Antibiotic resistance and drug modification: Synthesis, characterization and bioactivity of newly modified potent ciprofloxacin derivatives.

Development of new derivatives of commercial antibiotics using different organic reagents and testing these derivatives against different microorganisms are the main goals of this article. Thus, the antibiotic ciprofloxacin, CF, was acylated via reaction with ethyl cyanoacetate and ethyl acetoacetate in basic medium to give the cyanoacetylpiprazinyl dihydroquinoline derivative 3, and oxobutanoylpiprazinyl dihydroquinoline derivative 5, respectively. On the other hand, N-alkylated derivatives 8-10, were prepared through the reaction of CF with chloroacetonitrile, chloroacetyl acetone and chloroacetone in the presence of carbonate salt. In basic medium, both 3 and 10 were coupled with benzenediazonium chloride to afford hydrazono derivatives, which were then cyclized to give 4-(dihydropyridazinecarbonyl)piperazinyl-1,4-dihydroquinoline. Furthermore, compounds 3 and 10 were reacted with benylidenemalononitrile to produce 4H-pyan and pyrido[1,2-a]pyrazine derivatives, respectively. Both 3 and 10 were reacted with DMFDMA to give enaminone derivatives. These enaminones were cyclized to aminopyrimidine derivatives by reacting with urea or thiourea. X-ray, elemental analysis and spectral data were used to illustrate and confirm the structures of the isolated compounds. The bioactivities of the novel compounds were investigated against different gram-positive and gram-negative bacteria. In addition, these novel antibiotic derivatives were tested against ciprofloxacin-resistant bacteria isolated from patients aged 65-74 years. This study reveals that most of the modified drugs show high to moderate antibacterial activity. Additionally, these drugs show good effects against ciprofloxacin-resistant bacteria.

3,7-bis-benzylidene hydrazide ciprofloxacin derivatives as promising antiproliferative dual TOP I & TOP II isomerases inhibitors.

We report herein design and synthesis of a new series of 3,7-bis-benzylidenes of ciprofloxacin. Most of the target compounds revealed good cytotoxic activity; the most potent 4e and 4i achieved strong broad spectrum antiproliferative activity with comparable activity to Doxorubicin with IC50 (µM) of 1.21 ± 0.02, 0.87 ± 0.04, 1.21 ± 0.02; 0.41 ± 0.02, 0.57 ± 0.06, 1.31 ± 0.04 and 1.26 ± 0.01, 1.79 ± 0.04, 0.63 ± 0.01 against leukemia cancer cell line HL-60 (TB), colon cancer cell line HCT-116 and breast cancer cell line MCF7, respectively. Moreover, the most potent derivative 4i induced apoptosis at G2/M phase Investigating the mechanism of action of compounds 4e, 4 h and 4i exhibited promising dual TOP Iα and TOP IIB % inhibition comparable to Camptothecin and Etoposide; respectively. Docking of 4e, 4 h and 4i into the active site of topo I and II proteins compared to Camptothein and Etoposide revealed acceptable binding score and augmented enzyme assay data. Hence, 4e and 4i are promising targeted antiproliferative dual acting TOP Iα TOP IIB inhibitors that require further optimization.

Novel urea linked ciprofloxacin-chalcone hybrids having antiproliferative topoisomerases I/II inhibitory activities and caspases-mediated apoptosis.

A novel series of urea-linked ciprofloxacin (CP)-chalcone hybrids 3a-j were synthesized and screened by NCI-60 cancer cell lines as potential cytotoxic agents. Interestingly, compounds 3c and 3j showed remarkable antiproliferative activities against both colon HCT-116 and leukemia SR cancer cells compared to camptothecin, topotecan and staurosporine with IC50 = 2.53, 2.01, 17.36, 12.23 and 3.1 µM for HCT-116 cells, respectively and IC50 = 0.73, 0.64, 3.32, 13.72 and 1.17 µM for leukemia SR cells, respectively. Also, compounds 3c and 3j exhibited inhibitory activities against Topoisomerase (Topo) I with % inhibition = 51.19% and 56.72%, respectively, compared to camptothecin (% inhibition = 60.05%) and Topo IIß with % inhibition = 60.81% and 60.06%, respectively, compared to topotecan (% inhibition = 71.09%). Furthermore, compound 3j arrested the cell cycle of leukemia SR cells at G2/M phase. It induced apoptosis both intrinsically and extrinsically via activation of proteolytic caspases cascade (caspases-3, -8, and -9), release of cytochrome C from mitochondria, upregulation of proapoptotic Bax and down-regulation of Bcl-2 protein level. Thus, the new ciprofloxacin derivative 3j could be considered as a potential lead for further optimization of antitumor agent against leukemia and colorectal carcinoma.

Impact of Critical Material Attributes (CMAs)-Particle Shape on Miniature Pharmaceutical Unit Operations.

The U.S. Food and Drug Administration (FDA) emphasizes drug product development by Quality by Design (QbD). Critical material attributes (CMAs) are a QbD element that has an impact on pharmaceutical operations and product quality. Pharmaceutical drugs often crystallize as needle-shaped (a CMA) particles and affect the process due to poor flowability, low bulk density, and high compressibility, and eventually the product performance. In this study, the product obtained from crystallization was needle-shaped Ciprofloxacin HCl (CIPRO), formed lumps during drying, and compacted during processing through feeders. To delump small amounts of materials and break the needles, multiple available devices (mortar-pestle, Krups grinder) and custom-made grinder were assessed before formulation. The processed CIPRO powder was then used to make tablets in the miniature tablet manufacturing unit developed by the team at MIT. The critical quality attributes (CQA) of the tablets, set by the United States Pharmacopeia (USP), were then assessed for the drug powder processed with each of these devices. Powder properties comparable to commercial CIPRO were obtained when the custom MIT-designed grinder was used, leading to tablets that meet the USP criteria, with comparable dissolution profiles of those for marketed CIPRO tablets. This study demonstrates how needle-shaped crystals have an impact on pharmaceutical operations, even if it is on a miniature scale, and how proper shape and subsequent flow properties can be obtained by processing the particles through the MIT team-designed grinder.

In-vitro cytotoxic evaluation of newly designed ciprofloxacin-oxadiazole hybrids against human liver tumor cell line (Huh7).

Fluoroquinolones are targets of interest due to their broad spectrum antibacterial activity. Structure-activity relationship (SAR) of fluoroquinolones clearly indicates that substitution at C-7 position enhances the lipophilicity of these scaffolds resultantly affording pharmacologically significant compounds. Therefore, various ciprofloxacin-oxadiazole hybrids were synthesized and characterized by spectral analysis. Cytotoxic activity of these derivatives was assessed using human liver tumor cells (Huh7). One dose anticancer test results revealed moderate cytotoxicity of the newly synthesized compounds against this cell line. As the only compound 4a depicted comparatively lower cell viability value (81.91% using 100µg/mL concentration) than the other compounds.

Synthesis and biological assessment of ciprofloxacin-derived 1,3,4-thiadiazoles as anticancer agents.

The quinolone-3-carboxylic acid scaffold is essential structure for antibacterial activity of fluoroquinolones such as ciprofloxacin. Modification of 3-carboxylic functionality in this structure can be used for switching its activity from antibacterial to anticancer. Accordingly, a series of C-3 modified ciprofloxacin derivatives containing N-(5-(benzylthio)-1,3,4-thiadiazol-2-yl)-carboxamide moiety was synthesized as novel anticancer agents. Most of compounds showed significant activity against MCF-7, A549 and SKOV-3 cancer cells in the MTT assay. In particular, compounds 13a-e and 13g were found to be as potent as standard drug doxorubicin against MCF-7 cell line (IC50s = 3.26-3.90 µM). Furthermore, the 4-fluorobenzyl derivatives 13h and 14b with IC50 values of 3.58 and 2.79 µM exhibited the highest activity against SKOV-3 and A549 cells, being as potent as doxorubicin. Two promising compounds 13e and 13g were further tested for their apoptosis inducing activity and cell cycle arrest. Both compounds could significantly induce apoptosis in MCF-7 cells, while compound 13e was more potent apoptosis inducer resulting in an 18-fold increase in the proportion of apoptotic cells at the IC50 concentration in MCF-7 cells. The cell cycle analysis revealed that compounds 13e and 13g could increase cell portions in the sub-G1 phase, inducing oligonucleosomal DNA fragmentation and apoptosis confirmed by comet assay.

Synthesis, evaluation of thymidine phosphorylase and angiogenic inhibitory potential of ciprofloxacin analogues: Repositioning of ciprofloxacin from antibiotic to future anticancer drugs.

Over expression of thymidine phosphorylase (TP) in various human tumors compared to normal healthy tissue is associated with progression of cancer and proliferation. The 2-deoxy-d-ribose is the final product of thymidine phosphorylase (TP) catalyzed reaction. Both TP and 2-deoxy-d-ribose are known to promote unwanted angiogenesis in cancerous cells. Discovery of potent inhibitors of thymidine phosphorylase (TP) can offer appropriate approach in cancer treatment. A series of ciprofloxacin 2, 3a-3c, 4a-4d, 5a-5b, 6 and 7 has been synthesized and characterized using spectroscopic techniques. Afterwards, inhibitory potential of synthesized ciprofloxacin 2, 3a-3c, 4a-4d, 5a-5b, 6 and 7 against thymidine phosphorylase enzyme was assessed. Out of these twelve analogs of ciprofloxacin nine analogues 3a-3c, 4a-4c, 5a-5b and 6 showed good inhibitory activity against thymidine phosphorylase. Inhibitory activity as presented by their IC50 values was found in the range of 39.71 ± 1.13 to 161.89 ± 0.95 µM. The 7-deazaxanthine was used as a standard inhibitor with IC50 = 37.82 ± 0.93 µM. Furthermore, the chick chorionic allantoic membrane (CAM) assay was used to investigate anti-angiogenic activity of the most active ciprofloxacin-based inhibitor 3b. To enlighten the important binding interactions of ciprofloxacin derivatives with target enzyme, the structure activity relationship and molecular docking studies of chosen ciprofloxacin analogues was discussed. Docking studies revealed key π-π stacking, π-cation and hydrogen bonding interactions of ciprofloxacin analogues with active site residues of thymidine phosphorylase enzyme.

Design and synthesis of theranostic antibiotic nanodrugs that display enhanced antibacterial activity and luminescence.

We report the modular formulation of ciprofloxacin-based pure theranostic nanodrugs that display enhanced antibacterial activities, as well as aggregation-induced emission (AIE) enhancement that was successfully used to image bacteria. The drug derivatives, consisting of ciprofloxacin, a perfluoroaryl ring, and a phenyl ring linked by an amidine bond, were efficiently synthesized by a straightforward protocol from a perfluoroaryl azide, ciprofloxacin, and an aldehyde in acetone at room temperature. These compounds are propeller-shaped, and upon precipitation into water, readily assembled into stable nanoaggregates that transformed ciprofloxacin derivatives into AIE-active luminogens. The nanoaggregates displayed increased luminescence and were successfully used to image bacteria. In addition, these nanodrugs showed enhanced antibacterial activities, lowering the minimum inhibitory concentration (MIC) by more than one order of magnitude against both sensitive and resistant Escherichia coli The study represents a strategy in the design and development of pure theranostic nanodrugs for combating drug-resistant bacterial infections.

Design, Synthesis and Pharmacological Evaluation of Some C3 Heterocyclic-Substituted Ciprofloxacin Derivatives as Chimeric Antitubercular Agents.

A series of new C3 heterocyclic-substituted ciprofloxacin derivatives were prepared from ciprofloxacin acid hydrazide as possible chimeric molecules. They were evaluated for their possible in vitro antibacterial (agar cup/bore diffusion method) and antitubercular (Lowenstein-Jensen (LJ) slant method) activities. The results indicated that all the test compounds are highly effective against all the bacterial strains and have shown excellent anti-tubercular activity against normal, multidrug resistant and extensively drug resistant strains of Mycobacterium tuberculosis. They were found to be more potent antibacterial and antitubercular agents than the standard, ciprofloxacin. The minimum inhibitory concentration (MIC)'s of all the compounds against M. tuberculosis were found to be 0.0625 µg/mL as compared to ciprofloxacin (MIC = 2 to > 8 µg/mL). Molecular docking studies were performed by using AUTODOCK 4.2 on the new ciprofloxacin derivatives at the active site of crystal structure of fluoroquinolones target enzyme Mtb DNA gyrase GyrA N-terminal domain (PDB ID: 3ILW) and also on the active site of crystal structure of chosen heterocyclics target enzyme enoyl-acyl carrier protein (ACP) reductase enzyme (PDB ID: 4TZK). Interestingly, almost all the compounds have shown relatively greater binding affinity at both the active sites than ciprofloxacin. Compound 6 exhibited the highest affinity for 3ILW and 4TZK.

Targeted Antibacterial Activity Guided by Bacteria-Specific Nitroreductase Catalytic Activation to Produce Ciprofloxacin.

Fluoroquinolones (FQs) are among the front-line antibiotics used to treat severe infections caused by Gram-negative bacteria. However, recently, due to toxicity concerns, their use has been severely restricted. Hence, efforts to direct delivery of this antibiotic specifically to bacteria/site of infection are underway. Here, we report a strategy that uses a bacterial enzyme for activation of a prodrug to generate the active antibiotic. The ciprofloxacin-latent fluorophore conjugate 1, which is designed as a substrate for nitroreductase (NTR), a bacterial enzyme, was synthesized. Upon activation by NTR, release of Ciprofloxacin (CIP) as well as a fluorescence reporter was observed. We provide evidence for the prodrug permeating bacteria to generate a fluorescent signal and we found no evidence for activation in mammalian cells supporting selectivity of activation within bacteria. As a testament to its efficacy, 1 was found to have potent bactericidal activity nearly identical to CIP and significantly reduced the bacterial burden in a neutropenic mouse thigh infection model, again, at comparable potency with CIP, a clinically used FQ. Thus, together, we have developed a small molecule that facilitates bacteria-specific fluoroquinolone delivery.

A Consolidated and Continuous Synthesis of Ciprofloxacin from a Vinylogous Cyclopropyl Amide.

Ciprofloxacin is a broad-spectrum antibiotic that is recognized as one of the World Health Organization's Essential Medicines. It is particularly effective in the treatment of Gram-negative bacterial infections associated with urinary, respiratory, and gastrointestinal tract infections. A streamlined and high yielding continuous synthesis of ciprofloxacin has been developed, which employs a chemoselective C-acylation step that precludes the need for intermediate isolations, extractions, or purifications. The end-to-end process has a residence time of 4.7 min with a 15.8 g/h throughput at laboratory scale and an overall isolated yield of 83%.

Encapsulation of ciprofloxacin within modified xanthan gum- chitosan based hydrogel for drug delivery.

The aim of the present work was to investigate the preparation of polyelectrolyte hydrogel as potential drug carrier for antibacterial Ciprofloxacin drug (CFX), intended for controlled release formulation. Hydrogel of N-trimehtyl chitosan (TMC)/sodium carboxymethyl xanthan gum (CMXG) was prepared and ciprofloxacin was employed as a model drug to investigate the loading and release performance of the prepared hydrogel. FTIR, DSC, TGA and SEM analysis were used to characterize the TMC/CMXG hydrogel and its CFX loaded hydrogel. The results showed that the ciprofloxacin was successfully incorporated and released from the prepared hydrogel without the loss of structural integrity or the change in its functionality. The encapsulation efficiency of CFX within the prepared hydrogel was found to be increased with increasing the concentration of drug reaching about 93.8 ±â€¯2.1% with concentration of CFX 250 µg/ml. It was shown also that the drug is entrapped within the gel without significant interaction as confirmed from FTIR spectra and DSC analysis. In vitro release study in phosphate buffer saline (PBS), indicated the steady rise in cumulative drug release with the highest release amount, reaching about 96.1 ±â€¯1.8% up to 150 min, whereby the gel with high drug loading efficiency (3.52 ±â€¯0.07%) displayed faster and higher release rate than that of gel containing a smaller amount of drug (0.44 ±â€¯0.01%). The release kinetics of loaded drug followed zero-order kinetics. CFX drug loaded hydrogel showed high activity against the gram positive and gram negative bacterial strains due to the successful released of CFX from the CFX loaded hydrogel into the tested bacterial strains with the highest diameter of inhibition zone against Escherichia coli (67.0 ±â€¯1.0) as compared to reference antibiotic, Gentamicin (28 ±â€¯0.5). Cytotoxicity of the prepared hydrogel was examined in vitro using lung human normal cell lines and showed the highest cell viability (97 ±â€¯0.5%) at concentration up to 50 µg/ml. Consequently, TMC/CMXG hydrogel can be proposed as new controlled release drug delivery system.

Proof-of-concept preparation and characterization of dual-drug amorphous nanoparticle complex as fixed-dose combination of poorly soluble drugs.

OBJECTIVES: To carry out a proof-of-concept study on the development of dual-drug amorphous nanoparticle complex (nanoplex in short) as a potential formulation platform for fixed-dose combination (FDC) of poorly-soluble drugs. SIGNIFICANCE: FDC has been proven effective in improving patient compliance for treatment that requires complex multidrug regimen. Currently, there is growing interest to develop FDC of poorly-soluble drugs due to the increased number of drugs exhibiting poor solubility thus low bioavailability. METHODS: The dual-drug nanoplex was prepared by electrostatically-driven co-complexation of drug molecules with oppositely charged dextran sulfate, using ciprofloxacin (CIP) and itraconazole (ITZ) as the model poorly-soluble drugs. RESULTS: We first verified that the co-complexation products were dual-drug CIP-ITZ nanoplex, and not binary mixtures of the single-drug CIP and ITZ nanoplexes, by demonstrating their distinct thermal behaviors and dissolution characteristics. Depending on the preparation condition, the dual-drug nanoplex exhibited size and zeta potential of 160-410 nm and -35-50 mV, respectively. The individual drug payloads were readily manipulated by varying the CIP/ITZ mass ratio in the feed, resulting in CIP and ITZ payloads in the range of 60-30% and 15-45%, respectively. The CIP-ITZ nanoplex, however, exhibited diminished CIP supersaturation generation, thus lower CIP solubility enhancement, compared to the single-drug CIP nanoplex. The CIP-ITZ nanoplex, nonetheless, remained capable of generating high ITZ supersaturation level. CONCLUSION: Dual-drug nanoplex was successfully prepared with a high degree of control over its physical characteristics. Nevertheless, whether dual-drug nanoplex always exhibits diminished solubility enhancement compared to its single-drug counterparts needs to be investigated using different poorly-soluble drugs.

Advanced Continuous Flow Platform for On-Demand Pharmaceutical Manufacturing.

As a demonstration of an alternative to the challenges faced with batch pharmaceutical manufacturing including the large production footprint and lengthy time-scale, we previously reported a refrigerator-sized continuous flow system for the on-demand production of essential medicines. Building on this technology, herein we report a second-generation, reconfigurable and 25 % smaller (by volume) continuous flow pharmaceutical manufacturing platform featuring advances in reaction and purification equipment. Consisting of two compact [0.7 (L)×0.5 (D)×1.3 m (H)] stand-alone units for synthesis and purification/formulation processes, the capabilities of this automated system are demonstrated with the synthesis of nicardipine hydrochloride and the production of concentrated liquid doses of ciprofloxacin hydrochloride, neostigmine methylsulfate and rufinamide that meet US Pharmacopeia standards.

Design, Synthesis and Antibacterial Evaluation of 1-[(1R,2S)-2-Fluorocyclopropyl]ciprofloxacin-1,2,4-triazole-5(4H)-thione Hybrids.

A new class of 1-[(1R,2S)-2-fluorocyclopropyl]ciprofloxacin (CPFX)-1,2,4-triazole-5(4H)-thione hybrids 6a - 6o was designed, synthesized and evaluated for their in vitro antibacterial activities against a panel of clinically important drug-sensitive and drug-resistant Gram-positive and Gram-negative pathogens. Our results revealed that all hybrids 6a - 6o had great potency against the tested strains, especially Gram-negative pathogens. The synthesized hybrids were more potent than the parent 1-[(1R,2S)-2-fluorocyclopropyl]CPFX (1) and comparable to CPFX and levofloxacin against the majority of the tested pathogens, worth to be further investigated.

Biology-Oriented Drug Synthesis (BIODS) Approach towards Synthesis of Ciprofloxacin-Dithiocarbamate Hybrids and Their Antibacterial Potential both in Vitro and in Silico.

A novel series of ciprofloxacin-dithiocarbamate hybrids 7a - 7l were designed, synthesized, and evaluated against Gram-positive and Gram-negative bacteria. A significant part of the title compounds showed considerable antibacterial activity against Gram-positive species. The most potent compound against Gram-positive bacteria was 2-chloro derivative 7h and the most potent derivative against Gram-negative bacteria was 3-chloro compound 7i. In vitro antibacterial evaluation of compound 7h against clinically isolated bacteria methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive Staphylococcus aureus (MSSA) showed that this compound acted better than ciprofloxacin against the latter bacteria. Docking study of compound 7h in the active site of S. aureus DNA gyrase revealed that this ciprofloxacin-dithiocarbamate derivative interacted with the main components of the active site of the enzyme.

Comparing a Statistical Model and Bayesian Approach to Establish the Design Space for the Coating of Ciprofloxacin HCl Beads at Different Scales of Production.

The primary objective of this study was to compare two methods for establishing a design space for critical process parameters that affect ethylcellulose film coating of multiparticulate beads and assess this design space validity across manufacturing scales. While there are many factors that can affect film coating, this study will focus on the effects processing conditions have on the quality and extent of film formation, as evaluated by their impact coating yield and drug release. Ciprofloxacin HCl layered beads were utilized as an active substrate core, ethylcellulose aqueous dispersion as a controlled release polymer, and triethyl citrate as a plasticizer. Thirty experiments were conducted using a central composite design to optimize the coating process and map the response surface to build a design space using either statistical least squares or a Bayesian approach. The response surface was fitted using a linear two-factor interaction model with spraying temperature, curing temperature, and curing time as significant model terms. The design spaces established by the two approaches were in close agreement with the statistical least squares approach being more conservative than the Bayesian approach. The design space established for the critical process parameters using small-scale batches was tested using scale-up batches and found to be scale-independent. The robustness of the design space was confirmed across scales and was successfully utilized to establish process signature for the coating process.

Sulfonamide-Linked Ciprofloxacin, Sulfadiazine and Amantadine Derivatives as a Novel Class of Inhibitors of Jack Bean Urease; Synthesis, Kinetic Mechanism and Molecular Docking.

Sulfonamide derivatives serve as an important building blocks in the drug design discovery and development (4D) process. Ciprofloxacin-, sulfadiazine- and amantadine-based sulfonamides were synthesized as potent inhibitors of jack bean urease and free radical scavengers. Molecular diversity was explored and electronic factors were also examined. All 24 synthesized compounds exhibited excellent potential against urease enzyme. Compound 3e (IC50 = 0.081 ± 0.003 µM), 6a (IC50 = 0.0022 ± 0.0002 µM), 9e (IC50 = 0.0250 ± 0.0007 µM) and 12d (IC50 = 0.0266 ± 0.0021 µM) were found to be the lead compounds compared to standard (thiourea, IC50 = 17.814 ± 0.096 µM). Molecular docking studies were performed to delineate the binding affinity of the molecules and a kinetic mechanism of enzyme inhibition was propounded. Compounds 3e, 6a and 12d exhibited a mixed type of inhibition, while derivative 9e revealed a non-competitive mode of inhibition. Compounds 12a, 12b, 12d, 12e and 12f showed excellent radical scavenging potency in comparison to the reference drug vitamin C.

Synthesis and In Vitro Antimycobacterial and Antibacterial Activity of 8-OMe Ciprofloxacin-Hydrozone/Azole Hybrids.

A series of novel 8-OMe ciprofloxacin (CPFX)-hydrazone/azole hybrids were designed, synthesized, and evaluated for their in vitro biological activities. Our results reveal that all of the hydrozone-containing hybrids (except for 7) show potency against Mycobacterium tuberculosis (MTB) H37Rv (minimum inhibitory concentration (MIC): <0.5 µM), which is better than the parent drug CPFX, and comparable to moxifloxacin and isoniazid, some of the tested Gram-positive strains (MIC: 0.06-4 µg/mL), and most Gram-negative strains (MIC: ≤0.03-4 µg/mL).

A Rapid Total Synthesis of Ciprofloxacin Hydrochloride in Continuous Flow.

Within a total residence time of 9 min, the sodium salt of ciprofloxacin was prepared from simple building blocks via a linear sequence of six chemical reactions in five flow reactors. Sequential offline acidifications and filtrations afforded ciprofloxacin and ciprofloxacin hydrochloride. The overall yield of the eight-step sequence was 60 %. No separation of intermediates was required throughout the synthesis when a single acylation reaction was applied to remove the main byproduct, dimethylamine.