Glutamine analogues containing a keto function – novel inhibitors of fungal glucosamine-6-phosphate synthase

Figures & data

Figure 1 Structures of new inhibitors.

Scheme 1 Syntheses of (E)-4-oxo-2-butenoic acids (2). i) AlCl₃; ii) H⁺ for 2 f–q and 2 t; 1. H⁺2. OH⁻ for 2 r; OH⁻ for 2 s; iii) 1. BuLi, 2. H₂O, 3. MnO₂, 4. HBr_aq/dioxane.

Table I.  Properties of (E)-4-oxo-2-butenoic acids.

Co	R=	Method of synthesis (lit.)	Yield [%]	Crystal. solvents	Mp. [°C] (lit.)
2a	4-CH3C6H4	A1^[14]	75	toluene	135–137 (139–140)^[15]
2b	4-C2H5C6H4	A2^[15]	80	cyclohex/toluene	95–98 (105–106)^[15]
2c	4-n-C4H9C6H4	A2	82	cyclohex/toluene	76–78 (90–91)^[15]
2d	4-tert-C4H9C6H4	A2	63	cyclohex/toluene	119–123 (123–125)^[13]
2e	4-n-C10H21C6H4	A2	79	cyclohex/toluene	79–81 (82–83)^[15]
2f	2,4-(CH3)2C6H3	B1^[18]	39	cyclohex/toluene	105–106 (113–114)^[16]
2g	2,4,6-(CH3)3C6H2	B1	52	hex/diethyl ether*	139–141
2h	2-ClC6H4	B1	24	hex/benzene	78–81
2i	3-ClC6H4	B1	23	hex/benzene	151–153
2j	4-ClC6H4	B1	50	toluene/ethyl acetate	154–156 (156–157)^[15]
2k	2-BrC6H4	B1	28	hex/benzene	76–80
2l	3-BrC6H4	B1	54	hex/benzene	145–147
2m	4-BrC6H4	B1	36	hex/benzene	150–153 (154–155)^[13]
2n	4-FC6H4	B1	23	benzene	130–132 (133–135)^[16]
2o	3,4-(Cl)2C6H3	B1	32	hex/benzene	138–141
2p	3-Cl–4-NO2C6H3	B1	34	hex/benzene	154–156
2q	2-C10H7	B1	56	hex/benzene	158–161
2r	2-C4H3O	B2^[18]	10	2-propanol	153–155 (158–160)^[18]
2s	2-C4H3N	B3	30	hex/diethyl ether	> 160 (dec.)
2t	(CH3)3C	B1	14	hex/benzene	87–92 (68–86)^[21]
2u	CH3CH2	C^[19]	47	chloroform/acetic acid*	105-108 (110–112)^[22]
2w	(CH3)2CH	C	27	chloroform/acetic acid*	81–83 (83)^[23]
2x	CH3(CH2)3	C	27	chloroform/acetic acid*	98–100 (98–99,5)^[24]

* Purified by column chromatography on silica gel.

Scheme 2 Syntheses of keto protected derivatives of alkenoic acids.

Table II.  Properties of keto protected derivatives of alkenoic acids.

Comp	Yield (%)	M.p. (°C)
3u	75	63–64
3w	80	60–61
3x	72	82–84

Scheme 3 Syntheses of new compounds. i) HOSu, DCC, ii) 1. NH₂CH₂CH(NHBoc)COONa, 2. H⁺, iii) TFA, iv) 1. NH₂CH₂CH(BocNvaNH)COONa, 2. H⁺.

Table III.  Properties of N-succinimidoyl active esters.

Comp.	Yield (%)	M. p. (°C)	Comp.	Yield (%)	M. p. (°C)
4a	80	137–141	4m	66	150–155
4b	70	130–132	4n	60	160–162,5
4c	63	86–90	4o	70	140–143
4d	78	145–147	4p	52	176–181 (dec.)
4e	90	95–98	4q	77	181–183
4f	53	134–137	4r	81	172–178 (de.)
4g	90	157–161	4s	57	166–170 (dec.)
4h	56	92–95	4t	89	125–128
4i	83	127–131	4u	86	147–149
4j	67	141–144	4w	74	152–155
4k	81	97–100	4x	46	82–84
4l	82	145–149

Table IV.  Properties of protected inhibitors.

Comp.	R=	Yield (%)	M. p. (°C)
5a	4-CH3C6H4	63	125–128
5b	4-C2H5C6H4	49	128–132
5c	4-n-C4H9C6H4	60	129–131
5d	4-tert-C4H9C6H4	64	127–130
5e	4-n-C10H21C6H4	53	134–136
5j	4-ClC6H4	67	141–144 (dec.)
5n	4-FC6H4	60	136–140
5r	2-C4H3O	20	oil (chloroform/methanol)*
5s	2-C4H3N	66	oil (chloroform/methanol)*
5u	CH3CH2	65	oil (chloroform/acetic acid)*
5w	(CH3)2CH	74	oil (chloroform/acetic acid)*
5x	CH3CH2CH2CH2	80	oil (chloroform/acetic acid)*

* Purified by column chromatography on silica gel

Table V.  Properties of final inhibitors.

Comp.	R=	Yield (%)
1a	4-CH3C6H4	75	− 10.1
1b	4-C2H5C6H4	80	− 19.3
1c	4-n-C4H9C6H4	79	− 31.5
1d	4-tert-C4H9C6H4	80	− 41.7
1e	4-n-C10H21C6H4	86	− 26.7
1f	2,4-(CH3)2C6H3	53	− 44.7
1g	2,4,6-(CH3)3C6H2	87	− 35.3
1h	2-ClC6H4	75	− 13.3
1i	3-ClC6H4	29	− 24.8
1j	4-ClC6H4	75	− 36.7
1k	2-BrC6H4	57	− 10.0
1l	3-BrC6H4	36	− 31.3
1m	4-BrC6H4	45	− 30.1
1n	4-FC6H4	80	− 37.6
1o	3,4-(Cl)2C6H3	45	− 13.2
1p	3-Cl-4-NO2C6H3	19	− 14.9
1q	2-C10H7	67	− 21.3
1r	2-C4H3O	45*	− 32.1
1s	2-C4H3N	70	− 213
1t	(CH3)3C	70	− 28.2
1u	CH3CH2	90	− 60.4
1w	(CH3)2CH	82	− 36.2
1x	CH3CH2CH2CH2	89	− 40.3

Table VI.  Properties of new peptides.

Comp.	Yield (%)	Comp.	Yield (%)
6b	51	7b	91	− 1.4
6s	45	7s	90	+2.2
6u	61	7u	89	− 4.2

Table VII.  Inhibitory and inactivatory data for new compounds in respect to C. albicans GlcN-6-P synthase and affinity to biological membranes.

Compound	R=	IC50 [mM]	T [min]	k2	Kinact [μM]	k2/Kinact
1a	4-methylphenyl	0.45	0.30	2.31	921	41.8	− 0.09
1b	4-ethylphenyl	0.2	0.77	0.904	136	111	0.277
1c	4-n-butylphenyl	0.3	0.94	0.735	890	13.8	1.08
1d	4-tert-butylphenyl	1.5*	0.60	1.16	940	20.5	0.807
1e	4-n-decylphenyl	2.1*	–	–	–	–	–
1f	2.4-dimethylphenyl	0.45	1.80	0.384	1 880	3.4	0.236
1g	2.4.6-trimethylphenyl	1.5	1.00	0.693	80 800	0.1	0.410
1h	2-chlorophenyl	0.4	3.50	0.198	4 040	0.8	− 0.211
1i	3-chlorophenyl	0.2	1.00	0.693	1 730	6.7	0.028
1j	4-chlorophenyl	0.4	0.97	0.717	1 360	8.8	0.055
1k	2-bromophenyl	0.2	5.10	0.136	18 700	0.1	− 0.095
1l	3-bromophenyl	0.3	0.50	1.39	1 080	21.4	0.170
1m	4-bromophenyl	0.45	0.63	1.11	1 360	13.6	0.203
1n	4-fluorophenyl	0.65	1.63	0.425	2 230	3.2	− 0.409
1o	3.4-dichlorophenyl	0.6	1.20	0.578	3 320	2.9	0.581
1p	4-chloro-3-nitrophenyl	0.4	0.48	1.430	590	40.4	0.159
1q	2-naphthyl	0.3	1.05	0.657	640	17.0	0.755
1r	2-furyl	0.65	0.625	1.11	7 830	2.4	− 1.11
1s	2-pyrrolyl	1.65	2.12	0.326	13 500	0.4	− 0.813
1t	tert-butyl	4.0	1.49	0.467	1 480	5.3	− 0.883
1u	ethyl	0.6	1.75	0.396	6 060	1.1	− 1.24
1w	iso-propyl	3.0	1.55	0.448	3 000	2.5	− 1.11
1x	n-butyl	1.0	1.1	0.630	40 000	0.3	0.325
BADP	phenyl	0.2	0.38	1.84	327	93.8	− 0.477
AADP	methyl	0.65	1.5	0.462	13 500	0.6	− 1.42

* Difficult to measure due to poor water solubility.

Table VIII.  Antifungal activity of new inhibitors.

	MIC [μg/ml]
Compound	S. cerevisiae	C. glabrata	C. albicans	C. crusei
1a	125	250	250	250
1b	62.5	250	250	125
1c	62.5	250	125	125
1d	62.5	250	250	250
1e	> 500	> 500	> 500	> 500
1g	62.5	250	125	250
1f	125	250	125	250
1h	125	250	250	250
1i	62.5	250	250	250
1j	125	250	250	250
1k	62.5	250	125	250
1l	62.5	250	250	250
1m	62.5	250	250	125
1n	125	250	250	250
1o	62.5	250	250	250
1p	125	250	250	250
1q	62.5	250	250	250
1r	125	> 1000	> 1000	–
1s	> 250	> 250	> 250	–
1t	500	> 1000	> 1000	–
1u	125	> 1000	> 1000	–
1w	250	> 1000	> 1000	–
1x	> 250	> 250	> 250	–
BADP	–	250	125	250
AADP	15.64	> 1000	> 1000	–

Table IX.  Antifungal activity of new peptides.

	MIC [μg/ml]
Peptide	S. cerevisiae	C. glabrata	C. albicans
7b	> 1000	31.25	> 1000
7s	31.25	15.63	> 1000
7u	31.25	7.81	> 1000
LysNvaFMDP	31.25	7.81	3.13

Table X.  Peptides and inhibitors activity in cell-free extracts.

Peptide	IC50 (μM) in cell-free extract	Inhibitor	IC50 (μM) in cell-free extract	IC50 (μM) for purified enzyme
7b	150	1b	160	200
7s	160	1s	1600	1650
7u	12	1u	360	600
NvaFMDP	160*	FMDP	1	2

* Difficult to measure, probably due to hydrolysis of methyl ester group present in a peptide molecule, resulting in the loss of inhibitory activity.

Table XI.  Activity of tested peptides against HL-60 human promyelocytic leukaemia cells.

Compound	HL-60 IC50 ± SEM (μg/ml)
7s	30% inhibition at 250 μg/ml
7u	232 ± 2
NvaFMDP	15% inhibition at 250 μg/ml