Synthesis and Antiviral Activity of Conformational Analogues of Leucamide A

In order to study the effect of heterocyclic core conformational state of leucamide A on its anti-influenza virus A activity, five conformational analogues were prepared by replacing the Pro-Leu dipeptide in the molecule with various amino acids. The amino acids used were of 2 to 6 carbons. The results showed that these replacements not only changed the conformational relationship between the 4,2-bisheterocycle tandem pair and the third heterocycle, but also had dramatic effect on its activity against influenza virus A.


Introduction
Leucamide A (1, Figure 1) is a bioactive cyclic heptapeptide isolated from the Australian marine sponge Leucetta microraphis. It contains a unique mixed 4,2-bisheterocycle tandem pair consisting of a methyloxazole and thiazole subunit. It was found to be moderately cytotoxic toward several tumor cell lines [1]. Many highly bioactive compounds also contain similar heterocyclic tandem subunits [2,3].

OPEN ACCESS
It was reported that the heterocyclic tandem pair plays a pivotal role in their bioactivity through specific interaction(s) with DNA or other targets [4][5][6][7][8]. One such example is the anti-tumor drug bleomycin, which interacts with DNA by binding to the nucleic bases in a mode that it is partially intercalative, penetrates positively charged moieties distal to the bithiazole into the major grove of the DNA [9]. Therefore, the unique mixed 4,2-bisheterocyclic tandem pair in leucamide A provides us with a useful scaffold for searching for compounds with therapeutic potential.

Results and Discussion
Our efforts to search for novel bioactive compounds started with leucamide A [10]. We constructed a library of 4,2-bisheterocyclic tandem derivatives consisting of a methyloxazole and thiazole subunit. Based on the bleomycin example, we hypothesized the compounds to be synthesized would have the ability to interact with DNA or RNA. Thus, the bioassay target was focused on virus in relation with DNA or RNA replication, such as influenza virus and hepatitis B virus. As we reported before, several compounds containing tandem 2,4-disubstituted methyloxazole-thiazole subunits, showed moderate activity against influenza A virus [11,12], herpes simplex type 2 virus (HSV2) [12], and hepatitis B virus (HBV) [13], while leucamide A itself did not show any antiviral activity. We postulated the conformational state of heterocyclic core might play pivotal role in their bioactivity against influenza A virus. To prove this, we synthesized a series of conformational analogues using a series of amino acids in the place of the proline-leucine dipeptide fragment in leucamide A. The difference of anti-viral activity between leucamide A and conformational analogues 2-6, which have various ring sizes and different conformational states, was examined.

Chemistry
The analogues of leucamide A (1) were prepared in three steps from 7 [10] (Scheme 1). The coupling reaction of 7 with various amino acid methyl esters was accomplished using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) in the presence of 1-hydroxybenzotriazole (HOBt). This step produced the corresponding amide 8 in 73%-83% yield. The carboxylic acid group of 9 was obtained from 8 in almost quantitative yield by saponification with lithium hydroxide in aqueous methanol. The final ring-closure step was successfully implemented after deprotecting the Boc group using TFA in CH 2 Cl 2 followed by treatment of the resulted crude amino acid with HBTU and diisopropylethylamine in dry DMF. The yields of these macrocyclization reactions varied from 25% to 49%. The macrocyclization yields were generally lower compared to that of leucamide A, which was as high as 87% [10]. Leucamide A it contains a proline residue, and proline amide bonds are known to have cis/trans geometry [14] and facilitate cyclization [15].

Biological Activities
The analogues of leucamide A were evaluated for their ability to inhibit Influenza A virus H3N2 (A3 China/15/90) replication in Madine-Darby Canine kidney (MDCK) cells [11]. The results are summarized in Table 1. The compounds 3-6 exhibited a general progression in relative biological activity as the size of the linker increases, especially the compound 6 [12] with an IC 50 of 41 μM against influenza A virus, while leucamide A and compound 2 were inactive. The potency of compound 6 was comparable with that of ribavirin, which showed an IC 50 of 16 μM. Furthermore, compounds 4-6 showed low cytotoxicity, which nonetheless still exhibited a reasonable selectivity index (CC 50 /IC 50 ). The results indicated that the most salient difference between active and inactive analogues was their relative ability to achieve a biological conformational state.

Conformational Analysis of the Conformational Analogues of Leucamide A
To study the relationship between biological activity and the structures of leucamide A and its analogues, the molecular structures were refined by molecular dynamics (MD) simulation using the Standard Dynamics Cascade protocol of Discovery Studio 2.1 in CHARMm force field. First, these molecules were solvated in an orthorhombic solvent model with explicit periodic boundary conditions. The Standard Dynamics Cascade includes two stages of energy minimization followed by three stages of dynamics, including heating, equilibration and production. To carry out energy minimizations, the RMS gradients 0.1 kcal/mol/Å and 0.001 kcal/mol/Å were used for the steepest descent and conjugate gradient algorithms, respectively. The heating dynamics started from 50 K and gradually increased to reach the target temperature of 300 K in 50,000 steps with a time step of 1fs. Subsequently, the systems were equilibrated for 200 ps and the production dynamics were carried out for 500 ps at 300 K with the time step of 1 fs using the NPT ensemble. The conformations were obtained at every 1 ps. The root mean square deviation (RMSD) values of all the conformations were calculated by aligning all of the frames to the initial production structure. The potential energy and RMSDs of the collected snapshots of MD simulations are shown in Figure 2. The graph indicates that leucamide A and its analogues are relatively stable during the course of MD simulation. The conformations with lowest potential energy of the compounds were selected and aligned. As presented in Figure 3, the thiazole ring (A) and the oxazole ring (B) were almost coplanar in compounds 1-6. As far as the third oxazole ring (C) was concerned, increasing the size of the linker in the heterocycle core gradually distorted the conformational relationship between the 4,2-bisheterocycle tandem pair and the third heterocycle. This partially supported the corresponding data and suggested that the conformation changes of heterocycle core existing in the leucamide A and its analogues, had a great influence on the bioactivity against influenza virus A. To further understand the structure-activity relationships, we compared the most active compound 6 with the previously reported active compound 10 [11] (with an IC 50 value of 70 μM against influenza A virus). An overlay of 10 and 6 is shown in Figure 4. Obviously, these two compounds share a bulk hydrophobic group in a same space near the bisheterocyclic tandem pair, and are different from the other inactive compounds.

Chemistry
All chemicals were reagent grade and used as purchased. All reactions were performed under an inert atmosphere of dry argon or nitrogen using distilled dry solvents. 1 H (300 MHz) and 13 C (75 MHz) NMR spectra were recorded on a Varian Mercury-VX300 Fourier Transform spectrometer. The chemical shifts were reported in (ppm) using the 7.26 signal of CDCl 3 ( 1 H-NMR) and the 77.23 signal of CDCl 3 ( 13 C-NMR) as internal standards. High-resolution MS data were obtained on a Finnigan MAT-95 mass spectrometer.

Conclusions
In summary, we have prepared five conformational analogues of leucamide A, by replacing the Pro-Leu dipeptide of leucamide A with various amino acids. The compounds 3-6 showed activity against influenza virus A. The inability of leucamide A and compound 2 to exhibit comparable biological action underscores the importance of the topological conformation requirements for bisheterocyclic tandem pairs to interact with biological targets. From these results, combined with the results we reported previousely [11], the conclusion is that the bisheterocyclic tandem pairs with a third heterocyclic or even the bisheterocyclic tandem pairs alone present a useful scaffold for searching for novel antiviral compounds, and the introduction of a bulk hydrophobic group in certain positions might lead to enhancement of antiviral potency. Further efforts focused on the simple open chain bisheterocyclic tandem derivatives are in progress.