N-Methyl-β-carbolinium Salts and an N-Methylated 8-Oxoisoguanine as Acetylcholinesterase Inhibitors from a Solitary Ascidian, Cnemidocarpa irene.

New brominated β-carbolines irenecarbolines A (1) and B (4) along with known β-carbolines 2 and 3 and a new 8-oxoisoguanine derivative, 5, were isolated from a solitary ascidian, Cnemidocarpa irene. The structures of these compounds were determined on the basis of their spectral data. All, except for 3, inhibited the action of acetylcholinesterase (AchE). The activities of 1 and 5 were comparable to those of galantamine, a clinically used AchE inhibitor. Compounds 1 and 2 were found to be present in high concentrations in blood, and fluorescence was observed in certain types of cells found in the blood of the tunicate.

Introduction

Ascidians (phylum Urochordata) are known to be a rich source of important biologically active secondary metabolites, as represented by ecteinascidin 743, developed as the anticancer drug trabectedin and now used worldwide to treat soft-tissue sarcomas.[1] Morphologically, ascidians can be classified into two types, colonial and solitary. Although numerous secondary metabolites (more than 1200 compounds) have been isolated from ascidians, they have generally been obtained from colonial ascidians. To date, approximately a hundred compounds have been obtained from solitary species, with many metabolites with interesting structures and biological activities reported only recently.[2,3] In our continuing effort to discover molecules that interact with neuronal receptors,[4] ion channels, or neurotransmitters, we found that the aqueous crude extract of the solitary ascidian Cnemidocarpa irene collected in Hokkaido, Japan, inhibited acetylcholinesterase (AchE). Here, we report the isolation and structural determination of new β-carboline derivatives and a new purine derivative as bioactive principals of the tunicate. We also report our unexpected finding that some of those compounds are present in large amounts in the blood (coelomic fluid) of the animal.

Results and Discussion

A specimen of a solitary ascidian, C. irene, was collected from the Sea of Japan from an underwater cave in the Oshima-Kojima Islet off the Oshima Peninsula of Hokkaido, Japan.

Both water and methanol extracts of the tunicate exhibited inhibition of AChE, as measured by Ellman’s method,[5] inhibiting 73 and 63% of the enzyme activity at a final concentration of 0.2 mg/mL, respectively. Thus, we separated the extracts guided by the AChE inhibition activity and obtained four β-carbolines (1–4) and a purine derivative (5) (Figure ).

Figure 1

Compounds isolated from C. irene.

The frozen tunicate (130 g) was extracted with water, and the extract was dialyzed with a cellulose membrane to remove macromolecules. The low-molecular-weight fraction exhibited the bioactivity of a highly complex mixture of UV-absorbing compounds and so it was fractionated on a C18 reversed-phase flash column. The fraction was eluted with 40% aqueous methanol and further separated by a Sephadex LH-20 column to give 1 (55.3 mg, purity 96%) as a yellowish solid. Compound 1 showed two molecular ions at m/z 261 and 263 in its matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrum, indicating the presence of a bromine atom. A molecular formula of C12H10N2Br was established on the basis of its high-resolution electrospray ionization mass spectrometry (HRESIMS) data along with its 13C NMR data, in which 12 carbon signals were observed (Table ). The extended conjugation nature of the compound was suggested from UV data, showing characteristic absorptions at λ 247, 311, and 368 nm. In the 1H NMR spectrum of 1, two singlets and four doublets were observed in the aromatic region. Analysis of the J-values as well as correlation spectroscopy (COSY) data indicated that sets of protons that resonated at δH 8.59/8.49 (J = 5.9 Hz) and δH 8.24/7.53 (J = 8.3 Hz) were adjacent to each other. In addition to those aromatic protons, a heteroatom-bound methyl singlet was observed at δH 4.51. Two spin systems, for N-methyl pyridinium (C-9a/CH-1/N-CH3/CH-3/CH-4/C-4a) and trisubstituted benzene (C-5a/CH-5/CH-6/C-7/CH-8/C-8a) substructures, were established on the basis of NMR data, including COSY, heteronuclear single-quantum correlation, and heteronuclear multiple-bond correlation (HMBC) data (Figure ).

Table 1

NMR Spectroscopic Data for Irenecarbolines A (1) and B (4) in Methanol-d4

	1		4
#C	δC	δH, mult. (J in Hz)	δC	δH, mult. (J in Hz)
1	131.5, CH	9.19, s	142.6, C
3	134.9, CH	8.59, d (5.9)	136.0, CH	8.45, d (6.4)
4	118.8, CH	8.49, d (5.9)	116.8, CH	8.48, d (6.4)
4a	133.8, C		133.0, C
4b	119.7, C		120.5, C
5	125.7, CH	8.24, d (8.3)	125.7, CH	8.27, d (8.0)
6	126.5, CH	7.53, d (8.3)	126.6, CH	7.56, dd (8.4, 1.6)
7	127.4, C		127.1, C
8	116.9, CH	7.89, s	116.7, CH	7.92, d (1.6)
8a	146.3, C		146.0, C
9a	137.1, C		136.9, C
N-Me	48.6, CH3	4.51, s	45.5, CH3	4.38, s
1-Me			15.6, CH3	3.07, s

Figure 2

Key HMBC and nuclear Overhauser effect correlations for 1.

Cross HMBC correlations between H-5 and C-4a and H-4 and C-5a connected C-4a and C-5a (Figure ). A nitrogen atom was inserted between the aromatic C-8a (δC 146.3) and C-9a (δC 137.1) on the basis of chemical shifts of those carbons to form a pyrrole ring (Table ).

The remaining hydrogen and bromine atoms were connected to the pyrrole nitrogen and a benzene ring, respectively, to form an N-methyl-β-carbolinium structure. The bromine atom was assigned to C-7 (δC 127.4) because the nuclear Overhauser enhancement spectroscopy (NOESY) correlation between H-5 (δH 8.24) and H-4 (δH 8.49) indicated that spin system δ 8.24/7.53 (H-5/H-6) is in the upper half of the carboline ring (Figure ). The counterion was assigned to be a chloride ion on the basis of the negative-mode ESIMS data (Figure S7b in the Supporting Information (SI)). Thus, the structure of 1 was determined to be 7-bromo-N-methyl-β-carbolinium chloride. Although compound 1 is structurally related to 2-methyleudistomines D (6) and J (7), which are cytotoxic compounds that are found in Eudistoma gilboverde (Figure ),[6]1 itself has never been reported previously. It was named irenecarboline A after the species name of the tunicate.

Figure 3

Known halogenated β-carbolinium compounds.

Compounds 2 and 3, which emitted blue fluorescent light on thin-layer chromatography (TLC) upon irradiation with UV light, were also isolated from a bioactive eluent (30% aqueous methanol) of the above C18 flash column. The characteristic UV absorption patterns of these compounds, similar to those of 1, suggested that they are also β-carboline alkaloids. Thus, the fraction was further separated by Sephadex LH-20 column chromatography, and the fluorescent compound was finally purified by high-performance liquid chromatography (HPLC) to give 2. A minor fluorescent compound accompanying 2 was also purified to give 3. Compounds 2 and 3 were assigned to be N-methyl-β-carbolinium and N-methyl-β-carboline-3-carboxylate, respectively, on the basis of their spectroscopic and spectrometric data (Figures S8 and S9).

N-Methyl-β-carboline was first found in the bark of Desmodium pilchellum(7) and later in the Okinawan Haploscrerida sponge,[8] whereas N-methyl-β-carboline-3-caroxylate was first reported in the deep sea soft coral Lignopsis spongiosum as a weak antimicrobial compound.[9]

Compound 4 was isolated from a 2-propanol extract of the residual material after water extraction of the animal. The molecular formula of 4, C13H12N2Br, deduced from HRESIMS, along with NMR data (Table ), suggested that it was a higher homologue of 1. The overall profile of the 1H NMR data of 4 was very similar to that of 1, with a new methyl singlet at δH 3.07 replacing the signal for H-1. The NOESY correlations observed between this methyl (δH 3.07) and the N-methyl group (δH 4.38), along with the above data, indicate the position of the methyl to be at C-1. An ABX system for the trisubstituted benzene and an AB system for H-3/H-4 were evident from the 1H NMR data. The position of the bromine substitution was again determined to be C-7 because a NOESY correlation was observed between H-5 (δH 8.27) and H-4 (δH 8.48), as in the case of 1. Thus, the structure of 4 was determined to be 7-bromo-1-methyl-N-methyl-β-carbolinium chloride. As 4 had never been reported previously, we named it irenecarboline B.

Compound 5 was present in the 10% aqueous methanol eluate of the above C18 flash column. This fraction was further purified by combinations of gel-filtration chromatography on Sephadex LH-20 and HW-40 columns to give 5 as a major component. The HRESIMS of 5 along with the 13C NMR data suggest a formula of C8H11N5O2. Three singlets at δH 3.56, 3.66, and 3.82 in the 1H NMR spectrum were assignable to N-methyl groups. Compound 5 exhibited a characteristic UV absorption at 313 nm, which differs significantly from that observed for β-carbolines. These spectral characteristics were similar to those of the neuroactive methylated 8-oxoisoguanines (9–11) previously reported by us from marine sponges (Figure ).[10] The positions of the three methyl groups were assigned on the basis of the key HMBC correlations observed between CH3-3/CH3-9 (δH 3.82/3.66) and C-4 (δC 141.6), CH3-9 and C-8 (δC 154.0), CH3-1 (δH 3.56) and C-6 (δC 146.7), and CH3-1/CH3-3 and C-2 (δC 150.2) (Figure ). The heteroatom substitution pattern in the purine ring of 5 was assigned to be of the 8-oxoisoguanine type because the UV absorption at a λmax of 313 nm for 5 was closer to that of 9–11[8] than to that of the 8-oxoguanine type (∼290 nm).[11] Therefore, the structure of 5 was assigned to be 1,3,9-trimethyl-8-oxoisoguanine.

Figure 4

HMBC correlations observed in 5 and structures of related isoguanines from sponges.

Next, we tested the inhibition of AChE by compounds 1–5 at a fixed concentration of 0.1 μg/mL. All compounds except for 3 inhibited more than 50% of the enzyme activity (Table ). Thus, we generated concentration–response curves for 1, 2, 4, 5, and galantamine (GAL), a positive control (Figure ). All tested compounds exhibited concentration-dependent inhibitions. The IC50 values calculated from the curves are listed in Table .

Table 2

AChE Inhibition of Compounds 1–5

compound	inhibitiona	IC50b
1	87	0.67 (0.42–1.1)
2	78	6.6 (4.8–9.2)
3	0	not tested
4	92	0.47 (0.36–0.60)
5	62	24 (20–28)
GAL	80	0.41 (0.23–0.73)

Inhibition % at 0.1 μg/mL.

Mean of trials in triplicate in μM (95% confidence interval).

Figure 5

Concentration–response curves for compounds 1, 2, 4, and 5 and GAL.

It is well known that β-carbolines inhibit cholinesterases[7,12,13] and that β-carbolinium salts are better inhibitors than their nonionic counterparts.[7,14] The most recent example is a chlorinated nostocarboline (8) (Figure ) isolated from the cyanobacterial strain of Nostoc 78-12A, which was shown to inhibit AChE at an IC50 value of 5.3 μM.[15] Nevertheless, our finding added new information to the structure–activity relationship of β-carbolinium AChE inhibitors in that the bromine substituent on the benzene ring and an alkyl substituent at C-1 of the pyridine ring positively contributed to the activity, whereas the carboxyl group at C-3 reduced the activity. Moreover, we detected inhibitory activity in purine 5. To the best of our knowledge, this is the first example of a natural purine compound with anticholinesterase activity. Of note, however, synthetic theophylline derivatives that were designed and synthesized based on the structure of donepezil, a commercially used AChE inhibitor for the treatment of Alzheimer’s disease, exhibit inhibitory activity.[16]

To date, no chemical investigations on C. irene have been reported, although several interesting secondary metabolites, including pentacyclic pyridoacrydine, the cnemidines[17] and taurine amides of various heteroaromatics, and stolonines A–C,[18] were reported from Australian Cnemidocarpa stolonifera. 3-Bromotryptamine and 1,3-dimethylisoguanine were also reported from Cnemidocarpa bicornuta from New Zealand.[19] Thus, this genus of ascidians might be of interest due to its unique biosynthetic machinery for the production of bioactive aromatic molecules.

The presence of potent inhibitors of neurotransmitter biosynthesis in ascidians is intriguing in light of their physiological functions. We thus examined the localization of β-carbolines in the animal. A live animal was dissected, and the organs and blood were separately collected. Irradiation with UV light (360 nm) onto the dissected animal resulted in the emission of blue fluorescence, mainly from the blood (see the graphic in the abstract). Liquid chromatography (LC) analysis of the blood indicated that the concentrations in 1 and 2 were 250 and 210 μM, respectively, which are 340 and 30 times higher than their IC50 values.

We were able to keep the ascidian healthy for more than 5 months in a laboratory aquarium. Fortuitously, the animal spawned and larvae were collected. Interestingly, the entire body of the larva emitted fluorescence upon irradiation at 405 nm (Figure ). Fluorescent micrograph observations of the blood showed many types of morphologically distinguishable cells. Interestingly, the same types of cells reacted differently to the fluorescence (Figure ). Six to nine different cell types have been identified in ascidians, and their physiological roles have been reported to be involved in the immune response and vanadium concentration, although the details are largely still unknown.[20−22] It is known, however, that the tunichromes found in several species of both solitary and colonial ascidians form fluorescent complexes with vanadium ions, and certain blood cells emit fluorescence upon irradiation with blue light. The presence of vanadium and tunichromes in the present species has never been reported, and indeed we could not detect the presence of tunichromes in the LC/mass spectrometry (MS) analysis of the present sample.

Figure 6

Laboratory grown C. irene discharging eggs (an arrow) from the cloacal siphon (A). A larva of the ascidian (B) and the same shown under blue light (405 nm) (C). Photograph courtesy of Y. Tadokoro. Copyright 2016.

Figure 7

Cells found in the blood of C. irene. Light (A, C) and fluorescent (B, D) micrograph images. (A, B) and (C, D) each represent the same view field. Numbered cells (arrow) show morphologically different cells. Some morphologically identical cells that differ in fluorescence (e.g., 10 and 11) are numbered separately.

In solitary ascidians, defensive primary metabolites, such as antimicrobial peptides[23,24] and lectins,[25,26] are well known to be present in the blood. In some cases, however, small secondary metabolites are suspected to function as chemical defenses. For example, halocyamines, tetrapeptide-like metabolites with antimicrobial activity, were found in the blood “morula”-like cells, the most abundant cells in the hemocytes of the edible tunicate Halocynthia roretzi.[27]

The presence of β-carbolines in the blood of C. irene suggested some physiological functions of the compounds in this species. However, a possible defensive role of these compounds against bacterial infection is less likely, as 1 and 2 did not show antimicrobial activity against Escherichia coli or Bacillus subtilis (data not shown). Of note, the cholinergic neuron in Ciona intestinalis larvae was characterized and shown to govern the complex motor behavior of the larvae.[28] Interestingly, the settlement process of the metamorphosing larvae of C. intestinalis was stimulated by treatment with acetylcholine.[29] These observations supported the idea that the AChE inhibitors in ascidian blood have functions. Further investigations on the biosynthesis and physiological functions of the aromatic metabolites present in this ascidian species are in progress in our laboratory.

Experimental Section

General

Infrared spectra were recorded on FT/IR-4200 (JASCO, Tokyo) using KBr pellets. UV–vis data were recorded on SpectraMax M2 (Molecular Devices, Sunnyvale, CA) using water as a solvent. NMR data were recorded on a JNM 400, ECP 400, ECZS 400 (1H 400 MHz, 13C 100 MHz), or ECA600 (1H 600 MHz, 13C 150 MHz) (JEOL, Tokyo). The samples were dissolved either in D2O (99.990 atom % D) or methanol-d4 (99.8 atom % D). Chemical shifts were referenced in 1H NMR to methanol-d4 δH 3.30 and in 13C NMR to methanol-d4 δC 49.0. MALDI-TOF mass spectra were recorded on an AB4700 spectrometer (Sciex, Tokyo) using either α-cyano-4-hydroxycinamic acid or gentisic acid as a matrix. HRESIMS spectra were recorded on Exactive (Thermo-Fisher Scientific, Yokohama). An LC/MS experiment was performed in the positive ESI mode on a Sciex 5600 LC/MS system using an AM12S03 YMC reversed-phase column (C18, 4.6 × 75 mm2), eluting with a gradient of MeOH (0.05% formic acid)–H2O (0.1% formic acid). The purity of the compounds was established using a Corona ultra RS charged aerosol detector (CAD) (Thermo-Fisher). Normal-phase (silica gel 60F 254) or reversed-phase (RP18F 245S) plates were used for TLC using (A) pyridine/EtOAc/AcOH/H2O 150:70:33:60 or (B) CHCl3/MeOH/AcOH/H2O 40:20:2:4 (v/v) for normal-phase and MeOH/H2O 25:75 for reversed-phase TLC. HPLC was performed on a system comprising an LC-20AD pump and an SPD-M20A photodiode array detector (Shimadzu) using COSMOSIL (5C18-AR-II packed column, 10 mm i.d. × 250 mm), eluting with a gradient of MeOH–H2O 0.05% trifluoroacetic acid (TFA) (v/v).

Biological Material

Ascidians were collected using SCUBA in September 2013 and 2015 from a 20 m deep underwater cave in the Oshima-Kojima Islet of the Sea of Japan, off Oshima Peninsula, Hokkaido, Japan, during the cruise of the Hokkaido University research vessel, Ushio Maru. The specimens were stored at −20 °C pending use. Two live specimens were kept in an aquarium for 6 months by feeding with brine shrimp. A larva found in the aquarium was kept separately in a Petri dish. The animal was identified by one of the authors (T.N.) as C. irene (Hartmeyer, 1906), and reference specimens were deposited in the National Museum of Nature and Science, Tsukuba (registered as NSMT-Pc1124 to 1128).

Isolation of 1, 2, 3, and 5

A frozen specimen (HAK83, 130 g) was lyophilized, and the dried sample was extracted with water to give a crude extract after lyophilization (6 g). The crude extract was suspended in water and dialyzed using a cellulose (15 kDa cutoff) tube to give a macromolecular portion (0.28 g) and a low-molecular-weight portion (5.3 g). The small molecular portion (3.81 g) was separated on a C18 (Wako gel) flash column (5.5 × 20 cm2, 0.05% TFA aq. with increasing amounts of 10, 20, 30, 40, 50, 75, and 100% methanol, each 300 mL). The active fraction (40% methanol eluate) was separated on a Sephadex LH-20 column (2.5 × 120 cm2, 0.05% TFA, 1.5 mL/min, 10.5 mL/tube × 120 fr.). The fractions were combined according to TLC into nine fractions. Compound 1 was eluted in fraction 7. The purity of the fraction was determined to be 95% using a CAD detector. Irenecarboline A (1), pale yellow solid: UV (H2O) λmax (log ε) 204 (4.00), 247 (3.91), 311 (3.70), 368 (3.26); IR (KBr) νmax 3398, 2928, 2370, 1682, 1440, 1335, 1198, 1135, 813, 724, 594, 474; 1H NMR (methanol-d4, 400 MHz) and 13C NMR (methanol-d4, 100 MHz), see Table ; HRESIMS m/z 261.0026 [M]+ (calcd for C12H10N2Br, 261.0022). A set of chloride ions was detected in negative-mode ESIMS m/z 34.96/36.96 [M]−.

The 30% methanol eluent of the C18 column was further separated on a Sephadex LH-20 column (2.5 × 120 cm2, 0.05% TFA, 1.5 mL/min, 10.5 mL/tube × 120 fr.) to give a fraction containing 2 (540–620 mL eluate) and 3 (630–840 mL eluate). HPLC purification gave pure 2 (95.4%) and 3. 2-Methyl-β-carbolinium chloride 2, pale yellow solid: UV (H2O) λmax (log ε) 248 (4.08), 302 (3.75), 372 (3.26); IR (KBr) νmax 3428, 2370, 1684, 1519, 1337, 1200, 1134, 729, 475; 1H NMR (methanol-d4, 400 MHz) δ 9.14 (s, H-1), 8.60 (d, J = 6.8 Hz, H-4), 8.45 (dd, J = 6.4, 0.8 Hz, H-3), 8.37 (brd, J = 8.0 Hz, H-5), 7.79 (ddd, J = 8.4, 6.8, 1.0 Hz, H-7), 7.73 (d, J = 8.4 Hz, H-8), 7.45 (ddd, J = 8.0, 6.8, 0.8 Hz, H-6), 4.51 (3H, s, N-Me) and 13C NMR (methanol-d4, 100 MHz) δ 146.0 (C-8a), 136.9 (C-9a), 134.3 (C-3), 134.1 (C-7), 131.0 (C-1), 124.2 (C-5), 123.1 (C-6), 120.8 (C-4b), 118.6 (C-4), 113.9 (C-8), 48.5 (N-Me); HRESIMS m/z 183.09179 [M]+ (calcd for C12H10N2, 183.09167). 2-Methyl-β-carbolinium-3-carboxylate (3): UV (H2O) λmax (log ε) 262 (4.05), 306 (3.75), 375 (3.43); IR (KBr) νmax 3417, 2926, 2369, 1636, 1392, 1081, 663, 474; 1H NMR (D2O–methanol-d4 60:4, 400 MHz) δ 8.91 (s, H-1), 8.61 (s, H-4), 8.33 (d, J = 8.0 Hz, H-5), 7.80 (t, J = 8.0 Hz, H-6), 7.72 (d, J = 8.4 Hz, H-7), 7.60 (t, J = 7.6 Hz, H-8), 4.48 (s, 3H, N-Me); HRESIMS m/z 227.08146 [M + H]+, 249.06348 [M + Na]+ (calcd for C12H11N2O2, 227.08150; C12H10N2O2Na, 249.06345).

A fraction (266 mg) eluted with 10% MeOH using the C18 flash column was chromatographed on a Sephadex LH-20 column into nine fractions. The major fraction, 3, was further separated by gel-filtration chromatography on a TOYOPEARL HW-40 column (1.5 × 80 cm2, 0.05% TFA, 0.25 mL/min) to give pure 5 (22.2 mg). 1,3,9-Trimethyl-8-oxoisoguanine (5), colorless solid: UV (H2O) λmax (log ε) 211 (4.28), 247 (3.66), 313 (4.11); IR (KBr) 3416, 2370, 1756, 1684, 1545, 1429, 1200, 1140, 727, 514; 1H NMR (D2O–methanol-d4 60:4, 400 MHz) δ 3.56 (s, CH3-1), 3.66 (s, CH3-9), 3.82 (s, CH3-3); 13C NMR (D2O–methanol-d4 60:4, 100 MHz) δ 154.0 (C-8); 150.2 (C-2), 146.7 (C-6), 141.6 (C-4), 94.9 (C-5), 33.4, 32.4, 31.1; HRESIMS m/z 210.0990 [M + H]+ (calcd for C8H12N5O2, 210.0990).

Isolation of 4

A fresh specimen of the ascidian (448 g) was extracted with water, and then, the residual material was exhaustively extracted with 2-propanol and finally with methanol. The organic extract was concentrated, and the residue was partitioned between the upper and lower layers of hexane–ethyl acetate–methanol–water (4:7:4:3). The aqueous layer was further partitioned between butanol and water. HPLC analysis of the butanol layer indicated the presence of compounds 1 and 2 and a small amount of 4. Purification of the butanol layer (26 mg) by reversed-phase HPLC afforded 4 (1.96 mg). Irenecarboline B (4): UV (MeOH) λmax (log ε) 248 (4.00), 313 (3.92), 370 (3.52); IR (KBr) 3433, 2928, 2372, 1681, 1398, 1330, 1201, 1337, 806, 667, 472; 1H NMR (methanol-d4, 400 MHz) and 13C NMR (methanol-d4, 100 MHz), see Table ; HRESIMS m/z 275.01758 [M]+ (calcd for C12H12N2Br, 275.01784).

Inhibition of AChE

Ellman’s method was used to assess the inhibition of AChE.[30] Briefly, 5,5′-dithiobis[2-nitrobenzoic acid] (3 mM, 125 μL), acetylthiocholine (1.5 mM, 25 μL), Tris–HCl buffer (pH 8.0, 50 mM, 50 μL), and the sample solution (10 mg/mL, 5 μL) were mixed in a 96-well microtiter plate, and then, AChE from the electric eel Electrophorus electricus (0.28 U/mL, Sigma-Aldrich) was added to the mixture. Absorption with the sample (A), with control water (B), and without AChE (C) at 412 nm was measured after 5 min of incubation at room temperature. Inhibition was calculated using the following equationConcentration–inhibition curves (n = 3 at each data point) were generated, and IC50 values were calculated using the software GraphPad Prism.