Synthesis of 3-acyltetramates by side chain manipulation and their antibacterial activity

Abstract

An efficient approach for the introduction of 3-acyl side chain groups onto a core tetramate system, which are suitable for further manipulation by nucleophilic displacement or Horner–Wadsworth–Emmons coupling, provides access to a diverse library of substituted tetramates related to two distinct classes of natural products, equisetin and pramanicin. Assessment against S. aureus and E. coli indicated that some compounds exhibit significant antibacterial activity, providing unusual leads for further optimisation in the drug discovery process.

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3-Acyltetramic acids 1 are core structural skeletons found in a wide range of natural products, exhibiting diverse biological activity.^1–3 Several are of interest for their antibacterial activity, notable examples being equisetin,⁴ reutericyclin,⁵ kibdelomycin,⁶ and streptolodygin,⁷ and significant progress in the development of synthetic routes to such systems⁸ and their analogues has recently been made.³ Interestingly, methodology for the general preparation of β-tricarbonyl systems is scarce,⁹ and we have recently reported methodology providing access to highly substituted 3-acyltetramates, which relies upon Dieckmann cyclisation of templates 2 derived from serine,¹⁰ threonine¹¹ or cysteine,¹² the chemoselectivity of which can be controlled by judicious use of reaction conditions and substituents to give products 3 and/or 4 (Scheme 1).¹³ Moreover, we have established a reliable approach for the introduction of diverse 3-acyl groups into either of 3 or 4,¹⁴ and this permits rapid generation of chemical diversity around the core tetramate scaffold. In some cases, these compounds possess potent antibacterial activity,^15,16 even though the core tetramate system itself is generally devoid of such activity.¹⁷ Aiming to further extend this approach, we report here a modification of the strategy which permits selective and efficient 3-acyl side chain manipulation in tetramate systems, thereby permitting wider access to libraries of modified derivatives. Interest in the total synthesis of naturally occurring antibiotics and their analogues is increasing given the recognition of the urgent need for new generations of antibiotics.^18–23

Scheme 1

Key to the approach is the successful extension of our O-acylation/rearrangement procedure, which had been found to be most effective for aromatic carboxylic acids, for the introduction of substituted acyl derivatives;¹⁴ the successful rearrangement is readily seen by disappearance of the enol acetate signal at about δ 6.3 once the C-acyl product is formed. Thus, treatment of tetramate 3 (R¹ = H; X = O) with any of acetic acid, 4-pentenoic acid, chloroacetic acid and bromoacetic acid with DCC–DMAP gave the products 5a–d in good yield (Scheme 2), although reaction with vinylacetic acid gave a complex product mixture. Assignment of 5a existing in fact as the major tautomer 6b was made by ¹³C NMR analysis in which the respective tautomers could be readily identified by down-shifted carbonyl signals (6a, C(6) 195 ppm, C(8) 172 ppm; 6b, C(6) 188 ppm, C(8) 180 ppm, assigned by analogy to earlier work¹⁴) as a result of deshielding from H-bonding and we assume that the other analogues are the same; detailed analysis of related systems has indicated that such exo-enol forms generally predominate in these tetramate systems.^14,16 Successful access to these systems provided opportunity for side chain extension, and by way of exemplification, 5a when treated with aromatic aldehydes in the presence of mild base (piperidine) gave the enamine products 7a–c in good yields. The newly formed trans-double bond geometry was evident from the large coupling constant (ca. 16 Hz) and the exo-enamine tautomeric form is assumed from the above analysis, although the geometry about this double bond was not determined. Of interest is that these enamines could not be hydrolysed under acidic conditions (aq. HCl), as might have been expected, but instead required alkaline conditions (LiOH, THF, H₂O), giving the enone products 10a–c in excellent yield (93, 100 and 89%, respectively); presumably this process occurs by an addition-elimination reaction. In an alternative process, condensation of acetyl derivative 5a with p-methoxybenzaldehyde in the presence of dibenzylamine gave a mixture of enamines 7d and 6d (each 18%), the former of which could be converted to enone product 9 in a two-stage process consisting of hydrogenolysis to 8 and subsequent alkaline hydrolysis (Scheme 2, enamine and enol geometry of 7, 8, 9 and 10 not unequivocally assigned); the isolated product was consistent with structure 9, as indicated by LRMS and ¹H NMR spectrum (absence of enamine signals near 10 ppm). The identity of intermediate 8 was made from the presence of a peak at 415 m/z (ESI⁻; corresponding to [M − H]⁻) by LRMS analysis, and critically the ¹H NMR showed broad signals near 10 ppm, assumed to be the enamine –NH₂ protons. Of interest was that enamine 6d isolated from this process could be further condensed with more p-methoxybenzaldehyde, to give enamine 7d, in a conversion of 71% (based on ¹H NMR analysis), suggesting that the earlier reactions leading to 7a–c proceeded by the initial formation of enamine 6c, rather than by formation of the enolate of 5a. Although this process is only applicable to non-enolisable aldehydes, it was nonetheless more efficient than a single-step one involving formation of the dianion by deprotonation of acetyl 5a with excess LDA to generate the dianion and reaction with aldehydes, which returned only 12, 14, 31, 11 and 18% yield of enones 10a–e, respectively (Scheme 2).

Scheme 2 (a) RCH₂C(O)OH (1.1 eq.), DCC (1.1 eq.), DMAP (0.05 eq.), CH₂Cl₂, r.t.; (b) RC₆H₄CHO, C₅H₁₀NH, toluene, reflux; (c) MeOC₆H₄CHO, Bn₂NH, toluene, reflux; (d) LiOH (1.0 eq.), THF–H₂O (1 : 1), r.t., 1.5 h; (e) NH₄CO₂H, 10% Pd/C, MeOH, reflux, 28 h; (f) LDA (2.1 eq.) then RCHO.

Moreover, side chain manipulation of chloro- and bromo-derivatives by nucleophilic displacement also proved to be readily feasible. Thus, displacement of the halogen in chloroacetyl 5c with phenols gave ethers 11a–c very efficiently (Scheme 3) and this approach was readily extendible to reaction using bromoacetyl 5d with diethyl phosphite, to give phosphorane 12 in excellent yield. For this compound 11c, poor NMR spectral resolution was found in both CDCl₃ and CD₂Cl₂ as solvents, but detailed analysis by COSY-EXSY demonstrated that this was due to the presence of a tautomeric mixture. Condensation of the anion of phosphonate 12, generated by treatment with t-butoxide, with a series of aldehydes (Scheme 3), gave excellent yields of the enone products 10a–h, a process which was significantly more efficient than those involving direct condensation and shown in Scheme 2. Attempted deprotection of these systems, however, via Corey–Reichard protocol²⁴ proved to be problematic; for example, reaction of 10c under conditions which we have previously found to be universally successful in related substrates,^25–27 gave a low and irreproducible yield of the expected tetramate 13 (Scheme 4). In addition, use of other acidic conditions such as THF–H₂O–formic acid (3 : 1 : 1)²⁸ or BiBr₃–MeCN with catalytic H₂O²⁹ resulted in no conversion of the oxazolidine. We believe that this unexpected behaviour is due to unusual structural features in these rigid and highly functional systems,³⁰ and it is also worth highlighting that all tetramic acids with the exocyclic enol isolated via silica gel column chromatography are subsequently washed with dilute HCl to remove chelated metal cations in order to give a well resolved ¹H NMR spectra with distinct resonance signals; that this is necessary to obtain pure samples has been reported previously.^3,9,31

Scheme 3 (a) RC₆H₄OH, Cs₂CO₃, MeCN, r.t.; (b) NaH, then (EtO)₂P(O)H, THF; (c) tBuOK (2.1 eq.), THF, 0 °C, RCHO.

Scheme 4

These compounds could be considered to be hybrid mimics of pramanicin and equisetin, and when assayed against S. aureus and E. coli, the antibacterial profile shown in Table 1 was obtained. The phenotypic assay used here automatically selects for cell penetration activity, a property now recognised as being difficult to re-introduce into candidates identified by target screening approaches, for example.³² Although the parent systems 5a–d and ether derivatives 11a–c showed no activity of any significance, some of the enamines (7a and 7c) and most of the enones (10a, 10c–f and 10h), and especially those with long hydrophobic side chains, showed potent activity against S. aureus, although not against E. coli; this Gram-positive selectivity appears to be typical for tetramate systems.^15,17 Correlation with cheminformatic descriptors^33,34 suggests that enamine compounds with c log P values in the range 4.1–4.8 and %PSA values of about 11%, and enones with values of 2.3–4.7 and 14–20% exhibit optimal bioactivity, and this is similar behaviour to other tetramate libraries.^12,15,35 Although members of the library have a low hydrogen bond donor count (HBD), many of the most active (e.g.5b, 7c, 10a, 10c–f, 10h) have high numbers of rotatable bonds (5–10). These calculated physicochemical properties³⁶ are acceptable for good oral absorption as described by Lipinski,³⁷ although they do not match the physicochemical characteristics of existing antibacterial classes.¹⁹

Table 1 Antibacterials bioactivity and cheminformatic parameters for selected compounds

Compound	Conc. (mg ml^−1)	Bioactivity (mm)		c log P	c log D7.4	PSA	MSA	CMR	%PSA	HBD	rotB
		SA	EC
NA = not active; SA = S. aureus; EC = E. coli; PSA = polar surface area; MSA = molecular surface area; CMR = calculated molar refractivity; %PSA = (PSA/MSA) × 100; HBD = H-bonds donor; rotB = number of rotatable bonds.
5a	4	NA	NA	1.22	0.32	93.1	649.9	110.1	14.3	1	3
	2	NA	NA
	1	NA	NA
5b	4	20	13	2.33	1.29	93.1	496.6	86.2	18.8	1	6
	2	19	NA
	1	18	NA
5c	2	NA	NA	1.53	−1.11	93.1	448.2	77.0	20.8	1	4
	1	NA	NA
	0.5	NA	NA
5d	2	NA	NA	1.71	−0.64	93.1	451.7	80.0	20.6	1	4
	1	NA	NA
	0.5	NA	NA
7a	4	19	16	4.74	4.74	76.2	712.5	131.5	10.7	0	6
7b	4	NA	14	4.07	4.07	85.4	729.4	133.0	11.7	0	7
7c	4	28	15	4.83	4.82	76.2	697.7	131.3	10.9	0	6
7d	4	NA	NA	6.67	6.67	85.4	879.6	170.0	9.7	0	11
6c	4	11	NA	2.25	2.25	76.2	570.2	96.3	13.3	0	4
10a	4	25	NA	3.71	3.01	93.1	574.1	107.7	16.2	1	5
	2	23	—
10b	2	NA	NA	3.04	2.20	102.4	589.6	109.1	17.4	1	6
	1	NA	NA
	0.5	NA	NA
10c	4	33	15	3.80	2.76	93.1	558.4	107.4	16.7	1	5
	1	29	13
	0.5	19	NA
10d	2	30	14	4.00	3.52	93.1	603.5	111.8	15.4	1	7
	1	28.5	NA
	0.5	25	NA
10e	2	35	NA	4.65	4.16	93.1	650.0	110.1	14.3	1	10
	1	35	NA
	0.5	33	NA
10f	2	30.5	14	3.72	3.08	93.1	574.2	112.9	16.2	1	6
	1	28	NA
	0.5	25	NA
10g	2	13	NA	2.26	0.97	106.3	513.4	95.0	20.7	1	5
	1	NA	NA
	0.5	NA	NA
10h	2	25	13.5	3.11	2.10	93.1	521.8	99.5	17.8	1	5
	1	20	NA
	0.5	13.5	NA
11a	2	NA	NA	3.02	0.13	102.4	588.7	103.3	17.4	1	6
	1	NA	NA
	0.5	NA	NA
11b	2	NA	13.5	2.35	−0.56	111.6	604.6	104.7	18.5	1	7
	1	NA	12.5
	0.5	NA	NA
11c	2	NA	14	3.11	−0.04	102.4	572.5	103.1	17.9	1	6
	1	NA	NA
	0.5	NA	NA
12	2	NA	NA	0.83	−2.0	128.7	641.3	101.5	20.1	1	9
	1	NA	NA
	0.5	NA	NA

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The antibacterial activity observed for tetramic acids 10a–h is consistent with the known antibacterial activity of natural products possessing a tetramic acid skeletal core. However, the target responsible for the observed phenotypic activity still remains to be elucidated; our recent work has established that similar tetramic acids with pendant 3-acyl groups act as bacterial RNA polymerase (RNAP) and/or UPPS inhibitors,¹⁵ while similar systems have been reported either to have quorum sensing activity,³⁸ interfering with the bacterial proton gradient and membrane potential via non-specific interactions, or siderophoric activity.³⁹ Noteworthy, though, is that some 3-acyltetramic acids (analogous to 10) reported in these studies showed selectivity for bacterial cells over mammalian cells, suggesting the potential of such compounds to be developed for use as therapeutic agents for human use.

This work has shown that hybrid mimics of two related but distinct antimicrobial natural products, equisetin and the pramanicin (Fig. 1), comprising the tetramate core of the former⁴ and the enoyl side chain of the latter,^40,41 are readily accessible, and may exhibit high levels of antibacterial activity. It confirms our earlier results which indicate that small ring lactams devoid of side chain functionalization exhibit no or only modest activity, but that the introduction of longer chain appendages can significantly enhance such activity.¹⁷ These results serve to illustrate the validity of a recent call for the greater use of natural products in the drug discovery process,^42,43 particularly of antibiotics,⁴⁴ and provide one illustration of non-planar heterocyclic systems⁴⁵ suitable for “escaping from flatland”^46,47 of particular relevance to new antibacterial drug candidates.⁴⁸

Fig. 1 Relationship of natural products with the tetramate library.

Acknowledgements

S.W.B.T. gratefully acknowledges the award of an A*STAR Graduate Scholarship.

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Footnote

† Electronic supplementary information (ESI) available: Experimental details and NMR spectra are provided. See DOI: 10.1039/c4ob00095a

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