CN113773283A - Oxidobicycloheptene sulfonamide compound containing hydrophobic label and application thereof - Google Patents

Oxidobicycloheptene sulfonamide compound containing hydrophobic label and application thereof Download PDF

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CN113773283A
CN113773283A CN202111076698.1A CN202111076698A CN113773283A CN 113773283 A CN113773283 A CN 113773283A CN 202111076698 A CN202111076698 A CN 202111076698A CN 113773283 A CN113773283 A CN 113773283A
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周海兵
董春娥
辛丽兰
胡志烨
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Wuhan University WHU
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Abstract

An oxo-bridged bicycloheptene sulfonamide compound containing a hydrophobic label and application thereof, belonging to the technical field of medicines. The compound of the invention comprises an OBHSA compound containing a adamantane tag and having a structure shown in a general formula I and an OBHSA compound containing a hydrophobic amino acid tag and having a structure shown in a general formula II, wherein n in the general formula1、n2Is a natural number of 1-60, and R is hydrophobic amino acid. The compound can degrade estrogen receptors and inhibit human breast cancer cells, and can be used for preparing medicines for targeted degradation of the estrogen receptors and anti-breast cancer medicines.
Figure DDA0003262513350000011

Description

Oxidobicycloheptene sulfonamide compound containing hydrophobic label and application thereof
Technical Field
The invention belongs to the technical field of medicines, and relates to a synthetic method of an oxido bridged bicycloheptene sulfonamide (OBHSA) compound containing amantadine and a hydrophobic amino acid label and application of the compound in an anti-breast cancer medicine.
Background
The targeted protein degradation strategy as an effective means for overcoming drug resistance receives more and more attention today when drug resistance becomes a key influencing factor for treating a plurality of diseases. Whether the resistance can be overcome by aiming at an important target Estrogen Receptor (ER) of the breast cancer also becomes a key judgment standard of the therapeutic effect of the target drug. There are two main strategies for targeted degradation of ER targets. One is that the compound displaces the protein helix exposing the hydrophobic pocket to recognition degradation by the ubiquitin-protease system, such as RAD 1901. And PROTAC, a ternary complex E3 ligand, binds directly to E3 ubiquitin ligase to allow protein degradation by ubiquitin labeling, such as ARV-471 entering phase I clinical studies.
However, each of these approaches has its limitations, such as the first SERDs, which usually require access to the active pocket of the compound, require a certain binding affinity and occupy the binding pocket of the estrogen receptor molecule to change and expose the hydrophobic pocket for the effect, so that the rational design of the structure is very demanding and it is difficult to achieve a patterned design strategy. In the case of ProTAC technology, however, it has a high molecular weight, undesirable molecular rigidity and water solubility, and poor oral absorption and film permeability. Its poor pharmacokinetic properties are a major obstacle to its pharmacological properties. Therefore, it is important to discover new degradation strategies, compare the advantages and disadvantages of various strategies, and to investigate in-depth the mechanisms of action and biological activities of different types of molecules, such as reducing the size of the PROTAC, which is currently much larger than typical drugs (usually less than 500Da), to increase cell permeability and potency. As shown in FIG. 1, there is a class of compounds similar to the strategy of PROTAC, where the target protein is recognized by the ubiquitin protease system by the addition of a hydrophobic tag, which can be adamantane, a small molecule, such as Compound 1 targeting tyrosine kinase and targeting androgensCompound 2, the hormone receptor AR, can also be a single hydrophobic amino acid structure such as Boc3Arg, a successful example of which is compound 3 targeting glutathione S transferase. The molecules not only have good target protein degradation activity, but also have smaller molecular weight than that of the corresponding PROTAC, and the characteristic is also beneficial to the later-stage further research on the druggability.
Disclosure of Invention
In order to overcome the problems in the prior art, the compound of the present invention is designed by selecting an OBHSA compound having a degradation activity as a basic skeleton on a mother-nucleus structure, and then adding a hydrophobic tag to the compound by a side chain introduction method. The present invention contemplates that this tag may label the target protein by binding of OBHSA to the estrogen receptor, causing degradation of the target protein. In the selection of the hydrophobic tag, amantadine with a simple small molecular structure and Boc3Arg with a hydrophobic amino acid structure are respectively selected as the hydrophobic tags, and two types of molecules are designed and synthesized (figure 2). The invention hopes that the obtained compound not only can enhance the weaker ER degradation activity of the parent compound OBHSA, but also provides more reference for the modeling design for the degradation of estrogen receptors.
The invention aims to provide an OBHSA compound containing a hydrophobic label, and also aims to provide a preparation method and a pharmaceutical application of the compound.
The purpose of the invention is realized by the following technical scheme:
the OBHSA compound containing the hydrophobic tag comprises the OBHSA compound containing the adamantane tag and the OBHSA compound containing the hydrophobic amino acid tag.
The adamantane tag-containing OBHSA compound has a structure shown in a general formula I:
Figure BDA0003262513330000021
in the general formula I, n1Is a natural number of 1 to 60, preferably 1, 3, 5, 7.
Preferably, the adamantane tag-containing OBHSA compound is selected from the following compounds:
n- ((3s,5s,7s) -adamantan-1-yl) -2- (4- ((1R,4R) -5, 6-bis (4-hydroxyphenyl) -N- (2,2, 2-trifluoroethyl) -7-oxabicyclo [2.2.1] hept-5-ene) -2-sulfonamido) phenoxy) acetamide (20a),
N- ((3s,5s,7s) -adamantan-1-yl) -4- (4- ((1R,4R) -5, 6-bis (4-hydroxyphenyl) -N- (2,2, 2-trifluoroethyl) -7-oxabicyclo [2.2.1] hept-5-ene) -2-sulfonamido) phenoxy) butanamide (20b),
N- ((3s,5s,7s) -adamantan-1-yl) -6- (4- ((1R,4R) -5, 6-bis (4-hydroxyphenyl) -N- (2,2, 2-trifluoroethyl) -7-oxabicyclo [2.2.1] hept-5-ene) -2-sulfonamido) phenoxy) hexanamide (20c),
N- ((3s,5s,7s) -adamantan-1-yl) -8- (4- ((1R,4R) -5, 6-bis (4-hydroxyphenyl) -N- (2,2, 2-trifluoroethyl) -7-oxabicyclo [2.2.1] hept-5-ene) -2-sulfonamido) phenoxy) octanamide (20 d).
The OBHSA compound containing the hydrophobic amino acid tag has a structure shown in a general formula II:
Figure BDA0003262513330000022
Figure BDA0003262513330000031
in the general formula II, n2Is a natural number of 1-60, preferably 3, 6; r is hydrophobic amino acid, such as D-Arg, L-Phe, L-Leu, L-Lys, L-Trp, etc.
Preferably, the hydrophobic amino acid tag-containing OBHSA compound is selected from the following compounds:
(2S) -2-amino-N- (3- (4- ((5, 6-bis (4-hydroxyphenyl) -N- (2,2, 2-trifluoroethyl) -7) - [2.2.1] hept-5-ene) -2-sulfonamide (phenoxy) propyl) -5-guanidinopentaneamide derivatives (30a),
(2R) -2-amino-N- (6- (4- ((5, 6-bis (4-hydroxyphenyl) -N- (2,2, 2-trifluoroethyl) -7) - [2.2.1] hept-5-ene) -2-sulfonamide (phenoxy) hexyl) -5-guanidinopentaneamide derivatives (30b),
Tert-butyl ((2S) -1- ((3- (4- ((1R,4R) -5, 6-bis (4-hydroxyphenyl) -N- (2,2, 2-trifluoroethyl) -7-oxabicyclo [2.2.1] hept-5-ene) -2-sulfonamido) phenoxy) propyl) amino) -1-oxo-3-phen-yn-2-yl) carbamate (30c),
Tert-butyl ((2R) -1- ((6- (4- ((1R,4R) -5, 6-bis (4-hydroxyphenyl) -N- (2,2, 2-trifluoroethyl) -7-oxabicyclo [2.2.1] hept-5-ene) -2-sulfonamido) phenoxy) hexyl) amino) -1-oxo-3-phen-yn-2-yl) carbamate (30d),
Tert-butyl ((2S) -1- ((3- (4- ((1R,4R) -5, 6-bis (4-hydroxyphenyl) -N- (2,2, 2-trifluoroethyl) -7-oxa-cyclo [2.2.1] hept-5-en) -2-sulfanyl) phenoxy) propyl) amino) -4-methyl-1-oxopentan-2-yl) carbamate (30e),
Tert-butyl ((2R) -1- ((6- (4- ((1R,4R) -5, 6-bis (4-hydroxyphenyl) -N- (2,2, 2-trifluoroethyl) -7-oxabicyclo [2.2.1] hept-5-ene) -2-sulfonamido) phenoxy) hexyl) amino) -4-methyl-1-oxopentan-2-yl) carbamate (30f),
Di-tert-butyl ((5S) -6- ((3- (4- ((1R,4R) -5, 6-bis (4-hydroxyphenyl) -N- (2,2, 2-trifluoroethyl) -7-oxabicyclo [2.2.1] hept-5-ene) -2-sulfonamido) phenoxy) propyl) amino) -6-oxahexane-1, 5-diyl) dicarbamate (30g),
Di-tert-butyl ((5R) -6- ((6- (4- ((1R,4R) -5, 6-bis (4-hydroxyphenyl) -N- (2,2, 2-trifluoroethyl) -7-oxabicyclo [2.2.1] hept-5-ene) -2-sulfonamido) phenoxy) hexyl) amino) -6-oxohexane-1, 5-diyl) dicarbamate (30h),
Tert-butyl ((2S) -1- ((3- (4- ((1R,4R) -5, 6-bis (4-hydroxyphenyl) -N- (2,2, 2-trifluoroethyl) -7-oxabicyclo [2.2.1] hept-5-en) -2-sulfonamido) phenoxy) propyl) amino) -3- (1H-indol-2-yl) -1-oxopropan-2-yl) carbamate (30i),
Tert-butyl ((2R) -1- ((6- (4- ((1R,4R) -5, 6-bis (4-hydroxyphenyl) -N- (2,2, 2-trifluoroethyl) -7-oxabicyclo [2.2.1] hept-5-ene) -2-sulfonamido) phenoxy) hexyl) amino) -3- (1H-indol-2-yl) -1-oxopropan-2-yl) carbamate (30 j).
The OBHSA compound containing the hydrophobic label can degrade estrogen receptors and inhibit human breast cancer cells, and can be used for preparing drugs for targeted degradation of estrogen receptors and anti-breast cancer drugs.
The drug for targeted degradation of estrogen receptors or the anti-breast cancer drug comprises the OBHSA compound containing the hydrophobic tag and one or more pharmaceutically acceptable carriers or excipients.
Compared with the prior art, the invention has the advantages and beneficial effects that:
PROTAC has the limitation of too large molecular weight, and a series of estrogen receptor down-regulators which are based on an OBHSA framework and mediated by hydrophobic tags, namely adamantane or hydrophobic amino acids, are synthesized by the invention, so that the obtained target compound has certain ER alpha and ER beta binding capacity. The introduction of the hydrophobic label can not only lead the compound to obtain MCF-7 cell inhibitory activity which is better than that of a parent compound (OBHSA), but also lead the compound to have good estrogen receptor down-regulation activity.
The hydrophobic label is introduced, the synthesis is simple and convenient, the yield is high, and the obtained target molecular weight is smaller than that of PROTAC and is better in cell membrane permeability and pharmacy. The introduction of the hydrophobic label as a successful technical means capable of selectively degrading the target protein has a more extensive application of the modeled design strategy compared with the traditional SERDs aiming at the reasonable design of the binding pocket, and is possible to obtain active compounds aiming at the target points which cannot be drugged. The addition of the two hydrophobic labels can be used as a modeling strategy in the future, and provides ideas and references for developing novel estrogen receptor down-regulation agents.
Drawings
FIG. 1 is a representation of a hydrophobic tag mediated proteolytic degradation compound.
Figure 2 is an estrogen receptor down-regulator design mediated by a hydrophobic tag based on the OBHSA backbone.
FIG. 3 is a graph showing the results of example 17. (A) Western blot analysis of ER α in MCF-7 cells treated with 5 μ M of compounds Ful, 20a-d and 30 a; (B) comparison of the levels of ER α degradation by Compounds 20a-d and 30 a; in the figure, C is a blank control.
Detailed Description
The preparation method of the hydrophobic tag-containing OBHSA compound comprises the following steps:
(1) synthesis of 3, 4-bis (4-hydroxyphenyl) furan 9
The synthetic route is shown as the following reaction formula, and specifically comprises the following steps:
Figure BDA0003262513330000041
1) synthesis of 2- (4-methoxyphenyl) -2-carbonylethyl-2- (4-methoxyphenyl) acetate (Compound 6)
Weighing 2-bromo-1- (4-methoxyphenyl) ethane-1-ketone 4(1.5896g, 6.94mmol) and p-methoxyphenylacetic acid 5(1.1532g, 6.94mmol) in a 50mL round-bottomed flask, adding 25mL of anhydrous acetonitrile, slowly dropwise adding anhydrous triethylamine (702.3mg, 6.94mmol), continuing to react at room temperature for 12h, monitoring by TLC for reaction completion, concentrating under reduced pressure after the reaction is finished to remove acetonitrile and triethylamine, adding ethyl acetate to dissolve, washing with dilute hydrochloric acid (2M, 30mL), saturated sodium bicarbonate (2X 30mL) and saturated sodium chloride (30mL), drying the organic layer with anhydrous sodium sulfate, filtering, and spin-drying to obtain a crude product, and purifying by silica gel column chromatography (V)Methylene dichloride:VMethanolCompound 6 was obtained as a yellow solid in 88% yield after 500:1-400: 1).
2) Synthesis of 3, 4-bis (4-methoxy-phenyl) furan-2-one (Compound 7)
Baking 25mL of double-mouth bottle and magneton at 105 ℃ for 15min, putting the bottle in a hot device, performing anhydrous and anaerobic operation, weighing and adding compound 6(786.2mg and 2.5mmol) under Ar, adding 10mL of anhydrous DMSO, slowly dropwise adding 80% NaH (150.1mg and 5.0mmol), reacting at 25 ℃ for 3h, monitoring the reaction completion by TLC, adding 5mL of 2N HCl to quench the reaction, extracting with ethyl acetate (3X 25mL), and obtaining an organic layer of anhydrous NaSO4Drying and desolventizing under reduced pressure gave the crude product which was purified on silica gel column (petroleum ether/ethyl acetate 9:1, v/v) to give 475.9mg (64.3% yield) of compound 7.
3) Synthesis of 3, 4-bis (4-hydroxy-phenylfuran) -2-one (Compound 8)
Mixing 100mL single-mouth bottle with magnetBaking semen at 105 deg.C for 15min, loading compound 7(1.345g, 4.56mmol) under Ar, adding 25mL DCM, and adding BBr at-20 deg.C3(2.6mL, 27.33mmol) for 12h, the reaction was quenched with 10mL water, extracted with ethyl acetate (3X 20mL), and saturated NaHCO3The solution (15mL) was washed and the organic layer was anhydrous NaSO4Drying and desolventizing under reduced pressure gave the crude product which was purified on silica gel column (petroleum ether/ethyl acetate 7:3) to give 1.06g (86.7% yield) of compound 8.
4) Synthesis of 3, 4-bis (4-hydroxy-phenyl) furan (Compound 9)
Baking 50mL single-neck bottle and magneton at 105 deg.C for 15min, loading into a hot apparatus, adding compound 8(560mg, 1.98mmol) under Ar atmosphere, adding diisobutylaluminum hydride (DIBAL-H, 8mL, 7.93mmol) at-78 deg.C, reacting for 12H, adding 4% H2SO4The reaction was quenched, extracted with ethyl acetate (3X 25mL), washed with saturated NaCl solution (30mL), and the organic layer was dried over anhydrous NaSO4Drying and desolventizing under reduced pressure gave the crude product which was purified on silica gel column (petroleum ether/ethyl acetate 6:4) to give 203.1mg (40.7% yield) of compound 9.1H NMR(400MHz,CDCl3):δ7.41(s,2H),6.94(d,J＝8.4Hz,2H),6.87(d,J＝8.8Hz,2H).
(2) Synthesis of OBHSA derivatives with different length acid side chains
The synthesis route is shown as the following reaction formula, and the specific synthesis comprises the steps of stirring 4-methoxyaniline 10 and trifluoroacetic anhydride for 4 hours at room temperature to obtain a compound 11, reducing the compound 11 and borane dimethylsulfide at 60 ℃ to obtain a compound 12, and reacting the compound 12 and dichloroethane sulfonyl chloride at room temperature to obtain a compound 13, a compound 13 and BBr3Reacting at-20 ℃ to obtain a compound 14; under alkaline conditions, compound 14 is firstly reacted with ethyl bromoate (compounds 15a-d, n is respectively 1, 3, 5 and 7) with different lengths to obtain dienophile 16a-d with an ethyl ester side chain, then, the dienophile 16a-d and dihydroxyfuran compound 9 are subjected to Diels-Alder reaction to obtain a corresponding end product, and finally, the obtained compound is hydrolyzed by LiOH to obtain OBHSA derivative 18a-d containing an acidic side chain.
Figure BDA0003262513330000061
(3) Synthesis of the end product 20a-d with an adamantane Label
After obtaining the OBHSA derivatives 18a-d with different length acid side chains, the four compounds were condensed separately with commercially available amantadine molecules. The synthetic route is shown in the following reaction formula, DCC and HOBt are used as composite condensing agents, and under the action of DMF solvent, final products 20a-d with side chains of different lengths and containing adamantane labels are synthesized. Compared with PROTAC, the compound has the advantages of cheap and easily obtained raw materials, rapid synthesis and relatively simple and convenient operation.
Figure BDA0003262513330000062
(4) Synthesis of OBHSA derivative 25 with Boc amino side chain
The synthetic route is shown in the following reaction scheme, first, Boc protection of amino group in 3-hydroxy propylamine 21 to obtain compound 22, followed by activation of hydroxy group of compound 22 with p-toluenesulfonyl chloride to obtain compound 23, then nucleophilic substitution of dienophile 14 and compound 23 to obtain dienophile derivative 24 with Boc amino side chain, and finally Diels-Alder reaction of compound 24 and dihydroxyfuran ring 9 to obtain OBHSA derivative 25 with Boc amino side chain.
Figure BDA0003262513330000071
(5) Synthesis of amino acid-based active ester derivatives 28a-e (for example, 26a arginine)
The synthetic route is shown in the following reaction formula, firstly, Boc protection is carried out on the amino group of arginine 26a by using di-tert-butyl dicarbonate under alkaline condition to obtain an intermediate compound 27a, and then the carboxyl group of the compound 27a and N-hydroxysuccinimide react in the presence of EDC hydrochloride and DMAP to generate an active ester product 28 a. Other reactive intermediates 28b-e were synthesized in a similar manner.
Figure BDA0003262513330000072
(6) With Boc3Synthesis of Arg side-chain OBHSA-like target product 30a
The synthetic route is shown in the following reaction scheme, after obtaining OBHSA derivative 25 with Boc protected amino side chain, under the action of hydrochloric acid, deprotection is carried out to obtain amino compound 29. The amino group of compound 29 was then reacted with arginine active ester 28a in THF as a solvent and TEA as a base to form a compound with Boc3 OBHSA target product 30a of Arg side chain.
Figure BDA0003262513330000081
(7) Synthesis of OBHSA target product 30b-j with amino acid side chain
In a manner similar to the synthesis of compound 30a, after obtaining the OBHSA derivative 25 having a Boc-protected amino side chain, it is deprotected under the action of hydrochloric acid to obtain an amino compound 29. Then, the amino group of compound 29 and the amino acid active ester products 28b-f are reacted under THF as a solvent and TEA as a base to form OBHSA target products 30b-30j with Boc amino acid side chains.
Figure BDA0003262513330000082
The following examples are intended to further illustrate the invention but should not be construed as limiting it. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
Example 1: preparation of (2S) -2-amino-N- (3- (4- ((5, 6-bis (4-hydroxyphenyl) -N- (2,2, 2-trifluoroethyl) -7) - [2.2.1] hept-5-ene) -2-sulfonamide (phenoxy) propyl) -5-guanidinopentaneamide derivative (30 a):
Figure BDA0003262513330000083
compound 29(74.62mg, 0.126mmol) and compound 28a (85.95mg, 0.15mmol) were placed in a 25mL single-neck flask, dissolved in 2mL of THF, triethylamine (15.21mg, 0.15mmol) was added, after 2h of reaction, TLC confirmed the completion of the reaction, and extracted with ethyl acetate, the organic phase was concentrated to dryness, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography (eluent ratio v)Methylene dichloride/vMethanol120:1-30:1) to yield 108mg of compound 30a as a yellow solid in 77% yield.1H NMR(400MHz,MeOD-d6)δ7.19(d,J＝8.8Hz,2H),7.12(t,J＝8.4Hz,4H),6.78(t,J＝9.1Hz,4H),6.69(d,J＝8.7Hz,2H),5.45(s,1H),5.29(d,J＝4.2Hz,1H),4.40(q,J＝8.4Hz,2H),3.97(t,J＝6.1Hz,3H),3.84(td,J＝13.8,6.9Hz,2H),3.44-3.34(m,3H),2.21(dt,J＝11.8,4.3Hz,1H),2.01(dd,J＝7.0,4.5Hz,1H),1.99-1.92(m,2H),1.73-1.67(m,1H),1.69-1.57(m,3H),1.51(s,9H),1.45(s,9H),1.39(s,9H).HRMS(ESI)calcd for C50H65F3N6O13S[M+Na]+,1069.4175；found 1069.4168.
Example 2: preparation of (2R) -2-amino-N- (6- (4- ((5, 6-bis (4-hydroxyphenyl) -N- (2,2, 2-trifluoroethyl) -7) - [2.2.1] hept-5-ene) -2-sulfonamide (phenoxy) hexyl) -5-guanidinopentaneamide derivative (30 b):
Figure BDA0003262513330000091
compound 29(40mg, 0.068mmol) and compound 28b (57.3mg, 0.10mmol) were placed in a 25mL single-necked flask, dissolved in 2mL of THF, triethylamine (8.2mg, 0.08mmol) was added, after 2h of reaction, TLC confirmed the completion of the reaction, extracted with ethyl acetate, the organic phase was concentrated and dried, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography (eluent ratio v)Methylene dichloride/vMethanol100:1-15:1) to give 47mg of compound 30b as a pale yellow solid in 50% yield. Nuclear magnetic hydrogen spectrum of point 1:1H NMR(400MHz,Acetone-d6)δ7.55(s,1H),7.33(d,J＝2.2Hz,1H),7.30(d,J＝2.2Hz,1H),7.26(d,J＝4.3Hz,4H),7.20(dd,J＝8.5,4.3Hz,5H),6.88(d,J＝1.2Hz,1H),6.86(d,J＝1.2Hz,1H),6.83(d,J＝2.7Hz,1H),6.81(s,2H),6.79(s,1H),6.13(s,1H),5.53(s,1H),5.35(d,J＝3.9Hz,1H),4.58–4.47(m,2H),4.34(t,J＝6.0Hz,1H),3.94(d,J＝6.2Hz,2H),3.54(dd,J＝12.6,4.3Hz,1H),3.45–3.35(m,2H),3.14(dd,J＝13.7,6.1Hz,1H),2.95(dd,J＝13.6,8.3Hz,1H),2.24(dd,J＝13.7,6.0Hz,1H),2.10(d,J＝4.9Hz,1H),1.98-1.86(m,2H),1.39(d,J＝19.8Hz,2H),1.33(s,9H).HRMS(ESI)calcd for C43H46F3N3O9S[M+Na]+,860.2799；found 860.2789.
example 3: preparation of tert-butyl ((2S) -1- ((3- (4- ((1R,4R) -5, 6-bis (4-hydroxyphenyl) -N- (2,2, 2-trifluoroethyl) -7-oxabicyclo [2.2.1] hept-5-ene) -2-sulfonamido) phenoxy) propyl) amino) -1-oxo-3-phen-inph-2-yl) carbamate (30 c):
Figure BDA0003262513330000101
compound 29(40mg, 0.068mmol) and compound 28c (32mg, 0.10mmol) were placed in a 25mL single-neck flask, dissolved in 2mL of THF, triethylamine (8.2mg, 0.08mmol) was added, after 2h of reaction, TLC confirmed the completion of the reaction, extracted with ethyl acetate, the organic phase was concentrated to dryness, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography (eluent ratio v)Methylene dichloride/vMethanol100:1-15:1) to yield 47mg of compound 30c as a pale yellow solid in 86% yield. Nuclear magnetic hydrogen spectrum of point 1:1H NMR(400MHz,Acetone-d6)δ7.55(s,1H),7.33(d,J＝2.2Hz,1H),7.30(d,J＝2.2Hz,1H),7.26(d,J＝4.3Hz,4H),7.20(dd,J＝8.5,4.3Hz,5H),6.88(d,J＝1.2Hz,1H),6.86(d,J＝1.2Hz,1H),6.83(d,J＝2.7Hz,1H),6.81(s,2H),6.79(s,1H),6.13(s,1H),5.53(s,1H),5.35(d,J＝3.9Hz,1H),4.58–4.47(m,2H),4.34(t,J＝6.0Hz,1H),3.94(d,J＝6.2Hz,2H),3.54(dd,J＝12.6,4.3Hz,1H),3.45–3.35(m,2H),3.14(dd,J＝13.7,6.1Hz,1H),2.95(dd,J＝13.6,8.3Hz,1H),2.24(dd,J＝13.7,6.0Hz,1H),2.10(d,J＝4.9Hz,1H),1.98-1.86(m,2H),1.39(d,J＝19.8Hz,2H),1.33(s,9H).HRMS(ESI)calcd for C43H46F3N3O9S[M+Na]+,860.2799；found 860.2789.
example 4: preparation of tert-butyl ((2R) -1- ((6- (4- ((1R,4R) -5, 6-bis (4-hydroxyphenyl) -N- (2,2, 2-trifluoroethyl) -7-oxabicyclo [2.2.1] hept-5-ene) -2-sulfonamido) phenoxy) hexyl) amino) -1-oxo-3-phen-inp-2-yl) carbamate (30 d):
Figure BDA0003262513330000102
compound 29(40mg, 0.068mmol) and compound 28c (68mg, 0.169mmol) were placed in a 25mL single-necked flask, dissolved in 2mL of THF, triethylamine (8.2mg, 0.08mmol) was added, after 2h of reaction, TLC was performed to confirm completion of the reaction, extraction was performed with ethyl acetate, the organic phase was concentrated and dried, dried over anhydrous sodium sulfate, and separation and purification were performed by silica gel column chromatography (eluent ratio v)Methylene dichloride/vMethanol100:1-15:1) to yield 23.2mg of compound 30d as a yellow solid in 39% yield.1H NMR(400MHz,CDCl3)δ7.30–7.24(m,2H),7.23–7.19(m,2H),7.17(d,J＝6.1Hz,2H),7.15–7.08(m,5H),6.82(d,J＝8.0Hz,2H),6.74(dd,J＝16.8,8.0Hz,4H),6.40(s,1H),5.51(s,1H),5.38(s,1H),5.31(s,1H),4.29(d,J＝26.7Hz,2H),3.86(s,2H),3.40(s,1H),3.22(s,1H),3.11(s,1H),3.00(d,J＝14.1Hz,2H),2.38(s,1H),2.00(s,1H),1.68(s,2H),1.42(s,1H),1.39(s,9H),1.31(s,3H),1.20(s,2H).HRMS(ESI)calcd for C46H52F3N3O9S[M+Na]+,902.3268；found 902.3264.
Example 5: preparation of tert-butyl ((2S) -1- ((3- (4- ((1R,4R) -5, 6-bis (4-hydroxyphenyl) -N- (2,2, 2-trifluoroethyl) -7-oxa [2.2.1] hept-5-ene) -2-sulfonamido) phenoxy) propyl) amino) -4-methyl-1-oxopentan-2-yl) carbamate (30 e):
Figure BDA0003262513330000111
compound 29(80mg, 0.135mmol) and compound 28d (44.4mg, 0.135mmol) were placed in a 25mL single-neck flask, dissolved in 2mL of THF, triethylamine (16.4mg, 0.16mmol) was added, after 2h of reaction, TLC confirmed the completion of the reaction, extracted with ethyl acetate, the organic phase was concentrated to dryness, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography (eluent ratio v)Methylene dichloride/vMethanol150:1-30:1) to yield 92.6mg of compound 30e as a pale yellow solid in 85% yield.1H NMR(400MHz,Acetone-d6)δ7.59(s,1H),7.30(dd,J＝8.4,3.1Hz,2H),7.26(d,J＝4.3Hz,1H),7.19(d,J＝8.6Hz,4H),6.87(d,J＝8.5Hz,2H),6.83(dd,J＝9.0,2.5Hz,2H),6.79(d,J＝8.6Hz,2H),6.14(s,1H),5.50(s,1H),5.35(d,J＝4.3Hz,1H),4.51(dd,J＝10.0,7.7Hz,2H),4.17–3.98(m,2H),3.52(dd,J＝7.9,3.9Hz,1H),3.42(d,J＝6.1Hz,2H),2.24(dd,J＝11.9,4.4Hz,1H),2.15(dd,J＝39.0,9.3Hz,1H),1.97(dd,J＝13.6,5.5Hz,2H),1.71(dt,J＝13.2,6.5Hz,1H),1.63–1.52(m,2H),1.39(s,9H),1.33(d,J＝2.9Hz,2H),1.30(s,2H),1.28(d,J＝1.8Hz,1H),0.91(t,J＝6.2Hz,6H).HRMS(ESI)calcd for C40H48F3N3O9S[M+Na]+,826.2956；
found 826.2950.
Example 6: preparation of tert-butyl ((2R) -1- ((6- (4- ((1R,4R) -5, 6-bis (4-hydroxyphenyl) -N- (2,2, 2-trifluoroethyl) -7-oxabicyclo [2.2.1] hept-5-ene) -2-sulfonamido) phenoxy) hexyl) amino) -4-methyl-1-oxopentan-2-yl) carbamate (30 f):
Figure BDA0003262513330000112
compound 29(80mg, 0.127mmol) and compound 28d (44.4mg, 0.135mmol) were placed in a 25mL single-neck flask, dissolved in 2mL of THF, triethylamine (16.0mg, 0.16mmol) was added, after 2h of reaction, TLC confirmed the completion of the reaction, extracted with ethyl acetate, the organic phase was concentrated to dryness, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography (eluent ratio v)Methylene dichloride/vMethanol200:1-20:1) to yield 85.6mg of 30f as a pale yellow compound in 80% yield.1H NMR(400MHz,CDCl3)δ8.33(s,1H),7.11–7.03(m,6H),6.82(d,J＝7.3Hz,2H),6.73(d,J＝8.5Hz,2H),6.67(dd,J＝8.9,3.4Hz,2H),5.45(s,1H),5.30(d,J＝6.9Hz,1H),5.10(s,1H),4.27(dd,J＝13.9,7.0Hz,2H),4.07(s,1H),3.86(t,J＝5.8Hz,2H),3.36(d,J＝5.9Hz,1H),3.25(dd,J＝13.5,7.0Hz,2H),2.39(dd,J＝12.1,8.0Hz,1H),2.06–1.82(m,3H),1.74–1.66(m,2H),1.62(dd,J＝14.9,7.2Hz,2H),1.54–1.46(m,2H),1.41(s,9H),1.34(d,J＝6.2Hz,2H),0.89(t,J＝5.9Hz,6H).HRMS(ESI)calcd for C43H54F3N3O9S[M+Na]+,868.3425；found 868.3421.
Example 7: preparation of di-tert-butyl ((5S) -6- ((3- (4- ((1R,4R) -5, 6-bis (4-hydroxyphenyl) -N- (2,2, 2-trifluoroethyl) -7-oxabicyclo [2.2.1] hept-5-ene) -2-sulfonamido) phenoxy) propyl) amino) -6-oxahexane-1, 5-diyl) dicarbamate (30 g):
Figure BDA0003262513330000121
compound 29(45mg, 0.076mmol) and compound 28e (56.3mg, 0.127mmol) were placed in a 25mL single-necked flask, dissolved in 2mL of THF, triethylamine (9.3mg, 0.09mmol) was added, after 2h of reaction, TLC confirmed the completion of the reaction, extracted with ethyl acetate, the organic phase was concentrated and dried, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography (eluent ratio v)Methylene dichloride/vMethanol200:1-15:1) to give 36.5mg of compound 30g as a pale yellow solid with a yield of 52%.1H NMR(400MHz,Acetone-d6)δ8.99(s,1H),8.73(s,1H),7.62(s,1H),7.30(d,J＝7.7Hz,3H),7.19(d,J＝8.7Hz,5H),6.87(d,J＝8.6Hz,2H),6.84(dd,J＝9.0,2.3Hz,2H),6.79(d,J＝8.6Hz,2H),6.17(d,J＝6.9Hz,1H),6.02(s,1H),5.51(s,1H),5.36(d,J＝4.2Hz,1H),4.52(d,J＝8.5Hz,3H),3.53(dd,J＝7.4,3.8Hz,1H),3.43(d,J＝5.7Hz,3H),3.10(s,5H),3.06(dd,J＝12.9,6.6Hz,4H),2.23(dd,J＝11.9,4.5Hz,1H),2.10(d,J＝7.8Hz,1H),1.97(d,J＝5.9Hz,2H),1.79(dd,J＝11.8,7.3Hz,1H),1.68(dd,J＝17.8,9.0Hz,2H),1.54–1.43(m,5H),1.41(s,11H),1.39(s,10H).
Example 8: preparation of di-tert-butyl ((5R) -6- ((6- (4- ((1R,4R) -5, 6-bis (4-hydroxyphenyl) -N- (2,2, 2-trifluoroethyl) -7-oxabicyclo [2.2.1] hept-5-ene) -2-sulfonamido) phenoxy) hexyl) amino) -6-oxohexane-1, 5-diyl) dicarbamate (30 h):
Figure BDA0003262513330000131
compound 29(60mg, 0.10mmol) and compound 28e (50.5mg, 0.11mmol) were placed in a 25mL single-neck flask, dissolved in 2mL of THF, triethylamine (11.52mg, 0.11mmol) was added, after 2h of reaction, TLC confirmed the completion of the reaction, extracted with ethyl acetate, the organic phase was concentrated to dryness, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography (eluent ratio v)Methylene dichloride/vMethanol200:1-15:1) to yield 47.2mg of compound as a pale yellow solid in 30h, 52% yield.1H NMR(400MHz,Acetone-d6)δ8.91(s,1H),8.68(s,1H),7.32(d,J＝8.9Hz,2H),7.20(dd,J＝8.5,6.6Hz,4H),6.85(dd,J＝8.6,7.5Hz,4H),6.79(d,J＝8.6Hz,2H),6.17(d,J＝7.6Hz,1H),6.00(s,1H),5.53(s,1H),5.35(d,J＝4.0Hz,1H),4.52(q,J＝8.4Hz,2H),4.08–3.92(m,4H),3.52(dd,J＝9.5,5.0Hz,1H),3.26(dd,J＝13.2,6.6Hz,2H),3.06(dd,J＝12.8,6.5Hz,2H),2.31–2.21(m,1H),2.21–2.09(m,1H),1.84–1.73(m,4H),1.65(ddd,J＝18.1,9.3,4.9Hz,2H),1.57–1.46(m,6H),1.41(s,9H),1.41(s,9H),1.30(s,6H).HRMS(ESI)calcd for C48H63F3N4O11S[M+Na]+,969.4027；found 969.3961.
Example 9: preparation of tert-butyl ((2S) -1- ((3- (4- ((1R,4R) -5, 6-bis (4-hydroxyphenyl) -N- (2,2, 2-trifluoroethyl) -7-oxabicyclo [2.2.1] hept-5-ene) -2-sulfonamido) phenoxy) propyl) amino) -3- (1H-indol-2-yl) -1-oxopropan-2-yl) carbamate (30 i):
Figure BDA0003262513330000132
compound 29(40mg, 0.068mmol) and compound 28f (54mg, 0.134mmol) were placed in a 25mL single-neck flask, dissolved in 2mL of THF, triethylamine (8.2mg, 0.08mmol) was added, after 2h of reaction, TLC confirmed the completion of the reaction, extracted with ethyl acetate, the organic phase was concentrated to dryness, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography (eluent ratio v)Methylene dichloride/vMethanol120:1-15:1) to yield 47.5mg of 30i as a white solid in 80% yield.1H NMR(400MHz,MeOD)δ7.56(d,J＝7.8Hz,1H),7.30(d,J＝8.1Hz,1H),7.18(dd,J＝8.6,6.8Hz,2H),7.12(dd,J＝8.1,4.0Hz,4H),7.06(d,J＝5.8Hz,2H),6.98(d,J＝7.5Hz,1H),6.76(d,J＝8.6Hz,2H),6.72–6.66(m,4H),5.45(s,1H),5.29(d,J＝4.1Hz,1H),4.40(q,J＝8.3Hz,2H),4.27(t,J＝9.7Hz,1H),3.64(d,J＝9.1Hz,2H),3.45–3.40(m,1H),3.16(dd,J＝9.3,5.3Hz,2H),2.20(dd,J＝9.5,5.2Hz,1H),1.99(dd,J＝12.2,8.6Hz,1H),1.79–1.66(m,2H),1.49(dd,J＝17.6,10.5Hz,2H),1.36(s,9H).
Example 10: preparation of tert-butyl ((2R) -1- ((6- (4- ((1R,4R) -5, 6-bis (4-hydroxyphenyl) -N- (2,2, 2-trifluoroethyl) -7-oxabicyclo [2.2.1] hept-5-ene) -2-sulfonamido) phenoxy) hexyl) amino) -3- (1H-indol-2-yl) -1-oxopropan-2-yl) carbamate (30 j):
Figure BDA0003262513330000141
compound 29(40mg, 0.068mmol) and compound 28f (32mg, 0.10mmol) were placed in a 25mL single-necked flask, dissolved in 2mL of THF, triethylamine (8.2mg, 0.08mmol) was added, after 2h of reaction, TLC was performed to confirm completion of the reaction, extraction was performed with ethyl acetate, the organic phase was concentrated and dried, dried over anhydrous sodium sulfate, and separation and purification were performed by silica gel column chromatography (eluent ratio v)Methylene dichloride/vMethanol200:1-15:1) to yield 39.5mg of compound 30j as a white solid in 68% yield.1H NMR(400MHz,Acetone-d6)δ10.12(s,1H),9.00(s,1H),8.80(s,1H),7.62(d,J＝7.8Hz,1H),7.37(d,J＝8.1Hz,1H),7.30(d,J＝8.9Hz,2H),7.20(s,1H),7.17(d,J＝8.8Hz,4H),7.08(t,J＝7.4Hz,1H),7.00(t,J＝7.5Hz,1H),6.84(t,J＝8.7Hz,4H),6.78(d,J＝8.6Hz,2H),5.98(d,J＝7.6Hz,1H),5.52(s,1H),5.33(d,J＝4.2Hz,1H),4.50(q,J＝8.6Hz,2H),4.38(d,J＝7.0Hz,1H),3.94(t,J＝6.4Hz,2H),3.52(dd,J＝9.2,3.4Hz,1H),3.22(d,J＝6.2Hz,1H),3.15(d,J＝7.0Hz,1H),2.21(dd,J＝8.3,3.8Hz,1H),2.12(dd,J＝35.0,5.8Hz,1H),1.71(dd,J＝13.9,6.8Hz,2H),1.45–1.38(m,4H),1.35(s,9H),1.28(s,4H).
N- ((3s,5s,7s) -adamantan-1-yl) -3- (4- ((((((1R, 4R) -5, 6-bis (4-hydroxyphenyl) -N- (2,2, 2-trifluoroethyl) -7-oxabicyclo [ 2.2.1)]Synthesis of hept-5-ene) -2-sulfonylamino) phenoxy) amide end-products 20 a-d: under the protection of Ar, the compounds 18a-d, DCC and HOBT were respectively added into a double-mouth bottle and dissolved in DMF. After 10min, adding amantadine, stirring at room temperature for about 4h, TLC confirming reaction completion, adding water for quenching, EA extracting, washing organic phase with supersaturated sodium bicarbonate for multiple times, concentrating, spin drying, and separating and purifying by silica gel column chromatography (eluent ratio v)Methylene dichloride/vMethanol200:1-15:1) to give the final product 20a-d (65% -74% yield).
Example 11: preparation of N- ((3s,5s,7s) -adamantan-1-yl) -2- (4- ((1R,4R) -5, 6-bis (4-hydroxyphenyl) -N- (2,2, 2-trifluoroethyl) -7-oxabicyclo [2.2.1] hept-5-ene) -2-sulfonamido) phenoxy) acetamide (20 a):
Figure BDA0003262513330000151
under Ar protection, compound 18a (100mg, 0.17mmol), DCC (105mg, 0.51mmol) and HOBT (31mg, 0.20mmol) were added to a two-necked flask and dissolved in DMF. After 10min, adding amantadine (38mg, 0.25mmol), stirring at room temperature for about 4h, TLC confirming reaction completion, adding water for quenching, EA extracting, washing organic phase with supersaturated sodium bicarbonate for multiple times, concentrating, spin drying, and separating and purifying by silica gel column chromatography (eluent ratio v isMethylene dichloride/vMethanol200:1-15:1) to yield 125mg of yellow solid 20a in 68% yield.1H NMR(400MHz,MeOD-d6)δ7.27(d,J＝9.0Hz,2H),7.13(t,J＝8.8Hz,4H),6.88(d,J＝9.0Hz,2H),6.78(d,J＝8.6Hz,2H),6.70(d,J＝8.7Hz,2H),5.46(d,J＝0.7Hz,1H),5.29(d,J＝3.9Hz,1H),4.48–4.38(m,4H),3.46(dd,J＝8.4,4.4Hz,1H),2.24–2.20(m,1H),2.18(t,J＝4.5Hz,1H),2.03(s,9H),1.72(d,J＝13.3Hz,6H).
Example 12: preparation of N- ((3s,5s,7s) -adamantan-1-yl) -4- (4- ((1R,4R) -5, 6-bis (4-hydroxyphenyl) -N- (2,2, 2-trifluoroethyl) -7-oxabicyclo [2.2.1] hept-5-ene) -2-sulfonamido) phenoxy) butanamide (20 b):
Figure BDA0003262513330000152
under Ar protection, compound 18b (100mg, 0.16mmol), DCC (100mg, 0.48mmol) and HOBT (29mg, 0.19mmol) were dissolved in DMF in a two-necked flask. After 10min, adding amantadine (37mg, 0.24mmol), stirring at room temperature for about 4h, TLC confirming reaction completion, adding water for quenching, EA extracting, washing organic phase with supersaturated sodium bicarbonate for multiple times, concentrating, spin drying, and separating and purifying by silica gel column chromatography (eluent ratio v is v)Methylene dichloride/vMethanol200:1-15:1) to give 125mg of 20b as a yellow solid in 74% yield.1H NMR(400MHz,MeOD-d6)δ7.20(dd,J＝9.0,2.3Hz,2H),7.14(t,J＝9.3Hz,4H),6.81–6.76(m,4H),6.71(d,J＝8.7Hz,2H),5.46(s,1H),5.30(d,J＝4.0Hz,1H),4.42(q,J＝8.5Hz,2H),4.02–3.93(m,2H),3.44(dt,J＝8.6,4.4Hz,1H),2.29–2.15(m,1H),2.15–2.05(m,1H),1.98(s,9H),1.83(dd,J＝28.9,13.6Hz,2H),1.68(s,6H),1.40–1.34(m,2H).
Example 13: preparation of N- ((3s,5s,7s) -adamantan-1-yl) -6- (4- ((1R,4R) -5, 6-bis (4-hydroxyphenyl) -N- (2,2, 2-trifluoroethyl) -7-oxabicyclo [2.2.1] hept-5-ene) -2-sulfonamido) phenoxy) hexanamide (20 c):
Figure BDA0003262513330000161
under the protection of Ar, adding the compounds into a double-mouth bottle respectively18c (100mg, 0.15mmol), DCC (96mg, 0.46mmol), HOBT (28mg, 0.19mmol) were dissolved in DMF. After 10min, adding amantadine (35mg, 0.23mmol), stirring at room temperature for about 4h, TLC confirming reaction completion, adding water for quenching, EA extracting, washing organic phase with supersaturated sodium bicarbonate for multiple times, concentrating, spin drying, and separating and purifying by silica gel column chromatography (eluent ratio v is v)Methylene dichloride/vMethanol150:1-30:1) to yield 125mg of 20c as a pale yellow solid in 65% yield.1H NMR(400MHz,MeOD-d6)δ7.27(d,J＝9.0Hz,2H),7.13(t,J＝8.8Hz,4H),6.88(d,J＝9.0Hz,2H),6.78(d,J＝8.6Hz,2H),6.70(d,J＝8.7Hz,2H),5.46(d,J＝0.7Hz,1H),5.29(d,J＝3.9Hz,1H),4.48–4.38(m,4H),3.46(dd,J＝8.4,4.4Hz,1H),2.24–2.20(m,1H),2.18(t,J＝4.5Hz,1H),2.03(s,9H),1.72(d,J＝13.3Hz,6H).
Example 14: preparation of N- ((3s,5s,7s) -adamantan-1-yl) -8- (4- ((1R,4R) -5, 6-bis (4-hydroxyphenyl) -N- (2,2, 2-trifluoroethyl) -7-oxabicyclo [2.2.1] hept-5-ene) -2-sulfonamido) phenoxy) octanamide (20 d):
Figure BDA0003262513330000162
under Ar protection, compound 18d (100mg, 0.15mmol), DCC (92mg, 0.44mmol) and HOBT (27mg, 0.18mmol) were added to a two-necked flask and dissolved in DMF. After 10min, adding amantadine (34mg, 0.22mmol), stirring at room temperature for about 4h, TLC confirming reaction completion, adding water for quenching, EA extracting, washing organic phase with supersaturated sodium bicarbonate for multiple times, concentrating, spin drying, and separating and purifying by silica gel column chromatography (eluent ratio v is v)Methylene dichloride/vMethanol150:1-30:1) to yield 131mg of a pale yellow solid 20d, 70% yield.1H NMR(400MHz,MeOD-d6)δ7.20(dd,J＝9.0,2.3Hz,2H),7.14(t,J＝9.3Hz,4H),6.81–6.76(m,4H),6.71(d,J＝8.7Hz,2H),5.46(s,1H),5.30(d,J＝4.0Hz,1H),4.42(q,J＝8.5Hz,2H),4.02–3.93(m,2H),3.44(dt,J＝8.6,4.4Hz,1H),2.29–2.15(m,1H),2.15–2.05(m,1H),1.98(s,9H),1.83(dd,J＝28.9,13.6Hz,2H),1.68(s,6H),1.40–1.34(m,2H).
Example 15: relative affinity assay for Compounds
The affinity of the target compound for ER alpha and ER beta is determined by fluorescence polarization method, and the affinity of the compound is endogenous E2Relative value of affinity, set E2The value of the affinity for the receptor RBA is 100%. To a 384 well plate, 20. mu.L of a potassium phosphate buffer composed of 0.8. mu.MER. alpha. or ER. beta. protein, 150nM fluorescent ligand and 2.4. mu.g bovine immunoglobulin was added, and then 20. mu.L of a target compound solution was added, with a compound concentration gradient of 3.16X 10-4M、1×10-4M、3.16×10-5M、1×10-5M、3.16×10-6M、1×10-6M、3.16×10-7M、1×10-7M、3.16×10-8M、1×10-8M、3.16×10-9And M. Placing the plate at room temperature in a dark place for 2 hours, reading the plate on an enzyme labeling instrument, selecting a wavelength at 485nm as a main wavelength and a wavelength at 528nm as a reference wavelength, analyzing an experimental result, and determining the receptor affinity RBA (RBA) as a test substance K according to a formulaiEstradiol KiX 100 the RBA (relative binding affinity) value for each compound was calculated and the results are shown in Table 1.
TABLE 1 Relative Binding Affinities (RBA) of target compounds 20a-d and target compounds 30a-j for ER α and ER βa
Figure BDA0003262513330000171
Figure BDA0003262513330000172
aRelative binding affinity was tested by fluorescence polarization, expressed as IC50estradiol/IC50Compound x 100 ± standard deviation (binding affinity of estradiol is set as 100%).
This example tested the 14 compounds for binding affinity to era and the results are listed in table 1. In compounds 20a-d and 30a-j, either the introduction of the adamantane side chain or Boc3Introduction of Arg side chain, and the obtained final product is all directed to ER alphaHas certain binding force. Wherein, contains Boc3The RBA value for era for compound 30a of the Arg side chain reached 3.21, which may be related to the correct positioning of the compound in the era pocket, with the side chain moiety exposed outside the pocket. Introduction of other hydrophobic tags, e.g. L-Boc3-Arg、L-Boc-Phe、L-Boc-Leu、L-Boc2-Lys, L-Boc-Trp, all have a relatively good affinity, with 30j having the highest RBA for ER α of 9.76.
In addition, the obtained compounds 20a-d and 30a-j can keep the characteristic that the parent compound OBHSA is used as an ER ligand, and lay a foundation for the compounds to exert the biological effects.
Example 16: cell viability assay
MCF-7 cells were cultured in DMEM liquid medium with phenol red containing 10% fetal bovine serum. When the cell density reached 80% -90%, the cells were digested and the cell suspension was plated in 96-well cell culture plates with phenol red-free DMEM medium containing 10% FBS. After the cells were completely attached to the wall, the original culture solution was discarded, and 100. mu.L of fresh compound solution prepared in 10% FBS-containing DMEM medium was added to each well, with a compound concentration gradient of 1X 10-7.5M、1×10-7M、1×10-6.5M、1×10-6M、1×10- 5.5M、1×10-5M、1×10-4.5M、1×10-4And M. After 4 days of drug treatment, the plates were removed, the medium aspirated, 100. mu.L of CCK8 medium added to each well, and the plates were incubated at 37 ℃ with 5% CO2Incubating in the incubator for 1.5-2 hours. Reading the plate on a microplate reader, selecting the wavelength of 450nm as the reference wavelength, analyzing the experimental result, and calculating IC50The results are shown in Table 2.
TABLE 2 Effect of target Compounds 20a-d and 30a-j on Breast cancer cell MCF-7 cell viability
Figure BDA0003262513330000181
Figure BDA0003262513330000191
aIC50Is the mean ± standard deviation of at least three independent experiments.
This example evaluated the effect of these 14 compounds on MCF-7 cell viability. Compounds 20a-d with adamantane side chains all had some effect on MCF-7 cell viability, whereas, of these, compounds 20a and 20b had the greatest effect, both compounds having IC' s50Values were around 21 and 40 nM. While the inhibition of MCF-7 cell viability by the adamantane bearing OBHSA derivatives 20c and 20d was gradually diminished with further increase in the compound side chain length, indicating that the side chain length has a critical effect on the activity when a hydrophobic tag was introduced.
When Boc is reacted3The Arg label is introduced into the OBHSA side chain to obtain a target product 30a, and the compound 30a also has good cell inhibition activity on MCF-7 and IC thereof50The value was 16nM, which indicates Boc3Arg can also be used as an excellent group for the modification of a targeting ER compound to obtain a sprout head molecule with better activity for inhibiting MCF-7 cell viability. By testing the cell activity of hydrophobic amino acids with different side chains, it was found that the cell activity at a chain length of 6, where R is L-Boc, was generally superior to that of 32-Lys and L-Boc-Trp, the cellular activity of the compounds was at 4.17 and 5.77 nM.
Example 17: ER degradation Activity assay
Figure BDA0003262513330000192
In order to demonstrate the capability of the synthesized OBHSA derivatives with hydrophobic tags to degrade estrogen receptors, the influence of chain length and different types of hydrophobic tags on the protein degradation activity was analyzed. This example treated MCF-724 h with fulvestrant (Ful) and five compounds 20a-d, 30a (5. mu.M) and then extracted all proteins and analysed by western blotting for the effect of compounds 20a-d and also 30a on the degradation of ER α and compared with fulvestrant. The results are shown in FIG. 3.
As can be seen from FIG. 3, this seriesThe compounds have good ER alpha down-regulation activity, wherein the compounds 20a, 20b and 30a and fulvestrant can completely degrade ER alpha at 5 mu M. In addition, even though compound 20d had poor inhibitory activity on MCF-7 cell viability, it was able to degrade most ER α at 5 μ M. It can also be seen from the results that the degradation activity of compounds 20a-d on ER α is also related to the length of its side chain, and that the degradation activity of the target product decreases when the chain length increases to a certain length. Therefore, the appropriate side chain length is also important for the ER α degradation activity. By comparing adamantyl containing tags with Boc3Arg-tagged two classes of compounds, the introduction of Boc was found3The Arg tag also allows the compound to obtain better ER alpha degrading activity, and the compound 30a can completely degrade ER alpha at the same concentration.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims (9)

1. An oxo-bridged bicycloheptene sulfonamide compound containing a hydrophobic label is characterized in that: comprises an oxo-bridged bicycloheptene sulfonamide compound containing a amantadine label and an oxo-bridged bicycloheptene sulfonamide compound containing a hydrophobic amino acid label:
the oxo-bridged bicycloheptene sulfonamide compound containing the amantadine label has a structure shown in a general formula I:
Figure FDA0003262513320000011
in the general formula I, n1Is a natural number of 1-60;
the oxo-bridged bicycloheptene sulfonamide compound containing the hydrophobic amino acid label has a structure shown as a general formula II:
Figure FDA0003262513320000012
in the general formula II, n2Is a natural number of 1-60; r is hydrophobic amino acid.
2. The hydrophobic tag containing oxido-bicycloheptene sulfonamide compound of claim 1, wherein: n is1Is 1, 3, 5, 7, n2Are 3 and 6.
3. The hydrophobic tag containing oxido-bicycloheptene sulfonamide compound of claim 1, wherein: the hydrophobic amino acid comprises D-Arg, L-Phe, L-Leu, L-Lys and L-Trp.
4. The hydrophobic tag containing oxido-bicycloheptene sulfonamide compound of claim 1, wherein:
the oxo-bridged bicycloheptene sulfonamide compound containing the amantadine label is selected from the following compounds:
n- ((3s,5s,7s) -adamantan-1-yl) -2- (4- ((1R,4R) -5, 6-bis (4-hydroxyphenyl) -N- (2,2, 2-trifluoroethyl) -7-oxabicyclo [2.2.1] hept-5-ene) -2-sulfonamido) phenoxy) acetamide,
N- ((3s,5s,7s) -adamantan-1-yl) -4- (4- ((1R,4R) -5, 6-bis (4-hydroxyphenyl) -N- (2,2, 2-trifluoroethyl) -7-oxabicyclo [2.2.1] hept-5-ene) -2-sulfonamido) phenoxy) butanamide,
N- ((3s,5s,7s) -adamantan-1-yl) -6- (4- ((1R,4R) -5, 6-bis (4-hydroxyphenyl) -N- (2,2, 2-trifluoroethyl) -7-oxabicyclo [2.2.1] hept-5-ene) -2-sulfonamido) phenoxy) hexanamide,
N- ((3s,5s,7s) -adamantan-1-yl) -8- (4- ((1R,4R) -5, 6-bis (4-hydroxyphenyl) -N- (2,2, 2-trifluoroethyl) -7-oxabicyclo [2.2.1] hept-5-ene) -2-sulfonamido) phenoxy) octanamide;
the oxo-bridged bicycloheptene sulfonamide compound containing the hydrophobic amino acid label is selected from the following compounds:
(2S) -2-amino-N- (3- (4- ((5, 6-bis (4-hydroxyphenyl) -N- (2,2, 2-trifluoroethyl) -7) - [2.2.1] hept-5-ene) -2-sulfonamide (phenoxy) propyl) -5-guanidinopentaneamide derivatives,
(2R) -2-amino-N- (6- (4- ((5, 6-bis (4-hydroxyphenyl) -N- (2,2, 2-trifluoroethyl) -7) - [2.2.1] hept-5-ene) -2-sulfonamide (phenoxy) hexyl) -5-guanidinopentaneamide derivatives,
Tert-butyl ((2S) -1- ((3- (4- ((1R,4R) -5, 6-bis (4-hydroxyphenyl) -N- (2,2, 2-trifluoroethyl) -7-oxabicyclo [2.2.1] hept-5-ene) -2-sulfonamido) phenoxy) propyl) amino) -1-oxo-3-phend-en-2-yl) carbamate,
Tert-butyl ((2R) -1- ((6- (4- ((1R,4R) -5, 6-bis (4-hydroxyphenyl) -N- (2,2, 2-trifluoroethyl) -7-oxabicyclo [2.2.1] hept-5-ene) -2-sulfonamido) phenoxy) hexyl) amino) -1-oxo-3-phend-en-2-yl) carbamate,
Tert-butyl ((2S) -1- ((3- (4- ((1R,4R) -5, 6-bis (4-hydroxyphenyl) -N- (2,2, 2-trifluoroethyl) -7-oxa-cyclo [2.2.1] hept-5-en) -2-sulfonamido) phenoxy) propyl) amino) -4-methyl-1-oxopentan-2-yl) carbamate,
Tert-butyl ((2R) -1- ((6- (4- ((1R,4R) -5, 6-bis (4-hydroxyphenyl) -N- (2,2, 2-trifluoroethyl) -7-oxabicyclo [2.2.1] hept-5-ene) -2-sulfonamido) phenoxy) hexyl) amino) -4-methyl-1-oxopentan-2-yl) carbamate,
Di-tert-butyl ((5S) -6- ((3- (4- ((1R,4R) -5, 6-bis (4-hydroxyphenyl) -N- (2,2, 2-trifluoroethyl) -7-oxabicyclo [2.2.1] hept-5-ene) -2-sulfonamido) phenoxy) propyl) amino) -6-oxahexane-1, 5-diyl) dicarbamate,
Di-tert-butyl ((5R) -6- ((6- (4- ((1R,4R) -5, 6-bis (4-hydroxyphenyl) -N- (2,2, 2-trifluoroethyl) -7-oxabicyclo [2.2.1] hept-5-ene) -2-sulfonamido) phenoxy) hexyl) amino) -6-oxohexane-1, 5-diyl) dicarbamate,
Tert-butyl ((2S) -1- ((3- (4- ((1R,4R) -5, 6-bis (4-hydroxyphenyl) -N- (2,2, 2-trifluoroethyl) -7-oxabicyclo [2.2.1] hept-5-ene) -2-sulfonamido) phenoxy) propyl) amino) -3- (1H-indol-2-yl) -1-oxopropan-2-yl) carbamate,
Tert-butyl ((2R) -1- ((6- (4- ((1R,4R) -5, 6-bis (4-hydroxyphenyl) -N- (2,2, 2-trifluoroethyl) -7-oxabicyclo [2.2.1] hept-5-ene) -2-sulfonamido) phenoxy) hexyl) amino) -3- (1H-indol-2-yl) -1-oxopropan-2-yl) carbamate.
5. Use of the oxo-bridged bicycloheptene sulfonamides containing hydrophobic tags as claimed in any one of claims 1 to 4 in the manufacture of a medicament for the treatment of breast cancer.
6. Use of the oxido-bicycloheptenesulfonamide compounds containing hydrophobic tags according to any one of claims 1 to 4 for the preparation of a medicament for the targeted degradation of estrogen receptors.
7. An anti-breast cancer drug, which is characterized in that: a compound comprising the hydrophobic tag-containing oxido-bicycloheptenesulfonamide of any one of claims 1 to 4.
8. A drug for targeted degradation of an estrogen receptor, comprising: a compound comprising the hydrophobic tag-containing oxido-bicycloheptenesulfonamide of any one of claims 1 to 4.
9. The medicament according to claim 7 or 8, characterized in that: comprising one or more pharmaceutically acceptable carriers or excipients of a compound according to any one of claims 1 to 4.
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