Synthesis of budesonide conjugates and their anti-inflammatory effects: a preliminary study

Figures & data

Figure 1 (A) Synthesis of amino acid ester conjugates of budesonide and (B) synthesis of acetates conjugates of budesonide.

Notes: 1, budesonide; 2, budesonide-21-chloracetate; 2a–2c, N-Boc-amino acid ester conjugates of budesonide; 3a, budesonide-21-glycine ester; 3b, budesonide-21-phenylalanine ester; 3c, budesonide-21-alanine ester; 3d, budesonide-21-diethylamine acetate; 3e, budesonide-21-dimethylamine acetate; 3f, budesonide-21-N-methylpiperazine acetate; 3g, budesonide-21-morpholine acetate.
Abbreviations: DMAP, 4-dimethylaminopyridine; DMF, N,N-dimethylformamide; EDCI, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride.

Figure 2 ¹H NMR spectra (in CDCl₃) of Bud conjugates.

Notes: 3a, budesonide-21-glycine ester; 3b, budesonide-21-phenylalanine ester; 3c, budesonide-21-alanine ester; 3d, budesonide-21-diethylamine acetate; 3e, budesonide-21-dimethylamine acetate; 3f, budesonide-21-N-methylpiperazine acetate; 3g, budesonide-21-morpholine acetate.
Abbreviations: Bud, budesonide; CDCl₃, chloroform-d; ¹HNMR, proton nuclear magnetic resonance.

Figure 3 Equilibrium solubility of Bud conjugates at room temperature in distilled water for 72 hours.

Notes: **P<0.01 compared with Bud group. Data are expressed as mean ± SD, n=3. One-way ANOVA followed by the Fisher’s LSD test was used for data analysis. 3a, budesonide-21-glycine ester; 3b, budesonide-21-phenylalanine ester; 3c, budesonide-21-alanine ester; 3d, budesonide-21-diethylamine acetate; 3e, budesonide-21-dimethylamine acetate; 3f, budesonide-21-N-methylpiperazine acetate; 3g, budesonide-21-morpholine acetate.
Abbreviations: ANOVA, analysis of variance; Bud, budesonide; LSD, least significant difference.

Figure 4 IC₅ of Bud conjugates on A549 cell lines.

Notes: The cell viability was analyzed by MTT assay. Data are expressed as mean ± SD, n=8. 3a, budesonide-21-glycine ester; 3b, budesonide-21-phenylalanine ester; 3c, budesonide-21-alanine ester; 3d, budesonide-21-diethylamine acetate; 3e, budesonide-21-dimethylamine acetate; 3f, budesonide-21-N-methylpiperazine acetate; 3g, budesonide-21-morpholine acetate.
Abbreviations: Bud, budesonide; IC₅, 5% inhibiting concentration.

Figure 5 Bud conjugates (1 nM) inhibit the production of IL-6 on LPS-induced A549 cell lines.

Notes: **P<0.01 compared with Bud group. Data are expressed as mean ± SD, n=3. One-way ANOVA followed by the Fisher’s LSD test was used for data analysis. 3a, budesonide-21-glycine ester; 3b, budesonide-21-phenylalanine ester; 3c, budesonide-21-alanine ester; 3d, budesonide-21-diethylamine acetate; 3e, budesonide-21-dimethylamine acetate; ANOVA, analysis of variance; Bud, budesonide; IL-6, interleukin-6.
Abbreviations: ANOVA, analysis of variance; Bud, budesonide; IL-6, interleukin-6; LPS, lipopolysaccharide; LSD, least significant difference.

Figure 6 Effects of Bud conjugates on xylene-induced ear edema.

Notes: Ear edema was considered as the weight difference between the right ear and the left ear. Mice received 2% DMSO solution (control), 200 μg/kg of Bud, and different amounts of conjugates (equivalent to 200 μg/kg Bud), respectively. *P<0.05 vs control group; **P<0.01 vs control group. Data are expressed as mean ± SD, n=10. One-way ANOVA followed by the Fisher’s LSD test was used for data analysis. 3a, budesonide-21-glycine ester; 3c, budesonide-21-alanine ester; 3d, budesonide-21-diethylamine acetate; 3e, budesonide-21-dimethylamine acetate.
Abbreviations: ANOVA, analysis of variance; Bud, budesonide; DMSO, dimethyl sulfoxide; LSD, least significant difference.

Table 1 Regression equations of budesonide conjugates in different tissue samples for HPLC analysis

Samples	Equations	R^2	LOQ (μg/mL)
3a
Phosphate buffer	y=25,812x−138,166	0.9995	0.4
Rat plasma	y=7,485.4x−43,139	0.9974	0.4
Human plasma	y=1,709x+18,631	0.9992	0.4
Rat lung homogenate	y=902.3x+26,761	0.9919	0.4
3c
Phosphate buffer	y=33,600x+38,829	0.9998	0.4
Rat plasma	y=4,654.1x−45,180	0.9920	0.4
Human plasma	y=1,750.2x−9,513	0.9990	0.4
Rat lung homogenate	y=953.4x+7,973	0.9915	0.4
3d
Phosphate buffer	y=29,258x+99,124	0.9999	0.4
Rat plasma	y=3,162.2x−6,084	0.9924	0.4
Human plasma	y=4,399x+18,835	0.9980	0.4
Rat lung homogenate	y=3,306.7x−3,568.4	0.9953	0.4
3e
Phosphate buffer	y=24,838x+141,746	0.9997	0.4
Rat plasma	y=5,033.7x+31,322	0.9939	0.4
Human plasma	y=4,503.2x+143,087	0.9952	0.4
Rat lung homogenate	y=5,291.8x+30,999	0.9910	0.4

Notes: 3a, budesonide-21-glycine ester; 3c, budesonide-21-alanine ester; 3d, budesonide-21-diethylamine acetate; 3e, budesonide-21-dimethylamine acetate.

Abbreviations: HPLC, high-performance liquid chromatography; LOQ, limit of quantitation.

Table 2 Recovery and precision for the detection of budesonide conjugates in different tissue samples by HPLC

Samples	Concentration (μg/mL)	Recovery (%)	Intraday RSD (%)	Interday RSD (%)
3a
Rat plasma	5.0	100.97±1.27	0.48	1.28
	50.0	100.11±2.18	0.83	1.47
	500.0	100.05±0.73	1.26	2.15
Human plasma	5.0	101.94±1.12	1.23	1.36
	50.0	99.50±1.01	1.04	1.55
	500.0	99.08±1.68	1.48	2.29
Rat lung homogenate	5.0	99.54±1.55	0.78	2.42
	50.0	101.98±0.69	1.65	2.73
	500.0	100.59±0.35	1.35	2.51
3c
Rat plasma	5.0	101.95±1.32	0.62	1.28
	50.0	100.91±0.58	0.29	1.33
	500.0	100.12±0.37	1.24	2.44
Human plasma	5.0	100.22±1.17	2.15	1.36
	50.0	100.90±1.33	1.18	2.05
	500.0	99.92±1.32	1.73	2.93
Rat lung homogenate	5.0	101.03±1.85	2.25	2.35
	50.0	99.67±0.71	1.71	2.74
	500.0	101.75±0.93	1.91	2.87
3d
Rat plasma	5.0	99.98±1.76	1.25	1.25
	50.0	103.23±0.45	0.36	1.72
	500.0	101.09±1.85	1.45	2.58
Human plasma	5.0	100.67±1.33	2.27	1.30
	50.0	101.27±1.81	0.57	1.18
	500.0	100.40±0.44	0.23	1.63
Rat lung homogenate	5.0	101.70±1.16	1.02	2.09
	50.0	100.37±0.48	1.78	2.17
	500.0	101.30±1.22	1.72	2.37
3e
Rat plasma	5.0	101.75±1.22	0.55	1.28
	50.0	100.02±0.80	0.24	2.32
	500.0	101.66±1.58	1.12	2.30
Human plasma	5.0	100.79±2.37	2.67	1.31
	50.0	100.62±0.73	1.33	2.13
	500.0	100.23±0.56	1.94	2.55
Rat lung homogenate	5.0	99.63±1.63	0.74	1.42
	50.0	99.61±1.59	1.51	2.35
	500.0	100.45±1.26	2.25	2.73

Notes: Data are expressed as means ± SD, n=3. 3a, budesonide-21-glycine ester; 3c, budesonide-21-alanine ester; 3d, budesonide-21-diethylamine acetate; 3e, budesonide-21-dimethylamine acetate.

Abbreviations: HPLC, high-performance liquid chromatography; RSD, relative standard deviation.

Figure 7 Hydrolysis behavior of budesonide conjugates in different pH solutions.

Notes: The residual content of conjugates that decreased with time in 48 hours was measured by HPLC. Data are expressed as mean ± SD, n=3. 3a, budesonide-21-glycine ester; 3c, budesonide-21-alanine ester; 3d, budesonide-21-diethylamine acetate; 3e, budesonide-21-dimethylamine acetate.
Abbreviation: HPLC, high-performance liquid chromatography.

Figure 8 Hydrolysis behavior of budesonide conjugates in rat plasma (A), human plasma (B), and rat lung homogenate (C).

Notes: The residual content of conjugates that decreased with time in 24 hours was measured by HPLC. Data are expressed as mean ± SD, n=3. 3a, budesonide-21-glycine ester; 3c, budesonide-21-alanine ester; 3d, budesonide-21-diethylamine acetate; 3e, budesonide-21-dimethylamine acetate.
Abbreviation: HPLC, high-performance liquid chromatography.