Identification and Quantification of MIDD0301 metabolites

Abstract

Background:

MIDD0301 is an oral asthma drug candidate that binds GABA_A receptors on airway smooth muscle and immune cells.

Objective:

The objective of this study is to identify and quantify MIDD0301 metabolites in vitro and in vivo and determine the pharmacokinetics of oral, IP, and IV administered MIDD0301.

Methods:

In vitro conversion of MIDD0301 was performed using liver and kidney microsomes/S9 fractions followed by quantification using liquid chromatography-tandem mass spectrometry (LC-MS/MS). A LC-MS/MS method was developed using synthesized standards to quantify MIDD0301 and its metabolites in urine and feces. Blood, lung, and brain were harvested from animals that received MIDD0301 by oral, IP, and IV administration, followed by LC-MS/MS quantification. Imaging mass spectrometry was used to demonstrate the presence of MIDD0301 in the lung after oral administration.

Results:

MIDD0301 is stable in the presence of liver and kidney microsomes and S9 fractions for at least two hours. MIDD0301 undergoes conversion to the corresponding glucuronide and glucoside in the presence of conjugating cofactors. For IP and IV administration, unconjugated MIDD0301 together with significant amounts of MIDD0301 glucoside and MIDD0301 taurine were found in urine and feces. Less conjugation was observed following oral administration, with MIDD0301 glucuronide being the main metabolite. Pharmacokinetic quantification of MIDD0301 in blood, lung, and brain showed very low levels of MIDD0301 in the brain after oral, IV, or IP administration. The drug half-life in these tissues ranged between 4-6 hours for IP and oral and 1-2 hours for IV administration. Imaging mass spectrometry demonstrated that orally administered MIDD0301 distributes uniformly in the lung parenchyma.

Conclusion:

MIDD0301 undergoes no phase I and moderate phase II metabolism.

1. Introduction

The metabolic analysis of clinical drug candidates is essential to identify potential safety liabilities, especially for drugs with significant human metabolism. This analysis is of particular significance for metabolites that reach >10% of the area under the curve exposure [1]. Metabolites can form via phase I and phase II processes. The majority of xenobiotic phase I metabolites are formed by cytochrome P450 oxidases [2]. Other phase I modifications can be formed by monooxygenases [3], dehydrogenases [4], peroxidase [5], reductase [6], esterase [7], amidases and epoxyhydrolases [8]. Phase II xenobiotic metabolism, or conjugation, is catalyzed predominately by transferases that include UDP-glucosyltransferases [9], N-acetyltransferase [10], methyltransferase [11], glutathione transferase [12], and sulfotransferases [13]. Conjugations with amino acids or taurine occur via activation by acyl CoA synthetase [14], followed by conjugation catalyzed by the corresponding N-acyltransferase [15].

MIDD0301 is a clinical drug candidate being developed for asthma symptom control that was shown to attenuate airway hyperresponsiveness and reduce lung inflammation in rodents when administered orally [16] or nebulized [17]. The drug acts via binding to gamma-aminobutyric acid receptors (GABA_ARs) [18] that are expressed on airway smooth muscle [19] and inflammatory cells [20]. Relaxation of contracted human airway smooth muscle in the presence of MIDD0301 has been shown to occur within minutes using ex vivo tissue [21]. MIDD0301 did not cause any adverse effects or suppress systemic T-dependent antibody responses following repeated high dose exposure in rodents [22]. The compound contains a carboxylic acid and exhibits high aqueous solubility at neutral pH [23]. As a result, there is very little brain absorption and no observed central nervous system effects at high single or repeated dosing [16].

MIDD0301 is based on a benzodiazepine scaffold, which has been the subject of several decades of medicinal chemistry research to modify GABA_AR binding selectivity and pharmacokinetic properties. In this study, we describe the metabolism and excretion of MIDD0301. The substitution of the N-methyl amide by an imidazole ring impacts the sensitivity to demethylation [24]. Introduction of a chiral methyl group prevents C3-hydroxylation [25]. Finally, the introduction of a carboxylic acid group not only creates tissue selectivity but also prevents further phase I metabolism [26].

2. Materials and Methods

Experimental animals:

Seven-week-old female Swiss Webster mice (Charles River Laboratory, Wilmington, MA) were used for animal experiments. Mice were housed under specific pathogen-free conditions using standard conditions of humidity and temperature. A twelve hour light and dark cycle was implemented and all mice had ad libitum access to food and water.

Phase I microsomal stability assay:

MIDD0301 (4 μL of 1 mM solution in dimethyl sulfoxide (DMSO)) was added to 0.282 mL of water. To this solution, 0.08 mL of 0.5M phosphate buffer (pH 7.4), 0.02 mL NADPH Regenerating System Solution A, and 0.004 mL NADPH Regenerating Solution B (BD Bioscience) were added. The assay concentration of MIDD0301 was 10 μM. The solution was incubated for 5 min at 37°C on a heating shaking dry bath. A 0.05 mL aliquot was removed at the 0 min time point and added to 100 μl methanol that contained 10 μM 4,5-diphenylimidazole as internal standard (IS). The enzymatic reaction was initiated with 8.8 μL of human, mouse, rat, or dog liver microsomes (each from Xenotech, Kansas City, KS), resulting in a protein concentration of 0.5 mg/mL. Aliquots of 0.05 mL were taken at 10, 20, 30, 60, and 120 min. Each sample was transferred to 100 μL of cold methanol containing 10 μM IS. The solutions were sonicated for 10 s and centrifuged for 5 min at 10,000 x g. The supernatant was filtered with a spin-X HPLC filter tube (Corning Inc., Corning, NY) and centrifuged again for 30 s at 10,000 x g. The resulting supernatant was diluted 20-fold with water before LC-MS/MS analysis (Shimadzu 8040, Shimadzu, Kyoto, Japan). The peak area ratios relative to the internal standard were calculated and ln of the area ratio was graphed against time. The slope and half-life (t_1/2 = 0.693/k) were determined by linear regression. All determinations were carried out on two different days in triplicate (n =6).

Phase II microsomal stability assay (glucuronidation and glucosidation):

MIDD0301 (4 μL of 1 mM solution in 50:50 water:acetonitrile) was added to a mixture of 222 μL water, 80 μL phosphate buffer (0.5 M, pH 7.4), 40 μL cofactor (50 mM of UDP-glucuronic acid or UDP glucose), 40 μL inhibitor (50 mM of sacharic-1,4-lactone or gluco-1,5-lactone), 4 μL 100 mM MgCl₂, and 1.8 μL 5 mg/ml alamethicin in DMSO. The assay concentration of MIDD0301 was 10 μM. The solution was incubated for 5 min at 37°C on a heating shaking dry bath followed by removal of 50 μL at the 0 min time point, which was added to 0.1 ml of a 5 μM solution of compound 2 [27] in acetonitrile as IS. The enzymatic reaction was initiated with 8.8 μL of either human or mouse liver S9 fraction or mouse kidney S9 fraction (each from Xenotech, Kansas City, KS) resulting in a protein concentration of 0.5 mg/mL. Aliquots of 50 μL were taken at 10, 20, 30, 60, 90, and 120 min (for short assay) or 1, 2, 4, 8, and 24 h (for long assay). Each aliquot was added to 0.1 mL of cold acetonitrile with 5 μM IS. The solution was sonicated for 10 s and centrifuged at 16,000 x g for 5 min. The supernatant was transferred into a 0.22 μm nylon spin-X HPLC filter tube (Corning Inc., Corning, NY) and centrifuged at 10,000 x g for 30 s. The filtrate was diluted 10-fold (for short assay, no dilution for long assay) and analyzed by LC-MS/MS (Shimadzu 8040, Shimadzu, Kyoto, Japan). The peak area ratios relative to the internal standard were calculated and ln of the area ratio was graphed against time. The slope and half-life (t_1/2 = 0.693/k) were determined by linear regression. For the long assay, area ratios of internal standard and MIDD0301 glucuronide (or MIDD0301 glucoside) were plotted against time for first order kinetic analysis to determine rate constant k. All determinations were carried using two independent assays in triplicate (n = 6).

Quantification of MIDD0301 and MIDD0301 metabolites in urine and feces:

Mice were housed individually in metabolic cages (Techniplast, Varese, IT) for the indicated time periods (24 and 48 h) after MIDD0301 administration. Urine and feces were collected from three individual mice for each route of administration. The change of body weight and food and water consumption were recorded. Mice had ad libitum access to water and ground diet. All feces and urine samples were kept at −20°C. Three different routes of administration were investigated in this study. Oral: vehicle: 2% polyethylene glycol and 98% of a 2% aqueous hydroxypropyl-methyl cellulose solution; dose: 7.5 mg/mL; injection volume: 100 μL, and total administered amount 750 μg. Intraperitoneal: vehicle: 50% propylene glycol and 50% phosphate buffered saline pH 7.2; dose: 3.25 mg/mL; injection volume: 200 μL, and total administered amount 750 μg. Intravenous: phosphate buffered saline pH 7.2; dose: 5 mg/mL; injection volume: 50 μL, and total administered amount 250 μg. Urine samples were thawed on ice and vortexed for 5 s. Aliquots of 0.02 mL were blended with 0.02 mL acetonitrile including compound 2 [27] as IS1. The IS1 concentration was 0.5 ppm (24 h) and 0.05 ppm (24 - 48 h). Samples were vortexed for 15 s and centrifuged for 10 min at 10,000 x g. The supernatant was transferred into a clean tube, diluted ten times with 80:20 water:acetonitrile, and filtered by size exclusion (Amicon Ultra-0.5, 3000 NMWL centrifuge filter, Millipore, St. Louis, MO) at 15,000 x g for 30 min. A 0.1 mL aliquot was added to an autosampler vial with a 200 μL glass insert and 10 μL of 0.5 ppm XHE-III-74A [28] in acetonitrile added as a second internal standard (IS2). Fecal samples were collected during the initial 24 h and 24-48 h time periods into 50 mL Eppendorf tubes and diluted with 20 mL of a 50:50 solution of water:acetonitrile containing 2.5 ppm IS1 and 10 mL of a 50:50 solution of water:acetonitrile containing 0.25 ppm IS1. The samples were homogenized with a handheld homogenizer (Cole Palmer LabGen 7B Homogenizer) and centrifuged for 20 min at 2,700 x g at 4°C. An aliquot of 0.1 mL was diluted with 0.1 mL of an 80:20 solution of water:acetonitrile. The solution was filtered by size exclusion (Amicon Ultra-0.5, 3000 NMWL centrifuge filter, Millipore, St. Louis, MO) at 10,000 x g for 30 min at 4°C. A 100 μL sample of the filtrate was transferred into a HPLC vial with a 200 μL glass insert and 10 μL of 0.5 ppm IS2 in acetonitrile added as second internal standard. Analysis was conducted with a Shimadzu Nexera X2 LC30AD (Shimadzu, Kyoto, Japan) using a 5 μL injection volume. Separation was achieved by reverse phase chromatography using an Agilent RRHD Extend-C18. A 2.1 mm x 50 mm column with 1.8 μm particle size was used with a 0.5 mL/min flow rate. The mobile phase was methanol and water (both containing 0.1% formic acid). Program: 35% B (0 min) → 50% B (0.75 min), hold at 50% B (1.75 min), → 62% B (3.5 min) → 99% B (0.5 min), hold at 99% B (1 min), return to 35% B (0.25 min), hold at 35% B (2.25 min). The column temperature was 25 °C. Analysis was performed with positive ionization using MRM (multiple reaction monitoring) with a Shimadzu 8040 (Shimadzu, Kyoto, Japan) using electrospray, as well as atmospheric pressure ionization (DUIS). Ion pairs for MIDD0301 were m/z 415.95 > 304.90, m/z 415.95 > 397.95, m/z 413.95 > 302.90, m/z 413.95 > 395.95; XHE-III-74A [28] m/z 314.10 > 368.10, m/z 314.10 > 278.10; compound 2 [27] m/z 360.20 > 249.10, m/z 360.20 > 273.10, m/z 360.20 > 342.15, m/z 360.20 > 301.10; MIDD0301 glucoside m/z 578.10 > 416.05, m/z 578.10 > 414.05; MIDD0301 glucuronide m/z 592.10 > 416.05, m/z 592.10 > 305.00, m/z 590.10 > 342.15, m/z 590.10 > 303.00, MIDD0301 taurine m/z 523.10 > 398.05, m/z 523.05 > 356.95, m/z 521.10 > 396.05, m/z 521.05 > 354.95, MIDD0301 glycine m/z 473.00 > 398.00, m/z 471.00 > 396.00, m/z 473.00 > 356.80, m/z 471.00 > 354.80. The collision energy was adjusted for each ion transition. The heating block temperature was 400°C. The drying gas flow was 14 L/min and the desolvation line temperature was 250°C. The nebulizing gas flow was 1.5 L/min and voltage of 4.5 kV was used for the needle and interface. Data acquisition and processing were performed using LabSolutions software. Standard curves were constructed with analytical standards and analyzed by linear regression. The concentrations were calculated using individual calibration curves.

Pharmacokinetic studies in mice:

Six-week-old female Swiss Webster mice were housed in groups of four and received MIDD0301 doses of 25 mg/kg by intra-gastric gavage, 25 mg/kg by intraperitoneal injection, or 1 mg/kg by tail vein injection using the formulations described in Quantification of MIDD0301 and MIDD0301 metabolites in urine and feces. Blood, lung, and brain samples from four mice given oral and intraperitoneal dosing were collected for each of the 10, 20, 40, 60, 120, 240, 480, and 1,440 minute time points. For intravenous administration, blood, lung, and brain samples from eight mice for each of these time points were collected. The blood samples were vortexed for 10 s. Samples of 0.1 mL were combined with 0.4 mL methanol containing 0.3 μM IS2 (XHE-III-74A [28]). Samples were vortexed for 30 s and centrifuged for 10 min at 15,000 x g at 4°C followed by evaporation. Each sample was reconstituted with 0.4 mL methanol and filtered with a centrifugal filter (0.22 μm, Costar, Glendale, AZ), followed by dilution with methanol containing 4,5-diphenylimidazole as IS. For all samples, an injection volume of 5 μL was used. Brain and lung were weighed and homogenized in 0.4 mL or 0.6 mL methanol, which contained 0.3 μM IS2. Samples were centrifuged at 15,000 x g for 10 min at 4°C. Spin-filtration was accomplished with centrifugal filters followed by evaporation. Reconstitution was carried out with 0.2 mL or 0.4 mL methanol for brain and lung containing 4,5 diphenylimidazole as IS, followed by spin-filtration. Analysis was conducted as described above with the following changes. The time program was 20% B (0 min) → 45% B (2 min) → 99% B (4 min), hold at 99% B (4.75 min), return to 20% B (5 min), hold at 20% B (1.5 min). Transition pairs for 4,5-diphenylimidazole were m/z 220.80 > 193.10, m/z 220.80 > 166.90, m/z 220.80 >151.95 and m/z 220.80 > 116.10. Data were analyzed with PK solution 2.0 using a two compartment PK model. Half-lives were calculated based on elimination rates.

Imaging mass spectrometry (IMS):

Female Swiss Webster mice were administered 25 mg/kg MIDD0301 orally and sacrificed one hour after administration. Lungs were removed quickly and frozen on dry ice vapor for 5 s and then stored at −80°C. Before cryo-sectioning, lungs were warmed to −20°C (30 minutes). 20-micron sections were cut followed by thaw-mounting to metal analysis slides (Shimadzu Scientific, Kyoto, Japan). Slides were warmed in a desiccator to 25 °C for 5 minutes, followed by rinsing with a 0.1% TFA water solution. After drying for 1 h, 2,5-dihydroxyacetophenone was applied to the samples as matrix using a TLC sprayer (10 mL of a 50 mg/mL solution was sprayed with 3 psi nitrogen). Each layer was allowed to dry fully between applications. For IMS analysis, a 7090 MALDI-TOF/TOF mass spectrometer (Shimadzu Kyoto, Japan) was used in the reflectron MS/MS mode. The ion transitions for MIDD0301 were m/z 416 > 372 and m/z 416 > 331. The spatial laser resolution was 50 μm.

3. Results and Discussion

First, we investigated the metabolic stability of MIDD0301 in the presence of liver microsomes and S9 fractions derived from several species. The results are depicted in Figure 1.

Figure 1.

In vitro phase I metabolism of MIDD0301 in the presence of A) human liver S9 fraction; B) beagle dog liver microsomes; C) mouse liver microsomes; D) rat liver microsomes. All assays were performed as two independent assays with n = 3. Data are presented as averages (n = 6) with standard deviations. The percent MIDD0301 remaining at the 120-minute time point is shown on each graph.

In the presence of human, dog, mouse, and rat liver fractions, metabolic conversion of MIDD0301 over a period of two hours was not observed. A similar compound SH-053-2’F-R-CH₃ bearing an ester group instead of an acid group was metabolized fully within four hours, whereas the corresponding amide (MP-III-022) was stable during this time period [29]. Thus, the carboxylic acid group in the MIDD0301 imidazodiazepine structure is consistent with resistance of similar compounds to conversion by microsomal enzymes that use NADHP as cofactor. To investigate if MIDD0301 inhibits P450 enzyme activity and consequently appears to be stable in the presence of liver microsomes, we conducted a CYP3A4 enzyme inhibition assay (Figure 2).

Figure 2.

CYP3A4 inhibition assay. Recombinantly expressed CYP3A4 enzyme was incubated in the presence of NADPH, fluorescent substrate, and MIDD0301 (10 μM) or vehicle. Fluorescence intensity was determined over time and depicted as mean with standard deviation and rates determined by non-linear regression (first-order kinetics). % inhibition was defined as (1-((X-B)/(A-B)))*100, A is the rate observed for vehicle, B is the rate observed in the presence of nelfinavir.

A concentration of 10 μM MIDD0301 inhibited less than 1% activity of CYP3A4, a P450 enzyme that metabolizes more than 50% of all drugs. In contrast, nelfinavir at 100 μM completely inhibited the activity of CYP3A4. Accordingly, MIDD0301 does not inhibit phase I metabolism meditated by CYP3A4, consistent with the observed resistance of MIDD0301 to metabolism by liver microsomes. Furthermore, we expect the absence of drug-drug interactions for MIDD0301, which occurs when a co-administered drug alters the metabolism of a primary drug.

To identify and quantify phase II metabolic products of MIDD0301 using LC-MS/MS, glycine, glucuronide, glucoside, and taurine conjugates of MIDD0301 were synthesized as outlined in Scheme 1.

Scheme 1.

Synthesis of possible MIDD0301 phase II metabolites.

MIDD0301 taurine was generated by conversion of MIDD0301 to the corresponding acid chloride using thionyl chloride followed by the addition of taurine in the presence of trimethylamine. The methyl ester of glycine was coupled with MIDD0301 in the presence of O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU) in excellent yield followed by hydrolysis in the presence of sodium hydroxide to yield MIDD0301 glycine. MIDD0301 glucuronide was synthesized by adopting a procedure developed by the Stachulski group using allyl glucuronide [30]. After the coupling step in the presence of 1-[bis(dimethylamino) methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxid hexafluorophosphate (HATU), the MIDD0301 allyl glucuronide was converted to MIDD0301 glucuronide in the presence of Pd(PPh₃)₄ and morpholine. Glucose and MIDD0301 formed the corresponding MIDD0301 glucose in the presence of HATU in low yield as a mixture of α and β anomers.

In vitro assays using liver and kidney S9 fractions were conducted in the presence of appropriate cofactors to determine phase II metabolism kinetics. The results are depicted in Figure 3.

Figure 3.

In vitro phase II metabolism of MIDD0301. Glucuronidation of MIDD0301 was determined in the presence of A) human liver S9 and B) and C) mouse liver S9. For C), peak area ratios of internal standard and MIDD0301 glucuronide were plotted versus time and first order kinetic analysis was used to determine rate constant k. Glucosidation of MIDD0301 was determined in the presence of D) mouse liver S9 and E) and F) mouse kidney S9. For F), peak area ratios of internal standard and MIDD0301 glucoside were plotted versus time and first order kinetic analysis was used to determine rate constant k. All assays were performed by LC-MS/MS as two independent assays with n = 3. Data are presented as averages (n = 6) with standard deviations.

Using human or mouse liver S9 fractions, conversion of MIDD0301 to its glucuronide was observed in the presence of uridine 5’-diphosphoglucuronic acid (UDP-GlcUA) with half-lives of 240 and 143 min, respectively (Figure 3A and B). The reactions were carried out in the presence of alamethicin [31] and β-glucuronidase inhibitor saccharolactone [32]. Possible enzymatic conversion using mouse liver S9 fraction was monitored further for 8 h resulting in a reaction rate of 0.0042 min⁻¹ when analyzed by a first order kinetics (Figure 3C). Thus, MIDD0301 is readily converted by liver UDP-glucuronosyltransferases (UGT) and the MIDD0301 glucuronide formed is stable under physiological conditions for at least 8 h. It has been reported that benzoic acid acyl-glucuronides are more stable than aliphatic acyl-glucuronides, which can undergo intramolecular rearrangement and protein alkylation.[33]

Conjugation of xenobiotics by UGTs using uridine 5’-diphosphoglucose (UDP-Glc) has been reported infrequently and usually occurs to a lesser extent [9]. We used mouse liver S9 fraction initially but did not observe conversion of MIDD0301 (Figure 3D). However, in the presence of mouse kidney S9, a pronounced conversion of MIDD0301 was observed (Figure 3E). The half-life of MIDD0301 in mice for the glucosidation was significantly longer (296 min) than the glucuronidation (143 min). MIDD0301 glucoside was identified by LC-MS/MS, bearing the signature bromine isotope ratio. Quantification relative to an IS enabled us to analyze the formation of MIDD0301 glucoside, which occurred with a reaction rate of 0.0038 min⁻¹ (Figure 3F), thus slower than the corresponding glucuronidation reaction. The tissue selective glucosidation reaction of MIDD0301 could be mediated by UGT3A2 [9]. This specific UGT uses UDP-Glc and UDP-xylose instead of UDP-GlcUA and is not expressed in liver.

To investigate the extent of MIDD0301 conjugation in vivo, we quantified phase II metabolites in mouse feces and urine following oral administration. The results are summarized in Table 1.

Table 1.

Excretion of MIDD0301 and its metabolites after oral administration.

	0-24 hours		24-48 hours		0-48 hours
	Feces (%)1	Urine (%)1	Feces (%)1	Urine (%)1	Feces & Urine (%)1
MIDD0301	54.3 ± 5.2	0.1 ± 0.01	0.7 ± 0.4	<LOD	55.1 ± 5.7
MIDD0301 taurine	2.7 ± 0.7	0.2 ± 0.04	<LOD	<LOD	2.9 ± 0.8
MIDD0301 glucoside	<LOD	2.5 ± 0.1	<LOD	0.3 ± 0.3	2.8 ± 0.5
MIDD0301 glucuronide	<LOD	5.8 ± 0.4	<LOD	0.6 ± 0.3	6.4 ± 1.1
Total	57.0 ± 5.9	8.6 ± 0.55	0.7 ± 0.4	0.9 ± 0.6	67.2 ± 7.45

¹% is based on moles of MIDD0301 administered; LOD, lower limit of detection; data are presented as average (n = 3) ± StD

After 24 hours, 54.3% of unconjugated MIDD0301 was found in feces, whereas 0.1% of unconjugated MIDD0301 was found in urine. The major metabolite was MIDD0301 glucuronide followed by MIDD0301 taurine and glucoside. MIDD0301 glucuronide and glucoside were excreted exclusively in urine, whereas MIDD0301 taurine was predominately found in feces. During the 24-48 hour sample collection period, only 0.7% of MIDD0301 was recovered in feces and very low concentrations of MIDD0301 glucuronide and glucoside were observed in urine. Overall, 67.2% of the initial dose of MIDD0301 was recovered, thus, other metabolites such as MIDD0301 amino acid conjugates might be formed. However, MIDD0301 glycine was detected at less than 0.01% in urine and feces.

Next, we investigated the metabolic fate of MIDD0301 when administered intravenously. The results are summarized in Table 2.

Table 2.

Excretion of MIDD0301 and its metabolites after IV injection.

	0-24 hours		24-48 hours		0-48 hours
	Feces (%)1	Urine (%)1	Feces (%)1	Urine (%)1	Feces & Urine (%)1
MIDD0301	30.4 ± 3.4	0.4 ± 0.1	1.2 ± 0.2	<LOD	32.0 ± 3.7
MIDD0301 taurine	7.0 ± 3.1	0.6 ± 0.1	<LOD	<LOD	7.6 ± 3.2
MIDD0301 glucoside	<LOD	44.0 ± 3.4	<LOD	2.0 ± 0.7	46.0 ± 4.1
MIDD0301 glucuronide	<LOD	2.5 ± 0.5	<LOD	0.1 ± 0.04	2.6 ± 0.54
Total	37.4 ± 6.5	47.5 ± 4.1	1.2 ± 0.2	2.1 ± 0.74	88.2 ± 11.54

¹% is based on moles of MIDD0301 administered; LOD, lower limit of detection; data are presented as average (n = 3) ± StD

The recovery of unconjugated MIDD0301 during the first 24 h was 30.4% in feces and 0.4 % in urine. In contrast to oral administration of MIDD0301, MIDD0301 glucoside following IV administration was the major metabolite, followed by MIDD0301 taurine and glucuronide. We established that glucosidation occurs exclusively in the kidney, thus the absence of first pass liver metabolism might be responsible for more pronounced glucosidation. However, the formation of MIDD0301 taurine was still more than twice as high as in feces in comparison to oral administration. For IV administration, the recovery of MIDD0301 and its metabolites were below 4% for the 24-48 hour sample collection period. Interestingly, 88.2% of the initial dose was recovered as unconjugated MIDD0301 and MIDD0301 metabolites implying that formation of unknown MIDD0301 metabolites occurred to a lesser degree for IV administered MIDD0301 than orally administered MIDD0301. Because 32% of the initial IV dose was excreted as unconjugated MIDD0301 in comparison to 55.1% when given orally, bioavailability of MIDD0301 could be as high as 77%.

The recovery of MIDD0301 and formation of MIDD0301 conjugates were also investigated following IP injection of MIDD0301 (Table 3).

Table 3.

Excretion of MIDD0301 and its metabolites after IP injection.

	0-24 hours		24-48 hours		0-48 hours
	Feces (%)1	Urine (%)1	Feces (%)1	Urine (%)1	Feces & Urine (%)1
MIDD0301	43.2 ± 5.9	0.3 ± 0.2	0.3 ± 0.1	0.1 ± 0.04	43.9 ± 6.24
MIDD0301 taurine	16.2 ± 0.9	0.4 ± 0.1	<LOD	<LOD	16.6 ± 1.0
MIDD0301 glucoside	<LOD	13.9 ± 7.1	<LOD	2.4 ± 0.2	16.3 ± 7.3
MIDD0301 glucuronide	<LOD	2.8 ± 1.2	<LOD	0.3 ± 0.2	3.1 ± 1.4
Total	59.4 ± 6.8	17.4 ± 8.6	0.3 ± 0.1	2.8 ± 0.44	79.9 ± 15.94

¹% is based on moles of MIDD0301 administered; LOD, lower limit of detection; data is presented as average (n = 3) ± StD

After IP injection of MIDD0301, 43.2% of unconjugated MIDD0301 was found in feces, whereas 0.3% was detected in urine. In contrast to oral and IV administration, MIDD0301 taurine was the main metabolite for IP injected MIDD0301 followed by MIDD0301 glucoside and glucuronide. Similar to oral and IV injected MIDD0301, MIDD0301 taurine was found predominately in feces. Total MIDD0301 glucoside was five-fold greater than the glucuronide, much less than the 17.6-fold difference for IV injected MIDD0301. Also, the percentage of MIDD0301 taurine was twice as high as for IP injected MIDD0301. Less than 4% of unconjugated MIDD0301 and its metabolites were detected during the 24-48 hour sample collection period, which was consistent for all routes of administration. Total recovery of the MIDD0301 initial dose following IP administration was 79.9%, which was greater than for the oral administration but less than for the IV injection.

Next, we determined the blood concentrations of MIDD0301 following oral, IP, and IV administration. The blood concentrations were determined over a period of 24 h and are depicted in Figure 4.

Figure 4.

Pharmacokinetic analysis of MIDD0301. A) 25 mg/kg by oral gavage; B) 25 mg/kg by IP injection; C) 1 mg/kg by tail vein injection. Animals were sacrificed at indicated time points and concentrations of MIDD0301 quantified by LC-MS/MS. Data are shown as means (n = 4 for oral and IP and n = 8 for IV). A two-compartment PK model was used for the analysis. Determined elimination rates were used to calculate half-lives (t_1/2).

For orally administered MIDD0301, we observed a t_max of 20 min (Figure 4A). Even after 10 min, the blood and lung concentrations of MIDD0301 exceeded receptor affinity (EC₅₀ = 72 nM) at a dose of 25 mg/kg. Thus, orally administered MIDD0301 was absorbed very quickly. Based on calculated elimination rates, MIDD0301 half-lives for blood and lung were 4-5 h. MIDD0301 concentrations in the non-perfused brains were very low, usually less than 7% of the corresponding blood concentrations. After 24 h, lung and brain MIDD0301 concentrations were less than 3 nM, whereas 12 nM MIDD0301 was still found in blood. Intraperitoneal administration of the same dose of MIDD0301 in a 50:50 mixture of phosphate buffered saline and polypropylene glycol resulted in very high lung and blood concentrations of MIDD0301 during the first 40 min (Figure 4B). Mice exhibited no signs of toxicity throughout the study. Half-lives for MIDD0301 in blood, lung, and brain for IP injections were similar to oral administration. Tail vein injections of an aqueous MIDD0301 solution (1 mg/kg) were well-tolerated without signs of irritation at the injection side. Half-lives of MIDD0301 were shorter (1-2 h) for this administration, probably due to the more rapid formation and secretion of MIDD0301 glucoside (Table 2).

IMS was used to investigate the spatial distribution of MIDD0301 in lung tissue. For this study, mice were dosed orally with MIDD0301 and lung harvested after 40 minutes. After cryo-sectioning and deposition of dihydroxyacetophenone matrix, laser desorption mass spectrometry was carried out with resolution of 50 microns.

For IMS imaging of MIDD0301, MALDI-MS/MS with a transition of 416 m/z to ion fragment 372 m/z was used, which was slightly more pronounced than the 416 m/z to 331 m/z transition (Figure 5A). Rasterization resulted in a two-dimensional image that depicts the spatial intensity of this ion transition representative of the location of MIDD0301. In comparison with the bright field image of a lung section at 10x magnification (Figure 5B), the composition of lung tissue intersected by bronchiole, alveoli, and blood vessels is visible for in the IMS image. The intensity of the MS-MS signal for MIDD0301 in comparison with the background indicates a uniform distribution of MIDD0301 in the mouse lung when administered orally.

Figure 5.

Spatial distribution of MIDD0301 in mouse lung. A) IMS image of MIDD0301 in a mouse lung section. MIDD0301 was administered orally and mouse lungs were harvested after 1 h followed by freezing, sectioning, and matrix deposition, which enabled the detection of MIDD0301 using 416 m/z to 372 m/z transition by MALDI-MS/MS. Intensity of selective fragment ions was visualized by a transition from white to black. B) Bright field image lung section at 10x magnification.

4. Conclusion

In conclusion, MIDD0301 is metabolized solely via conjugation, with no observable phase I oxidation. The route of administration significantly influences the percent of MIDD0301 and its metabolites, indicating that the sequence of tissue exposure, including first-pass metabolism, affects the formation and rates of discrete phase II metabolites. However, the elimination rates of parent MIDD0301 for oral and IP administration are similar. More detailed studies are necessary to quantify the in vivo conjugation rates for all identified MIDD0301 metabolites as well as their rates of excretion via feces and urine. It is possible that concentrations of MIDD0301 conjugates are higher in blood than observed in feces and urine due to the lability of glucuronide and glucoside conjugates in aqueous media [34]. Deconjugation can occur enzymatically or via hydrolysis at elevated temperatures. We demonstrated that MIDD0301 metabolites can be synthesized and used to develop accurate LC-MS/MS methods for identification and quantification. Furthermore, we developed an IMS method for detecting MIDD0301 in lung samples, which can be extended to detect MIDD0301 metabolites in the lung.

Appendix

Chemistry:

All reactions were performed in round-bottom flasks with magnetic stirrer under an argon atmosphere. Chemicals and solvents were purchased from either Millipore Sigma, Oakwood Chemical, Alfa Aesar, Matrix Scientific, or Acros Organic and used as received. Reaction progress was monitored by silica gel TLC (Dynamic Adsorbents Inc.) with fluorescence indicator. ¹H, ¹³C and ¹⁹F-NMR spectra were obtained on Bruker 500 MHz instruments with the chemical shifts in δ (ppm) reported by reference to the deuterated solvents as an internal standard DMSO-D₆: δ = 2.50 ppm (¹H-NMR) and δ = 39.52 ppm (¹³C-NMR) and CDCl₃: δ = 7.20 ppm (¹H-NMR) and δ = 77.00 ppm (¹³C-NMR). HRMS spectral data were recorded using a LCMS-IT-TOF spectrometer (Shimadzu). High performance liquid chromatography (Shimadzu Nexara series HPLC) coupled with a Photo Diode Array detector (PDA, Shimidzu SPD-M30A) and a single quadrupole mass analyzer (LCMS 2020, Shimadzu, Kyoto, Japan) was used for purity analysis (absolute area %). Analytes were separated using a Restek Pinnacle-C18 (4.6 mm x 50 mm, 5 μm particle size) column with gradient elution of water and methanol (0.1% formic acid) at a flow rate of 0.8 mL/min. Optical purity was determined with an Agilent 1100 HPLC system using a DAD detector. The mobile phase consisted of HPLC grade ethanol and n-hexane and the stationary phase was a Chiralpak IB-N3 column (4.6 mm x 15 cm, 3 μm) for MIDD0301. Trifluoroacetic acid (0.1%) was used as modifier for the analysis of MIDD0301. MIDD0301 was synthesized using a published procedure [35].

MIDD0301 taurine:

MIDD0301 (400.0 mg, 0.97 mmol) was dissolved in 30 mL dry dichloromethane along with 5 drops of dimethylformamide and 12 equivalents of thionyl chloride followed by reflux for 5 h. After the starting material was fully consumed as determined by thin layer liquid chromatography, the reaction mixture was cooled and concentrated under reduced pressure. The residue was stripped three times with 20 mL of dichloromethane by dissolvation and concentration until the acid chloride was obtained as yellow solid. To the solid was added to 20 mL of dry dichloromethane and the solution was cooled to 0°C. A suspension was prepared consisting of 3 eq taurine, 5 eq Et₃N, and 10 mL DMF. The suspension was added dropwise to the reaction mixture. The reaction was removed from the ice bath and stirred at room temperature for 30 min before being gently heated to 40°C. After conversion by TLC (12-15 h), the reaction was cooled and quenched with 30 mL water. The product was extracted with chloroform (3x 30 mL) and the organic layers combined and concentrated. The product was purified by column chromatography (0.4% acetic acid in methanol:0.4% acetic acid in chloroform, isocratic at 1:5) to yield MIDD0301 taurine as an off-white powder (154.7 mg, 31%); Rf 0.60 (1.8 mL methanol + 0.1 mL acetic acid + 4.3 mL chloroform); ¹H-NMR (500 MHz, MeOD) δ 8.20 (s, 1H), 7.86 (d, J = 8.5 Hz, 1H), 7.76 (d, J = 8.6 Hz, 1H), 7.57-7.51 (m, 2H), 7.35 (s, 1H), 7.32-7.30 (m, 1H), 7.14 (t, J = 7.31 Hz, 1H), 6.58 (q, J = 7.17 Hz, 0.89H), 4.43-4.35 (m, 0.11H), 3.85-3.77 (m, 2H), 3.09 (t, J = 6.4 Hz, 2H), 2.11-2.01 (m, 0.35H), 1.27 (d, J = 7.2 Hz, 2.65H); ¹³C-NMR: (126 MHz, MeOD) δ 165.36, 165.09, 161.40 (d, 1JCF = 247.7 Hz), 139.62, 136.63, 136.47, 135.32, 134.16, 133.60 (d, 3JCF = 8.7 Hz), 132.36, 132.09, 132.07, 129.61 (d, 2JCF = 16.5 Hz), 125.84, 125.72, 121.73, 117.08 (d, 2JCF = 21.5 Hz), 51.56, 51.08, 36.08, 15.08; m/z [M + H]⁺ calculated for C₂₁H₁₈⁷⁹BrFN₄O₄S: 521.02 found 521.2.

MIDD0301 glycine methyl ester:

MIDD0301 (0.5 mmol, 206 mg) and HBTU (0.226 mg, 0.6 mmol) were stirred in dry acetonitrile (5 mL) and N-methylmorpholine (0.220 mL, 2 mmol) under nitrogen at RT. A solution of glycine methyl ester HCl (0.7 mmol, 87.8 mg) in acetonitrile (2 ml) and N-methylmorpholine (0.220 mL, 2 mmol) was added to the solution. The reaction was monitored by TLC. After the completion of the reaction (TLC, silica gel) the solvent was removed under reduced pressure, treated with aq. NH₄Cl and brine, and dried over MgSO₄. The residue was loaded on a precolumn with ethyl acetate and separated by Biotage: 20% ethyl acetate in hexanes (3 CV), 20-100% (20 CV) EA, 100% EA (3 CV). 232 mg of a white solid was obtained (96%). ¹H NMR (500 MHz, CDCl₃) δ 7.82 (s, 1H), 7.68 (d, J = 7.5 Hz, 1H), 7.62-7.58 (m, 1H), 7.45-7.38 (m, 3H), 7.23 (td, J = 7.5, 1.1 Hz, 1H), 7.01 (d, J = 7.5 Hz, 1H), 6.83 (q, J = 7.3 Hz, 0.71H), 4.25-4.13 (m, 2.29H), 3.76 (s, 2H), 2.79 (s, 3H), 2.22-2.18 (m, 0.48H), 1.25 (d, J = 7.0 Hz, 2.52H); ¹³C NMR (126 MHz, CDCl₃) δ 170.42, 162.86, 162.39, 160.20 (d, 1JCF = 256.7 Hz), 139.36, 134.78, 133.88, 133.71, 133.12, 132.10 (d, 3JCF = 8.2 Hz), 131.44, 131.38, 131.25, 128.70 (d, 2JCF = 17.4 Hz), 124.62, 123.65, 120.88, 116.23 (d, 2JCF = 22.1 Hz), 52.41, 50.01, 40.86, 15.12; ¹⁹F NMR (471 MHz, d₆ DMSO) δ −112.29; m/z [M + H]⁺: calculated for C₂₁H₁₆⁷⁹BrFN₄O₃ 485.03 found 485.1.

MIDD0301 glycine:

MIDD0301 glycine ethyl ester (200 mg, 0.41 mmol) was dissolved in 2 ml THF, 0.2 ml of water, and NaOH (40 mg, 1 mmol). The solution was heated for 18 hours at 60°C. After full conversion was achieved, 60 μL of acetic acid was added and the solution was evaporated to dryness. The solid was dissolved in hot ethanol and filtered to yield the pure acid with 85% yield (163 mg). ¹H NMR (500 MHz, d₆ DMSO) δ 8.40 (s, 1H), 7.96-7.85 (m, 2H), 7.58 (t, J = 7.5, 1H), 7.53 (d, J = 7.5 Hz, 1H), 7.34-7.28 (m, 2H), 7.21 (t, J = 7.5, 1H), 6.66 (q, J = 7.3 Hz, 0.92H), 4.31-4.24 (m, 0.08H), 3.51 (s, 2H), 2.02-1.96 (m, 0.44H), 1.15 (d, J = 7.3 Hz, 2.56H). ¹³C NMR (126 MHz, d₆ DMSO) δ 171.07, 161.73, 161.34, 159.38 (d, 1JCF = 255.5 Hz), 137.18, 135.24, 134.91, 133.68, 132.15 (d, 3JCF = 7.6 Hz), 131.96, 131.43, 130.92, 130.39, 128.42 (d, 2JCF = 17.4 Hz), 125.04, 124.69, 119.58, 115.92 (d, 2JCF = 21.2 Hz), 49.01, 43.44, 14.80; ¹⁹F NMR (471 MHz, d₆ DMSO) δ −114.26; m/z [M + H]⁺: calculate for C₂₁H₁₆⁷⁹BrFN₄O₃ 471.3 found 471.1.

MIDD0301 allyl glucuronide:

A round bottom flask was charged with MIDD0301(100 mg, 0.24 mmol), allyl glucoronate [30] (140 mg, 0.6 mmol), HATU (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b] pyridinium 3-oxide hexafluorophosphate, 182.5 mg, 0.48 mmol), and acetonitrile (20 mL) under argon atmosphere. N-methylmorpholine (0.075 mL, 0.72 mmol) was added and the reaction mixture stirred at room temperature for 4 h and analyzed by thin layer liquid chromatography (5% methanol in dichloromethane). Upon conversion of MIDD0301, Amberlyst A-15 (H+, 3 eq) was added and the reaction evaporated and purified by column chromatography (silica gel) with 5% methanol in dichloromethane to give 69 mg (46% yield) of the allyl protected MIDD0301 glucuronide as white foamy solid. Rf = 0.26 (5% methanol in dichloromethane) ¹H NMR (500 MHz, MeOD) δ 8.43 (s, 1H), 7.91 (d, J = 8.6 Hz, 1H), 7.83 (d, J = 8.7 Hz, 1H), 7.61-7.56 (m, 2H), 7.40 (s, 1H), 7.34 (td, J = 7.6, 0.9 Hz, 1H), 7.15 (dd, J = 16.5, 7.5 Hz, 2H), 6.59 (q, J = 7.2 Hz, 0.74H), 5.96 (ddd, J = 19.2, 10.6, 5.4 Hz, 1H), 5.70 (d, J = 8.0 Hz, 0.9 Hα), 5.37 (d, J = 10.5 Hz, 1H), 5.24 (dd, J = 10.5 Hz, 1H), 4.69 (d, J = 5.5 Hz, 2H), 4.47 (d, J = 9.8 Hz, 0.1Hβ), 4.08 (d, J = 9.7 Hz, 1H), 3.70-3.60 (m, 2H), 3.55 (t, J = 9.1 Hz, 1H), 2.92 (s, 3H), 1.30 (d, J = 7.2 Hz, 3H); ¹³C NMR (126 MHz, MeOD) δ 168.46, 164.21, 161.10, 159.99 (d, 1JCF = 248.7 Hz), 142.14, 136.62, 135.84, 135.43 (α and β), 133.55, 133.39 (s, α and β), 132.82, 132.36 (d, 3JCF = 8.3 Hz), 131.60, 130.98, 130.78 (α and β), 127.94 (d, 2JCF = 12.9 Hz), 127.38, 127.36 (α and β), 127.22, 127.16 (α and β), 124.62, 124.48 (d, 4JCF = 3.3 Hz), 121.04, 120.94 (α and β), 117.42, 115.71 (d, 2JCF = 21.4 Hz), 95.00, 94.82 (α and β), 76.12, 75.64, 72.08, 71.55, 65.67, 49.78, 13.39, 13.00 (α and β).¹⁹F NMR (471 MHz, MeOD) δ −72.19 (conformer α), −77.52 (conformer β). m/z [M + H]⁺: calculate for C₂₈H₂₅⁷⁹BrFN₃O₈ 630.1 found 630.1.

MIDD0301 glucuronide:

To MIDD0301 allyl glucuronide (50 mg, 0.08 mmol) in THF (4 mL) was added Pd(PPh₃)₄ (12 mg, 0.0096 mmol) and morpholine (0.01 g, 0.096 mmol) at 0°C under vigorous stirring. The reaction was monitored by thin layer liquid chromatography (methanol:chloroform:acetic acid 15:84.6:0.4) for 45 min. Evaporation of solvent left a yellow gum, which was purified by chromatography. A gradient of 5 to 20 % methanol in chloroform in the presence of 0.4% acetic acid gave 34 mg (72% yield) of MIDD0301 glucuronide as an off-white powder. ¹H NMR (500 MHz, MeOD) δ 8.44 (s, 1H), 7.92 (dd, J = 11.1, 4.4 Hz, 1H), 7.85 (d, J = 7.1 Hz, 1H), 7.57 (m, 2H), 7.43-7.36 (m, 1H), 7.33 (t, J = 7.2 Hz, 1H), 7.16 (dd, J = 18.5, 8.9 Hz, 1H), 6.59 (q, 1H), 5.71 (d, J = 7.3 Hz, 0.9 H α), 4.45 (d, J = 7.3 Hz, 0.1H β), 3.64 (dd, J = 7.9 Hz, 1H), 3.59 (t, J = 6.9 Hz, 2H), 3.33 (t, J = 3.2 Hz, 1H), 2.09 (d, J = 5.4 Hz, 0.4 H), 1.30 (d, J = 7.6 Hz, 2.6 H). ¹³C NMR (126 MHz, MeOD) δ 174.61, 164.04, 161.37, 160.01 (d, 1JCF= 249.1 Hz), 142.04, 141.17 (α and β), 136.49, 136.33 (α and β), 135.39, 133.56, 133.44 (s, α and β), 132.80, 132.33 (d, 3JCF = 8.7 Hz), 131.20, 131.00 (α and β), 130.81, 130.69 (s, a and b), 128.03 (d, 2JCF = 12.2 Hz), 127.46, 124.59, 124.48 (d, 4JCF = 3.2 Hz), 120.92, 115.71 (d, 2JCF = 21.5 Hz), 94.87, 94.78 (s, α and β), 76.41, 76.11, (s, α and β), 75.35, 73.16, 73.08 (α and β), 72.60, 72.28 (α and β), 49.84, 49.74 (α and β), 20.42; ¹⁹F NMR (471 MHz, MeOD) δ −73.44 (conformer α) −76.52 (conformer β). HRMS [M-H]⁻ Calculated for C₂₅H₂₁⁷⁹BrFN₃O₈ 588.0462, found 588.0423.

MIDD0301 glucoside:

503.0 mg (1.21 mmol) of MIDD0301 was dissolved in 10 mL of dimethylformamide. 5 eq of 4-methyl morpholine was added dropwise followed by the addition of HATU (460.0 mg, 1.21 mmol). The mixture was stirred for 10 min at room temperature. D(+)-Glucose (218.0 mg, 1.21 mmol) was dissolved in 15 mL of dimethylformamide and added dropwise to the reaction mixture. The reaction was heated to 60°C for 24 h. Upon consumption of the starting material by TLC (1.8 mL methanol + 0.1 mL acetic acid + 4.3 mL chloroform) the solvent was removed under reduced pressure before stripping the product with 5 mL of toluene four times. The crude product was divided into 200 mg portions before being individually loaded onto a Biotage Sfär C18 D precolumn before being separated by Biotage: MeOH:H2O 35-50% (15 CV), 50-60% (10 CV), 60-90% (10 CV). A mixture of isomers of the desired product was collected as a white solid (103.79 mg, 14.8%). Rf first isomer: 0.30, second isomer: 0.26 (1.8 mL methanol + 0.1 mL acetic acid + 4.3 mL chloroform); ¹H-NMR (500 MHz, MeOD) δ 8.30-8.25 (m, 1H), 7.82-7.77 (m, 1H), 7.70 (t, J = 8.65 Hz, 1H), 7.49-7.42 (m, 2H), 7.28-7.26 (m, 1H), 7.22 (dt, J = 8.67, 1.21 Hz, 1H), 7.09-7.04 (m, 1H), 6.47 (m, 1H), 5.56 (d, J = 7.94 Hz, 0.3H), 5.34 (d, J = 3.50 Hz, 0.1H), 4.99 (d, J = 3.74 Hz, 0.3H), 4.57-4.54 (m, 0.3H), 4.50-4.45 (m, 0.3H), 4.42-4.33 (m, 0.8H), 4.03-3.98 (m, 0.3H), 3.80-3.70 (m, 0.7H), 3.61-3.50 (m, 1.1H), 3.44 (t, J = 8.57 Hz, 0.4H), 3.38 (t, J = 8.75 Hz, 0.4H), 3.32-3.17 (m, 1.6H), 3.07-3.04 (m, 0.2H), 1.97 (m, 0.6H), 1.19 (dd, J = 3.07, 2.04 Hz, 3H); ¹³C-NMR: (126 MHz, MeOD) δ 164.29, 164.18 (d, 1JCF = 9.91 Hz), 162.57, 162.48, 161.26, 161.00, 159.01, 143.63, 161.89, 141.03, 140.91 (α and β), 136.54, 136.46, 136.40 (α and β), 135.41 (d, 3JCF = 2.99 Hz), 133.60, 133.47, 132.81, 132.38, 132.32 (α and β), 130.96, 130.78, 130.68, 130.64 (α and β), 128.53, 128.39, 128.32 (α and β), 128.02 (d, 2JCF = 12.98 Hz), 127.54, 124.54 (d, 4JCF = 12.33 Hz), 120.91, 120.78 (α and β), 115.81, 115.64 (α and β), 96.89, 84.95, 92.65, 89.82, 77.58, 76.59, 76.39, 74.88, 73.90, 73.46, 72.48, 70.86, 70.61, 70.36, 69.70, 69.27, 64.01, 63.88 (α and β), 60.97, 49.96, 49.78 (α and β), 13.55, 13.41 (α and β). HRMS calculate for C₂₅H₂₄⁷⁹BrFN₃O₇ 576.0776, found 576.0818.

9. Availability of data and materials

Data can be shared upon request by email.