Synthesis and Antimicrobial Activity of Novel Substituted Ethyl 2-(Quinolin-4-yl)-propanoates

Substituted 4-hydroxyquinolines were synthesized from anilines and diethyl 2-(ethoxymethylene)malonate by the Gould-Jacobs reaction via cyclization of the intermediate anilinomethylenemalonate followed by hydrolysis and decarboxylation. The 4-hydroxyquinolines reacted with phosphorous oxychloride to form 4-chloroquinolines, which reacted on heating with diethyl sodiomethylmalonate in DMF to yield moderate yields of substituted ethyl 2-(quinolin-4-yl)propanoates, many of which showed potent antimicrobial activity against Helicobacter pylori.


Introduction
Many natural products in the plant kingdom contain the quinoline nucleus, such as quinine, which has well known anti-malarial and anti-inflammatory activities [1,2]. During World War II an enormous effort was realised by synthetic organic chemists to develop new anti-malarials based on quinine as the supply of quinine from natural sources was insufficient for the Allied and US forces fighting in South East Asia. This intense synthetic attention resulted in a large number of substituted 4-chloroquinolines which were subsequently reacted with a variety of alkylamines to form 4-alkylaminoquinolines as OPEN ACCESS substitution products [3][4][5][6], from which the new potent synthetic anti-malarial chloroquin resulted. Quinoline compounds have also had a history of being potential anti-inflammatory compounds [7][8][9].
In the last twenty five years there has been a steep decline in the commercial output and research and development of antimicrobial agents by the major pharmaceutical companies due to the more attractive commercial returns that can be made for treatments of chronic human diseases. At the same time there has been an explosion both in the numbers of pathogenic bacteria that have become resistant to antibiotics and of immuno-compromised patients that are particularly susceptible to opportunistic pathogens. More recently there has also been rapid emergence of new resistance mechanisms to antibiotics recently introduced to the market, including linezolid and daptomycin. The discovery of new therapeutic agents with novel modes of action and no cross-resistance with current antibiotics will be vital to meet the threats created by the emergence of bacteria resistant to current therapeutic agents [10].
Helicobacter pylori (formerly Campylobacter pylori) and its clinical association with the development of peptic ulcers has led to the development of various chemotherapeutic agents to eliminate infection caused by this pathogen [11][12][13][14][15]. H. pylori is also implicated in the development of acute and chronic gastritis, gastric adenocarcinoma and gastric lymphoma (MALT), and has been classified as a class I carcinogen in humans and is a major contributing factor in the development of gastric cancer [16]. Infection with H. pylori is treated with a combination of clartithromycin, ampicillin and a proton pump inhibitor, but this triple therapy approach is costly [17]. The infection is typically eradicated in up to 90% of patients but side effects, poor compliance and the development of antimicrobial resistance are common causes of treatment failure [18]. H. pylori infection has been implicated with increased COX-2 expression in gastric antral mucosa for both NSAID users and non-users [19][20][21]. In this paper we report a set of novel quinoline derived propionic acid esters 10a-p with H. pylori activity. These are novel compounds that to the best of our knowledge have not been made before.

Chemistry
Classically a variety of methods have existed for the synthesis of quinolines [22]. In this work we have used the Gould-Jacobs reaction [23][24][25] of anilines 1 and diethyl 2-(ethoxymethylene) malonate (2) for the synthesis of 4-hydroxyquinolines 7 as key intermediates (Scheme 1). Thus, commercially available substituted anilines 1 were condensed with 2 to yield the anilinodiesters 3 in high yields. Compounds 3 underwent a thermally induced intramolecular cyclisation in diphenyl ether to form the quinolones 4 in high yields. Hydrolysis followed by thermal decarboxylation of enols 5 furnished the requisite 4-hydroxyquinolines 7 in good yields. The 4-chloroquinolines 8, obtained from 7 by reaction with phosphorus oxychloride [26], underwent nucleophilic aromatic substitution reactions with diethyl sodiomethylmalonate in DMF to yield products 9 which subsequently de-ethoxycarbonylated by the Krapcho reaction [27] to produce the target propionic esters 10 in moderate to good yields (Scheme 2).   Although nucleophilic substitution reactions of halopyridines and haloquinolines with a variety of nucleophiles are well documented [28], this is the first time such a reaction has been reported on substituted 4-chloroquinolines to form substituted ethyl 2-(quinolin-4-yl)propanoates. A literature search showed that ethyl 2-(quinilin-4-yl)propanoate (10, X = H) has been reported [29]. Synthesis of a pyridine equivalent ethyl 2-(4-pyridyl)propionate (11) by titanium enolate addition to the 4-position of 1-phenoxycarbonylpyridinium salts to give 1,4-dihydropyridines which on subsequent aromatization provided 4-(2-oxoalkyl) pyridines has been reported [30]. A 3-substituted(4-pyridyl) propionic acid 12 has also been reported as a key intermediate in the synthesis of the potent and long-acting histamine H 2 -receptor antagonist SK&F 93574 [31].
During the chromatographic purification of the two compounds 10k and 10m we isolated the compounds 4-ethoxyquinolines 13 and 14 as side products. 4-Ethoxyquinolines have previously been reported as side products in the preparation of ethyl 7-chloro-4-hydroxyquinoline-3-carboxylate (4e) from diethyl ethoxymethylmalonate (2) and 3-chloroaniline [32]. One possible explanation is that in our case sodium hydroxide had formed in small amounts when we were adding sodium hydride to the DMF as a result of some moisture in the DMF and this caused partial hydrlolysis of diethyl methylmalonate to form ethanol. The ethanol reacted with sodium hydride to form sodium ethoxide which then produced the 4-ethoxyquinolines 13 and 14 by nucleophilic substitution reactions (Scheme 3).

Microbiological Results and Discussion
All of the quinoline compounds 10b-p exhibited antimicrobial effects against our panel of organisms (Table 1). There was modest antimicrobial activity against H. pylori when compared with the standard anti-Helicobacter agents, however, there appears to be no Gram-specificity which makes these agents promising for broad-spectrum anti-infective development. There is quite a significant contrast in inhibitory activity for the different quinolines using the two different types of H. pylori strains. Thus, in Figures 1 and 2 it can be seen that overall many of the compounds have inhibitory effects, but there are some differences in the concentration-responses for the inhibition of H. pylori strain 3339 obtained with compounds 10a-p, 13 and 14 after 7 days. Compounds 10b, 13 and 14 show relatively potent inhibition in the low concentration range 3-6 μmol/mL toward H. pylori 3339, whilst compounds 10e, 10g and 10n were the least potent at both low (3-6 μg/mL) and high concentrations (25-50 μg/mL). The decline in absorbance at concentrations of 25-50 µg/mL below zero probably represents the lysis of the cells by many of the compounds.  In contrast to the situation with H. pylori strain 3339, the results with strain 26695 were somewhat different. Thus, only compounds 10h and 10p show inhibitory activity at low concentration (6.25 μg/mL) while compounds 10g, 10m and 13 failed to show any inhibitory effects (Figures 3 and 4). However, compounds 10p and 14 show a dramatic potency in the concentration range 6-12 μmol/mL. At the concentration of 25 μg/mL all of the four compounds 10k, 10m, 10p and 14 show equal and complete inhibitory activity.  In the context of quantitative structure activity relationship (QSAR) the following conclusions can be made. An alkyl group in positions 6, 7 and 8 on the quinoline ring leads to lack of reactivity as shown by the reactivity profile of compounds 10i, 10j and 10m on H. pylori 26695 at 25 μg/mL concentration. A methyl group in the position-6 of quinoline showed lack of potency against H. pylori 3339 at 25 μg/mL concentration, as uniquely illustrated by compound 10i. However, an alkyl group in position-8 such as a methyl or n-butyl group with an alkoxy group such as ethoxy seems to enhance potency, as shown by compounds 13 and 14 against H. pylori 3339 at low concentration. Furthermore, the presence of a halogen atom in position-6 caused lack of reactivity against H. pylori 26695 at 25 μg/mL concentration as shown by compounds 10a, 10d and 10g.

Chemistry
Melting points were recorded on Stuart SMP3 digital apparatus; 1 H-NMR and 13 C-NMR spectra were recorded in CDCl 3 on a Bruker AC 250 MHz and a Bruker Avance III 400 MHz spectrometer, respectively. GC-MS analyses of quinoline derivatives 10a-p were performed with an Alignment Technology 5975C VLMSD mass spectrometer interfaced with a GC7890A gas chromatography system with triple-axis detector having an auto-sampler set-up system and a splitless injection (inlet temperature 250 °C) HP5MS capillary column (30 m, 0.25 mm i.d., 0.25 um film thickness) was selected. The oven temperature was programmed from 150 °C (initial hold time of 1 min) to 280 °C at a rate of 10-20 °C min −1 ; this final temperature was maintained for 15 min. Mass spectra (MS) were obtained on VG 770E spectrometer operated in EI mode at 70 eV. High resolution accurate mass (HRMS) of compounds was detected using an Applied Biosystems/MDS Sciex Hybrid quadrupole time-of-flight instrument (Q-Star Pulsar-i) fitted with an orthogonal MALDI ion source and an ND: VAG Laser. TLC analyses were done using Merck silica gel coated aluminium sheets and flash chromatography was performed using BDH flash silica gel and the eluents are indicated in parenthesis for each compound. The two commonly used eluents are abbreviated: ethyl acetate (EtOAc), petroleum ether (Pet.).
We used the exact procedures in the literature to synthesise all the 4-hydroxyquinolines 7 [24] (Scheme 1) and for the conversion of 4-hydroxyquinolines 7 to 4-chloroquinolines 8 we used another reported procedure [26].

Conclusions
We have a simple method for the synthesis of novel 4-quinolylpropanoates 10a-p as potential antimicrobial compounds. These 4-quinolylpropanoates have shown antimicrobial activities against a panel of microorganisms, but without Gram-specificity. However, the compounds showed potent antimicrobial activity against two strains of H. pylori. No evidence of membrane damage was observed in BacLight fixed time-point assays following exposure to any of the compound in this study (data not shown) suggesting an intracellular target. Quinoline derivatives have been associated with inhibition of DNA supercoiling in Mycobacterial species [40] and, although there is significant structural differences betweeen those molecules and the series presented in this study, this is a point of further investigation.