Peng
Liu
ab,
Bo
Li
*ab,
Mengyu
Xi
ab,
Zhaoqiang
Chen
ab,
Haiguo
Sun
ab,
Xiajuan
Huan
a,
Xuejun
Xu
a,
Yong
Zhang
a,
Kun
Zou
a,
Xiangrui
Jiang
ab,
Zehong
Miao
ab,
Jinggen
Liu
ab,
Jingshan
Shen
ab,
Kaixian
Chen
abc and
Weiliang
Zhu
*abc
aKey Laboratory of Receptor Research, Drug Discovery and Design Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China. E-mail: boli@simm.ac.cn; wlzhu@simm.ac.cn
bSchool of Pharmacy, University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing 100049, China
cOpen Studio for Druggability Research of Marine Natural Products, Pilot National Laboratory for Marine Science and Technology (Qingdao), 1 Wenhai Road, Aoshanwei, Jimo, Qingdao 266237, China
First published on 12th July 2019
The chemical modification of the primary amino groups of amino acid derivatives and peptides is an important process in the pharmaceutical industry and the field of chemical biology. However, suitable reactions that can be carried out under mild and environmentally friendly conditions are limited. We present a versatile method to selectively modify primary amino groups using novel dihydrooxazolo[3,2-a]quinoliniums in 1-butanol as solvent under mild and metal-free conditions. The application of this method to peptides with primary amino, secondary amino, amide, alcoholic hydroxyl, phenolic hydroxyl, disulfide bond, ester and cyano groups revealed that only the primary amino groups were selectively modified, suggesting that this method is compatible with other reactive moieties. We also demonstrated that the quinolylation of existing peptides affected peptide bioactivity and stability, indicating that the novel dihydrooxazolo[3,2-a]quinoliniums can be widely applied, especially in medicinal chemistry and chemical biology.
Several strategies have been developed for the chemical modification of primary amino groups in amino acid derivatives and peptides, including acetylation,21–23 alkylation,24 oximation,25–27 arylation28 and quinonylation.29 The quinoline skeleton is a prevalent structure in small-molecule drugs, including quinine sulfate (antimalarial),30 montelukast sodium (asthma),31 and saquinavir (anti-HIV).32 Furthermore, the quinolylation of primary amino groups can also be used to generate peptide–drug conjugates (PDCs) and is thus important in the development of both peptide and PDC drugs. To the best of our knowledge, Buchwald–Hartwig amination is currently the only available quinolinylation method and requires metal catalysis, expensive ligands and high temperature (Fig. 1a).28 Furthermore, no suitable method is available for the direct quinolylation of amino acid derivatives and peptides. Accordingly, new metal-free strategies with mild reaction conditions and good group compatibility are needed to selectively modify the primary amino groups of amino acid derivatives and peptides with the goal of promoting the development of peptide drugs with green chemistry characteristics.
Fig. 1 N-Quinolylation of amino acid residues: (a) palladium-catalyzed Buchwald–Hartwig reaction; and (b) metal-free click reaction (this work). |
Previously, we disclosed a novel oxazoline[3,2-a]pyridinium that was treated with different nucleophiles for performing regioselective and metal-free C–O and C–N bond-cleaving to afford heterocyclic N-substituted pyridones and 2-substituted pyridines.33 These results inspired us to investigate whether the oxazoline[3,2-a]pyridinium is suitable for coupling with amino acid residues. In this study, we found that the quinoline quaternary ammonium salt can be used to modify the primary amino group (Fig. 1b and S1†) under mild reaction conditions without the need for a heavy-metal catalyst. Therefore, we further optimized this reaction.
Entry | Base | Eq. of amino amide | Solvent | Yieldb (%) |
---|---|---|---|---|
a All reactions were performed at ambient temperature. 5a was dissolved in different solvents and treated with 2-amino-N-methylacetamide and base. b The yields were determined by HPLC. | ||||
1 | DABCO | 1.0 | 1-Butanol | 47 |
2 | DMAP | 1.0 | 1-Butanol | 57 |
3 | DBU | 1.0 | 1-Butanol | 47 |
4 | CO(NH2)2 | 1.0 | 1-Butanol | 11 |
5 | DIPEA | 1.0 | 1-Butanol | 59 |
6 | Arginine | 1.0 | 1-Butanol | 22 |
7 | Et3N | 1.0 | 1-Butanol | 62 |
8 | Et3N | 1.0 | DMSO | 6 |
9 | Et3N | 1.0 | THF | 6 |
10 | Et3N | 1.0 | 1,4-Dioxane | 9 |
11 | Et3N | 1.0 | Acetone | Trace |
12 | Et3N | 1.0 | MeCN | 10 |
13 | Et3N | 1.0 | PhMe | 20 |
14 | Et3N | 1.0 | EtOH | 20 |
15 | Et3N | 1.0 | DMF | 17 |
16 | Et3N | 1.0 | CCl4 | 18 |
17 | Et3N | 1.0 | ClCH2CH2Cl | 18 |
18 | Et3N | 1.0 | CH2Cl2 | 26 |
19 | Et3N | 1.5 | 1-Butanol | 71 |
20 | Et 3 N | 2.0 | 1-Butanol | 95 |
21 | Et3N | 3.0 | 1-Butanol | 95 |
Using the optimized reaction conditions (Table 1, entry 20), we explored a variety of amino substrates bearing different substituents (Table 2). We found that α-amino amides with different substitutions at the α position gave the quinolylated products in 63%–92% yields over two steps (6a–6d, 6f). Among these, (S)-2-amino-N-methylpropanamide afforded the (S)-N-methyl-2-(quinolin-2-ylamino)propanamide 6a in 92% yield. By switching to 2-amino-N-3,3-trimethylbutanamide, which bears a hindered tertiary butyl at the α position of the amino group, the yield of 6b decreased to 66%, suggesting that steric hindrance had an obvious effect on the reaction. Similarly, the N-quinolylation of (S)-2-amino-N-methyl-2-phenylacetamide and (S)-2-amino-N-methyl-3-phenylpropanamide proceeded smoothly to provide products 6c and 6d in 63% and 72% yields, respectively. Specially, the amino group of 2-aminobutanamide was preferentially N-quinolylated rather than the acylamino to afford 6f in 85% yield. In addition, products 6g and 6e were obtained from glycine methyl ester and phenylalanine methyl ester in 95% and 61% yields, respectively. Accordingly, more amino acid esters were explored. Alanine methyl ester, valine methyl ester and isoleucine methyl ester gave the quinolylated products 6h, 6i and 6j in high yields of 82%–93%. Serine methyl ester, threonine methyl ester and tyrosine methyl ester gave 6k, 6l and 6m in moderate yields (63%–88%) without the interference of hydroxyl groups. Moreover, tryptophan methyl ester was converted into the quinolylated product 6n in 70% yield, while the indole amino group was not N-quinolylated. Neither proline methyl ester nor formamide formed quinolylated products (6o1 and 6o2), and the di-n-propylamine did not form the quinolylated product (6t). These results indicate that the quinoline quaternary ammonium salt selectively reacted with primary amino groups rather than secondary amino groups. Interestingly, methionine methyl ester furnished product 6p in 96% yield, while quinolylated product 6q was not observed with cysteine methyl ester, indicating that the thiol group needs to be protected before the quinolylation reaction. Particularly, N-quinolylation mainly occurred at the ε amino group rather than the α amino group of lysine methyl ester and amide (6r and 6s).
To demonstrate the utility of this developed protocol, we investigated the N-quinolylation of 2-amino-N-methylacetamide with a variety of substituted 2-(2,2-dimethoxyethoxy) quinolines and isoquinolines (Table 3). Both electron-donating (Me–, phenyl) and withdrawing (Cl–, Br–) groups at different positions of the quinoline or isoquinoline moiety gave the quinolylated products 6A–6K in 42%–91% yields. Among these, methyl group-substituted quinoline derivatives smoothly afforded 6A and 6B in 91% and 45% yields, respectively. The quinoline substrates substituted with chloro and bromo groups were also compatible under our experimental protocol, giving 6C and 6D in 53% and 42% yields, respectively. The halogens can be applied in further coupling reactions under Suzuki conditions.35,36 Moreover, when a 4-methoxyphenyl group was installed at the 6-position of the quinoline moiety, the substrate provided 6G in 54% yield. Substrates in which phenyl groups were substituted by electron-withdrawing groups (CF3–, CN–, CO2Me–) also afforded the desired products 6H, 6I and 6J in 65%, 78% and 57% yields, respectively. When the quinoline moiety was simultaneously substituted with 2-fluorophenyl and methyl, it provided 6K in 70% yield.
The proposed mechanism of the reaction is shown in Scheme 1. First, dihydrooxazolo[3,2-a]quinolinium 5a is generated from 4 after treatment with hydrogen chloride in diethyl ether. The nucleophilic attack of 2-amino-N-methylacetamide then affords the corresponding intermediate 9. Quantum chemistry calculation at the M06-2X/6-311+G(d) level shows that the activation energy of the transition state (TS) is 14.5 kcal mol−1, indicating that the reaction occurs easily. The subsequent removal of hydrogen chloride facilitated by triethylamine provides the intermediate 10, which ultimately undergoes aromatization to give product 6 with 2-butoxy-2-methoxyethan-1-ol as a possible byproduct.
As mentioned above, 2-(2,2-dimethoxyethoxy) quinoline was treated with hydrogen chloride in diethyl ether after distilling the solvent and without any complicated purification step to give the 1-methoxy-1,2-dihydrooxazolo[3,2-a]quinolinium 5a (Table 4). 5a was reacted with 2-amino-N-methylacetamide to afford 6 in 95% yield. However, the N-quinolylation of other reported quaternary ammonium salts, viz., 1-methylquinolin-1-ium (11), quinoline 1-oxide (12) and 1-acetylquinolin-1-ium (13), with 2-amino-N-methylacetamide did not occur, indicating that the novel dihydrooxazolo[3,2-a]quinolinium has unique reaction characteristics.
The established method was further used for the N-quinolylation of the primary amino groups of seven molecules (Table 5). Two drug molecules used to treat hypotension and type 2 diabetes, midodrine hydrochloride37 and saxagliptin,38 were successfully coupled with quinoline quaternary ammonium salt to provide the quinolylated products 8a and 8b in 72% and 46% yields, respectively. Five peptide molecules, methyl tyroserleutide (liver cancer),39,40 Val-Cit-PAB-OH (a linker for antibody–drug-conjugation),41–43 oxytocin (improvement of uterine contractions),44 dermorphin (a μ-opioid receptor agonist),45 and octreotide (functional gastrointestinal pancreatic endocrine tumors),46 were reacted to give the desired site-selectively N-quinolylated peptides 8c–8i under the same protocol.
a Used 2.0 equiv. quinoline quaternary ammonium salt and 1.0 equiv. peptide. |
---|
These structural modifications of peptide drugs indicate that reactive or sensitive groups such as secondary amino groups (6n), amides (6f, 8d, 8e, 8f, 8g, 8h and 8i), alcoholic hydroxyls (6k, 6l, 8a–8e, 8g, 8h and 8i), phenolic hydroxyls (6m, 8c, 8f and 8g), disulfide bonds (8f, 8h and 8i), ester groups (6e, 6g–6n, 6p, 6r, 8c) and cyano groups (8b) are well tolerated, and the site-selective N-quinolylation occurs at the primary amino group position under metal-free reaction conditions.
Among these peptides, tyroserleutide (YSL) has been studied in phase III clinical trials for the treatment of liver cancer. Therefore, YSL, YSL-M (tyroserleutide methyl ester) and 8c were evaluated for anticancer activity against hepatocarcinoma BEL-7402 and SMMC-7721 cells. Bioassay results revealed that 8c exhibited the highest cytotoxicity against both BEL-7402 and SMMC-7721 cells (ESI Table S1†), indicating that the introduction of a quinolyl group into the peptide changed its bioactivity. Dermorphin is a clinically used μ-opioid receptor agonist. Its quinolylated compound 8g exhibited a slightly decreased activity compared to dermorphin (ESI Table S2†), again suggesting that the modification of active peptides is of significance. 7-(2-Fluorophenyl)-4-methylquinolin-2(1H)-one was found to be a tankyrase inhibitor with an IC50 value of 0.052 μM.47 This compound can be coupled with Val-Cit-PAB-OH to afford 8evia our method. A preliminary in vitro liver stability assessment (ESI Fig. S2†) of 8e indicated good metabolic stability, suggesting the potential to develop PDC drugs based on the target peptide using our new method. N-Quinolylation mainly occurred at the α amino group of octreotide with both lysine ε amino and α amino groups (8h and 8i). Small amounts of the quinolylation products of both the α and ε amino groups were detected for this reaction, but no single ε amino group coupling product was observed.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c9gc01442j |
This journal is © The Royal Society of Chemistry 2019 |