide at the C-terminal is generally per tide obtained in the precipitation step, formed in a solvent that does not influence the reaction. For 0263 (6) a step of condensing the N-terminal of peptide example, when Y is a halogen atom (e.g., a bromo group, a obtained in the above-mentioned step with N-protected chloro group, an iodo group, preferably a bromo group and a amino acid or N-protected peptide, and chloro group), the reaction can be performed in the presence of a base. As the base, etheramine, ethylamine, trimethy 0264 (7) a step of precipitating the peptide obtained in the lamine, triethylamine, triethanolamine, diisopropylethy above-mentioned step. lamine and the like can be mentioned. When Y is a hydroxyl group, the reaction can be performed in the presence of a Step 5 condensing agent. Examples of the condensing agent include dicyclohexylcarbodiimide, diisopropylcarbodiimide, 0265. This step is performed in the same manner as in the N-ethyl-N'-3-dimethylaminopropylcarbodiimide and hydro N-terminal deprotection step of step 2. chloride thereof (EDC-HCl) and the like. Where necessary, a condensing agent may be used together with a promoter Such Step 6 as N-hydroxysuccinimide, 1-hydroxybenzotriazole (HOBt) 0266 This step is performed in the same manner as in the and the like. peptide chain elongation step of step 3. 0253) As the solvent to be used for this step, halogenated hydrocarbon such as chloroform, dichloromethane and the Step 7 like; and nonpolar organic solvent such as 1,4-dioxane, tet rahydrofuran and the like can be mentioned. These solvents 0267. This step is performed in the same manner as in the may be used in a mixture of two or more kinds at an appro precipitation step of step ii. priate ratio. It is preferably chloroform. The amount of the 0268. The method of producing a peptide of the present solvent to be used is generally 2- to 50-fold volume relative to invention can further comprise, after the precipitation step of the fluorene compound of the present invention. step 4 or step 7, a step of deprotecting the C-terminal of 0254 The reaction temperature is generally 10°C. to 100° peptide which is protected with a fluorene compound. For C., preferably 20°C. to 70° C. The reaction time is generally example, the step is performed according to the step of 1 to 70 hours. removing the protecting reagent of the present invention men Step 2 (N-Terminal Deprotection Step) tioned above. 0255. In this step, the protecting group at the N-terminal of 4. Kit for Liquid Phase Synthesis of Peptide the amino acid or peptide obtained in the above-mentioned step is removed. 0269. The present invention also provides a kit for liquid 0256 Deprotection is appropriately selected according to phase synthesis of peptide, which contains the above-men the kind of the N-protecting group. A group that can be tioned compound of the present invention as an essential removed under conditions different from those for the constituent component. The kit may contain, besides the removal of the protecting reagent derived from the compound compound of the present invention, other components neces of the present invention is preferable. For example, for an sary for liquid phase synthesis reaction of peptide, for Fmoc group, it is removed by treating with a base. The reac example, various solvents used for the reaction, amino acid tion is generally performed in a solvent that does not influence (or peptide) to be the starting material and the like. When the reaction. desired, a manual of liquid phase synthesis of peptide using 0257. As the base, dimethylamine, diethylamine and the the compound of the present invention can also be attached. like can be mentioned. Examples of the solvent include halo 0270. Other features of the invention will become appar genated hydrocarbon Such as chloroform, dichloromethane ent in the course of the following descriptions of exemplary and the like; aromatic hydrocarbon Such as toluene, Xylene embodiments which are given for illustration of the invention and the like; ether such as diethyl ether, tetrahydrofuran, and are not intended to be limiting thereof. dioxane and the like: nitrile such as acetonitrile and the like, and a mixture thereof can be mentioned. EXAMPLES Step 3 (Peptide Chain Elongation Step) 0271 The reagents, apparatuses and materials used in the present invention are commercially available unless other 0258. In this step, the N-terminal of amino acid or peptide wise specified. In the present specification, when amino acid deprotected in step 2 is condensed with N-protected amino and is the like are indicated by abbreviation, each indication acid or N-protected peptide. is based on the abbreviation of the IUPAC-IUB Commission 0259. This step is performed according to the method of on Biochemical Nomenclature or conventional abbreviations the above-mentioned step 1 wherein Y is a hydroxyl group. in the art. US 2014/0046022 A1 Feb. 13, 2014 21

Example 1 0279 Under a nitrogen atmosphere, 2-docosyloxy-9-fluo renone (250 mg, 0.50 mmol) prepared in the above-men 1-1: Synthesis of 2-docosyloxy-9-fluorenone tioned 1-1 was suspended in THF (2.5 ml), and dissolved at 50° C. 4-Chlorophenylmagnesium bromide solution was 0272 added dropwise, and the mixture was stirred. After comple tion of the reaction, the reaction mixture was cooled to room temperature, 1N hydrochloric acid was added dropwise thereto in a water bath until foaming stopped to quench the reaction. The mixture was extracted with chloroform (15 ml), and washed 3 times with 1N hydrochloric acid (5 ml), 3 times with 5% NaHCO, solution (5 ml), and once with 20% NaCl solution (5 ml). The organic layer was dried over NaSO, and the solvent of the filtrate was evaporated. The obtained resi due was isolated and purified by silica gel column chroma tography, and precipitated with methanol (3 ml) to give 2-docosyloxy-9-(4-chlorophenyl)-9-fluorenol (270 mg, 0.44 mmol, 88%). 0280 'H-NMR (400 MHz) (0281 0.88 (3H, t, J–7.0, CH-Me) 1.20-1.40 (36H, br. 0273 2-Hydroxy-9-fluorenone (196 mg, 2.04 mmol) was alkyl-H) 1.45 (2H, br. —O—CH-CH CoHo.) 1.75 dissolved in DMF (8 ml), potassium carbonate (423 mg, 3.06 (2H, m, O CH-CH ) 2.39 (1H, s, OH)3.91 (2H, mmol), and docosyl bromide (96%,785 mg, 1.93 mmol) were -O-CH ) 6.82 (1H, fluorene C3-H) 6.89 (1H, fluorene added. The mixture was heated to 80° C. and stirred over C1-H) 7.18 (1H, fluorene C7-H) 7.21-7.35 (6H, Ph-H, fluo night. After completion of the reaction, the reaction mixture renone-H) 7.56 (2H, fluorenone-H) was cooled to room temperature, and 1N hydrochloric acid (4 0282. MS ml) was added dropwise thereto in a water bath to quench the 0283 599 M OH reaction. The mixture was extracted with chloroform (23 ml), and washed once with 1N hydrochloric acid (7.5 ml) and 4 1-3: Synthesis of times with pure water (7.5 ml). The solvent of the organic 2-docosyloxy-9-(4-chlorophenyl)-9-bromofluorene layer was evaporated, and the residue was precipitated with methanol (10 ml) to give 2-docosyloxy-9-fluorenone (934 0284 mg, 1.85 mmol, 96%). (0274 'H-NMR (400 MHz) (0275 0.88 (3H, t, J=7.0, CH-Me) 1.20-1.40 (36H, br. alkyl-H) 1.45 (2H, br. —O CH, CH, CH,) 1.79 (2H, m, —O—CH2—CH2—) 4.00 (2H, t, J=6.6, —O C H ) 6.97 (1H, fluorenone C3-H) 7.19 (2H, fluorenone C1,7-H) 7.38-7.43 (3H, fluorenone C4.5,6-H) 7.59 (1H, fluo renone C8-H) 0276 MS 0277 505 M+H 1-2: Synthesis of 2-docosyloxy-9-(4-chlorophenyl)-9-fluorenol 0278

C -()– MgBr 0285 2-Docosyloxy-9-(4-chlorophenyl)-9-fluorenol (200 mg, 0.33 mmol) prepared in the above-mentioned 1-2 C22H45O QC) was dissolved in chloroform (2 ml), acetyl bromide (48 ul, 0.65 mmol) was added dropwise and the mixture was stirred for 1 hour. After completion of the reaction, the solvent was evaporated, and the residue was precipitated with acetonitrile (2 ml) to give 2-docosyloxy-9-(4-chlorophenyl)-9-bromof luorene (209 mg, 0.31 mmol, 95%. 0286 'H-NMR (400 MHz) (0287 0.88 (3H, t, J–7.0, CH-Me) 1.20-1.40 (36H, br. alkyl-H) 1.44 (2H, br. —O—CH-CH CoHo.) 1.77 (2H, m, —O—CH2—CH2—) 3.95 (2H, —O CH ) 6.92 (1H, fluorene-H)6.98 (1H, fluorene-H) 7.21-7.26 (3H) 7.34 (1H) 7.43 (1H) 7.46-7.49 (2H) 7.55-7.59 (2H) US 2014/0046022 A1 Feb. 13, 2014 22

Example 2 temperature, 1N hydrochloric acid (30 ml) was added drop wise thereto in a water bath to quench the reaction. The 2-1: Synthesis of 2,7-didocosyloxy-9-fluorenone mixture was extracted with chloroform (100 ml), and washed 3 times with 1N hydrochloric acid (30 ml), 3 times with 5% 0288 aqueous Sodium hydrogen carbonate solution (30 ml), and once with 20% aqueous sodium chloride solution (30 ml). The organic layer was dried over Sodium Sulfate, and the solvent of the filtrate was evaporated. The residue was iso lated and purified by silica gel column chromatography and precipitated with methanol (70 ml) to give 12-docosyloxy HO OO or C22H45B. dodecyl bromide (yield 78%). 0298 H-NMR (300 MHz) O 0299 0.88 (3H, t, J=6.9, CH-Me) 1.1.0-1.65 (76H, br. alkyl-H) 1.85 (2H, m, —CH2—CH2—Br) 3.39 (6H. m. CH-CH O—CH CoHo CH-Br) 0300 MS C22H45O QC) OC22H45 0301 573 M+H, 575 M+H+2) 3-2: Synthesis of O 2-(12-docosyloxy-dodecanoxy)-9-fluorenone 0289 2,7-Dihydroxy-9-fluorenone (1 g, 4.71 mmol) was 0302) In the same manner as in Example 1, 1-1 except that dissolved in DMF (40 ml), and potassium carbonate (1.95 g, 12-docosyloxy-dodecyl bromide prepared in the above-men 14.1 mmol) and docosyl bromide (96%, 4.02 g, 9.91 mmol) tioned 3-1 was used instead of docosyl bromide, 2-(12-doco were added. The mixture was stirred overnight at 80° C. Syloxy-dodecanoxy)-9-fluorenone was obtained. docosyl bromide (0.40 g, 0.99 mmol) was further added and (0303 Ö=0.88 (3H, t, OCH C22-H) 1.1-1.6 (58H, br. the mixture was stirred overnight. After completion of the Alkyl-H) 1.77 (2H, m, fl-OCHCH ) 3.39 (4H, t, —C reaction, the reaction mixture was cooled to room tempera HOCH ) 4.00 (2H, t, fl-OCH ) 6.97 (1H, m, fl C3-H) ture, 1N hydrochloric acid (120 ml) was added thereto in a 7.18 (2H, m, fl C1, 7-H) 7.37-7.45 (3H, m, fl C4,5,6-H) 7.60 water bath to allow precipitation. The crystals were collected (1H, d, fl C8-H) by filtration and slurry washed once with 1N hydrochloric acid (40 ml), once with pure water (40 ml) and once with 3-3: Synthesis of 2-(12-docosyloxy-dodecanoxy)-9- methanol (40 ml) to give 2,7-didocosyloxy-9-fluorenone (3-fluorophenyl)-9-bromofluorene (4.20 g, docosyl bromide mixture). 0304. In the same manner as in Example 1, 1-2, then 1-3 0290 H-NMR (300 MHz) except that 2-(12-docosyloxy-dodecanoxy)-9-fluorenone 0291 0.88 (6H, t, J=6.6, CH-Me) 1.15-1.60 (80H, br. prepared in the above-mentioned 3-2 was used instead of alkyl-H) 1.78 (4H. m. —O CH-CH )3.98 (4H, t, J=6. 2-docosyloxy-9-fluorenone and 3-fluorophenylmagnesium 6, —O CH ) 6.92 (2H, fluorenone C3,6-H) 7.14 (2H, d, bromide instead of 4-chlorophenylmagnesium bromide, J=2.1, fluorenone C1,8-H) 7.26 (2H, d, J–7.8, fluorenone 2-(12-docosyloxy-dodecanoxy)-9-(3-fluorophenyl)-9-bro C4,5-H) 7.59 (1H, fluorenone C8-H) mofluorene was obtained. 0292. MS 0305 ö=0.88 (3H, t, OCHC22-H) 1.2-1.6 (58H, br. 0293 829 M+H Alkyl-H) 1.77 (2H, m, fl-OCHCH ) 3.38 (4H, t, - C HOCH ) 3.95 (2H, m, fl-OCH ) 6.91 (1H, m, fl C3-H) 2-2: Synthesis of 2,7-didocosyloxy-9-(4-chlorophe 6.94 (1H, m, fl C1-H) 7.00 (1H, d, Ph C6-H) 7.20-7.38 (5H, nyl)-9-bromofluorene m, PhC2,4,5-H, flC6,7-H) 7.44 (1H, d, flC8-H) 7.58 (2H, d, fl C4.5-H) 0294. In the same manner as in Example 1, 1-2, then 1-3 except that 2,7-didocosyloxy-9-fluorenone prepared in the Example 4 above-mentioned 2-1 was used instead of 2-docosyloxy-9- fluorenone, 2,7-didocosyloxy-9-(4-chlorophenyl)-9-bro 4-1: Synthesis of mofluorene was obtained. 1,12-bis-(12-bromododecyloxy)-dodecane 0295 &=0.88 (6H, t, OCHC22-H) 1.1-1.6 (76H, br. OCH's C3-21-H) 1.75 (4H, m, OCHC2-H) 3.92 (4H, (0306 m, OCHC1-H)6.87 (2H, m, flC3,6-H) 6.94 (2H, d, flC1, Br—C12H2—Br--HO C12H2 OH->Br 8-H) 7.20-7.40 (2H, m, Ph C2,6-H) 742-749 (4H, m, Ph C2H2 O—CH2 O—CH2 Br C3,5-H, flC4,5-H) (0307 Sodium hydride (60%, 652 mg, 16.3 mmol) washed Example 3 with hexane was suspended in toluene (3 ml), 1,12-dode canediol (1.50 g, 7.41 mmol) was added, 1,12-dibromodode 3-1: Synthesis of 12-docosyloxy-dodecyl bromide cane (6.08 g. 18.5 mmol) was added, and the mixture was stirred at 80°C. for 4 days. The reaction mixture was cooled 0296 to room temperature, and pure water (30 ml) was added dropwise thereto in a water bath to quench the reaction. The mixture was extracted with chloroform (100 ml), and washed 3 times with 1N hydrochloric acid (30 ml), 3 times with 5% 0297 Sodium hydride (60%, 0.98g, 24.5 mmol) washed aqueous Sodium hydrogen carbonate solution (30 ml), and with hexane was Suspended in toluene (16 ml), 1-docosanol once with 20% aqueous sodium chloride solution (30 ml). (4.00 g, 12.2 mmol) was added, 1,12-dibromododecane (8.04 The organic layer was dried over sodium sulfate and the g, 24.5 mmol) was added, and the mixture was stirred at 85° solvent of the filtrate was evaporated. The residue was iso C. for 2 days. The reaction mixture was cooled to room lated and purified by silica gel column chromatography, and US 2014/0046022 A1 Feb. 13, 2014 precipitated with methanol (50 ml) to give 1,12-bis-(12-bro Eldecyloxy) dodecane (yield 56%). 0308 "H-NMR (300 MHz) 0309 120-1.65 (56H, br, alkyl-H) 1.85 (4H, dt, C H CH-Br) 3.39 (12H, m, —O—CH2, —CH2—Br) 4-2: Synthesis of 1,12-bis-12-(2'-O-9-fluorenone)- dodecyloxy-dodecane 0310

O OO O --OOO

0311 2-Hydroxy-9-fluorenone (577 mg, 2.94 mmol) was nol (20 ml) to give 1,12-bis-12-(2'-O-9-fluorenone)-dodecy loxy-dodecane (1.28 g., 96%). dissolved in DMF (10 ml), and potassium carbonate (595 mg. 0312) 'H-NMR (300 MHz) 4.37 mmol) and 1,12-bis-(12-bromododecyloxy)-dodecane 0313 1.20-1.65 (56H, br, alkyl-H) 1.79 (4H, m, fluo (1 g, 1.44 mmol) prepared in the above-mentioned 4-1 were renone-O CH, CH, ) 3.38 (8H, t, J=6.6, —CH C added. The mixture was stirred at 80° C. overnight, 2-hy H. O—CH2—CH2—) 4.00 (4H, t, J-6.6, fluorenone O CH-) 6.97 (2H, dd, J=2.4, 8.1, fluorenone C3-H) 7.19 droxy-9-fluorenone (56 mg, 0.29 mmol) was added, and the (4H, dt, J=1.8, 6.9 fluorenone C1,7-H) 7.40 (6H, m, fluo mixture was stirred overnight. After completion of the reac renone C4,5,6-H) 7.59 (1H, d, J=7.2, fluorenone C8-H) tion, the reaction mixture was cooled to room temperature, 4-3: Synthesis of 1,12-bis-12-(2'-O-9-(4-chlorophe and 1N hydrochloric acid (15 ml) was added dropwise thereto nyl)-9-fluorenol)-dodecyloxy-dodecane in a water bath to quench the reaction. The mixture was 0314) OCO O 92 S-2 extracted with chloroform (50 ml), and washed 3 times with wherein—R— is —CHOCHOCH2—. 1N hydrochloric acid (15 ml), 3 times with 5% aqueous 0315 Under a nitrogen atmosphere, 1,12-bis-12-(2'-O-9- fluorenone)-dodecyloxy-dodecane (350 mg, 0.38 mmol) Sodium hydrogen carbonate Solution (30 ml) and once with was suspended in THF (3.5 ml) and dissolved at 50° C. 20% aqueous sodium chloride solution (30 ml). The organic 4-Chlorophenylmagnesium bromide solution (5.65 ml, 5.65 layer was dried over sodium sulfate, the solvent of the filtrate mmol) was added dropwise thereto, and the mixture was was evaporated, and the residue was precipitated with metha stirred for 2 hours. After completion of the reaction, the US 2014/0046022 A1 Feb. 13, 2014 24 reaction mixture was cooled to room temperature, and 1N fl-OCH ) 6.92 (2H, d, fl C3-H) 6.98 (2H, s, fl C1-H) 7.19 hydrochloric acid was added dropwise thereto in a water bath 7.27 (6H, m, fl C7-H, Ph C2,6-H,) 7.34 (2H, t, fl C6-H) 7.43 until foaming stopped to quench the reaction. The mixture (2H, d, fl C8-H) 7.48 (4H, d, Ph C3.5-H) 7.55 (4H, d, fl C4.5- was extracted with chloroform (15 ml), and washed 3 times with 1N hydrochloric acid (5 ml), 3 times with 5% NaHCO H) solution (5 ml) and once with 20% NaCl solution (5 ml). The organic layer was dried over NaSO, and the solvent of the 4-5: Synthesis of Z-alanine-fluorene anchor filtrate was evaporated. The obtained residue was purified by compound silica gel column chromatography to give 1,12-bis-12-(2'-O- 9-(4-chlorophenyl)-9-fluorenol)-dodecyloxy-dodecane 0318

Z-Ala-OH O-R-O iPrNEt OSC) OSC) Ho

r C rol QC) Z-Ala-O O Z-Ala-O O C C

(214 mg., 0.19 mmol, 49%). 8–1.2-1.6 (56H, br. Alkyl-H) wherein—R— is —CH2OCHOCH2. 1.73 (4H, q, fl-OCHCH ) 2.54 (2H, s, OH)3.36 (8H, t, 0319. 1,12-bis-12-(2'-O-9-(4-Chlorophenyl)-9-bromof —CHOCH )3.90 (4H, t, fl-OCH ) 6.82 (2H, s, flC1-H) luorene)-dodecyloxy-dodecane (200 mg, 0.16 mmol) was 6.89 (2H, d, fl C3-H) 7.15-7.45 (14H, m, fl C6,7,8-H, Ph dissolved in chloroform (2 ml), Z-alanine (210 mg, 0.94 C2,3,5,6-H) 7.54 (4H, d, flC4,5-H) mmol) was added and N-ethyldiisopropylamine (216 ul, 1.25 4-4: Synthesis of 1,12-bis-12-(2'-O-9-(4-chlorophe mmol) was added, and the mixture was stirred at 50° C. nyl)-9-bromofluorene)-dodecyloxy-dodecane overnight. After completion of the reaction, the solvent was dissolved in chloroform (4 ml), and the mixture was washed 0316 once with pure water (2 ml), 5 times with 10% aqueous sodium carbonate solution (2 ml), twice with pure water (2 ml) and once with 20% aqueous sodium chloride solution (2 ml), dried over Sodium Sulfate, and the solvent was evapo rated to give Z-alanine-fluorene anchor compound (221 mg, ---OOO . 0.14 mmol, 90%). 0320 8=1.2-1.6 (59H, br, Ala CH, Alkyl-H) 1.76 (4H, q, HO O C fl-OCHCH ) 3.38 (8H, t, —CHOCH ) 3.94 (4H, m, fl-OCH ) 4.50 (2H, Br, Ala C-H) 5.07 (4H, s, Z. Ph-C HO ) 5.16 (2H, Br, NH) 6.77 (2H, d, flC3-H) 6.88 (2H, d, flC1-H) 7.15-7.45 (16H, m, flC6,7,8-H, Z. Ph-H) 7.59 (4H, d, fl C4.5-H) rol rol Example 5 5-1: Synthesis of trioctadecanoxypentaerythritol wherein—R— is —CHOCHOCH2—. 0317. 1,12-bis-12-(2'-O-9-(4-Chlorophenyl)-9-fluore 0321 nol)-dodecyloxy-dodecane (214 mg., 0.19 mmol) was dis solved in chloroform (2 ml), acetyl bromide (69 ul, 0.93 mmol) was added dropwise thereto, and the mixture was OH stirred for 3 hours. After completion of the reaction, the Solvent was evaporated, and the residue was decanted with acetonitrile (2 ml) to give 1,12-bis-12-(2'-O-9-(4-chlorophe Br-C18H3 + HO OH nyl)-9-bromofluorene)-dodecyloxy-dodecane (200 mg, 0.16 mmol, 84%). 8=1.2-1.6 (56H, br. Alkyl-H) 1.73 (4H, Br, OH fl-OCHCH ) 3.38 (8H, t, —CHOCH ) 3.88 (4H. m. US 2014/0046022 A1 Feb. 13, 2014 25

-continued 5-3: Synthesis of 2-(3-octadecyloxy-2.2-bis-octade OH cyloxymethyl-propoxy)-fluorenone 0329 C18H370 OC18H37

OC18H37 OC18H37

C18H3O I -- 0322 To pentaerythritol (1.5 g., 11.0 mmol) were added DMF (100 ml), 1-bromooctadecane (11.4g, 34.2 mmol) and NaH (60 wt %, 1.54 g. 38.5 mmol), and the mixture was C18H370 stirred at 100° C. for 22 hours. The reaction mixture was O cooled to room temperature, chloroform (150 ml) was added, 1N hydrochloric acid (150 ml) was further added dropwise thereto. After stirring for a while, the aqueous layer was discarded, and the organic layer was further washed with 1N hydrochloric acid (100 ml) and water (100 ml). The organic OC18H37 layer was evaporated under reduced pressure, the residue was precipitated with methanol (150 ml), and the obtained crys tals were slurry washed with methanol (150 ml). Crude crys tals were dried and purified by silica gel column chromatog raphy (hexane: chloroform=1:1->hexane:ethyl acetate=10:1) to give trioctadecanoxypentaerythritol (2.21 g, 2.47 mmol. C18H370 O yield 23%). 0323 'H-NMR (300 MHz) 0330 1-(3-Iodo-2,2-bis-octadecanoxymethyl-propoxy) octadecane (500 mg, 102 umol) and 2-hydroxy-9-fluorenone 0324 0.88 (3H, t, J–6.9, OCH-Me) 1.10-1.65 (96H, (30 mg, 152 umol) were dissolved in DMF (2 ml), potassium br, C18Alkyl-H)3.12 (1H, t, J=6.0, OH)3.38 (6H, t, J=6.3, carbonate (21 mg, 152 umol) was added, and the mixture was —C -(CH2—O—CH. C.Hs)) 3.43 (6H. S. —C- (C stirred at 130° C. After completion of the reaction, 0.5N H. O. C.H.)) 3.70 (2H, d. J=5.7, HO CH ) hydrochloric acid (6 ml) was added, the precipitate was fil tered, and the precipitate was washed with acetonitrile and 5-2: Synthesis of 1-(3-iodo-2,2-bis-octadecanoxym water to give 2-(3-octadecyloxy-2.2-bis-octadecyloxym ethyl-propoxy)octadecane ethyl-propoxy)-9-fluorenone (101 mg, 94 umol). 0325 5-4: Synthesis of 2-(3-octadecyloxy-2.2-bis-octade cyloxymethyl-propoxy)-9-(4-chlorophenyl)-9-fluore nol OC18H37 OC18H37 0331 HO OC18H37 --> I OC18H37

C18H3O C18H3O OC18H37 C-KX-MB; 0326 Trioctadecanoxypentaerythritol (500 mg, 560 cally. OC umol) prepared in the above-mentioned 5-1 was dissolved in C18H3O O toluene (10 ml), triphenylphosphine (294 mg, 1.12 mmol), OC18H37 imidazole (76.2 mg, 1.12 mmol) and iodine (284 mg, 1.12 mmol) were added, and the mixture was stirred at 100° C. overnight. The reaction mixture was cooled to room tempera C18H370 O OSC) ture, toluene (10 ml) was added, and the mixture was washed HO with water (5 mlx3). The organic layer was separated and C18H370 evaporated under reduced pressure, and the residue was pre cipitated with acetonitrile (10 ml) to give 1-(3-iodo-2,2-bis C octadecanoxymethyl-propoxy)octadecane (555 mg, 553 umol, yield 98%). 0332 Under a nitrogen atmosphere, 2-(3-octadecyloxy-2, 2-bis-octadecyloxymethyl-propoxy)-9-fluorenone (75 mg. 0327 'H-NMR (300 MHz) 73 umol) was suspended in THF (1 ml) to allow dissolution at 0328 0.88 (3H, t, J–6.9, OCH-Me) 1.10-1.65 (96H, 50° C. 4-Chlorophenylmagnesium bromide solution (0.15 br, C18Alkyl-H) 3.33 (6H, s, —C (CH, O CH7).) ml, 0.15 mmol) was added dropwise, and the mixture was 3.38 (6H, t, J=6.3, —C (CH, O CH, CHs)) 3.48 stirred for 30 minutes. After completion of the reaction, the (2H. S. I—CH2—) reaction mixture was cooled to room temperature, extracted US 2014/0046022 A1 Feb. 13, 2014 26 with chloroform (3 ml), and washed 3 times with 1N hydro 6.93 (1H, d, fl C3-H)6.95 (1H, s, fl C1-H) 7.13-7.57 (9H, m, chloric acid (1 ml), 3 times with 5% NaHCO solution (1 ml) Ph C2,3,5,6-H, flC4,5,6,7,8-H) and once with 20% NaCl solution (1 ml). The organic layer was dried over NaSO, the solvent of the filtrate was evapo Example 6 rated, and the obtained residue was separated and purified by silica gel column chromatography and crystallized from Introduction of Z-Ala into Fluorene Anchor methanol (1 ml) to give 2-(3-octadecyloxy-2.2-bis-octadecy Compound loxymethyl-propoxy)-9-(4-chlorophenyl)-9-fluorenol (66 mg, 56.1 mmol. 77%). 0337 0333 Ö=0.88 (9H, t, J=6.9 Hz, OCH C18-H) 1.1-1.6 (96H, br, OCH, C2-17-H) 2.36 (1H, s, -OH)3.35 (6H, t, OCsHC1-H)3.45 (6H, s, —CH OCsII2)3.92 (2H, m, fl-OCH ) 6.84 (1H, s, fl C1-H) 6.91 (1H, d, flC3-H) 7.13 7.35 (7H, m, Ph C2,3,5,6-H, fl C6,7,8-H) 7.54 (2H, m, fl C22H45O OO HerZ-Ala-OH C4,5-H) 5-5: Synthesis of 2-(3-octadecyloxy-2.2-bis-octade Br O cyloxymethyl-propoxy)-9-(4-chlorophenyl)-9-bro C mofluorene 0334 C22H45O OSC) OC18H37 Z-Ala-O O C18H370 O OO AcBr C HO 0338 2-Docosyloxy-9-(4-chlorophenyl)-9-bromofluo C18H3O rene (200 mg, 0.29 mmol) obtained in Example 1 was dis C solved in chloroform (2 ml), Z-alanine (197 mg, 0.88 mmol) OC18H37 was added and N-ethyldiisopropylamine (202 ul, 1.17 mmol) was added, and the mixture was stirred at 50° C. overnight. After completion of the reaction, the solvent was evaporated, C18H370 O OSC) and the residue was precipitated with acetonitrile (2 ml) to Br give Z-alanine-fluorene anchor compound (178 mg, 0.22 C18H370 mmol. 74%). C 0339) 'H-NMR (400 MHz) (0340 0.88 (3H, t, J–7.0, CH-Me) 1.20-1.40 (39H, br. alkyl-H, Alanine Me) 1.45 (2H, br. —O—CH C 0335 2-(3-Octadecyloxy-2,2-bis-octadecyloxymethyl H. C.H.) 1.73 (2H, br. —O CH, CH ) 3.89 (2H, propoxy)-9-(4-chlorophenyl)-9-fluorenol (66 mg, 56 umol) -O-CH ) 4.51 (1H, br. Z. NH-CH) 5.07 (2H, s, ben was dissolved in chloroform (1 ml), acetyl bromide (41 ul, Zyl-H) 5.19 (1H, br, s, Z. NH ) 6.78 (1H, d, J=13.5, fluo 0.55 mmol) was added dropwise thereto and the mixture was rene-H) 6.90 (1H, d, J–6.3, fluorene-H) 7.14-7.37 (12H) stirred for 2 hours. After completion of the reaction, the 7.57-7.60 (2H) Solvent was evaporated, and the residue was crystallized from acetonitrile (1 ml) to give 2-(3-octadecyloxy-2.2-bis-octade Example 7 cyloxymethyl-propoxy)-9-(4-chlorophenyl)-9-bromofluo rene (55 mg. 44 mol, 79%). Introduction of Fmoc-Ala-OH into Fluorene Anchor 0336 &=0.88 (9H, t, J=6.9 Hz, OCH C18-H) 1.1-1.6 Compound (96H, br, OCsh, C2-17-H)3.37 (6H, t, OCH C1-H) 3.47 (6H, s, —CH OCsI) 3.97 (2H, m, fl-OCH ) 0341 CH-O-C2H4-O OSO F + Fmoc-Ala-OH -- US 2014/0046022 A1 Feb. 13, 2014 27

-continued CH-O-C12H24-O OSC) F Fmoc-Ala-O O

(Fmoc-Ala-O-fl) fl: CHs - O - C12H2 - O QSO Or

0342. The fluorene anchor prepared in Example 3: 2-(12 Ph-H) 7.10-7.30 (3H, m, fluorene-H or Ph-H) 7.35 (1H, t, docosyloxy-dodecanoxy)-9-(3-fluorophenyl)-9-bromofluo J–7.4, fluorene-H or Ph-H) 7.60 (2H, m, fluorene-H or Ph-H) rene (600 mg. 0.71 mmol) was dissolved in chloroform (6 0351. MS ml), Fmoc-Ala-OH. HO (699 mg, 2.12 mmol) and diisopro 0352 784 M-(H-Ala-O) pylethylamine (366 ul, 2.12 mmol) were added, and the mix ture was stirred at 70° C. overnight. After completion of the Example 9 reaction, the solvent was evaporated, the residue was precipi tated with acetonitrile (6 ml) to give Fmoc-Ala-O-fl (726 mg, Condensation of Fmoc-Pro-OH 0.67 mmol, 95%). (0343 'H-NMR (300 MHz) 0353 0344 0.88 (3H, t, J-6.9, OCH-Me) 1.20-1.65 (63H, H-Ala-O-f->Fmoc-Pro-Ala-O-f br, Alkyl-H, Ala Me) 1.71 (2H, br, fl-O CH-CH )3.38 0354) H-Ala-O-fl (530 mg, 0.62 mmol) prepared in (4H, t, J–6.6, —CH O—CH CHA) 3.89 (2H, br, fl Example 8 was dissolved in chloroform (7 ml), HOBt (18 mg, O—CH ) 4.16 (1H, t, J=6.9, fluorene (Fmoc) C9-H) 4.34 0.13 mmol) and Fmoc-Pro-OH (230 mg, 0.68 mmol) were (2H, d. J=6.3, fluorene (Fmoc)-CH O) 4.53 (1H, br, Ala added, EDC.HCl (144 mg. 0.75 mmol) was added at 0°C., C-H) 5.25 (1H, br., Fmoc-NH) 6.80 (1H, br, m, fluorene-Hor and the mixture was stirred at room temperature overnight. Ph-H) 6.85-7.05 (4H, br, m, fluorene-H or Ph-H) 7.10-7.30 After completion of the reaction, the solvent was evaporated, (2H, br, m, fluorene-H or Ph-H) 7.37 (3H, t, J=7.2, fluorene and the residue was precipitated with methanol (7ml) to give H or Ph-H) 7.52 (2H, br, d. J=6.9, fluorene-H or Ph-H) 7.60 Fmoc-Pro-Ala-O-fl (695 mg, 0.59 mmol, 95% (total yield (2H, br, fluorene-Hor Ph-H)7.73 (2H, d, J–7.2, fluorene-Hor 89% vs fl-Br)). Ph-H) 0355 H-NMR (300 MHz) (0345 MS 0356 0.88 (3H, t, J–6.9, OCH-Me) 1.20-2.00 (67H, 0346) 784 M-(Fmoc-Ala) br, Alkyl-H, Pro N CH, CH, CH, CH, Ala Me) 3.38 (4H, t, J–6.6, —CH2—O—CH CHA) 3.48 (2H, br. Example 8 Fmoc-N CH ) 3.88 (2H, br, fl-O CH ) 4.10-4.50 (4H, br, fluorene (Fmoc) C9-H, fluorene (Fmoc)-CH, O, Removal of Fmoc from Fmoc-Ala-O-fl Ala C-H) 4.68 (1H, br. Pro C-H) 6.70-7.60 (16H, br, m, 0347 fluorene-H or Ph-H) 7.75 (2H, d, J=7.2, fluorene-H or Ph-H) Fmoc-Ala-O-fl->H-Ala-O-f Example 10 0348 Fmoc-Ala-O-fl (700 mg, 0.65 mmol) prepared in Example 7 was dissolved in chloroform-acetonitrile (1:1, 7 Removal of Fmoc from Fmoc-Pro-Ala-O-fl ml), diethylamine (1.36 ml, 13.0 mmol) was added dropwise 0357 thereto at 0°C., and the mixture was stirred at room tempera ture for 2 hours. Diethylamine (1.36 ml, 13.0 mmol) was Fmoc-Pro-Ala-O-fil->H-Pro-Ala-O-f further added dropwise thereto, and the mixture was stirred 0358 Fmoc-Pro-Ala-O-fl (660 mg, 0.56 mmol) prepared for 1 hour. After completion of the reaction, the solvent was in Example 9 was dissolved in chloroform-acetonitrile (7:6. evaporated, and the residue was precipitated with acetonitrile 6.5 ml), diethylamine (1.16 ml, 11.1 mmol) was added drop (6 ml) to give H-Ala-O-fl (542 mg, 0.63 mmol. 971 (total wise thereto at 0°C., and the mixture was stirred at room yield 931 vs fl-Br)). temperature for 2 hours. After completion of the reaction, the (0349 'H-NMR (300 MHz) Solvent was evaporated, and the residue was precipitated with 0350 0.88 (3H, t, J=6.9, OCH-Me) 1.20-1.65 (63H, acetonitrile (7 ml) to give H-Pro-Ala-O-fl (528 mg, 0.55 br, Alkyl-H, Ala Me) 1.74 (2H, br, fl-O CH-CH )3.38 mmol, 98% (total yield 88% vs fl-Br)). (4H, t, J–6.6, —CH2—O—CH2—CH)3.63 (1H, br, Ala 0359 'H-NMR (300 MHz) C-H) 3.90 (2H, d, J=6.6, fluorene-O-CH ) 6.80 (1H, d, 0360 0.88 (3H, t, J–6.9, OCH-Me) 1.15-1.80 (67H, J=2.1, fluorene-H or Ph-H) 6.85-7.07 (4H, m, fluorene-H or br. Alkyl-H, Pro HN CH, CH, CH, CH, Ala Me) US 2014/0046022 A1 Feb. 13, 2014 28

1.87 (1H, m, Pro HN CH-CH CH-CH) 2.11 (1H, fluorene-H or Ph-H) 7.01 (2H, m, fluorene-H or Ph-H) 7.10 m, Pro HN CH-CH CH-CH) 2.88 (2H, m, Pro 7.40 (4H, m, fluorene-H or Ph-H) 7.58 (2H, m, fluorene-Hor HN—CH) 3.38 (4H, t, J=6.6, —CH2—O CH CH) Ph-H) 3.69 (1H, m, Pro C-H) 3.90 (2H, d, J=6.6, fluorene-O-C H ) 4.72 (1H, m, Ala C-H) 6.79 (1H, m, fluorene-Hor Ph Example 13 H) 6.85-6.95 (2H, m, fluorene-Hor Ph-H) 7.01 (2H, d, J=8.1, fluorene-H or Ph-H) 7.10-7.30 (2H, m, fluorene-H or Ph-H) Condensation of Fmoc-Ser(tBu)-OH 7.34 (1H, m, fluorene-H or Ph-H) 7.59 (2H, m, fluorene-Hor 0369 Ph-H) 7.96 (1H, br, d. J=7.2, fluorene-H or Ph-H) H-Ile-Pro-Ala-O-fl->Fmoc-Ser(tBu)-Ile-Pro-Ala-O-f(SEQID NO: 1) Example 11 0370 H-Ile-Pro-Ala-O-fl (490 mg, 0.46 mmol) prepared in Example 12 was dissolved in chloroform (6 ml), HOBt (13 Condensation of Fmoc-Ile-OH mg, 0.10 mmol) and Fmoc-Ser(tBu)-OH (194 mg. 0.51 mmol) were added, EDC-HCl (107 mg 0.56 mmol) was 0361 added at 0°C., and the mixture was stirred at room tempera H-Pro-Ala-O-fil->Fmoc-Ile-Pro-Ala-O-f ture overnight. After completion of the reaction, the solvent 0362 H-Pro-Ala-O-fl (510 mg, 0.54 mmol) prepared in was evaporated, and the residue was precipitated with metha Example 10 was dissolved in chloroform (7 ml), HOBt (15 nol (7 ml) to give Fmoc-Ser(tBu)-Ile-Pro-Ala-O-n (SEQ ID mg, 0.11 mmol) and Fmoc-Ile-OH (206 mg, 0.58 mmol) were NO: 1) (610 mg, 0.43 mmol, 94% (total yield 74% vs fl-Br)). added, EDC.HCl (123 mg, 0.64 mmol) was added at 0°C., and the mixture was stirred at room temperature overnight. Example 14 After completion of the reaction, the solvent was evaporated, Removal of anchor from and the residue was precipitated with methanol (7ml) to give Fmoc-Ile-Pro-Ala-O-fl (642 mg, 0.50 mmol, 93% (total yield Fmoc-Ser(thBu)-Ile-Pro-Ala-O-fl (SEQID NO: 1) 82% vs fl-Br)). 0371 0363 'H-NMR (300 MHz) Fmoc-Ser(tEu)-Ile-Pro-Ala-O-fl->Fmoc-Ser(tEu)-Ile 0364 0.88 (3H, t, J–6.9, OCH-Me) 1.20-2.20 (75H, Pro-Ala-OH (SEQ ID NOS: 1 and br, m, Alkyl-H, Ile Me-CH CH-Me, Pro N CH, C 2, respectively, in order of appearance) H CH, CH, Ala Me) 2.30 (1H, br, s, Ile Me-CH C 0372 Fmoc-Ser(tBu)-Ile-Pro-Ala-O-fl (SEQ ID NO: 1) H-Me)3.38 (4H, t, J–6.6, —CH, O CH, CH)3.58 (600 mg, 0.42 mmol) prepared in Example 13 was dissolved (1H, br. Pro CO N CH )3.70 (1H, br. Pro CO. N. C in TFA-chloroform (1%, 6 ml, TFA: about 2 eq), and the H )3.88 (2H, br, t, J=6.6, fl-O CH ) 4.19 (1H, t, J=6.6, mixture was stirred at room temperature. After 30 minutes, fluorene (Fmoc) C9-H)4.25-4.45 (3H, m, fluorene (Fmoc)-C TFA (60 ul, about 2 eq) was added dropwise thereto, 1.5 hr H. O. Ile or Pro or Ala C.-H)4.50 (1H, br, m, Ile or Pro or Ala later, TFA (180 ul, about 6 eq) was added dropwise thereto, C-H) 4.66 (1H, br, m, Ile or Pro or Ala C-H) 5.40 (1H, d, and the mixture was stirred for 1.5 hour. After completion of J=9.3, Fmoc-NH ) 6.75-7.45 (12H, m, fluorene-Hor Ph-H) the reaction, the solvent was evaporated, acetonitrile (6 ml) 7.57 (4H, br, fluorene-H or Ph-H) 7.75 (2H, d. J–7.8, fluo was added to the residue and the precipitate was removed by rene-H or Ph-H) filtration. The filtrate was concentrated and washed with hex ane (6 ml) to give Fmoc-Ser(tBu)-Ile-Pro-Ala-OH (SEQ ID Example 12 NO: 2) (224 mg., 0.36 mmol, 86%). 0373) MS Removal of Fmoc from Fmoc-Ile-Pro-Ala-O-fl 0374 Fmoc-S(tEu)IPA-OH (SEQID NO: 2): 665 M+H 0365 Fmoc-S(H)IPA-OH: 609 M+H 0375 Similarly, Fmoc-Ser(tPu)-Ile-Pro-Ala-O-fl (SEQ Fmoc-Ile-Pro-Ala-O-fil->H-Ile-Pro-Ala-O-f ID NO: 1) (100 mg, 0.07 mmol) prepared in Example 13 was 0366 Fmoc-Ile-Pro-Ala-O-fl (620 mg, 0.48 mmol) pre dissolved intrifluoroethanol and chloroform, and the mixture pared in Example 11 was dissolved in chloroform-acetoni was stirred at 50° C. After completion of the reaction, the trile (7:6, 6.5 ml), diethylamine (1.00 ml, 9.57 mmol) was solvent was evaporated, methanol (3 ml) was added to the added dropwise thereto at 0°C., and the mixture was stirred at residue, and the precipitate was removed by filtration. The room temperature for 1 hour. Diethylamine (1.00 ml, 9.57 filtrate was concentrated, and hexane (6 ml) was added. The mmol) was added dropwise thereto, and the mixture was precipitate was collected by filtration to give Fmoc-Ser(tBu)- stirred for 2 hours. After completion of the reaction, the Ile-Pro-Ala-OH (SEQID NO: 2) (30 mg). Solvent was evaporated, and the residue was precipitated with acetonitrile (6 ml) to give H-Ile-Pro-Ala-O-fl (500 mg 0.47 Example 15 mmol, 98% (total yield 80% vs fl-Br)). 15-1: Synthesis of 0367 'H-NMR (300 MHz) (3,4,5-tris(octadecyloxy)-cyclohexyl)-methanol 0368 0.90 (9H, m, OCH-Me, Ile Me) 1.20-2.20 (75H, br, m, Alkyl-H, Ile Me-CH CH-Me, Pro 0376 Methyl trioctadecyloxy-cyclohexylcarboxylate N—CH CH-CH CH, Ala Me) 2.35 (1H, br, s, Ile (2.87 g, 3.03 mmol) was dissolved in dehydrating THF (30 Me-CH CH-Me) 3.38 (5H, m, —CH, O C mL). DIBAL-H (9 mmol) was added and the mixture was H. C. H., Ile C-H)3.52 (2H, dd, J=6.0. 7.8, Pro N—CH) stirred at room temperature for 30 minutes. 1N Hydrochloric 3.90 (2H, t, J=6.6, fluorene-O-CH ) 4.50-4.70 (2H, m, acid (10 mL) was added, and THF was evaporated under Pro, Ala C-H) 6.82 (1H, m, fluorene-H or Ph-H) 6.91 (2H, m, reduced pressure. Chloroform (30 mL) and 1N hydrochloric

US 2014/0046022 A1 Feb. 13, 2014 30 to give 9-(4-chlorophenyl)-2-(3,4,5-tris(octadecyloxy)-cy Reference Example 2 clohexylmethoxy)-9-bromofluorene (63.9 mg, 50.2 umol. 98%). Synthesis of Z-alanine-trity1 anchor (0387 &=0.88 (9H, t, J=6.6 Hz, OCHC18-H) 1.1-19 (3,5-distearyloxy) compound (101H, br. Cyclohexyl C1.2.6-H, OCH,C2-17-H) 3.16 (2H, d, J=10.3 Hz, Cyclohexyl C3.5-H) 3.46 (4H, m, 3, 0390 5-OCsh;C1-H) 3.68 (2H, t, J=6.7 Hz, 4-OC.H.C1-H) 3.82 (2H, m, fl-O CH ) 3.92 (1H, s, Cyclohexyl C4-H) C C 6.90 (1H, d, J=8.3 Hz, flC3-H)6.96 (1H, s, flC1-H)7.18-7.26 (3H, m, Ph C3,5-H, fl C7-H) 7.34 (1H, t, J=7.1 Hz, fl C6-H) 7.43 (1H, d, J=7.6 Hz, fl C8-H) 7.48 (2H, d, J=8.7 Hz, Ph O C C2,6-H) 7.57 (2H, m, fl C4.5-H) C18H370 Reference Example 1 O C Synthesis of Z-alanine-trity1 anchor (3,4,5-tristearyloxy) compound OC18H37 0388 C C C C O. SC O CS - OC18H37 C18H370 C

C18H3O OC18H37 OC18H37

0391) bis-(4-Chlorophenyl)-(3,5-distearyloxyphenyl)- C C methyl chloride (Calculated 404 mg., 0.46 mmol) prepared according to the method described in WO2007/122847 was dissolved in chloroform (4 ml), Z-alanine (204 mg. 0.91 mmol) was added, N-ethyldiisopropylamine (173 ul. 1.00 OC18H37 mmol) was added, and the mixture was stirred at room tem perature for 3 hours. After completion of the reaction, the Z-Ala-O O Solvent was evaporated, and the residue was precipitated with acetonitrile (8 ml) to give Z-alanine-trityl anchor compound OC18H37 (548 mg, 0.51 mmol. 100% 2 steps). OC18H37 Experimental Example 0392 (1) Stability Evaluation of Fluorene Type Anchor Condensed Product and Trityl Type Anchor-Condensed Prod uct, with Z-Alanine 0389 bis-(4-Chlorophenyl)-(3,4,5-tristearyloxyphenyl)- 0393 A Z-alanine-anchor-condensed product (35 mg) methyl chloride (150 mg, 0.13 mmol) prepared according to was dissolved or suspended in methanol and 10% acetic the method described in WO2007/122847 was dissolved in acid/chloroform solution (0.7 ml), and the amount of Z-ala chloroform (1.5 ml), Z-alanine (58 mg, 0.26 mmol) was nine (Z-Ala-OH) liberated in a predetermined time was quan added, N-ethyldiisopropylamine (49 ul, 0.28 mmol) was tified. added, and the mixture was stirred at room temperature for 1 0394 The trityl type anchor showed liberation of Z-ala hour. After completion of the reaction, the solvent was evapo nine in methanol even in 1 hour and the bond to the anchor rated, and the residue was precipitated with acetonitrile (1.5 was cut. On the other hand, it was confirmed that the fluorene ml) to give Z-alanine-trityl anchor compound (169 mg, 0.13 type anchor was not cut even in methanol or under acidic mmol, 97%). conditions of 10% solution (see Table 1). US 2014/0046022 A1 Feb. 13, 2014

TABLE 1 Amount of liberated Z-Ala-OH after stirring at room temperature for 1 hr MeOH 10% AcOHCHCl C C 70% 100% O Z-Ala-O O OC18H37 OC18H37 OC18H37

C C 20% 75%

Z-Ala-O O OC22H45

OC22H45

C 296 4%

Z-Ala-O C2H4s-O COO O-CHAs C O%

Z-Ala-O C2H45-O-C12H24-O COO

INDUSTRIAL APPLICABILITY with high quality. Moreover, the anchor enables selective removal thereof while maintaining the protecting group. 0395. A particular compound having a fluorene skeleton provided substance which functions as a Superior protecting Using the method of the present invention, various active group and anchor can be produced. The anchor can be pharmaceutical ingredients (API), intermediates and final removed under weak acidic conditions, and selectively products are easily obtained. removed even when other protecting groups (peptideside 0396. Where a numerical limit or range is stated herein, chain protecting group etc.) still remain in the compound the endpoints are included. Also, all values and Subranges obtained by organic synthesis reaction. Namely, the com within a numerical limit or range are specifically included as pound is a Substance that enables easy precipitation in metha if explicitly written out. nol and the like, since it dissolves only in halogen solvents, THF and the like and scarcely dissolves in polar organic 0397. Obviously, numerous modifications and variations Solvents. In the process of elongation of peptide chain length of the present invention are possible in light of the above wherein the Substance is used as a protecting group for C-ter teachings. It is therefore to be understood that, within the minal or side chain in the peptide synthesis and an operation Scope of the appended claims, the invention may be practiced including reaction in a halogen solvent, followed by precipi otherwise than as specifically described herein. tation with methanol and the like to remove impurity is 0398 All patents and other references mentioned above repeated, a side reaction to yield diketopiperazine is Sup are incorporated in full herein by this reference, the same as if pressed and peptide chain can be elongated in a highyield and set forth at length. US 2014/0046022 A1 Feb. 13, 2014 32

SEQUENCE LISTING

< 16 Os NUMBER OF SEO ID NOS: 2

SEQ ID NO 1 LENGTH: 4 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide FEATURE: NAME/KEY: MOD RES LOCATION: (1) ... (1) OTHER INFORMATION: Fmoc-Ser (tsu) FEATURE: NAME/KEY: MOD RES LOCATION: (4) ... (4) OTHER INFORMATION: Ala-O-f

<4 OOs, SEQUENCE: 1

Ser Ile Pro Ala 1.

SEQ ID NO 2 LENGTH: 4 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide FEATURE: NAME/KEY: MOD RES LOCATION: (1) ... (1) OTHER INFORMATION: Fmoc-Ser (tsu)

<4 OOs, SEQUENCE: 2

Ser Ile Pro Ala 1.

1. A fluorene compound represented by formula (I): (I)

(I)

wherein ring A is an aromatic ring; Y is a group reactive with one or more groups selected from an amino group, a carboxyl group, and a mercapto group; at least one of rings A, B, and C has an organic group having an ali phatic hydrocarbon group; and rings A, B, and C each independently optionally have an electron-withdrawing group, are connected via an organic group having an aliphatic hydrocarbon group, wherein ring A is an aromatic ring; Y is a group reactive which is contained in the unit structure. with one or more groups selected from an amino group, 3. A fluorene compound represented by formula (I):

a carboxyl group, and a mercapto group; at least one of (I) rings A, B, and C has an organic group having an ali phatic hydrocarbon group; and rings A, B, and C each independently optionally have an electron-withdrawing group. 2. A fluorene compound in which 2 to 20 divalent unit structures derived from a compound represented by formula (I): US 2014/0046022 A1 Feb. 13, 2014

wherein ring A is an aromatic ring; Y is a group reactive 8. The fluorene compound according to claim 4, wherein with one or more groups selected from an amino group, the divalent organic group having an aliphatic hydrocarbon a carboxyl group, and a mercapto group; Ra, Rb, and Rc group for Ric' is a group represented by formula (ii): are each independently an organic group having an ali phatic hydrocarbon group, a hydrogenatom, or an elec tron-withdrawing group, at least one of Ra, Rb, and Rc is (ii) an organic group having an aliphatic hydrocarbon --O-Re--O- group; and rings A, B, and C each independently option ally have an electron-withdrawing group. 4. A fluorene compound represented by formula (II): wherein Re is an aliphatic hydrocarbon group having a carbon number of 5 to 60; k is an integer of 1 to 3; and when a plurality of Re is present, respective Re may be (II) the same or different.

9. The fluorene compound of according to claim 1, wherein the organic group having an aliphatic hydrocarbon group is one or more kinds of group selected from the groups consist ing of a group represented by formula (a):

(a) wherein ring A is an aromatic ring; Y is a group reactive : --X-R-i-H with one or more groups selected from an amino group, a carboxyl is group, and a mercapto group; n is an integer of 1 to 19; Ric' is a divalent organic group having an aliphatic hydrocarbon group; rings A, B, and C each wherein * indicates the position of a bond; X is absent or independently optionally have one or more Substituents O— —S , NHCO—, or —CONH : R is an ali selected from an organic group having an aliphatic phatic hydrocarbon group having a carbon number of not less hydrocarbon group and an electron-withdrawing group; than 5; m is an integer of 1 to 10; when a plurality of X is when a plurality of ring A is present, respective rings A present, respective X may be the same or different; and when may be the same or different; when a plurality of Y is a plurality of R is present, respective R may be the same or present, respective Y may be the same or different; and when a plurality of Rc' is present, respective Rc' may be different: the same or different. a group represented by the formula (b): 5. The fluorene compound of according to claim 1, wherein the organic group having an aliphatic hydrocarbon group and/or the divalent organic group having an aliphatic hydro (b) carbon group is bonded on the ring via —O— —S , —NHCO—, or —CONH present in the organic group, or directly bonded to form a carbon-carbon bond. 6. The fluorene compound of according to claim 1, wherein the organic group having an aliphatic hydrocarbon group and/or the divalent organic group having an aliphatic hydro carbon group has a structure comprising 2 to 10 connections of an aliphatic hydrocarbon group bonded via —O— —S , —NHCO—, or —CONH , or an aliphatic hydrocarbon group directly bonded to form a carbon-carbon bond. 7. The fluorene compound according to claim 4, wherein the divalent organic group having an aliphatic hydrocarbon is wherein * indicates the position of a bond; X, X', X" and group for Ric' is a group represented by formula (i): X" are each independently absent or —O— —S- —NHCO—, or —CONH : R and Ra are each indepen dently a hydrogenatom, an aliphatic hydrocarbon group hav ing a carbon number of not less than 5 or a methyl group, R --Xa-Rd.--Xa is an organic group having an aliphatic hydrocarbon group having a carbon number of not less than 5: n, n., n and n are each independently an integer of 0 to 2: m is an integer of 1 wherein Xa is absent or —O— —S , —NHCO—, or or 2; when a plurality of any of n, n., n and n is present, —CONH-; Rd is an aliphatic hydrocarbon group hav each respective n, n., n and na may be the same or different: ing a carbon number of not less than 5; k is an integer of when a plurality of any of X, X" and X" is present, each 1 to 10; when a plurality of Rd is present, respective Rd respectiveX, X" and X" may be the same or different; and may be the same or different; and when a plurality of Xa when a plurality of any of R and R is present, each respec is present, respective Xa may be the same or different. tive R and R may be the same or different; and US 2014/0046022 A1 Feb. 13, 2014 34

a group represented by the formula (e): 19. The fluorene compound according to claim3, wherein the organic group having an aliphatic hydrocarbon group is present at the 2-position and/or the 7-position of the fluorene compound. (XR12)m3 20. The fluorene compound according to claim3, wherein : 8 5 ng Rb and/or Rc arefis -s (s a group represented by the formula (a) wherein m is 1: X is —O—; and R is an aliphatic hydrocarbon group wherein * indicates the position of a bond; X is absent or having a carbon number of 5 to 60; O —S , NHCO , or – CONH ; m is an integer a group represented by the formula (b) wherein X. X", of 0 to 15; ns is an integer of 0 to 11; n is an integer of 0 ti5: X" and X" are each —O—; R and Ra are each inde X, is absent or —O —S , —COO ... —OCONH . pendently an aliphatic hydrocarbon group having a car —NHCO-or—CONH : R is a hydrogenatom, a methyl bon number of 5 to 60; R is an organic group having an group or an aliphatic hydrocarbon group having a carbon aliphatic hydrocarbon group having a carbon number of number of not less than 5; when a plurality of X, is present, 5 to 60; n., n., n and n are each 1; and m2 is 1; or respective X, may be the same or different; and when a a group represented by the formula (e) wherein Xs is plurality of R is present, respective R may be the same or —O—: m is 2 or 3: ns is 1: n is 3: X, is —O—; and R. different. in the number of ms are each independently an alkyl 10. The fluorene compound according to claim 9, wherein: group having a carbon number of 14 to 30. 21. The fluorene compound according to claim3, wherein in the formula (a), X is —O—; R is an aliphatic hydro ring A is a benzene ring; Y is a hydroxyl group, a bromo carbon group having a carbon number of 5 to 60; m is 1: group, or a chloro group; Raisahalogenatom; and an organic in the formula (b), X is —O—, or —CONH : X, X." group having an aliphatic hydrocarbon group is a group rep and X" are each independently absent or —O—; R. resented by the formula (a) wherein m is 1: X is —O—, and and Rare each independently an aliphatic hydrocarbon R is an aliphatic hydrocarbon group having a carbon number group having a carbon number of 5 to 60 or a methyl of 5 to 60, a group represented by the formula (b) wherein X, group; R is an organic group having an aliphatic hydro X, X" and X" are each —O—; R and Ra are each inde carbon group having a carbon number 5 to 60; n, n., n pendently an aliphatic hydrocarbon group having a carbon and n are the same or different and each is an integer of number of 5 to 60; R is an organic group having an aliphatic 0 or 1; m is 1; and hydrocarbon group having a carbon number 5 to 60; n, n., n in the formula (e), Xs is —O—; m is 2 or 3; ns is 1; no is 2 and n are each 1; and m is 1, or a group represented by the or 3; X, is —O—; and R in the number of m are each formula (e) wherein X is —O—; m is 2 or 3; ns is 1: n is 3: independently an alkyl group having a carbon number of X, is —O—; and R2 in the number of ms are each indepen 8 to 6O. dently an alkyl group having a carbon number of 14 to 30, 11. The fluorene compound of according to claim 1, each of which formulas is present at the 2-position and/or the wherein the aliphatic hydrocarbon group of the organic group 7-position of the fluorene compound. having an aliphatic hydrocarbon group and/or the divalent 22. The fluorene compound according to claim 4, wherein organic group having an aliphatic hydrocarbon group is an n is 1. aliphatic hydrocarbon group having a carbon number of not 23. The fluorene compound according to claim 4, wherein less than 5. ring A has an electron-withdrawing group. 12. The fluorene compound of according to claim 1, 24. The fluorene compound according to claim 23, wherein wherein the aliphatic hydrocarbon group of the organic group the electron-withdrawing group is a halogen atom. having an aliphatic hydrocarbon group and/or the divalent 25. The fluorene compound according to claim 4, wherein organic group having an aliphatic hydrocarbon group is an Rc' is a group represented by the formula (i) wherein Xa is aliphatic hydrocarbon group having a carbon number of 5 to —O—: Rd in the number of k are each independently an 6O. aliphatic hydrocarbon group having a carbon number of 5 to 13. The fluorene compound of according to claim 1, 60, and k is an integer of 1 to 3. wherein the total carbon number derived from an aliphatic 26. The fluorene compound according to claim 4, wherein hydrocarbon group in one molecule is not less than 20. ring A is a benzene ring; Y is a hydroxyl group, a bromo group, or a chloro group; n is 1; ring A has a halogenatom as 14. The fluorene compound of according to claim 1, is the electron-withdrawing group; Ric' is a group represented by wherein the total carbon number derived from an aliphatic the formula (i) wherein Xa is —O—; Rd is an aliphatic hydrocarbon group in one molecule is 20 to 200. hydrocarbon group having a carbon number of 5 to 60, and ki 15. The fluorene compound of according to claim 1, is an integer of 1 to 3. wherein Y is a hydroxyl group, a bromo group, a chloro 27. The fluorene compound according to claim3, which is group, an iodo group, a thiol group, or an amino group. selected from the group consisting of 16. The fluorene compound of according to claim 1, 2-docosyloxy-9-(4-chlorophenyl)-9-fluorenol; wherein Y is a hydroxyl group, a bromo group, or a chloro 2-docosyloxy-9-(4-chlorophenyl)-9-bromofluorene; group. 2,7-didocosyloxy-9-(4-chlorophenyl)-9-bromofluorene; 17. The fluorene compound of according to claim 1, 2-(12-docosyloxy-dodecanoxy)-9-(3-fluorophenyl)-9- wherein the aromatic ring of ring A is a benzene ring. bromofluorene; 18. The fluorene compound according to claim3, wherein 1,12-bis-12-(2'-O-9-(4-chlorophenyl)-9-fluorenol)- Ra is a halogen atom. dodecyloxy-dodecane; US 2014/0046022 A1 Feb. 13, 2014 35

1,12-bis-12-(2'-O-9-(4-chlorophenyl)-9-bromofluo rene)-dodecyloxy-dodecane; 2-(3-octadecyloxy-2.2-bis-octadecyloxymethyl-pro poxy)-9-(4-chlorophenyl)-9-fluorenol; 2-(3-octadecyloxy-2.2-bis-octadecyloxymethyl-pro poxy)-9-(4-chlorophenyl)-9-bromofluorene: 9-(4-chlorophenyl)-2-(3,4,5-tris(octadecyloxy)-cyclo hexylmethoxy)-9-fluorenol; and 9-(4-chlorophenyl)-2-(3,4,5-tris(octadecyloxy)-cyclo hexylmethoxy)-9-bromofluorene. 28. A protecting reagent for organic synthesis reaction, comprising a fluorene compound according to claim 1. 29. A protecting reagent for a carboxyl group of an amino acid or a peptide, comprising a fluorene compound according to claim 1. 30. A protecting reagent for the C-terminal of an amino acid or a peptide, comprising a fluorene compound according to claim 1. 31. A method of producing a peptide by a liquid phase synthesis process, comprising: (a) binding a fluorene compound according to claim 1 to an amino acid or a peptide, to obtain a bonded product; and (b) precipitating said bonded product. k k k k k