N-取代馬來海松酸酰亞胺的合成及抗菌活性研究

李　慧, 王　殷, 代　立, 廖本仁, 楊旭石, 吳　劼, 揭元萍

(1.上海華誼集團技術研究院 精細化工研究所,上海 200241; 2.復旦大學 化學系,上海 200433)

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N-取代馬來海松酸酰亞胺的合成及抗菌活性研究

李慧1,2, 王殷1, 代立1, 廖本仁1, 楊旭石1, 吳劼2, 揭元萍1

(1.上海華誼集團技術研究院 精細化工研究所,上海 200241; 2.復旦大學 化學系,上海 200433)

摘要:以馬來海松酸與苯胺或其衍生物為原料,在聚乙二醇600(PEG-600)作為溶劑、甲苯作為帶水劑的條件下,采用一步法合成了8個N-取代馬來海松酸酰亞胺化合物;對化合物的結構進行了確認,并進行了初步的抗菌活性研究.

關鍵詞:馬來海松酸; 苯胺或其衍生物; 有機合成; 抗菌活性

松香是我國的天然豐產資源,利用豐富資源的優勢,開發與應用其深加工產品是松香化學研究的主要方向.N-取代馬來海松酸酰亞胺是一類重要的化工、醫藥產品,主要應用于防污劑、耐熱改性劑、合成藥物中間體、殺菌劑、殺蟲劑等[1-6]方面.目前文獻中主要報道的是與馬來酸酐直接合成的酰亞胺類化合物殺菌活性研究,而以天然產物松香為原料合成具有生物活性的酰亞胺類化合物鮮有報道[7].

本實驗報道了一種簡便、高效、環境友好的新方法:以松香為原料,首先進行異構化,再與馬來酸酐經Diels-Alder反應得到中間體馬來海松酸,繼而與苯胺或其衍生物采用一步法合成N-取代馬來海松酸酰亞胺.然后對其產物的結構進行了表征,并進行了初步的抗菌活性研究.合成路線如圖1所示.

圖1　N-取代馬來海松酸酰亞胺的合成

1實驗部分

1.1主要儀器與試劑

主要儀器:Bruker AV-400核磁共振波譜儀(以DMSO-d6為溶劑,TMS為內標,Bruker 公司);自動毛細管熔點測定儀(FP-62,梅特勒);84-1 型磁力加熱攪拌電熱套等.

試劑:松香(純度為75%);其他所有試劑均為市售化學純或分析純,除特別注明外,未經進一步處理.

1.2合成方法

1.2.1松香的異構化及馬來海松酸的合成[8-14]

馬來海松酸為白色的固體化合物,收率:91%.m.p.223~224.5℃.1H NMR (400 MHz,CDCl3) δ 5.54 (s,1H),3.07~3.16 (m,2H),2.74 (d,J=8.6 Hz,1H),2.51~2.56 (m,1H),2.23~2.30 (m,1H),1.62~1.80 (m,6H),1.45~1.58 (m,4H),1.37~1.41 (m,2H),1.23~1.29 (m,1H),1.17 (s,3H),1.01(d,J=1.5 Hz,3H),0.99 (d,J=1.4 Hz,3H),0.61 (s,3H).13C NMR (100 MHz,CDCl3) δ 185.21,172.74,170.96,148.17,125.15,53.33,53.08,49.10,46.86,45.65,40.45,37.98,37.58,36.74,35.68,34.79,32.77,27.23,21.67,20.57,19.95,16.93,16.44,15.51.

1.2.2N-取代馬來海松酸酰亞胺的合成[15-16]

向1000 mL的三頸瓶中加入0.5 mol的馬來海松酸,將其溶于300 mL PEG-600和400 mL甲苯中,再向反應體系中緩慢滴加0.55 mol的苯胺或其衍生物,滴加完畢后升溫至回流狀態4h.TLC檢測反應完全后,冷卻至室溫,將反應體系再用100 mL甲苯稀釋后,經過H2O洗滌、飽和NaCl溶液洗滌,無水Na2SO4干燥,抽濾,減壓蒸餾得到粗品.將粗品進行重結晶得到白色的目標化合物a～h.

化合物a為白色固體,收率:92%.m.p.289.6~290.5℃.1H NMR (400 MHz,DMSO-d6) δ 12.20(brs,1H,-COOH),7.44~7.47(m,2H),7.36~7.40(m,1H),7.01~7.06(m,2H),5.52(s,1H),3.49~3.51(m,4H),3.04(dd,J=2.8,8.0 Hz,1H),2.91~2.96(m,1H),2.71~2.73(m,1H),2.41~2.44(m,1H),2.15~2.18(m,1H),1.36~1.71(m,7H),1.15~1.18(m,2H),1.05(s,3H),0.94(d,J=2.4 Hz,6H),0.54(s,3H).13C NMR (100 MHz,DMSO-d6) δ 179.70,177.39,176.39,146.50,132.38,128.91(×2),128.22,126.80(×2),124.34,53.27,51.84,48.90,45.93,44.62,40.45,37.78,37.16,36.36,35.60,35.01,32.30,27.08,26.27,21.35,20.67,20.10,16.72,15.36.ESI-HRMS (m/z) [M+Na]+:calcd for C30H37NNaO4498.2615;found 498.2645.

化合物b為白色固體,收率93%.m.p.182.9~184.0℃.1H NMR (400 MHz,DMSO-d6) δ 12.15 (brs,1H),7.30~7.20 (m,3H),7.18~7.11 (m,2H),5.31 (s,1H),4.48~4.33 (m,2H),2.96~2.84 (m,2H),2.61 (d,J=7.8 Hz,1H),2.39 (d,J=13.3 Hz,1H),2.02~1.90 (m,1H),1.75~1.29 (m,13H),1.04 (s,3H),0.79 (d,J=6.6 Hz,3H),0.66 (d,J=6.6 Hz,3H),0.51 (s,3H).13C NMR (100 MHz,DMSO-d6) δ 179.68,177.97,177.06,146.11,136.27,128.30(×2),127.62(×2),127.30,124.18,69.80,53.41,51.69,48.90,45.92,44.43,41.13,40.23,37.78,37.10,36.35,34.99,31.94,27.06,21.31,20.20(×2),19.48,16.68,15.31.ESI-HRMS (m/z) [M+Na]+:calcd for C31H39NNaO4512.2771;found 512.2799.

化合物c為白色固體,收率94%.m.p.249.6~251.0℃.1H NMR (400 MHz,DMSO-d6) δ 12.13 (brs,1H),7.32~7.25 (m,2H),7.23~7.13 (m,3H),5.37 (s,1H),3.45~3.36 (m,2H),2.93~2.81 (m,2H),2.60 (t,J=7.7 Hz,2H),2.54 (d,J=8.1 Hz,1H),2.44~2.37 (m,1H),2.16~2.02 (m,1H),1.75~1.26 (m,13H),1.04 (s,3H),0.88 (t,J=7.0 Hz,6H),0.53 (s,3H).13C NMR (100 MHz,DMSO-d6) δ 179.68,177.99,176.90,146.15,137.99,128.49(×2),128.44(×2),126.47,124.24,53.31,51.54,48.90,45.92,44.30,40.16,38.83,37.79,37.09,36.33,35.12,34.95,33.22,32.16,27.06,21.32,20.53,19.91,16.71,16.68,15.32.ESI-HRMS (m/z) [M+Na]+:calcd for C32H41NNaO4526.2928;found 526.2936.

化合物d為白色固體,收率83%.m.p.250.5~252.0℃.1H NMR (400 MHz,DMSO-d6) δ 12.15(brs,1H,-COOH),7.24(d,J=8.4 Hz,2H),6.92(d,J=8.4 Hz,2H),5.51(s,1H),2.68~3.02(m,2H),2.68~2.70(m,1H),2.32~2.49(m,1H),2.31(t,J=4.4 Hz,3H),2.11~2.23(m,1H),2.15~2.18(m,1H),1.36~1.71(m,9H),1.15~1.19(m,3H),1.05(s,3H),0.94(d,J=2.4 Hz,6H),0.54(s,3H).13C NMR (100 MHz,DMSO-d6) δ 179.69,177.45,176.47,146.50,137.71,131.67,129.35(×2),126.55(×2),124.37,53.28,51.81,48.91,45.94,44.58,40.44,38.64,37.80,37.17,36.36,35.59,35.04,32.30,27.11,21.36,20.69(×2),20.10,16.70,15.35.ESI-HRMS (m/z) [M+Na]+:calcd for C31H39NNaO4512.2771;found 512.2798.

化合物e為白色粉末狀固體,收率78%.m.p.289.1~289.9℃.1H NMR (400 MHz,DMSO-d6) δ 12.17(brs,1H,-COOH),7.54(d,J=8.0 Hz,2H),7.10(d,J=8.0 Hz,2H),5.51(s,1H),3.03~3.05(m,1H),2.90~3.10(m,2H),2.95~3.00(m,1H),2.71~2.73(m,1H),2.40~2.44(m,1H),2.14~2.17(m,1H),1.47~1.73(m,9H),1.15~1.20(m,2H),1.05(s,3H),0.94(d,J=2.4 Hz,6H),0.54(s,3H).13C NMR (100 MHz,DMSO-d6) δ 180.72,178.23,177.22,147.57,133.68,132.17,130.04(×2),129.50(×2),125.43,54.21,52.90,49.88,46.94,45.69,41.47,38.78,38.17,37.36,36.60,35.99,33.31,28.08,22.36,21.69,21.16,17.72,16.36.ESI-HRMS (m/z) [M+Na]+:calcd for C30H36ClNNaO4532.2225;found 532.2260.

化合物f為白色粉末狀固體,收率89%.m.p.281.5~282.2℃.1H NMR (400 MHz,DMSO-d6) δ 12.17(brs,1H,-COOH),7.83(d,J=8.0 Hz,2H),6.88(d,J=8.0 Hz,2H),5.50(s,1H),2.90~3.10(m,2H),2.65~2.80(m,1H),2.40~2.43(m,1H),2.13~2.16(m,1H),1.40~1.80(m,11H),1.15~1.20(m,2H),1.05(s,3H),0.94(d,J=2.4 Hz,6H),0.54(s,3H).13C NMR (100 MHz,DMSO-d6) δ 179.68,177.13,176.13,146.50,137.80(×2),132.02,128.79(×2),124.39,94.23,53.18,51.87,48.85,45.91,44.67,40.43,37.75,37.14,36.51,36.34,35.56,34.96,32.29,27.06,21.33,20.67,20.13,19.45,16.69.ESI-HRMS (m/z) [M+Na]+:calcd for C30H36INNaO4624.1581;found 624.1587.

化合物g為白色固體,收率76%.m.p.285.0~285.5℃.1H NMR (400 MHz,DMSO-d6) δ 12.20(brs,1H,-COOH),7.00~7.07(m,4 H),5.57(s,1H),3.82(s,3H),3.02~3.08(m,2H),2.74~2.76(m,1H),2.40~2.50(m,1H),2.15~2.30(m,1H),1.55~1.80(m,11H),1.15~1.20(m,2H),1.05(s,3H),0.94(d,J=2.4 Hz,6H),0.54(s,3H).13C NMR (100 MHz,DMSO-d6) δ 179.71,177.57,176.57,158.83,146.51,127.95(×2),124.97,124.38,114.29,114.17,55.37,53.32,51.79,50.96,48.93,45.95,44.54,38.94,37.83,37.18,36.39,35.60,35.07,32.30,27.12,21.37,20.68,20.12,16.73,15.36.ESI-HRMS (m/z) [M+Na]+:calcd for C31H39NNaO5528.2720;found 528.2753.

化合物h為白色粉末狀固體,收率84%.m.p.244.1~244.8℃.1H NMR (400 MHz,DMSO-d6) δ 12.17(s,1H,-COOH),8.92(s,1H,-OH),6.76(s,1H),6.63(d,J=8.0 Hz,2H),6.52(d,J=8.0 Hz,2H),5.27(s,1H),4.22~4.31(m,2H),3.70(s,3H),2.86~2.88(m,2H),2.57~2.59(m,1H),2.37~2.41(m,1H),1.93~1.96(m,1H),1.38~1.70(m,9H),1.15~1.20(m,2H),1.05(s,3H),0.75(d,J=2.4 Hz,3H),0.60(d,J=2.4 Hz,3H),0.52(s,3H).13C NMR (100 MHz,DMSO-d6) δ 179.75,178.00,177.00,147.28,146.09,145.94,127.23,124.00,120.45,115.06,112.35,55.50,53.41,51.66,48.93,45.95,44.39,41.01,39.50,38.88,37.80,37.11,36.35,35.01(×2),32.07,27.11,21.35,20.28,19.49,16.71,15.34.ESI-HRMS (m/z) [M+Na]+:calcd for C32H41NNaO6558.2826;found 558.2840.

2實驗結果與討論

2.1實驗結果

松香是一種混合物,它的主要成分樅酸、長葉松酸和新樅酸在加熱的條件下易發生異構化生成左旋海松酸,而左旋海松酸與馬來酸酐容易發生Diels-Alder反應得到馬來海松酸.一般N-取代馬來海松酸酰亞胺由馬來海松酸與苯胺或其衍生物經酰胺化、催化脫水環合兩步反應合成.本文作者以馬來海松酸和苯胺或其衍生物作為原料,以聚乙二醇600(PEG-600)作為溶劑,以甲苯作為帶水劑,在溶劑和帶水劑同時存在時,回流反應4 h,合成了N-取代馬來海松酸酰亞胺.酰胺化、脫水環合只需一步反應,無需催化劑,溶劑低毒環保,反應溫度較低,帶水劑可回收,操作簡單、方便,且原料松香價廉易得,利于工業化生產.

2.2討論

酰亞胺化反應包括酰胺化和脫水兩步反應.最初,采用上海華誼(集團)技術中心N-苯基馬來酰亞胺(NPMI)成熟的工藝方法[17]來嘗試合成此類化合物,即使用混合酸作為催化劑,甲苯作為溶劑,采用馬來海松酸和苯胺(以苯胺為例,下同)通過一鍋法進行反應,結果沒有拿到所需要的目標化合物N-苯基馬來海松酸酰亞胺.分析原因,可能是馬來海松酸與馬來酸酐相比,化合物的活性較低,很難與苯胺進行酰亞胺化反應.后來,考慮到馬來海松酸反應活性較低,采用沸點較高的二甲苯等作為溶劑,其他反應條件不變,仍沒有得到N-苯基馬來海松酸酰亞胺.說明采用N-苯基馬來酰亞胺(NPMI)成熟的工藝方法不適合N-苯基馬來海松酸酰亞胺的合成.

接下來,嘗試采用分步反應的方法來合成N-苯基馬來海松酸酰亞胺,方法如下:(1)醋酸酐法[18-19]:在溶劑(丙酮,乙酸乙酯或乙酸)存在下,將馬來海松酸與苯胺在常溫下反應生成中間體N-苯基馬來酰胺酸,然后以醋酸酐作脫水劑,醋酸鈉為催化劑,在一定的溫度下進行脫水閉環反應希望生成目標化合物.實驗結果發現,此方法在該類化合物的合成上行不通;(2)共沸法[20-23]:該方法是由馬來海松酸和苯胺為原料,使用一種或兩種惰性有機溶劑(甲苯類、N,N-二甲基甲酰胺、氯烷類、四氫呋喃類等)和酸催化劑(如:磷酸、濃硫酸、對甲基苯磺酸、五氧化二磷等)在溶劑的沸點下進行酰亞胺化反應,通過合適的分水設備將反應生成的水不斷從反應體系中分離除去.令人遺憾的是,此方法也沒有得到目標化合物.

然后,嘗試采用2009年伍勇等人[7]報道的方法:以N,N-二甲基甲酰胺(DMF)為溶劑,155℃左右的條件下,將馬來海松酸與苯胺采用一步法反應直接得到目標化合物.按照文獻報道的方法進行嘗試,結果仍沒有得到目標化合物.

后來,又嘗試了Kumar等人[24]報道的方法:以馬來海松酸與苯胺為原料,以PEG-600作溶劑,60℃左右與苯胺反應.實驗結果發現:仍然沒有目標化合物生成.分析原因可能是馬來松香的位阻較大、反應活性較低使反應根本無法進行.

最后,在上述反應的基礎上,通過對溶劑和溫度的篩查,并經過大量的實驗探索,最終發現了在以聚乙二醇600(PEG-600)作為溶劑,以甲苯作為帶水劑,在溶劑和帶水劑同時存在時,回流狀態下就可以順利得到N-苯基馬來海松酸酰亞胺化合物,并且此方法也適用于其他苯胺衍生物.

2.3目標化合物的抑菌活性

參照美國臨床實驗室標準化委員會最小抑菌濃度(MIC)的測定方法,實驗方法如下:首先,單克隆挑起搖過夜后,OD600稀釋至0.01,繼續搖5~8 h;然后,菌液稀釋至OD 0.6,再稀釋400倍后,等比例加入含化合物的培養液中,37℃生長12 h左右觀察菌的生長狀況;最后,引入更多化合物濃度,經倍半稀釋后縮小區間.

以N-2,4,6-三氯苯基馬來酰亞胺(TCPM)作為參照活性,因為TCPM作為海洋防污劑目前已在日本涂料制造商協會(JPMA)登記,符合國際環保要求,并被開發成多種配方進行使用,說明其抑菌效果較好,已經得到公認,對其修飾,希望進一步提高其抑菌活性,所以選擇TCPM作為活性參照.對所有目標化合物進行了初步的抑菌活性測試,受試菌株為金黃色葡萄球菌Newman,測定結果如表1所示.

表1　化合物a～h的體外最低抑菌濃度

結果表明,化合物b(MIC=12.5~15μg/mL)和化合物c(MIC=7.5~10μg/mL)對金黃色葡萄球菌Newman有一定的抑制作用,并且這兩個化合物的抑菌活性都高于參照化合物TCPM的抑菌活性.

通過分析目標化合物的結構和抑菌活性,在順酐雙鍵上引入松香后,可初步得到如下結果:(1)苯環上有取代基的化合物不利于抑菌活性的增強;(2)兩環之間碳鏈的長度有利于化合物活性的增加,并且隨著兩環之間碳鏈的增長,化合物的抑菌活性增強.

3結論

以商業化的天然產物松香為原料,經過異構化、Diels-Alder反應和與苯胺或其衍生物采用一步法合成了N-取代馬來海松酸酰亞胺,開發了一種無需催化劑,溶劑低毒環保,操作簡單、方便的合成方法,且原料松香價廉易得,利于工業化生產.經初步的抑菌活性測試結果顯示,有兩個化合物對金黃色葡萄球菌表現出一定的抑菌活性,并且高于參照化合物TCPM.總之,在順酐雙鍵上引入松香后,進一步的增長兩環之間碳鏈的長度有可能獲得更高殺菌活性的化合物,這對將來設計、合成出抑菌活性更好的化合物具有一定的借鑒意義.

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(責任編輯：郁慧)

Synthesis and antibacterial activities ofN-substituted maleopimarimide

LI Hui1,2, WANG Yin1, DAI Li,LIAO Benren1, YANG Xushi1, WU Jie2, JIE Yuanping1

(1.Fine Chemistry R&D Institute,Technology Research Institute of Shanghai Huayi Group,Shanghai 200241,China;2.Department of Chemistry,Fudan University,Shanghai 200433,China)

Abstract:With Macrogol 600(PEG-600) as solvent and toluene as dehydrant,eight N-Substituted maleopimarimide compounds were synthesized from maleopimaric acid and aniline or its derivatives by one-step reaction;Their structures were confirmed and the antimicrobial activities of these compounds were also preliminarily investigated.

Key words:maleopimaric acid; aniline or its derivatives; organic synthesis; antimicrobial activity

中圖分類號:Q 643.32

文獻標志碼:A

文章編號:1000-5137(2016)01-0058-06

基金項目:上海市科技人才計劃項目(14R21421700)

收稿日期:2015-05-27

通信簡介: 廖本仁,中國上海市閔行區龍吳路4600號,上海華誼集團技術研究院精細化工研究所,郵編:200241,E-mail:liaobenren@shhuayi.com