JPWO2002059141A1 - Peptide derivatives - Google Patents
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Abstract
下式(1)(式中、例えば、nは0〜6の整数であり、Arは置換されていてもよく他の環と縮合していてもよい芳香環であり、R1は置換されていてもよい直鎖または分岐鎖状の低級アルキル基であり、R2は置換されていてもよい直鎖または分岐鎖状の低級アルキル基であり、R3は置換されていてもよい直鎖もしくは分岐鎖状の低級アルキル基であり、Xはアミノ基、ヒドロキシ基または低級アルコキシ基である。)で表されるペプチド誘導体は、平滑筋収縮作用を有する。In the following formula (1) (for example, n is an integer of 0 to 6, Ar is an aromatic ring which may be substituted or may be condensed with another ring, and R1 is R2 is a linear or branched lower alkyl group which may be substituted, and R3 is a linear or branched lower alkyl group which may be substituted. Is a lower alkyl group, and X is an amino group, a hydroxy group or a lower alkoxy group.) Has a smooth muscle contraction action.
Description
技術分野
本発明は、平滑筋収縮作用を有する新規なペプチド誘導体に関する。
背景技術
モチリンは、22個のアミノ酸残基を有する単一鎖ペプチドであり、様々な種類の動物の消化管(GI)粘膜における内分泌細胞から単離された(文献1)(Ito,Z.Motilin and ClinicalApplication.Peptides1997,18,593−608)。モチリンの生物学的な機能はまだ十分に確認されていないが、モチリンは多くの種類の動物におけるGI運動を刺激することが知られていた(前掲書)。
1973年にブタモチリン(pMTL)のアミノ酸配列が初めて確認された(FVPIF TYGEL QRMQE KERNK GQ)(文献2および3)(Brown,J.C.; Cook,M.A.; Dryburgh,J.R. Motilin,a Gastric Motor ActivityStimulating Polypeptide: The Complete Amino Acid Sequence. Can.J.Biochem.,1973,51,533−537.Schubert,H.; Brown,J. Correction to Amino Acid Sequence of Porcine Motilin. Can.J.Biochem.,1974,52,7−8.)。ヒトのモチリンは1985年に確認され、ブタのモチリンと同じであるが、イヌのモチリンとは7番目、8番目、12番目、13番目および14番目の5つの残基が異なることが見出された(文献1および4)。
モチリンと幾つかのGI症状(初期満腹(early satiety)、腹部膨満(abdominal distension)、吐き気、嘔吐および食欲不振)との生理学的関連が示唆されている。モチリン類似化合物(文献5−7)およびエリスロマイシン誘導体(文献8−10)のようなモチリンアゴニストがこのような症状に有効であることが既に示唆されていた。しかし、モチリンレセプター(MTL−R)に結合した後のモチリンアゴニスト作用の詳細なメカニズムは未だ解明されていなかった。モチリンの生物学的および生理学的メカニズムを解明し、モチリンに関係する疾患の治療に応用するため、低分子のモチリンアゴニストの発見が望まれていた。
本発明者らが報告したpMTLの構造活性相関(SAR)において(文献11)、N末端部分がその作用(agonism)にとって重要な役割を果たしていることが示唆された。
発明の開示
これらのことから、本発明者らは、N末端の小ペプチドでもアゴニスト作用を有することになると仮定した。本発明者らは、モチリンのN末端部分のテトラペプチドの収縮効果の増大を目的として、テトラペプチドの修飾に努力を集中した。その結果、本発明者らは、式1で表される新規なテトラペプチドのモチリンアゴニストを合成することに成功した。本発明は、この知見に基づいて完成したものである。
本発明は、下式(1)
(式中、
nは0〜6の整数であり、
Arは置換されていてもよく他の環と縮合していてもよい芳香環または置換されていてもよく他の環と縮合していてもよい複素環であり、
R1は置換されていてもよい直鎖または分岐鎖状の低級アルキル基であり、
R2は置換されていてもよい直鎖または分岐鎖状の低級アルキル基であり、
R3は水素原子または置換されていてもよい直鎖もしくは分岐鎖状の低級アルキル基であり、
Xはアミノ基、ヒドロキシ基または低級アルコキシ基であり、
また、Xがアミノ基である場合、Xのアミノ基はR2の低級アルキル基と一緒になって環を形成してもよい。)
で表されるペプチド誘導体を提供する。
また、本発明は、下式(2)
(式中、
nは0〜1の整数であり、
R1は置換されていてもよい直鎖または分岐鎖状の低級アルキル基であり、
R2は置換されていてもよい直鎖または分岐鎖状の低級アルキル基であり、
R3は水素原子または直鎖もしくは分岐鎖状の低級アルキル基であり、
Xはアミノ基であり、
また、Xのアミノ基はR2の低級アルキル基と一緒になって環を形成してもよい。)
で表されるペプチド誘導体を提供する。
さらに、本発明は、下式(3)
(式中、R1は置換されていてもよい直鎖または分岐鎖状の低級アルキル基である)
で表されるペプチド誘導体を提供する。
さらに加えて、本発明は、下式(4)
(式中、R1は置換されていてもよい直鎖または分岐鎖状の低級アルキル基である)
で表されるペプチド誘導体を提供する。
また、本発明は、上記式(1)〜(4)で表される化合物を1種または2種以上、および薬剤学的に許容される担体を含む薬剤組成物を提供する。
発明を実施するための最良の形態
本発明において、低級アルキル基の炭素数は1〜8である。本発明において、置換されていてもよい直鎖または分岐鎖状の低級アルキル基に関して、置換基を有する直鎖または分岐鎖状の低級アルキル基の置換基の位置および数は特に限定されず、任意の1以上の炭素原子に置換基を有することができる。また、同一の炭素原子が2以上の同一若しくは異なる置換基を有していてもよい。
置換基の種類は特に限定されないが、例えば、ヒドロキシ基、アルキル基で炭素数1〜6の1個若しくは2個のアルキル基で置換されていてもよいアミノ基、カルボキシル基等が挙げられる。特に好ましい置換基はヒドロキシ基である。
本発明において、置換基を有しない好ましい直鎖または分岐鎖状の低級アルキル基としては、例えば、メチル基、エチル基、n−プロピル基、イソプロピル基、n−ブチル基、イソブチル基、sec−ブチル基、tert−ブチル基、n−ペンチル基、n−ヘキシル基、n−オクチル基等が挙げられる。
本発明において、置換基を有する好ましい直鎖または分岐鎖状の低級アルキル基としては、ヒドロキシメチル基、1−ヒドロキシエチル基、2−ヒドロキシエチル基、3−ヒドロキシ−n−プロピル基、4−ヒドロキシ−n−ブチル基等を例示することができる。
以下に本発明のペプチド誘導体の一般合成法を記す。
一般合成法
本発明のペプチド誘導体は、たとえば「ペプチド合成の基礎と実験」(丸善:1985年)等に記載された、固相若しくは液相による方法(固相法若しくは液相法)により合成される。
インドール環の2‘位チオアルキル化は、インドール環に、トリクロロ酢酸、塩酸、酢酸などの酸類、酢酸ナトリウム等の酸金属類、トリエチルアミン等の塩基類、等の存在下、非存在下で、塩化チオアルキル等のハロゲン化チオアルキル化合物を反応することにより、
もしくは、N−ブロモサクシイミド等のハロゲン化試薬の存在下、スルフヒドリルアルキル化合物を反応することにより、
もしくは、酢酸水銀、銀トリフラート等の重金属化合物存在下、スルフヒドリルアルキル化合物やビスアルキルジスルフィド化合物を反応することによって、行うことができる。
溶媒は、テトラヒドロフラン(THF)、クロロホルム、ジオキサン、メタノール、ジクロルメタン等の有機溶媒、もしくは、酢酸、トリフルオロ酢酸(TFA)等の酸類等、当該化合物が溶解しうる溶媒であればよい。
望ましい反応条件としては、トリフルオロ酢酸中にて、銀トリフラート存在下、ビスアルキルジスルフィド化合物またはアルキルチオールまたはアルキルチオハライドとの反応が挙げられる(下記反応図式1を参照)。
反応図式1
(上記反応図式において、R1は置換されていてもよい直鎖または分岐鎖の低級アルキル基を表し、R2は置換されていてもよい直鎖または分岐鎖状の低級アルキル基を表し、R3は水素原子または置換されていてもよい直鎖もしくは分岐鎖状の低級アルキル基を表し、Xはアミノ基、ヒドロキシ基または低級アルコシキ基を表し、halogenはフッ素、塩素、臭素またはヨウ素を表し、
また、Xがアミノ基である場合、Xのアミノ基はR2の低級アルキル基と一緒になって環を形成してもよい。)
固相法(文献16および17)によるペプチドの合成は、例えば、MBHA−樹脂上でNα−Boc(Nα−t−ブチルオキシカルボニル)基若しくはNα−Fmoc(Nα−9−フルオレニルメチルオキシカルボニル)基を用いて行なうことができる(文献18および19)。
Nα−Boc基に関しては、次の保護アミノ酸を結合する前に、33−50%(v/v)トリフルオロ酢酸(TFA)/ジクロロメタン(DCM)溶液を用いてNα−Boc基の脱保護を行なう。アシル化種(acylating species)としては予め調製したNα−Bocアミノ酸対称酸無水物を利用することができる。アミノ酸対称酸無水物は、0.5当量のジシクロヘキシルカルボジイミド(DCC)をDCMに溶解した1.0当量のNα−Bocアミノ酸と反応させることにより得られる。
Nα−Fmoc基に関しては、次の保護アミノ酸を結合する前に、20%(v/v)ピペリジン/N,N−ジメチルホルムアミド(DMF)溶液を用いてNα−Fmoc基を除去する。すべてのNα−Fmocアミノ酸はN−ヒドロキシベンゾトリアゾール(HOBt)を用いてそのペンタフルオロフェニル(Pfp)エステルとして結合することができる。
結合反応の完了はカイザー試験(Kaiser test)(文献20)により確かめる。上述の固相法により合成されたペプチドは、側鎖を保護している基に依存して、TFA/トリメチルシリルトリフラート(TMSOTf)/m−クレゾール/チオアニソール(100:28.6:7.47:16.7;v/v)またはTFA/TMSOTf/m−クレゾール/チオアニソール/エタンジチオール(100:12:6.5:15:5;v/v)を用いて0℃若しくは室温で1−4時間処理することによって、同時に脱保護し、樹脂から回収する。Trpのインドール環の修飾は対応するジスルフィドを用いて銀トリフラートの存在下で行なう(反応図式2を参照)。
反応図式2
(上記反応図式2において、nは0〜6の整数を表し、
Arは置換されていてもよく他の環と縮合していてもよい芳香環または置換されていてもよく他の環と縮合していてもよい複素環を表し、
R1は置換されていてもよい直鎖または分岐鎖状の低級アルキル基を表し、R2は置換されていてもよい直鎖または分岐鎖状の低級アルキル基を表し、R3は水素原子または置換されていてもよい直鎖もしくは分岐鎖状の低級アルキル基を表し、
Xはアミノ基、ヒドロキシ基または低級アルコシキ基を表し、
また、Xがアミノ基である場合、Xのアミノ基はR2の低級アルキル基と一緒になって環を形成してもよい。)
粗ペプチドは次いで、アセトニトリルに溶解した0.1%水性TFAのリニアグラジエント(linear gradient)を用いて溶離する分取逆相高速液体クロマトグラフィー(RP−HPLC)によって精製する。分析RP−HPLCにより均質であると認められたペプチドフラクションは一緒にし、濃縮し、そして凍結乾燥する。このようにして得られたペプチドはNMRおよび質量分析計(FAB−MS)等によって構造確認を行う事ができる(以下の表3を参照)。
本発明のペプチド誘導体を1種または2種以上、および薬学的に許容し得る希釈剤、湿潤剤、乳化剤、分散剤、補助剤、防腐剤、緩衝剤、結合剤、安定剤などの担体を適宜含む薬学的組成物として、目的とする投与経路に応じ、適当な任意の形態にして投与することができる。
実施例
以下に本発明の実施例を示すが、本発明はこれらの実施例に何ら限定されるものではない。
使用材料
Nα−Fmocアミノ酸、またはそのPfpエステル、および固相合成用樹脂はMilliGen/Biosearch(Burlington,MA)または渡辺化学(Watanabe Chemical)(日本)のいずれかから購入した。TFA、TMSOTf、1,3−ジイソプロピルカルボジイミド(DIC)およびHOBtは渡辺化学(Watanabe Chemical)(日本)から購入した。ジ−n−プロピルジスルフィド、n−オクチルジチオジグリコール酸、トリフルオロメタンスルホン酸銀、トリエチルアミン、NH4F、2−メルカプトエタノール、m−クレゾール、エタンジチオールおよびチオアニソールは東京化成工業(Tokyo Chemical Industry)(日本)から購入した。ジメチルジスルフィドは半井テスク(Nakarai Tesque(日本)から購入した。DMF、ジクロロメタン(DCM)およびN−メチル−2−ピロリジノン(NMP)は国産化学(Kokusan Chemical)(日本)から購入した。メタノール、ジエチルエーテル、n−ヘキサン、NaOHおよび酢酸は純正化学(Junsei Chemical)(日本)から購入した。HPLC用アセトニトリルおよび水は関東化学(Kanto Chemical)(日本)または純正化学(Junsei Chemical)(日本)から購入した。
固相ペプチド合成
ペプチドはFmoc固相合成法に従って合成した(文献16及び17)。反応は震盪機によって実施した。
ペプチド精製
粗ペプチドはRP−HPLCによって精製した。このRP−HPLCは、C18 YMC−Pack S−343−15(YMC、日本)、15μm、120オングストローム、20×250mmを用いた、分取HPLC(Waters semi−prep syetem)を用いて行ない、一定濃度のTFA(0.1%、v/v)を含む水中でリニアアセトニトリルグラジエント(linearacetonitrile gradient)(0−60%)を用い、10ml/分の流速で60分間にわたって溶離した。分析HPLCによって精製されたペプチドのフラクションを凍結乾燥し、得られた粉末はそれらの生物学的アッセイまで−20℃以下で保存した。
ペプチド分析
最終生成物の純度は、2つの異なるカラムとリニアグラジエント溶媒による方式で日立D−7000HPLCシステムを使用してRP−HPLCにより確認した。一方の溶媒システム(A)は、YMC−Pack A−302分析カラム(4.6×150mm、120オングストローム,5μm粒子サイズ、YMC、日本)を用いて、1ml/分の速度で25分間にわたって100:0〜30:70(0.1%水性TFA:0.1%TFA含有アセトニトリル)まで変化させた。他方の溶媒システム(B)は、μ BONDASPHERE 5μ C18 300オングストローム(3.9×150mm、Waters)を用いて、10ml/分の速度で25分間にわたって100:0〜30:70または100:0〜2:98(0.1%水性TFA:メタノール)まで変化させた。
NMRは、JEOL JNM−A500、Varian Mercury 300、およびJEOL JNM−EX270上で実施した。スペクトルは、DMSO−d6で測定した。化合物の分子量は、FAB−MS(VG70−250SEQ,VG Analytical,英国)により測定した。値はMH+で示した。
参考例1:H−Phe−Val−Trp−Ile−NH2の合成
20%(v/v)ピペリジン/DMF溶液でMBHA−樹脂(0.79mmol/g;3.418g)を2回処理し、DMFで8回洗浄した。次いでNα−Fmoc−Ile−OPfp(3.5当量)加えて、それからHOBt(3.5当量)を加えた。結合反応混合物を震盪機上で室温で3時間震盪した。次いで樹脂をDMFで6回洗浄した。続いて対応するNα−Fmocアミノ酸Pfpエステル(Nα−Fmoc−Trp−OPfp、Nα−Fmoc−Val−OPfp、Nα−Fmoc−Phe−OPfp)を順次結合し、ペプチド鎖を伸長した。各結合サイクルにおいて次のような操作を行なった。
(1)20%(v/v)ピペリジン/DMF溶液による30分間の処理を2回実施。
(2)DMFによる8回の洗浄。
(3)アミノ酸活性エステルの結合。
(4)DMFによる6回の洗浄。
最後のアミノ酸を上記の通り結合した後、以下の工程を付加した。
(5)20%(v/v)ピペリジン/DMF溶液による30分間の処理を2回実施。
(6)DMFによる8回の洗浄。
ペプチド鎖の伸長は、Nα−Fmocアミノ酸Pfpエステル(3当量)とHOBt(3当量)とを用いDMF中で行なった。すべての結合反応は震盪機上で室温下3−30時間震盪することで行なった。最後のNα−Fmoc基の脱保護の後に、ペプチド樹脂をメタノールで洗浄し、真空乾燥して、H−Phe−Val−Trp−Ile−MBHA−樹脂(4.725g)を得た。
このペプチド−樹脂をTFA/TMSOTf/m−クレゾール/チオアニソール/エタンジチオール(100:12:6.5:15:5;v/v)を用いて、0℃で2時間および室温で更に2時間処理した。溶液を濾過し、冷ジエチルエーテル(500ml)およびn−ヘキサン(1500ml)に滴下した。沈殿したペプチドを遠心分離によって集め、ジエチルエーテルとn−ヘキサン(1:3)で洗浄し、乾燥した。残渣を0℃でメタノール(10ml)および水(6ml)中で2−メルカプトエタノール(0.2ml)で処理した。得られた溶液を0.5時間かけてトリエチルアミンでpH8.0に調整し、次いで、酢酸を用いてpH4.0まで酸性化した。粗ペプチドを分取HPLCによって精製した。この分取HPLCは、0.1%水性TFAに対して0−60%の0.1%TFA含有アセトニトリルのリニアグラジエントを用い、10ml/分の流速で60分間にわたって行なった。凍結乾燥した生成物は白色の非晶質固体であった。
収量:0.134g(9%)
1H−NMR(DMSO−d6)δ0.83(12H,m,4×CH3),1.06,1.37(2H,mm,CH2),1.65(1H,m,CH),1.98(1H,m,CH),2.72−3.24(4H,m,2×CH2),4.13(2H,m,2×CH),4.29(1H,m,CH),4.67(1H,m,CH),6.94−7.30(10H,m,aromatic−H),7.62(1H,d,J=6.9Hz,NH),8.25(2H,m,2×NH),8.35(1H,d,J=9.2Hz,NH)
実施例1:H−Phe−Val−Trp(2’−SCH2CH2OH)−Ile−NH2(1)の合成
または
参考例1の化合物(H−Phe−Val−Trp−Ile−NH2)(0.191g,0.339mmol)を氷冷下でビス−(2−アセトキシ−エチル)−ジスルフィド(0.82g,3.44mmol)のTFA(20ml)溶液に加え、その後トリフルオロメタンスルホン酸銀塩(0.8449g,3.29mmol)を加えた。この溶液を氷冷下で8時間、室温で120時間撹拌した。減圧下で溶媒を除去し、残渣をメタノール(10ml)中に溶解した。この溶液を1N−NaOH水溶液で0.5時間かけてpH9に調整して濾過し、次いで酢酸を用いてpH4に酸性化した。減圧下で溶媒を除去し、得られた残渣をRP−HPLCで精製した。凍結乾燥した生成物は白色の非晶質固体であった。
収量:0.108g(50%)
1H−NMR(DMSO−d6)δ0.80(12H,m,4×CH3),1.03,1.38(2H,mm,CH2),1.66(1H,m,CH),1.92(1H,m,CH),2.77,2.87(4H,mm,2×CH2),3.04,3.20(2H,dd,dd,J=5.9,8.3,14.2Hz,CH2),3.47(2H,m,CH2),4.12(1H,dd,J=7.3,8.6Hz,CH),4.26(1H,dd,J=6.9,8.2Hz,CH),4.68(1H,dt,J=6.6,7.9Hz,CH),4.91(1H,t,J=5.6Hz,CH),6.95(2H,m,aromatic−H),7.03(1H,t,J=7.4Hz,aromatic−H),7.12(5H,s,aromatic−H),7.20(1H,d,J=7.6Hz,aromatic−H),7.66(1H,d,J=8.9Hz,NH),7.69(1H,d,J=7.9Hz,NH),8.19(1H,d,J=7.9Hz,NH),8.40(1H,d,J=8.9Hz,NH)
実施例2:H−Phe−Val−Trp(2’−SCH3)−Ile−NH2(2)の合成
または
参考例1の化合物(H−Phe−Val−Trp−Ile−NH2)(0.589g,1.05mmol)を氷冷下でジメチルジスルフィド(1.028g,10.9mmol)のTFA(20ml)溶液に加え、その後トリフルオロメタンスルホン酸銀塩(1.308g,5.09mmol)を加えた。この溶液を冷ジエチルエーテル(400ml)およびn−ヘキサン(100ml)に滴下した。沈殿したペプチドを遠心分離によって集め、ジエチルエーテルで洗浄した。残渣をメタノール(30ml),酢酸(30ml)および水(20ml)に溶かし、ジチオスレイトール(0.8g)を加え1時間処理した。沈殿物を遠心分離によって除去した。減圧下で溶媒を除去し、得られた残渣を分取RP−HPLCによって精製した。凍結乾燥した生成物は白色の非晶質固体であった。
収量:0.322g(43%)
1H−NMR(DMSO−d6)δ0.81(12H,m,4×CH3),1.03,1.34(2H,mm,CH2),1.64(1H,m,CH),1.93(1H,m,CH),2.40(3H,s,CH3),2.87(2H,m,CH2),3.03,3.24(2H,dd,dd,J=6.3,8.0,13.9Hz,CH2),4.12(2H,m,2×CH),4.23(1H,dd,J=6.9,8.3Hz,CH),4.69(1H,dd,J=7.1,14.4Hz,CH),6.99−7.06(4H,m,aromatic−H),7.17(5H,m,aromatic−H),7.60(1H,d,J=8.9Hz,NH),7.67(1H,d,J=7.9Hz,NH),8.21(1H,d,J=7.9Hz,NH),8.44(1H,d,J=8.9Hz,NH)
実施例3:H−Phe−Val−Trp(2’−SCH2CH2CH3)−Ile−NH2(3)の合成
または
標題の化合物は、参考例1の化合物(H−Phe−Val−Trp−Ile−NH2)(0.592g,1.05mmol)およびジ−n−プロピルジスルフィドから実施例2に記載された方法に従い合成された。
収量:0.369g(47%)
1H−NMR(DMSO−d6)δ0.76(12H,m,4×CH3),0.93(3H,t,J=7.3Hz,CH3),1.05,1.41(2H,mm,CH2),1.48(2H,six,J=7.3Hz,CH2),1.64(1H,m,CH),1.95(1H,m,CH),2.79−2.95(4H,m,2×CH2),3.04,3.26(2H,dd,dd,J=6.3,8.0,14.2Hz,CH2),4.12(2H,m,2×CH),4.33(1H,dd,J=7.3,8.2Hz,CH),4.66(1H,dd,J=7.3,14.9Hz,CH),6.90−7.06(4H,m,aromatic−H),7.17(5H,m,aromatic−H),7.55(1H,d,J=8.9Hz,NH),7.66(1H,d,J=7.6Hz,NH),8.19(1H,d,J=8.2Hz,NH),8.44(1H,d,J=8.9Hz,NH)
実施例4:H−Phe−Val−Trp(2’−SCH2CH2NMe2)−Ile−NH2(4)の合成
標題の化合物は、参考例1の化合物(H−Phe−Val−Trp−Ile−NH2)(0.592g,1.05mmol)およびN,N,N‘,N’−テトラメチルシスタミン(文献21)から実施例2に記載された方法に従い合成された。
収量:0.589g(63%)
1H−NMR(DMSO−d6)δ0.82(12H,m,4×CH3),1.02,1.38(2H,mm,CH2),1.65(1H,m,CH),1.94(1H,m,CH),2.77(6H,s,2×CH3),2.87−3.00(6H,m,3×CH2),4.11(2H,m,2×CH),4.27(1H,dd,J=6.8,8.4Hz,CH),4.73(1H,dt,J=7.6,6.8Hz,CH),6.91−7.27(9H,m,aromatic−H),7.69(1H,d,J=9.2Hz,NH),7.74(1H,d,J=8.2Hz,NH),8.28(1H,m,NH),8.45(1H,d,J=8.9Hz,NH)
実施例5:H−Phe−Val−Trp(2’−SCH2COOH)−Ile−NH2(5)の合成
標題の化合物は、参考例1の化合物(H−Phe−Val−Trp−Ile−NH2)(0.584g,1.04mmol)およびジチオジグリコール酸から実施例2に記載された方法に従い合成された。
収量:0.335g(42%)
1H−NMR(DMSO−d6)δ0.79(12H,m,4×CH3),1.00,1.39(2H,mm,CH2),1.66(1H,m,CH),1.91(1H,m,CH),2.81,2.93(2H,dd,dd,J=4.6,8.3,14.2Hz,CH2),3.09,3.20(2H,dd,dd,J=5.9,8.3,14.2Hz,CH2),3.63(2H,d,J=1.7Hz,CH2),4.11(2H,m,2×CH),4.25(1H,dd,J=6.9,8.2Hz,CH),4.71(1H,dt,J=6.9,7.4Hz,CH)6.90−7.23(9H,m,aromatic−H),7.65(1H,d,J=10.2Hz,NH),7.68(1H,d,J=8.6Hz,NH),8.12(1H,d,J=8.2Hz,NH),8.51(1H,br,NH)
上記実施例1〜5で製造されたペプチド誘導体の物性データ(HPLCによる保持時間およびFAB−MS)を表1に示す。
参考例2:H−Phe−c[−Nε−Lys−Trp−βAla−]の固相ペプチド合成
アルコキシベンジルアルコール−樹脂(0.78mmol/g;3.41g)をDMF中に入れて、一晩中膨潤させた。Nα−Fmoc−βAla−OH(8当量)をDMFで溶解し、この溶液にDIC(4当量)を加えた。DMAP(1当量)をDMFで溶解した。このアミノ酸/DIC溶液およびDMAP溶液をDMF中で樹脂に加えて、ゆっくりと震盪し、そして室温で一晩中放置した。
この樹脂をDMFで2回、メタノールで2回、酢酸で2回、メタノールで2回、そしてDMFで3回洗浄した。続いて対応するNα−Fmocアミノ酸Pfpエステル(Nα−Fmoc−Trp−OPfp、Nα−Fmoc−Lys(Boc)−OPfp、Nα−Fmoc−Phe−OPfp)を順次結合させ、ペプチド鎖を伸長した。各結合サイクルにおいて次のような操作を行なった。
(1)20%(v/v)ピペリジン/DMF溶液による30分間の処理を2回実施。
(2)DMFによる6回の洗浄。
(3)アミノ酸活性エステルの結合。
(4)DMFによる6回の洗浄。
ペプチド鎖の伸長は、Nα−Fmocアミノ酸Pfpエステル(2.5当量)とHOBt(2.5当量)とを用いDMF中で行なった。すべての結合反応は震盪機上で室温下3−12時間震盪することで行なった。最後のNα−Fmocアミノ酸の結合の後に、ペプチド樹脂をメタノールで洗浄し、真空乾燥して、保護されたFmoc−Phe−Lys(Boc)−Trp−βAla−樹脂(5.93g)を得た。
この保護されたペプチド−樹脂をTFA/アニソール/エタンジチオール(94:5:1;v/v)を用いて、0℃で3時間処理した。溶液を濾過し、減圧濃縮し、過剰の冷ジエチルエーテルに滴下した。沈殿したペプチドを濾過し、ジエチルエーテルで洗浄し、乾燥した。このペプチドをDMF(3L)およびピリジン(3L)で溶解し、Bop試薬を添加して、室温で24時間放置した。減圧下で溶媒を除去し、残渣に水を加えた。濾過により沈殿物を集め、水で洗浄して、乾燥した。こうして得られたFmoc−環状ペプチドは20%(v/v)ピペリジン/DMF溶液で室温下0.5時間処理し、Fmoc基を除去した。溶媒を減圧下で除去し得られた粗ペプチドを分取HPLCによって精製した。この分取HPLCは、0.1%水性TFAに対して0.1%TFA含有アセトニトリルのリニアグラジエントを用い、10ml/分の流速で60分間にわたって行なった。凍結乾燥した生成物は白色の非晶質固体(0.133g)であった。
総括収率:57%
FAB−MS:533.2876,
HPLC RT:(A)13.36(98%),(B)18.29(100%)
実施例6:H−Phe−c[−Nε−Lys−Trp(2’−SCH2CH2OH)−βAla−](6)の合成
または
H−Phe−c[−Nε−Lys−Trp(2’−SCH2CH2OH)−βAla−]は、H−Phe−c[−Nε−Lys−Trp−βAla−](参考例2)から実施例1に記載した方法と同様な方法で合成した。
収量:104mg(収率:22%)
FAB−MS:609.2859,
HPLC RT:(A)13.33(96%),(B)18.73(100%)
試験例
以下に、本発明化合物を用いた2つの試験内容(レセプター結合試験および収縮試験)並びにその結果を記す。
試験内容
下記試験においては、表1に示す合成ペプチドが、MTL−Rに対する結合活性およびウサギ平滑筋収縮活性について試験された。
1.レセプター結合試験
雄性日本白色家兎(約3kg)を用いた、MTL−Rに対する結合試験は、Bormans et al.によって発表された方法(文献22)をわずかに改変し実施した。ウサギをチオペンタールナトリウム(30mg/kg,静脈注射)で麻酔した後に、放血し安楽死させた。その後、上部小腸(約50cm)を速やかに摘出し、氷冷した0.9%生理食塩液中で夾雑物を除去した。50mMTris−HCl緩衝液(pH=7.4)中で、結合組織および粘膜を除去し平滑筋層を得た。50mMのTris−HCl緩衝液(pH=7.4)中でテーパー型ホモゲナイザー(Wheaten,Millville,NJ)を用い、2000rpmの速度で約30秒間、平滑筋組織をホモゲナイズした。得られた懸濁液を1500rpmで5分間遠心分離し、上澄を捨て沈殿に新鮮な緩衝液を加え洗浄した。このホモゲナイズの操作を2回行い、沈殿を回収した。得られた沈殿を50mM Tris−HCl緩衝液(pH=8.0、MgCl210mMおよび、1.5% ウシ血清アルブミンを添加)に再度懸濁させたものを試料として以下の結合試験を行った。以上の操作は全て氷冷下で行った。試料(約1.0mg蛋白質/アッセイ)を25pMの125I−pMTL(比活性、33−66kBq/pmol)および各種濃度の被検化合物とともに25℃で120分間インキュベートした。インキュベーション後に、氷冷した緩衝液を加え反応を停止させ、1500rpmで5分間遠心分離し、遊離の125I−MTLを除去した。得られた沈殿を氷冷緩衝液で2回洗浄したものの放射活性を、ガンマカウンター(ARC−300、アロカ、東京、日本)で測定した。125I−pMTLの結合を50%に減弱させる各被検化合物の濃度をIC50とし、MTL−R結合活性の指標とした。
2.収縮試験
雄性日本白色家兎(約3kg)を用いた。このウサギをチオペンタールナトリウム(30mg/kg,静脈注射)麻酔下で、放血し安楽死させた後、上部小腸を速やかに摘出した。十二指腸を改良型クレブス液(組成(単位mM):NaCl 120.0,KCl 4.7,CaCl22.4,KH2PO41.0,MgSO41.2,NaHCO324.5およびグルコース5.6(pH=7.4))中で洗浄し、腸間膜などの結合組織を除去し、長軸に沿って切り、およそ長さ10mm、幅3mmの標本を作成した。混合物ガス(95%O2:5%CO2)を通気した28℃の改良型クレブス液で満たした10mlのマグヌス管中に、この標本を懸垂した(文献24)。1.0gの荷重を加え、等張性の収縮反応を、トランスデューサー(ME−4012、Medical Electronics Co.,東京)で測定し、記録器(Type 3066、Yokogawa−Decs、東京)で記録した。100μMのAChに対し安定した収縮が得られることを確認した後に、被検化合物の検討を行った。被検化合物は低濃度から累積的に添加した。各被検化合物の収縮活性は、100μMのAChによって誘発される収縮力に対する百分率で表し、50%の収縮をを与える添加濃度をEC50として表した。
試験結果
上記2つの試験結果を表2および表3に示す。
重要なことは、実施例1、2、3、および6の化合物がいずれも収縮試験に関しては効力を示しており(これらの化合物のEC50は、それぞれ、14.2、58.1、33.2、および63.0μMである)、N−末端テトラペプチドはin vitroでモチリンアゴニストとして作用し得ることが立証された。収縮作用に関して活性を有する実施例1、2、3および6の誘導体の構造的特徴は、トリプトファン環上における脂肪族側鎖の存在である。
産業上の利用の可能性
本発明の新規なテトラペプチド誘導体は、モチリンレセプターに対する結合活性を有し、ウサギの平滑筋を収縮させる作用を有するので、モチリンが関連する疾患の治療に応用することが期待される。
文献リスト
(1) Itoh,Z. Motilin and Clinical Application.Peptides 1997,18,593−608.
(2) Brown,J.C.;Cook,M.A.;Dryburgh,J.R. Motilin,a Gastric Motor Activity Stimulating Polypeptide:The Complete Amino Acid Sequence.Can.J.Biochem.,1973,51,533−537.
(3) Schubert,H.;Brown,J. Correction to Amino Acid Sequence of Porcine Motilin.Can.J.Biochem.1974,52,7−8.
(4) McIntosch,C.H.S.;Brown,J.C. Purification and Chemical Structure of Porcine and Canine Motilins and Evidence for the Existence of Motilin in Other Species.In Motilin Itoh,Z.,Ed;Academic Press:San Diego,CA,1990;pp 13−30.
(5) Kitazawa,T.;Ichikawa,S.;Yokoyama,T.;Ishii,A.;Shuto,K. Stimulating action of KW−5139 (Leu13−motilin) on gastrointestinal motility in the rabbit.Br.J.Pharmacol.1994,111,288−294.
(6) Kitazawa,T.;Taneike,T.;Ohga,A. Excitatory Action of [Leu13]Motilin on the Gastrointestinal Smooth Muscle Isolated From the Chicken.Peptides 1995,16,1243−1252.
(7) Assche,G.V.;Depoortere,I.;Thijs,T.;Janssens,J.J.;Peeters,T.L. Concentration−dependent stimulation of cholinergic motor nerves or smooth muscle by [Nle13]motilin in the isolated rabbit gastric antrum.Eur.J.Pharmacol.1997,337,267−274.
(8) Shiba,Y.;Mizumoto,A.;Satoh,M.;Inui,A.;Itoh,Z. Effect of Nonpeptide Motilin Agonist EM523 on Release of Gut and Pancreatic Hormones in Conscious Dogs.Gastroenterology 1996,110,241−250.
(9) Yamada,K.;Chen,S.;Abdullah,N.A.;Tanaka,M.;Ito Y.;Inoue,R. Electrophysiological.characterization of a motilin agonist,GM611,on rabbit duodenal smooth muscle.Am.J.Physiol.1996,271,G1003−G1016.
(10) Verhagen,M.A.;Samsom,M.;Mase,B.;Geypens,B.J.;Ghoos,B.J.;Smout,A.J.Effects of a new motilide,ABT−229,on gastric emptying and postprandial antrocluodenal motility in healthy volunteers.Aliment.Pharmacol.Ther.1997,11,1077−1086.
(11) Haramura,M.;Tsuzuki,K.;Okamachi,A.;Yogo,K.;Ikuta,M.;Kozono,T.;Takanashi,H.;Murayama,E. Structure−Activity Study of Intact porcine Motilin.Chem.Pharni.Bull.1999,47,1555−1559.
(16) The Practice of Peptide Synthesis;Bodansky,M.,Bodansky,A.,Eds.;Springer−Verlag:Berlin,Heidelberg,1984.
(17) Stewart,J.M.;Young,J.D. Solid Phase Peptide Synthesis;2nd ed.,Pierce ChemicalCo.:Rockford,IL,1984.
(18) Matsueda,G.R.;Stewart,J.M. A p−Methylbenzhydrylamine Resin for Improved Solid−Phase Synthesis of Peptide Amides.Peptides 1981,2,45−50.
(19) Carpino,L.A.;Han,G.Y. The 9−Fluorenylmethoxycarbonyl Amino−Protecting Group.J.Org.Cliem.1972,37,3404−3409.
(20) Kaiser,E.;Colescott,R.L.;Brossinger,C.D.;Cook,P.I. Color test for detection of free terminal amino groups in the solid−phase synthesis of peptides.Anal.Biochem.1970,34,595−598.
(21) Loibner,H.;Pruchner,A.;Stutz,A.Reduktive Methylierung Primarer und Sekundarer Amine mit Hilfe von Formaldehyd und Salzen Der Phosphorigen Saure.Tetrahedron Lett.1984,25,2535−2536.
(22) Bormans,V.;Peeters,T.L.;Vantrappen,G. Motilin receptors in rabbit stomach and small intestine.Regul.Pept.1986,15,143−153.
(24) Adachi,H.;Toda,H,;Hayashi,S.;Noguchi,M.;Suzuka,T.;Torizuka,K.;Yajima,H.;Kodama,K. Mechanism of the excitatory action of motilin on isolated rabbit intestine.Gastroenterology 1981,80,783−788. Technical field
The present invention relates to a novel peptide derivative having a smooth muscle contraction action.
Background art
Motilin is a single-chain peptide with 22 amino acid residues and was isolated from endocrine cells in the gastrointestinal (GI) mucosa of various types of animals (1) (Ito, Z. Motilin and Clinical Application). Peptides 1997, 18, 593-608). Although the biological function of motilin has not yet been fully identified, motilin was known to stimulate GI motility in many types of animals (ibid.).
The amino acid sequence of swine motilin (pMTL) was confirmed for the first time in 1973 (FVPIF TYGEL QRMQE KENK GQ) (References 2 and 3) (Brown, JC; Cook, MA; Dryburgh, JR Motilin). , a Gastric Motor ActivityStimulating Polypeptide:.... The Complete Amino Acid Sequence Can.J.Biochem, 1973,51,533-537.Schubert, H .; Brown, J Correction to Amino Acid Sequence of Porcine Motilin Can.J. Biochem., 1974, 52, 7-8.). Human motilin was identified in 1985 and is identical to porcine motilin, but is found to differ from canine motilin by five residues at positions 7, 8, 12, 13, and 14. (References 1 and 4).
Physiological associations with motilin and several GI symptoms (early satiety, abdominal distention, nausea, vomiting and anorexia) have been suggested. It has already been suggested that motilin agonists such as motilin analogs (References 5-7) and erythromycin derivatives (References 8-10) are effective in such conditions. However, the detailed mechanism of motilin agonist action after binding to the motilin receptor (MTL-R) has not been elucidated yet. In order to elucidate the biological and physiological mechanisms of motilin and to apply it to the treatment of diseases related to motilin, it has been desired to find a small molecule motilin agonist.
In the structure-activity relationship (SAR) of pMTL reported by the present inventors (Reference 11), it was suggested that the N-terminal part plays an important role for its action (agonism).
Disclosure of the invention
From these, the present inventors hypothesized that a small peptide at the N-terminus would also have an agonistic effect. The present inventors concentrated their efforts on the modification of the tetrapeptide in order to increase the contractile effect of the tetrapeptide on the N-terminal part of motilin. As a result, the present inventors succeeded in synthesizing a novel tetrapeptide motilin agonist represented by Formula 1. The present invention has been completed based on this finding.
The present invention provides the following formula (1)
(Where
n is an integer of 0 to 6,
Ar is an aromatic ring which may be substituted or condensed with another ring or a heterocyclic ring which may be substituted or condensed with another ring,
R1Is a linear or branched lower alkyl group which may be substituted,
R2Is a linear or branched lower alkyl group which may be substituted,
R3Is a hydrogen atom or a linear or branched lower alkyl group which may be substituted,
X is an amino group, a hydroxy group or a lower alkoxy group;
When X is an amino group, the amino group of X is R2May form a ring together with the lower alkyl group. )
And a peptide derivative represented by the formula:
Further, the present invention provides the following formula (2)
(Where
n is an integer of 0 to 1,
R1Is a linear or branched lower alkyl group which may be substituted,
R2Is a linear or branched lower alkyl group which may be substituted,
R3Is a hydrogen atom or a linear or branched lower alkyl group,
X is an amino group;
The amino group of X is R2May form a ring together with the lower alkyl group. )
And a peptide derivative represented by the formula:
Furthermore, the present invention provides the following formula (3)
(Where R1Is an optionally substituted linear or branched lower alkyl group)
And a peptide derivative represented by the formula:
In addition, the present invention provides the following formula (4)
(Where R1Is an optionally substituted linear or branched lower alkyl group)
And a peptide derivative represented by the formula:
The present invention also provides a pharmaceutical composition comprising one or more compounds represented by the above formulas (1) to (4) and a pharmaceutically acceptable carrier.
BEST MODE FOR CARRYING OUT THE INVENTION
In the present invention, the lower alkyl group has 1 to 8 carbon atoms. In the present invention, with respect to the optionally substituted straight-chain or branched lower alkyl group, the position and number of substituents of the substituted straight-chain or branched lower alkyl group are not particularly limited, and may be arbitrary. May have a substituent at one or more carbon atoms. Further, the same carbon atom may have two or more same or different substituents.
Although the type of the substituent is not particularly limited, examples thereof include a hydroxy group, an amino group which may be substituted with one or two alkyl groups having 1 to 6 carbon atoms by an alkyl group, a carboxyl group, and the like. Particularly preferred substituents are hydroxy groups.
In the present invention, preferred linear or branched lower alkyl groups having no substituent include, for example, methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl. Group, tert-butyl group, n-pentyl group, n-hexyl group, n-octyl group and the like.
In the present invention, preferred linear or branched lower alkyl groups having a substituent include a hydroxymethyl group, a 1-hydroxyethyl group, a 2-hydroxyethyl group, a 3-hydroxy-n-propyl group, and a 4-hydroxy -N-butyl group and the like.
Hereinafter, a general synthesis method of the peptide derivative of the present invention will be described.
General synthesis method
The peptide derivative of the present invention is synthesized by a solid phase or liquid phase method (solid phase method or liquid phase method) described in, for example, “Basics and Experiments of Peptide Synthesis” (Maruzen: 1985).
The thioalkylation at the 2'-position of the indole ring is carried out by adding an thioalkyl chloride to the indole ring in the presence or absence of an acid such as trichloroacetic acid, hydrochloric acid, or acetic acid, an acid metal such as sodium acetate, or a base such as triethylamine. By reacting a thioalkyl halide compound such as
Alternatively, by reacting a sulfhydrylalkyl compound in the presence of a halogenating reagent such as N-bromosuccinimide,
Alternatively, the reaction can be carried out by reacting a sulfhydrylalkyl compound or a bisalkyldisulfide compound in the presence of a heavy metal compound such as mercury acetate or silver triflate.
The solvent may be any organic solvent such as tetrahydrofuran (THF), chloroform, dioxane, methanol, and dichloromethane, or any solvent capable of dissolving the compound, such as acids such as acetic acid and trifluoroacetic acid (TFA).
Desirable reaction conditions include a reaction with a bisalkyldisulfide compound or an alkylthiol or an alkylthiohalide in trifluoroacetic acid in the presence of silver triflate (see Reaction Scheme 1 below).
Reaction scheme 1
(In the above reaction scheme, R1Represents an optionally substituted linear or branched lower alkyl group;2Represents an optionally substituted linear or branched lower alkyl group;3Represents a hydrogen atom or a linear or branched lower alkyl group which may be substituted, X represents an amino group, a hydroxy group or a lower alkoxy group, halogen represents fluorine, chlorine, bromine or iodine;
When X is an amino group, the amino group of X is R2May form a ring together with the lower alkyl group. )
Synthesis of peptides by the solid-phase method (References 16 and 17) can be performed, for example, by using NHAα−Boc (Nα-T-butyloxycarbonyl) group or Nα-Fmoc (Nα-9-fluorenylmethyloxycarbonyl) group (References 18 and 19).
NαFor the -Boc group, use a 33-50% (v / v) solution of trifluoroacetic acid (TFA) / dichloromethane (DCM) before coupling the next protected amino acidαDeprotection of the -Boc group is performed. As the acylating species, previously prepared Nα-Boc amino acid symmetrical anhydrides can be utilized. Amino acid symmetric anhydrides are obtained by reacting 0.5 equivalents of dicyclohexylcarbodiimide (DCC) with 1.0 equivalents of Nα-Boc amino acid dissolved in DCM.
NαFor the -Fmoc group, use a 20% (v / v) piperidine / N, N-dimethylformamide (DMF) solution before coupling the next protected amino acid.α-Remove the Fmoc group. All Nα-Fmoc amino acids can be linked as their pentafluorophenyl (Pfp) ester using N-hydroxybenzotriazole (HOBt).
Completion of the binding reaction is confirmed by the Kaiser test (Reference 20). The peptide synthesized by the solid phase method described above depends on TFA / trimethylsilyl triflate (TMSOTf) / m-cresol / thioanisole (100: 28.6: 7.47 :) depending on the group protecting the side chain. 16.7; v / v) or TFA / TMSOTf / m-cresol / thioanisole / ethanedithiol (100: 12: 6.5: 15: 5; v / v) at 0 ° C. or room temperature for 1-4. By treating with time, it is simultaneously deprotected and recovered from the resin. Modification of the indole ring of Trp is performed using the corresponding disulfide in the presence of silver triflate (see Scheme 2).
Reaction scheme 2
(In the above reaction scheme 2, n represents an integer of 0 to 6,
Ar represents an aromatic ring which may be substituted or condensed with another ring or a heterocyclic ring which may be substituted or condensed with another ring,
R1Represents an optionally substituted linear or branched lower alkyl group;2Represents an optionally substituted linear or branched lower alkyl group;3Represents a hydrogen atom or a linear or branched lower alkyl group which may be substituted,
X represents an amino group, a hydroxy group or a lower alkoxy group;
When X is an amino group, the amino group of X is R2May form a ring together with the lower alkyl group. )
The crude peptide is then purified by preparative reversed-phase high-performance liquid chromatography (RP-HPLC), eluting with a linear gradient of 0.1% aqueous TFA in acetonitrile. The peptide fractions found to be homogeneous by analytical RP-HPLC are combined, concentrated and lyophilized. The structure of the peptide thus obtained can be confirmed by NMR and mass spectrometry (FAB-MS) (see Table 3 below).
One or more of the peptide derivatives of the present invention may be used in combination with carriers such as pharmaceutically acceptable diluents, wetting agents, emulsifiers, dispersants, adjuvants, preservatives, buffers, binders, stabilizers and the like. The pharmaceutical composition may be administered in any appropriate form depending on the intended route of administration.
Example
Examples of the present invention will be described below, but the present invention is not limited to these examples.
Materials used
Nα-Fmoc amino acids, or their Pfp esters, and resins for solid phase synthesis were purchased from either MilliGen / Biosearch (Burlington, MA) or Watanabe Chemical (Japan). TFA, TMSOTf, 1,3-diisopropylcarbodiimide (DIC) and HOBt were purchased from Watanabe Chemical (Japan). Di-n-propyldisulfide, n-octyldithiodiglycolic acid, silver trifluoromethanesulfonate, triethylamine, NH4F, 2-mercaptoethanol, m-cresol, ethanedithiol and thioanisole were purchased from Tokyo Chemical Industry (Japan). Dimethyl disulfide was purchased from Nakai Tesque (Japan). DMF, dichloromethane (DCM) and N-methyl-2-pyrrolidinone (NMP) were purchased from Kokusan Chemical (Japan). Methanol, diethyl ether , N-hexane, NaOH and acetic acid were purchased from Junsei Chemical (Japan), and acetonitrile and water for HPLC were purchased from Kanto Chemical (Japan) or Junsei Chemical (Japan). .
Solid phase peptide synthesis
The peptide was synthesized according to the Fmoc solid phase synthesis method (References 16 and 17). The reaction was performed with a shaker.
Peptide purification
The crude peptide was purified by RP-HPLC. This RP-HPLC was performed using preparative HPLC (Waters semi-prep system) using C18 YMC-Pack S-343-15 (YMC, Japan), 15 μm, 120 angstroms, 20 × 250 mm, and a constant concentration. Using a linear acetonitrile gradient (0-60%) in water containing 0.1% TFA (0.1%, v / v) for 60 minutes at a flow rate of 10 ml / min. Fractions of the peptide purified by analytical HPLC were lyophilized and the resulting powders were stored at -20 ° C or below until their biological assay.
Peptide analysis
The purity of the final product was confirmed by RP-HPLC using a Hitachi D-7000 HPLC system in a manner with two different columns and a linear gradient solvent. One solvent system (A) uses a YMC-Pack A-302 analytical column (4.6 × 150 mm, 120 Å, 5 μm particle size, YMC, Japan) at a rate of 1 ml / min for 25 minutes: 0-30: 70 (0.1% aqueous TFA: acetonitrile containing 0.1% TFA). The other solvent system (B) was a 100: 0-30: 70 or 100: 0-2 over 25 minutes at a rate of 10 ml / min using a μBONDASPHERE 5μ C18 300 Å (3.9 × 150 mm, Waters). : 98 (0.1% aqueous TFA: methanol).
NMR was performed on a JEOL JNM-A500, Varian Mercury 300, and JEOL JNM-EX270. The spectrum is DMSO-d6Was measured. The molecular weight of the compound was measured by FAB-MS (VG70-250SEQ, VG Analytical, UK). Value is MH+Indicated by
Reference Example 1: H-Phe-Val-Trp-Ile-NH2Synthesis of
The MBHA-resin (0.79 mmol / g; 3.418 g) was treated twice with a 20% (v / v) piperidine / DMF solution and washed eight times with DMF. Then Nα-Fmoc-Ile-OPfp (3.5 equiv) was added, followed by HOBt (3.5 equiv). The ligation reaction mixture was shaken on a shaker at room temperature for 3 hours. The resin was then washed six times with DMF. Then the corresponding Nα-Fmoc amino acid Pfp ester (Nα-Fmoc-Trp-OPfp, Nα-Fmoc-Val-OPfp, Nα-Fmoc-Phe-OPfp) were sequentially bonded to extend the peptide chain. The following operations were performed in each binding cycle.
(1) Two 30-minute treatments with 20% (v / v) piperidine / DMF solution.
(2) 8 washes with DMF.
(3) Bonding of amino acid active ester.
(4) 6 washes with DMF.
After coupling the last amino acid as described above, the following steps were added.
(5) Two 30-minute treatments with a 20% (v / v) piperidine / DMF solution were performed.
(6) 8 washes with DMF.
The elongation of the peptide chain is NαPerformed in DMF using -Fmoc amino acid Pfp ester (3 eq) and HOBt (3 eq). All binding reactions were performed by shaking on a shaker at room temperature for 3-30 hours. Last NαAfter deprotection of the -Fmoc group, the peptide resin was washed with methanol and dried under vacuum to obtain H-Phe-Val-Trp-Ile-MBHA-resin (4.725 g).
The peptide-resin was treated with TFA / TMSOTf / m-cresol / thioanisole / ethanedithiol (100: 12: 6.5: 15: 5; v / v) for 2 hours at 0 ° C. and another 2 hours at room temperature. Processed. The solution was filtered and added dropwise to cold diethyl ether (500 ml) and n-hexane (1500 ml). The precipitated peptide was collected by centrifugation, washed with diethyl ether and n-hexane (1: 3) and dried. The residue was treated with 2-mercaptoethanol (0.2 ml) in methanol (10 ml) and water (6 ml) at 0 ° C. The resulting solution was adjusted to pH 8.0 with triethylamine over 0.5 hours and then acidified to pH 4.0 with acetic acid. The crude peptide was purified by preparative HPLC. The preparative HPLC was performed at a flow rate of 10 ml / min for 60 minutes using a linear gradient of acetonitrile containing 0-60% 0.1% TFA to 0.1% aqueous TFA. The lyophilized product was a white amorphous solid.
Yield: 0.134 g (9%)
1H-NMR (DMSO-d6) Δ 0.83 (12H, m, 4 × CH3), 1.06, 1.37 (2H, mm, CH2), 1.65 (1H, m, CH), 1.98 (1H, m, CH), 2.72-3.24 (4H, m, 2 × CH)2), 4.13 (2H, m, 2 × CH), 4.29 (1H, m, CH), 4.67 (1H, m, CH), 6.94-7.30 (10H, m, aromatic) −H), 7.62 (1H, d, J = 6.9 Hz, NH), 8.25 (2H, m, 2 × NH), 8.35 (1H, d, J = 9.2 Hz, NH)
Example 1: H-Phe-Val-Trp (2′-SCH2CH2OH) -Ile-NH2Synthesis of (1)
Or
Compound of Reference Example 1 (H-Phe-Val-Trp-Ile-NH2) (0.191 g, 0.339 mmol) was added to a solution of bis- (2-acetoxy-ethyl) -disulfide (0.82 g, 3.44 mmol) in TFA (20 ml) under ice-cooling, followed by silver trifluoromethanesulfonate Salt (0.8449 g, 3.29 mmol) was added. The solution was stirred under ice cooling for 8 hours and at room temperature for 120 hours. The solvent was removed under reduced pressure, and the residue was dissolved in methanol (10 ml). The solution was adjusted to pH 9 with 1 N NaOH aqueous solution over 0.5 hours, filtered, and then acidified to pH 4 with acetic acid. The solvent was removed under reduced pressure, and the obtained residue was purified by RP-HPLC. The lyophilized product was a white amorphous solid.
Yield: 0.108 g (50%)
1H-NMR (DMSO-d6) Δ 0.80 (12H, m, 4 × CH3), 1.03, 1.38 (2H, mm, CH2), 1.66 (1H, m, CH), 1.92 (1H, m, CH), 2.77, 2.87 (4H, mm, 2 × CH)2), 3.04, 3.20 (2H, dd, dd, J = 5.9, 8.3, 14.2 Hz, CH2), 3.47 (2H, m, CH2), 4.12 (1H, dd, J = 7.3, 8.6 Hz, CH), 4.26 (1H, dd, J = 6.9, 8.2 Hz, CH), 4.68 (1H, dt, J = 6.6, 7.9 Hz, CH), 4.91 (1H, t, J = 5.6 Hz, CH), 6.95 (2H, m, aromatic-H), 7.03 (1H) , T, J = 7.4 Hz, aromatic-H), 7.12 (5H, s, aromatic-H), 7.20 (1H, d, J = 7.6 Hz, aromatic-H), 7.66 ( 1H, d, J = 8.9 Hz, NH), 7.69 (1H, d, J = 7.9 Hz, NH), 8.19 (1H, d, J = 7.9 Hz, NH), 8.40 (1H, d, J = 8.9 Hz, NH)
Example 2: H-Phe-Val-Trp (2′-SCH3) -Ile-NH2Synthesis of (2)
Or
Compound of Reference Example 1 (H-Phe-Val-Trp-Ile-NH2) (0.589 g, 1.05 mmol) was added to a solution of dimethyl disulfide (1.028 g, 10.9 mmol) in TFA (20 ml) under ice-cooling, and then silver trifluoromethanesulfonate (1.308 g, 5.09 mmol). ) Was added. This solution was added dropwise to cold diethyl ether (400 ml) and n-hexane (100 ml). The precipitated peptide was collected by centrifugation and washed with diethyl ether. The residue was dissolved in methanol (30 ml), acetic acid (30 ml) and water (20 ml), and treated with dithiothreitol (0.8 g) for 1 hour. The precipitate was removed by centrifugation. The solvent was removed under reduced pressure, and the obtained residue was purified by preparative RP-HPLC. The lyophilized product was a white amorphous solid.
Yield: 0.322 g (43%)
1H-NMR (DMSO-d6) Δ 0.81 (12H, m, 4 × CH3), 1.03, 1.34 (2H, mm, CH2), 1.64 (1H, m, CH), 1.93 (1H, m, CH), 2.40 (3H, s, CH)3), 2.87 (2H, m, CH2), 3.03, 3.24 (2H, dd, dd, J = 6.3, 8.0, 13.9 Hz, CH2), 4.12 (2H, m, 2 × CH), 4.23 (1H, dd, J = 6.9, 8.3 Hz, CH), 4.69 (1H, dd, J = 7.1, 14.4 Hz, CH), 6.99-7.06 (4H, m, aromatic-H), 7.17 (5H, m, aromatic-H), 7.60 (1H, d, J = 8.9 Hz) , NH), 7.67 (1H, d, J = 7.9 Hz, NH), 8.21 (1H, d, J = 7.9 Hz, NH), 8.44 (1H, d, J = 8. 9Hz, NH)
Example 3: H-Phe-Val-Trp (2′-SCH2CH2CH3) -Ile-NH2Synthesis of (3)
Or
The title compound is the compound of Reference Example 1 (H-Phe-Val-Trp-Ile-NH2) (0.592 g, 1.05 mmol) and di-n-propyl disulfide according to the method described in Example 2.
Yield: 0.369 g (47%)
1H-NMR (DMSO-d6) Δ 0.76 (12H, m, 4 × CH3), 0.93 (3H, t, J = 7.3 Hz, CH3), 1.05, 1.41 (2H, mm, CH2), 1.48 (2H, six, J = 7.3 Hz, CH2), 1.64 (1H, m, CH), 1.95 (1H, m, CH), 2.79-2.95 (4H, m, 2 × CH)2), 3.04, 3.26 (2H, dd, dd, J = 6.3, 8.0, 14.2 Hz, CH2), 4.12 (2H, m, 2 × CH), 4.33 (1H, dd, J = 7.3, 8.2 Hz, CH), 4.66 (1H, dd, J = 7.3, 14.9 Hz, CH), 6.90-7.06 (4H, m, aromatic-H), 7.17 (5H, m, aromatic-H), 7.55 (1H, d, J = 8.9 Hz) , NH), 7.66 (1H, d, J = 7.6 Hz, NH), 8.19 (1H, d, J = 8.2 Hz, NH), 8.44 (1H, d, J = 8. 9Hz, NH)
Example 4: H-Phe-Val-Trp (2′-SCH2CH2NMe2) -Ile-NH2Synthesis of (4)
The title compound is the compound of Reference Example 1 (H-Phe-Val-Trp-Ile-NH2) (0.592 g, 1.05 mmol) and N, N, N ′, N′-tetramethylcystamine (Reference 21) according to the method described in Example 2.
Yield: 0.589 g (63%)
1H-NMR (DMSO-d6) Δ 0.82 (12H, m, 4 × CH3), 1.02, 1.38 (2H, mm, CH2), 1.65 (1H, m, CH), 1.94 (1H, m, CH), 2.77 (6H, s, 2 × CH)3), 2.87-3.00 (6H, m, 3 × CH)2), 4.11 (2H, m, 2 × CH), 4.27 (1H, dd, J = 6.8, 8.4 Hz, CH), 4.73 (1H, dt, J = 7.6, 6.8 Hz, CH), 6.91-7.27 (9 H, m, aromatic-H), 7.69 (1 H, d, J = 9.2 Hz, NH), 7.74 (1 H, d, J = 8.2 Hz, NH), 8.28 (1H, m, NH), 8.45 (1H, d, J = 8.9 Hz, NH)
Example 5: H-Phe-Val-Trp (2'-SCH2COOH) -Ile-NH2Synthesis of (5)
The title compound is the compound of Reference Example 1 (H-Phe-Val-Trp-Ile-NH2) (0.584 g, 1.04 mmol) and dithiodiglycolic acid according to the method described in Example 2.
Yield: 0.335 g (42%)
1H-NMR (DMSO-d6) Δ 0.79 (12H, m, 4 × CH3), 1.00, 1.39 (2H, mm, CH2), 1.66 (1H, m, CH), 1.91 (1H, m, CH), 2.81, 2.93 (2H, dd, dd, J = 4.6, 8.3, 14.3). 2Hz, CH2), 3.09, 3.20 (2H, dd, dd, J = 5.9, 8.3, 14.2 Hz, CH2), 3.63 (2H, d, J = 1.7 Hz, CH2), 4.11 (2H, m, 2 × CH), 4.25 (1H, dd, J = 6.9, 8.2 Hz, CH), 4.71 (1H, dt, J = 6.9, 7.4 Hz, CH) 6.90-7.23 (9H, m, aromatic-H), 7.65 (1H, d, J = 10.2 Hz, NH), 7.68 (1H, d, J = 8.6 Hz, NH), 8.12 (1 H, d, J = 8.2 Hz, NH), 8.51 (1 H, br, NH)
Table 1 shows the physical property data (retention time by HPLC and FAB-MS) of the peptide derivatives produced in Examples 1 to 5 above.
Reference Example 2: H-Phe-c [-Nε-Lys-Trp-βAla-] solid-phase peptide synthesis
The alkoxybenzyl alcohol-resin (0.78 mmol / g; 3.41 g) was placed in DMF and allowed to swell overnight. Nα-Fmoc-βAla-OH (8 eq) was dissolved in DMF and DIC (4 eq) was added to the solution. DMAP (1 equivalent) was dissolved in DMF. The amino acid / DIC solution and DMAP solution were added to the resin in DMF, shaken gently, and left at room temperature overnight.
The resin was washed twice with DMF, twice with methanol, twice with acetic acid, twice with methanol, and three times with DMF. Then the corresponding Nα-Fmoc amino acid Pfp ester (Nα-Fmoc-Trp-OPfp, Nα-Fmoc-Lys (Boc) -OPfp, Nα-Fmoc-Phe-OPfp) were sequentially bonded to extend the peptide chain. The following operations were performed in each binding cycle.
(1) Two 30-minute treatments with 20% (v / v) piperidine / DMF solution.
(2) 6 washes with DMF.
(3) Bonding of amino acid active ester.
(4) 6 washes with DMF.
The elongation of the peptide chain is NαPerformed in DMF using -Fmoc amino acid Pfp ester (2.5 eq) and HOBt (2.5 eq). All binding reactions were performed by shaking on a shaker at room temperature for 3-12 hours. Last NαAfter coupling of the -Fmoc amino acid, the peptide resin was washed with methanol and dried under vacuum to obtain a protected Fmoc-Phe-Lys (Boc) -Trp-βAla-resin (5.93 g).
The protected peptide-resin was treated with TFA / anisole / ethanedithiol (94: 5: 1; v / v) at 0 ° C. for 3 hours. The solution was filtered, concentrated under reduced pressure, and added dropwise to excess cold diethyl ether. The precipitated peptide was filtered, washed with diethyl ether and dried. This peptide was dissolved in DMF (3 L) and pyridine (3 L), Bop reagent was added, and the mixture was allowed to stand at room temperature for 24 hours. The solvent was removed under reduced pressure, and water was added to the residue. The precipitate was collected by filtration, washed with water and dried. The Fmoc-cyclic peptide thus obtained was treated with a 20% (v / v) piperidine / DMF solution at room temperature for 0.5 hour to remove the Fmoc group. The solvent was removed under reduced pressure and the resulting crude peptide was purified by preparative HPLC. The preparative HPLC was performed at a flow rate of 10 ml / min for 60 minutes using a linear gradient of acetonitrile containing 0.1% TFA to 0.1% aqueous TFA. The lyophilized product was a white amorphous solid (0.133 g).
Overall yield: 57%
FAB-MS: 533.2876,
HPLC RT: (A) 13.36 (98%), (B) 18.29 (100%)
Example 6: H-Phe-c [-Nε-Lys-Trp (2'-SCH2CH2OH) -βAla-] (6)
Or
H-Phe-c [-Nε-Lys-Trp (2′-SCH2CH2[OH) -βAla-] was synthesized from H-Phe-c [-Nε-Lys-Trp-βAla-] (Reference Example 2) in the same manner as described in Example 1.
Yield: 104 mg (22% yield)
FAB-MS: 609.22859,
HPLC RT: (A) 13.33 (96%), (B) 18.73 (100%)
Test example
Hereinafter, two test contents (receptor binding test and shrinkage test) using the compound of the present invention and the results thereof will be described.
contents of the test
In the tests described below, the synthetic peptides shown in Table 1 were tested for binding activity to MTL-R and rabbit smooth muscle contraction activity.
1. Receptor binding test
A binding test for MTL-R using male Japanese white rabbits (about 3 kg) was performed as described in Bormans et al. Was slightly modified and implemented. Rabbits were anesthetized with sodium thiopental (30 mg / kg, intravenous injection) and then exsanguinated and euthanized. Thereafter, the upper small intestine (about 50 cm) was promptly excised, and impurities were removed in ice-cooled 0.9% physiological saline. The connective tissue and mucous membrane were removed in a 50 mM Tris-HCl buffer (pH = 7.4) to obtain a smooth muscle layer. Using a taper homogenizer (Wheaten, Millville, NJ) in 50 mM Tris-HCl buffer (pH = 7.4), the smooth muscle tissue was homogenized at a speed of 2000 rpm for about 30 seconds. The obtained suspension was centrifuged at 1500 rpm for 5 minutes, the supernatant was discarded, and a fresh buffer was added to the precipitate for washing. This homogenization operation was performed twice, and the precipitate was collected. The resulting precipitate is washed with 50 mM Tris-HCl buffer (pH = 8.0, MgCl 2210 mM and 1.5% bovine serum albumin) were added to the suspension, and the following binding test was carried out. All of the above operations were performed under ice cooling. Samples (about 1.0 mg protein / assay) were prepared at 25 pM125The cells were incubated with I-pMTL (specific activity, 33-66 kBq / pmol) and various concentrations of the test compound at 25 ° C. for 120 minutes. After the incubation, the reaction was stopped by adding an ice-cold buffer, and centrifuged at 1500 rpm for 5 minutes.125I-MTL was removed. The obtained precipitate was washed twice with an ice-cold buffer, and the radioactivity was measured with a gamma counter (ARC-300, Aloka, Tokyo, Japan).125The concentration of each test compound that attenuates the binding of I-pMTL to 50% is determined by IC50And used as an index of MTL-R binding activity.
2. Shrinkage test
Male Japanese white rabbits (about 3 kg) were used. The rabbit was euthanized by exsanguination under anesthesia with sodium thiopental (30 mg / kg, intravenous injection), and the upper small intestine was immediately removed. The duodenum was improved with Krebs solution (composition (unit: mM): NaCl 120.0, KCl 4.7, CaCl 222.4, KH2PO41.0, MgSO41.2, NaHCO3Wash in 24.5 and glucose 5.6 (pH = 7.4) to remove connective tissue such as mesentery, cut along the long axis to make a specimen approximately 10 mm long and 3 mm wide did. Mixture gas (95% O2: 5% CO2) Was suspended in a 10 ml Magnus tube filled with a modified Krebs solution at 28 ° C. through which the air was passed (Reference 24). A 1.0 g load was applied and the isotonic contractile response was measured with a transducer (ME-4012, Medical Electronics Co., Tokyo) and recorded with a recorder (Type 3066, Yokogawa-Decs, Tokyo). After confirming that a stable contraction was obtained with respect to 100 μM of ACh, the test compound was examined. Test compounds were added cumulatively from low concentrations. The contractile activity of each test compound is expressed as a percentage of the contractile force induced by 100 μM ACh, and the concentration of the additive giving 50% contraction is expressed by EC50Expressed as
Test results
Tables 2 and 3 show the results of the above two tests.
Importantly, all of the compounds of Examples 1, 2, 3, and 6 show efficacy for shrinkage tests (EC of these compounds).50Are 14.2, 58.1, 33.2 and 63.0 μM, respectively), demonstrating that the N-terminal tetrapeptide can act as a motilin agonist in vitro. A structural feature of the derivatives of Examples 1, 2, 3 and 6, which are active with regard to contractile action, is the presence of an aliphatic side chain on the tryptophan ring.
Industrial potential
INDUSTRIAL APPLICABILITY The novel tetrapeptide derivative of the present invention has a binding activity to a motilin receptor and has an action of contracting rabbit smooth muscle, so that it is expected to be applied to the treatment of a disease related to motilin.
Reference list
(1) Itoh, Z. Motilin and Clinical Application. Peptides 1997, 18, 593-608.
(2) Brown, J. et al. C. Cook, M .; A. Dryburgh, J .; R. Motilin, a Gastric Motor Activity Stimulating Polypeptide: The Complete Amino Acid Sequence. Can. J. Biochem. , 1973, 51, 533-537.
(3) Schubert, H .; Brown, J .; Collection to Amino Acid Sequence of Porcine Motilin. Can. J. Biochem. 1974, 52, 7-8.
(4) McIntosch, C.I. H. S. Brown, J .; C. Purification and Chemical Structure of Porcine and Canine Motileins and Evidence for the Existence of Motilin in Other Species. In Motilin Itoh, Z .; Academic Press: San Diego, CA, 1990; pp 13-30.
(5) Kitagawa, T .; Ichikawa, S .; Yokoyama, T .; Ishii, A .; Shuto, K .; Stimulating action of KW-5139 (LeuThirteen-Motilin) on gastrointestinal mobility in the rabbit. Br. J. Pharmacol. 1994, 111, 288-294.
(6) Kitagawa, T .; Taneike, T .; Ohga, A .; Excitatory Action of [LeuThirteenMotilin on the Gastrointestinal Smooth Muscle Isolated From the Chicken. Peptides 1995, 16, 1243-1252.
(7) Assche, G .; V. Deportore, I .; Thijs, T .; Janssens, J .; J. Peters, T .; L. Concentration-dependent stimulation of cholinergic motor neighbors or smooth muscle by [NleThirteenMotilin in the isolated rabbit gastric antrum. Eur. J. Pharmacol. 1997, 337, 267-274.
(8) Shiba, Y .; Mizumoto, A .; Satoh, M .; Inui, A .; Itoh, Z .; Effect of Nonpeptide Motivin Agonist EM523 on Release of Gut and Pancreatic Hormones in Conscious Dogs. Gastroenterology 1996, 110, 241-250.
(9) Yamada, K .; Chen, S .; Abdulah, N .; A. Tanaka, M .; Ito Y .; Inoue, R .; Electrophysiological. Characterization of a motorin agonist, GM611, on rabbit duodenal smooth muscle. Am. J. Physiol. 1996, 271, G1003-G1016.
(10) Verhagen, M .; A. Samsom, M .; Mase, B .; Geypens, B .; J. Ghoos, B .; J. Smout, A .; J. Effects of a new motility, ABT-229, on gastric emptying and postprandial antrochlorodental mobility in health volunters. Alignment. Pharmacol. Ther. 1997, 11, 1077-1086.
(11) Haramura, M .; Tsuzuki, K .; Okamachi, A .; Yogo, K .; Ikuta, M .; Kozono, T .; Takanashi, H .; Murayayama, E .; Structure-Activity Study of Intact porcine Motilin. Chem. Pharni. Bull. 1999, 47, 1555-1559.
(16) The Practice of Peptide Synthesis; Bodansky, M .; Bodansky, A .; , Eds. Springer-Verlag: Berlin, Heidelberg, 1984.
(17) Stewart, J .; M. Young, J .; D. Solid Phase Peptide Synthesis; 2nd ed. , Pierce Chemical Co .; Rockford, IL, 1984.
(18) Matsuda, G .; R. Stewart, J .; M. Ap-Methylbenzhydramine Resin for Improved Solid-Phase Synthesis of Peptide Amides. Peptides 1981, 2, 45-50.
(19) Carpino, L .; A. Han, G .; Y. The 9-Fluorenylmethycarbonyl Amino-Protecting Group. J. Org. Cliem. 1972, 37, 3404-3409.
(20) Kaiser, E .; Colescott, R .; L. Brossinger, C .; D. Cook, P .; I. Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides. Anal. Biochem. 1970, 34, 595-598.
(21) Loibner, H .; Pruchner, A .; Stutz, A .; Reduced Methyllierung Primer and Sekunder Amine mit Hill von Formaldehyd and Salzen Der Phosphorigen Saure. Tetrahedron Lett. 1984, 25, 2535-2536.
(22) Bormans, V .; Peters, T .; L. Vantrappen, G .; Motilin receptors in rabbit stomachi and small intestine. Regul. Pept. 1986, 15, 143-153.
(24) Adachi, H .; Toda, H, Hayashi, S .; Noguchi, M .; Suzuka, T .; Torizuka, K .; Yajima, H .; Kodama, K .; Mechanism of the excitatory action of motilin on isolated rabbit intestine. Gastroenterology 1981, 80, 783-788.
Claims (6)
(式中、
nは0〜6の整数であり、
Arは置換されていてもよく他の環と縮合していてもよい芳香環または置換されていてもよく他の環と縮合していてもよい複素環であり、
R1は置換されていてもよい直鎖または分岐鎖状の低級アルキル基であり、
R2は置換されていてもよい直鎖または分岐鎖状の低級アルキル基であり、
R3は水素原子または置換されていてもよい直鎖もしくは分岐鎖状の低級アルキル基であり、
Xはアミノ基、ヒドロキシ基または低級アルコキシ基であり、
また、Xがアミノ基である場合、Xのアミノ基はR2の低級アルキル基と一緒になって環を形成してもよい。)
で表されるペプチド誘導体。The following formula (1)
(Where
n is an integer of 0 to 6,
Ar is an aromatic ring which may be substituted or condensed with another ring or a heterocyclic ring which may be substituted or condensed with another ring,
R 1 is a linear or branched lower alkyl group which may be substituted,
R 2 is a linear or branched lower alkyl group which may be substituted,
R 3 is a hydrogen atom or a linear or branched lower alkyl group which may be substituted,
X is an amino group, a hydroxy group or a lower alkoxy group;
Also, when X is an amino group, an amino group of X may form a ring together with the lower alkyl group for R 2. )
A peptide derivative represented by the formula:
(式中、
nは0〜1の整数であり、
R1は置換されていてもよい直鎖または分岐鎖状の低級アルキル基であり、
R2は置換されていてもよい直鎖または分岐鎖状の低級アルキル基であり、
R3は水素原子または直鎖もしくは分岐鎖状の低級アルキル基であり、
Xはアミノ基であり、
また、Xのアミノ基はR2の低級アルキル基と一緒になって環を形成してもよい。)
で表されるペプチド誘導体。The following formula (2)
(Where
n is an integer of 0 to 1,
R 1 is a linear or branched lower alkyl group which may be substituted,
R 2 is a linear or branched lower alkyl group which may be substituted,
R 3 is a hydrogen atom or a linear or branched lower alkyl group;
X is an amino group;
Further, the amino group of X may form a ring together with the lower alkyl group of R 2 . )
A peptide derivative represented by the formula:
(式中、R1は置換されていてもよい直鎖または分岐鎖状の低級アルキル基である)
で表されるペプチド誘導体。The following formula (3)
(Wherein, R 1 is a linear or branched lower alkyl group which may be substituted)
A peptide derivative represented by the formula:
(式中、R1は置換されていてもよい直鎖または分岐鎖状の低級アルキル基である)
で表されるペプチド誘導体。The following formula (4)
(Wherein, R 1 is a linear or branched lower alkyl group which may be substituted)
A peptide derivative represented by the formula:
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