CN117447471A - 一种吲哚二酮哌嗪类化合物及其制备方法和在制备破骨细胞分化抑制剂中的应用 - Google Patents
一种吲哚二酮哌嗪类化合物及其制备方法和在制备破骨细胞分化抑制剂中的应用 Download PDFInfo
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- CN117447471A CN117447471A CN202311457989.4A CN202311457989A CN117447471A CN 117447471 A CN117447471 A CN 117447471A CN 202311457989 A CN202311457989 A CN 202311457989A CN 117447471 A CN117447471 A CN 117447471A
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- indole
- diketopiperazine compound
- indole diketopiperazine
- prenylcyclotryprostatin
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- A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
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- A61P35/00—Antineoplastic agents
- A61P35/04—Antineoplastic agents specific for metastasis
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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Abstract
本发明提供一种吲哚二酮哌嗪类化合物及其制备方法和在制备破骨细胞分化抑制剂中的应用，本发明公开了一种吲哚二酮哌嗪类化合物，其命名为prenylcyclotryprostatinB，其结构式如式(Ⅰ)所示，其在10μM浓度下对LPS诱导的NF‑κB荧光素酶具显著抑制作用，能显著抑制RANKL诱导的破骨前体细胞BMMs分化成破骨细胞，且无明显的细胞毒性，是开发成为新型破骨细胞分化抑制剂类药物的理想候选化合物。
Description
技术领域
本发明涉及海洋药物领域，具体涉及一种吲哚二酮哌嗪类化合物及其制备方法和在制备破骨细胞分化抑制剂中的应用。
背景技术
骨质疏松症是一种常见的骨骼退行性疾病，主要表现为骨脆性增加、骨量减少和骨微结构破坏，严重威胁老龄化人群和绝经后女性的健康。破骨细胞(osteoclasts，OCs)是由单核细胞/巨噬细胞造血谱系前体细胞融合形成的特殊细胞，是人体内唯一具有骨吸收功能的细胞。破骨细胞活性缺陷导致骨硬化和骨髓衰竭，过度激活可导致骨质疏松症、类风湿性关节炎、肿瘤骨转移等溶骨性疾病，抑制OCs的形成和再吸收功能是治疗骨质疏松症的主要策略之一。
破骨细胞的形成是一个逐步过程，由核因子-κB受体活化因子配体(Receptoractivator of nuclear kappa B ligand，RANKL)的受体激活剂与其在单核细胞/巨噬细胞前体上的受体RANK结合而启动。目前临床上运用的破骨细胞分化相关抑制剂主要为地诺昔单抗和双膦酸盐类药物，但都具有一定的并发症和副作用。关于海洋来源的抗破骨细胞分化抑制剂的研究报道较少，仅仅公开报道有部分化合物具有抗破骨细胞分化作用，如中国专利申请1：CN201610478740.5，“海洋来源硝基苯酯倍半萜类化合物在制备破骨细胞分化抑制剂的应用”；中国专利申请2：CN202310241947.0，“海洋真菌来源开环吲哚二萜类化合物peniditerpenoid A及其制备方法和应用”；中国专利申请3：CN202210093211.9，“氯代间苯二酚醛类化合物及其在制备破骨细胞分化抑制剂中的应用”，中国专利申请4：CN201910176291.2，“一种萜类衍生物及其制备方法与应用”。目前尚未发现有吲哚二酮哌嗪类化合物具有抗破骨细胞分化的作用，也无公开报道有相关的应用。因此，进一步发掘更多的，具有安全、有效、质量可控、经济的靶向抑制破骨细胞形成和骨吸收的药物，对解决临床上对于破骨细胞分化抑制剂的需求具有巨大的意义，同时，还可提高吲哚二酮哌嗪类化合物的应用价值。
发明内容
本发明针对上述问题，提供一种吲哚二酮哌嗪类化合物及其制备方法和和在制备破骨细胞分化抑制剂中的应用。
为达到上述目的，本发明所采用的技术方案是：
一种吲哚二酮哌嗪类化合物，其命名为prenylcyclotryprostatin B；所述prenylcyclotryprostatin B的结构式如式(Ⅰ)所示：
本发明的另一个目的还在于保护一种用于制备上述的吲哚二酮哌嗪类化合物的菌株布雷菲德氏青霉(Penicillium brefeldianum)GXIMD 02511，于2022年09月27日保藏于广东省微生物菌种保藏中心(GDMCC)，地址：广东省广州市先烈中路100号广东省微生物研究所59号楼五楼，广东省微生物研究所，保藏编号为：GDMCC No.62843。
本发明的另一个目的还在于保护一种制备吲哚二酮哌嗪类化合物的方法，所述的吲哚二酮哌嗪类化合物是从菌株布雷菲德氏青霉(Penicillium brefeldianum)GXIMD02511的发酵培养物中制备分离得到的。
进一步的，所述方法包括如下步骤：
S1发酵培养物的制备：制备菌株布雷菲德氏青霉(Penicillium brefeldianum)GXIMD 02511的发酵培养物；
S2提取：用乙酸乙酯浸泡发酵培养物，再将发酵培养物切碎或粉碎后进行超声提取，过滤分别得到滤渣和滤液；滤液和滤渣分别用乙酸乙酯提取后浓缩去除乙酸乙酯，合并提取获得的浸膏；
S3分离和纯化：将浸膏经中压正相液相色谱，用石油醚/二氯甲烷作为洗脱剂，从体积比100：0～0：10进行梯度洗脱，收集石油醚：二氯甲烷体积比为90：10梯度洗脱下来的流份，继续过中压反相C18柱色谱，用甲醇/水作为洗脱剂，从体积比10：90～100：0进行梯度洗脱，收集甲醇：水体积比30：70梯度洗脱下来的流份，流份再经纯化后得到吲哚二酮哌嗪类化合物。
进一步的，S1中，所述发酵培养物由如下方法发酵制备得到：将活化的菌株布雷菲德氏青霉(Penicillium brefeldianum)GXIMD 02511接入种子培养基中，于25℃，180rpm条件下动态培养72h制得种子液；将所述种子液以5％的接种量接入到发酵培养基中，于25℃温度下，静态培养30天制得发酵培养物；所述种子培养基配方为每1L培养基中含有：麦芽提取粉15g，余量为水，pH 7.5；所述发酵培养基的配方为每1L三角瓶培养基中含有：大米200g，海盐2g，水200mL，pH 7.5。
本发明的另一个目的还在于保护所述的吲哚二酮哌嗪类化合物在制备破骨细胞分化抑制剂中的应用。
进一步的，所述的破骨细胞分化抑制剂为治疗破骨细胞过度活化造成的溶骨性疾病的药物。
进一步的，所述的破骨细胞分化抑制剂为治疗骨质疏松、类风湿性关节炎、肿瘤转移骨破坏的药物。
本发明的另一个目的还在于保护所述吲哚二酮哌嗪类化合物在制备NF-κB核因子表达抑制剂药物中的应用。
本发明的另外一个目的是提供一个药物组合物，药物组合物可以为NF-κB核因子表达抑制剂或破骨细胞分化抑制剂药物，所述药物组合物包括有效量的吲哚二酮哌嗪类化合物和/或药用盐和药学上可以接受的载体或辅剂。
本发明通过试验得到：吲哚二酮哌嗪类化合物prenylcyclotryprostatin B在10μM浓度下对LPS诱导的NF-κB荧光素酶具有抑制作用(p<0.001)，可以作为研制NF-κB核因子表达抑制剂的先导化合物，并且可以显著抑制RANKL诱导的破骨前体细胞BMMs(Bonemarrow macrophage cells，骨髓巨噬细胞)分化成破骨细胞，且无明显细胞毒性，因此可望开发成为安全有效的新型破骨细胞分化抑制剂药物。
由于采用上述技术方案，本发明具有以下有益效果：
本发明在对广西北仑河口红树林保护区根际底泥来源布雷菲德氏青霉(Penicillium brefeldianum)GXIMD 02511的次生代谢产物的研究过程中，分离获得一种吲哚二酮哌嗪类化合物prenylcyclotryprostatin B，该化合物对LPS诱导的NF-κB荧光素酶具显著抑制作用，呈剂量依赖地显著抑制RANKL诱导的破骨前体细胞BMMs分化成破骨细胞，无细胞毒性。因此是开发成为新型NF-κB核因子表达抑制剂或破骨细胞分化抑制剂药物的理想候选化合物。
附图说明
图1是吲哚二酮哌嗪类化合物prenylcyclotryprostatin B在10μM浓度下对在RAW264.7细胞经脂多糖(LPS)诱导的NF-κB荧光素酶的抑制活性对比图，BAY是阳性对照，###p<0.001vs.control group；***p<0.001vs.LPS group；
图2是吲哚二酮哌嗪类化合物prenylcyclotryprostatin B对小鼠骨髓巨噬细胞(BMMs)的细胞活力影响；
图3是吲哚二酮哌嗪类化合物prenylcyclotryprostatin B对破骨前体细胞BMMs分化成破骨细胞的影响的实验结果，其中：RANKL为激活核因子NF-κB受体的配体，与空白对照组比较，###P<0.001；与RANKL组比较，*P<0.05，**P<0.01；
具体实施方式
为了使本发明的目的、技术方案及技术效果更加清晰，以下结合实施例和附图，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅用以解释本发明，并不用于限定本发明。
实施例1
一种吲哚二酮哌嗪类化合物，所述吲哚二酮哌嗪类化合物的命名为prenylcyclotryprostatin B；所述prenylcyclotryprostatin B的结构式如式(Ⅰ)所示：
实施例2菌株布雷菲德氏青霉(Penicillium brefeldianum)GXIMD 02511
从采自中国广西北仑河口红树林保护区根际底泥分离得到菌株布雷菲德氏青霉(Penicillium brefeldianum)GXIMD 02511，于2022年09月27日保藏于广东省微生物菌种保藏中心(GDMCC)，地址：广东省广州市先烈中路100号广东省微生物研究所59号楼五楼，广东省微生物研究所，保藏编号为：GDMCC No.62843。
本实施例中的菌株布雷菲德氏青霉(Penicillium brefeldianum)GXIMD 02511可以用于制备实施例1中的吲哚二酮哌嗪类化合物prenylcyclotryprostatin B。
实施例3吲哚二酮哌嗪类化合物prenylcyclotryprostatin B的制备和分离
1、培养基
1.1、种子培养基：每1L升培养基中含有麦芽提取粉15g，余量为水，pH 7.5。按上述组份和含量混合均匀，然后121℃，灭菌30min备用。
1.2、发酵培养基：每1L三角瓶培养基中含有：大米200g，海盐2g，水200mL，pH 7.5。按上述组份和含量混合均匀，然后121℃，灭菌30min备用。
2、发酵
2.1、种子培养：将活化的菌株布雷菲德氏青霉(Penicillium brefeldianum)GXIMD 02511接入每瓶含有300mL种子培养基的1L的三角培养瓶中，25℃，180rpm，培养72h制得种子液。
2.2、发酵培养：将种子液以5％的接种量(体积百分比)接入到120瓶发酵培养基三角瓶中，25℃，静态培养100d，制得发酵培养物。
3、提取：用2倍体积乙酸乙酯浸泡发酵培养物近24h，然后将其切碎或捣碎后置超声仪中超声提取20min，用8层纱布进行过滤分别得到滤液和滤渣。滤液经旋转蒸发仪浓缩除去有机溶剂后用乙酸乙酯萃取多次至水相颜色变浅，浓缩除去乙酸乙酯。滤渣继续用乙酸乙酯提取多次后浓缩除去乙酸乙酯，最后合并两份棕黄色浸膏约132g。
4、吲哚二酮哌嗪类化合物prenylcyclotryprostatin B的分离纯化
将浸膏(132g)经中压正相液相色谱，用石油醚/二氯甲烷作为洗脱剂，从体积比(100：0)～(0：100)进行梯度洗脱，收集石油醚/二氯甲烷体积比90：10梯度洗脱下来的流份，继续过中压反相C18柱色谱，用甲醇/水作为洗脱剂，从体积比(10：90)～(100：0)进行梯度洗脱，收集甲醇/水体积比30：70梯度洗脱下来的流份，该流份最后用半制备高效液相精细分离，在洗脱体系为甲醇/水(体积比50：50，YMC-pack ODS-A色谱柱,10×250mm,5μm,2mL/min)进行纯化后得到吲哚二酮哌嗪类化合物prenylcyclotryprostatin B(3mg)。
对上述提取分离得到的吲哚二酮哌嗪类化合物prenylcyclotryprostatin B进行结构鉴定prenylcyclotryprostatin B为白色粉末，1H NMR谱图中可推测出该化合物含一个1,2,4-三取代苯环(δH7.46,6.81,6.74)，4个甲基(δH 2.14,2.00,1.83,1.76)，2个甲氧基(δH 3.84,3.67)，以及4个亚甲基和甲胺基团的信号。13C NMR谱图显示28个碳信号，包括6个甲基，3个芳香碳，4个亚甲基，5个次甲基和10个非质子化碳。核磁数据归属如下：
1H NMR(700MHz,CDCl3)δH 7.46(1H,dd,J＝8.6,2.7Hz,H-4),6.81(1H,dd,J＝8.6,2.2Hz,H-5),6.74(1H,d,J＝2.2Hz,H-7),6.73(1H,d,J＝9.7Hz,H-18),5.56(1H,m,H-19),5.12(1H,d,J＝5.9Hz,H-24),4.72(1H,s,H-8),4.62(1H,dd,J＝16.0,6.4Hz,H-23a),4.52(1H,dd,J＝16.0,6.4Hz,H-23b),4.48(1H,br s,9-OH),4.41(1H,dd,J＝10.8,6.2Hz,H-12),3.84(3H,s,6-OCH3),3.75(2H,m,H2-15),3.67(3H,s,8-OCH3),2.49(1H,m,H-13a),2.14(1H,m,H-14a),2.00(3H,s,H3-22),1.98(1H,m,H-13b),1.98(1H,m,H-14b),1.83(3H,s,H3-27),1.76(3H,s,H3-21),1.71(3H,s,H3-26)；13C NMR(175MHz,CDCl3)δC 165.7(C-11),164.7(C-17),156.8(C-6),138.2(C-20),137.9(C-7a),134.9(C-25),133.4(C-2),123.9(C-19),120.4(C-3a),120.0(C-24),118.7(C-4),109.9(C-5),104.5(C-3),94.5(C-7),85.7(C-9),77.2(C-8),60.2(C-12),59.2(OCH3-8),55.9(OCH3-6),48.3(C-18),45.9(C-15),42.3(C-23),30.0(C-13),26.2(C-21),25.6(C-26),22.04(C-14),18.62(C-22),18.35(C-27).
上述数据与文献(Chemistry&Biodiversity,2012,9:385-393.)报道的基本一致，故鉴定为异戊烯基化的吲哚二酮哌嗪类化合物prenylcyclotryprostatin B。
在上述基础上，本发明还进行如下实验：
实验一：吲哚二酮哌嗪类化合物prenylcyclotryprostatin B对LPS诱导的NF-κB荧光素酶抑制活性测定
NF-κB荧光素酶抑制活性测定主要参考文献(Fitoterapia,2022,159:105201.)。
取稳定转染NF-κB荧光素酶报告基因的RAW264.7细胞接种于96孔板中(1×104个/孔)，每孔加入含10％胎牛血清、100IU/mL青霉素和链霉素和0.1μg/mL的G418的DMEM培养基200μL，待细胞贴壁稳定后，加入吲哚二酮哌嗪类化合物prenylcyclotryprostatin B，设置6个复孔。继续孵育4h后，除阴性对照组外，每个化合物组(3孔)和阳性对照组(NF-κB抑制剂，BAY11-7082，5μM)分别加入LPS和RANKL，使其每孔终浓度为100ng/mL，两者刺激8h后，弃掉上清液，每孔加入细胞裂解液25μL，低速震荡10min以充***解细胞，然后取20μL转移至白板中，每孔加入荧光素溶液50μL，用多功能酶标仪检测Luciferase值。
试验结论：结果如图1所示，研究发现与LPS空白组对比，吲哚二酮哌嗪类化合物prenylcyclotryprostatin B在10μM对LPS诱导的NF-κB荧光素酶具有中等抑制作用(p<0.001)。
实验二：吲哚二酮哌嗪类化合物prenylcyclotryprostatin B对破骨前体细胞BMMs活力影响
采用CCK-8法检测吲哚二酮哌嗪类化合物prenylcyclotryprostatin B对小鼠骨髓巨噬细胞(BMMs)的活力影响
a.小鼠骨髓巨噬细胞(BMMs)的制备：在无菌条件下，取8-12周龄C57BL/6雌性小鼠的股骨，剪断股骨两端的关节部位，用无酚红α-MEM培养基(含10％胎牛血清、100IU/mL青霉素和100IU/mL链霉素)反复冲洗股骨，直至股骨腔发白。将冲洗出的股骨骨髓腔细胞置于37℃、5％CO2细胞培养箱孵育2h，吸取上清，用裂解液裂解红细胞，离心，重悬即得BMMs。
b.CCK-8法检测细胞存活情况：
取步骤a制备得到的BMMs(1×105个/孔)于96孔板中，每孔加入无酚红α-MEM培养基至200μL(含10％胎牛血清、100IU/mL青霉素和100IU/mL链霉素)，同时每孔加入巨噬细胞集落刺激因子(M-CSF，终浓度为50ng/mL)，然后将96孔板置于37℃、5％CO2的细胞培养箱进行孵育过夜。待细胞贴壁稳定后，分别加入不同浓度的吲哚二酮哌嗪类化合物prenylcyclotryprostatin B，使孔中的吲哚二酮哌嗪类化合物prenylcyclotryprostatinB终浓度为10μM和15μM，每组设3个复孔，孵育4天。孵育完成后，弃掉上清(100μL)，每孔加入5μL的CCK-8试剂(Cell Counting Kit-8细胞计数试剂)，摇匀，置于37℃、5％CO2环境下继续孵育3h，用TECANGENiosPro多功能酶标仪测定450nm波长处的光密度值(OD值)，计算各组细胞生存率。
结果如图2所示，在加入10μM和15μM的吲哚二酮哌嗪类化合物prenylcyclotryprostatin B后，BMMs细胞存活率未发生显著差异，说明在体外试验中吲哚二酮哌嗪类化合物prenylcyclotryprostatin B对BMMs无细胞毒性。
实验三：吲哚二酮哌嗪类化合物prenylcyclotryprostatin B对RANKL诱导的破骨前体细胞BMMs分化成破骨细胞的影响
RANKL诱导的破骨前体BMMs细胞分化抑制活性测定主要参考文献(Fitoterapia,2022,159:105201.)。
取上述步骤a制备得到的BMMs(2×104个/孔)于96孔板中，每孔加入无酚红α-MEM培养基至200μL(含10％胎牛血清、100IU/mL青霉素和100IU/mL链霉素)，同时每孔加入巨噬细胞集落刺激因子(M-CSF，终浓度为50ng/mL)，然后将96孔板置于37℃、5％CO2的细胞培养箱进行孵育过夜。待细胞贴壁稳定后，分别加入不同浓度的吲哚二酮哌嗪类化合物prenylcyclotryprostatin B，使孔中的吲哚二酮哌嗪类化合物prenylcyclotryprostatinB终浓度为5μM，10μM和15μM，每组设3个复孔，孵育4h。孵育完成后，加入RANKL(终浓度为100ng/mL)，培养3-4d。对孵育完成的细胞进行TRAP染色，在倒置显微镜下拍照并计数，其中细胞核大于5个的TRAP阳性细胞即为破骨细胞。
结果如图3所示，吲哚二酮哌嗪类化合物prenylcyclotryprostatin B能显著抑制RANKL诱导BMMs生成破骨细胞。在10μM有效浓度时即可显著抑制RANKL诱导破骨前体细胞BMMs生成破骨细胞。
综合上述结果可知，吲哚二酮哌嗪类化合物prenylcyclotryprostatin B对LPS诱导的NF-κB荧光素酶具显著抑制作用，且在10μM浓度下能显著抑制RANKL诱导的破骨前体细胞BMMs分化成破骨细胞，能体外能显著抑制破骨细胞的生成和活化，且无明显毒性作用，可以作为新型破骨细胞分化抑制剂或NF-κB核因子表达抑制剂进行开发，用于防治骨质疏松症等骨溶性疾病。
基于上述实验可以证明本发明的两个吲哚二酮哌嗪类化合物prenylcyclotryprostatin B可以在制备抗破骨细胞分化抑制剂或NF-κB核因子表达抑制剂药物中进行应用。所述的破骨细胞分化抑制剂或NF-κB核因子表达抑制剂药物为口服制剂、注射剂型或外用剂型；包括活性成分吲哚二酮哌嗪类化合物prenylcyclotryprostatinB和医学上可接受的药用辅料。抗破骨细胞分化类药物可以治疗包括但不限于：绝经后骨质疏松、肿瘤转移骨破坏等已知的破骨细胞抑制剂类药物如双磷酸盐、地诺昔单抗被批准治疗的临床适应症。
通常而言，作为药物，均是在制备成制剂后才临床应用。本发明所述的有效活性成分吲哚二酮哌嗪类化合物，作为有效活性成分吲哚二酮哌嗪类化合物可根据本领域公知的方法制备。可通过将本发明有效活性成分吲哚二酮哌嗪类化合物与一种或多种药学上可接受的固体或液体赋形剂和/或辅剂结合，制成适于人或动物使用的任何剂型。本发明有效活性成分吲哚二酮哌嗪类化合物或含有它的有效活性成分吲哚二酮哌嗪类化合物可以单位剂量形式给药，给药途径可为肠道或非肠道，如口服、静脉注射、肌肉注射、皮下注射、鼻腔、口腔粘膜、眼、肺和呼吸道、皮肤、***、直肠等。
口服制剂优先为胶囊剂。为了将给药单元制成胶囊剂，可以将有效成分本发明有效活性成分苯乙酸类化合物与稀释剂、助流剂混合，将混合物直接置于硬胶囊或软胶囊中。也可将有效成分本发明有效活性成分吲哚二酮哌嗪类化合物先与稀释剂、黏合剂、崩解剂制成颗粒或微丸，再置于硬胶囊或软胶囊中。用于制备本发明有效活性成分吲哚二酮哌嗪类化合物片剂的各稀释剂、黏合剂、润湿剂、崩解剂、助流剂品种也可用于制备本发明有效活性成分吲哚二酮哌嗪类化合物的胶囊剂。
为将本发明有效活性成分吲哚二酮哌嗪类化合物制成注射剂，可以用水、乙醇、异丙醇、丙二醇或它们的混合物作溶剂并加入适量本领域常用的增溶剂、助溶剂、pH调剂剂、渗透压调节剂。增溶剂或助溶剂可以是泊洛沙姆、卵磷脂、羟丙基-β-环糊精等；pH调剂剂可以是磷酸盐、醋酸盐、盐酸、氢氧化钠等；渗透压调节剂可以是氯化钠、甘露醇、葡萄糖、磷酸盐、醋酸盐等。如制备冻干粉针剂，还可加入甘露醇、葡萄糖等作为支撑剂。
综上，本发明为研制新型破骨细胞分化抑制剂或NF-κB核因子表达抑制剂药物提供了新的候选化合物，对中国自主知识产权的新药开发具有重要的意义。
上述说明是针对本发明较佳可行实施例的详细说明，但实施例并非用以限定本发明的专利申请范围。凡本发明所提示的技术构思下所完成的同等变化或修饰变更，均应属于本发明所涵盖专利范围。
Claims (10)
1.一种吲哚二酮哌嗪类化合物，其特征在于，所述的吲哚二酮哌嗪类化合物的命名为prenylcyclotryprostatin B；所述prenylcyclotryprostatin B的结构式如式(Ⅰ)所示：
2.一种用于制备权利要求1所述的吲哚二酮哌嗪类化合物的菌株布雷菲德氏青霉(Penicillium brefeldianum)GXIMD 02511，其保藏编号为：GDMCC NO.62843。
3.一种制备权利要求1所述的吲哚二酮哌嗪类化合物的方法，其特征在于，所述的吲哚二酮哌嗪类化合物是从菌株布雷菲德氏青霉(Penicillium brefeldianum)GXIMD 02511的发酵培养物中制备分离得到的。
4.根据权利要求3所述的方法，其特征在于，所述方法包括如下步骤：
S1发酵培养物的制备：制备菌株布雷菲德氏青霉(Penicillium brefeldianum)GXIMD02511的发酵培养物；
S2提取：用乙酸乙酯浸泡发酵培养物，再将发酵培养物切碎或粉碎后进行超声提取，过滤分别得到滤渣和滤液；滤液和滤渣分别用乙酸乙酯提取后浓缩去除乙酸乙酯，合并提取获得的浸膏；
S3分离和纯化：将浸膏经中压正相液相色谱，用石油醚/二氯甲烷作为洗脱剂，从体积比100：0～0：10进行梯度洗脱，收集石油醚：二氯甲烷体积比为90：10梯度洗脱下来的流份，洗脱下来的流份继续过中压反相C18柱色谱，用甲醇/水作为洗脱剂，从体积比10：90～100：0进行梯度洗脱，收集甲醇：水体积比30：70梯度洗脱下来的流份，流份再经纯化后得到所述吲哚二酮哌嗪类化合物。
5.根据权利要求4所述的方法，其特征在于，S1中，所述发酵培养物由如下方法发酵制备得到：将活化的菌株布雷菲德氏青霉(Penicillium brefeldianum)GXIMD 02511接入种子培养基中，于25℃，180rpm条件下动态培养72h制得种子液；将所述种子液以5％的接种量接入到发酵培养基中，于25℃温度下，静态培养30天制得发酵培养物；所述种子培养基配方为每1L培养基中含有：麦芽提取粉15g，余量为水，pH 7.5；所述发酵培养基的配方为每1L三角瓶培养基中含有：大米200g，海盐2g，水200mL，pH 7.5。
6.根据权利要求1所述的吲哚二酮哌嗪类化合物在制备破骨细胞分化抑制剂中的应用。
7.根据权利要求6所述的应用，其特征在于：所述的破骨细胞分化抑制剂为治疗破骨细胞过度活化造成的溶骨性疾病的药物。
8.根据权利要求6所述的应用，其特征在于：所述的破骨细胞分化抑制剂为治疗骨质疏松、类风湿性关节炎、肿瘤转移骨破坏的药物。
9.根据权利要求1所述的吲哚二酮哌嗪类化合物在制备NF-κB核因子表达抑制剂药物中的应用。
10.一种药物组合物，其特征在于，其包括有效量的prenylcyclotryprostatin B和/或其药用盐，和药学上可以接受的载体或辅剂。
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