Synthesis of a Non-Symmetrical Disorazole C1-Analogue and Its Biological Activity

The synthesis of a novel disorazole C1 analogue is described, and its biological activity as a cytotoxic compound is reported. Based on our convergent and flexible route to the disorazole core, we wish to report a robust strategy to synthesize a non-symmetrical disorazole in which we couple one half of the molecule containing the naturally occurring oxazole heterocycle and the second half of the disorazole macrocycle containing a thiazole heterocycle. This resulted in a very unusual non-symmetrical disorazole C1 analogue containing two different heterocycles, and its biological activity was studied. This provided exciting information about SAR (structure-activity-relationship) for this highly potent class of antitumor compounds.


Introduction
The disorazoles are a family of 39 macrolides isolated so far, showing macrolide ring sizes between 26 and 32 (Figure 1) [1][2][3].They are secondary metabolites from the Myxobacterium Sorangium cellulosum (So ce12) and were isolated by the research groups of Höfle and Reichenbach in 1994 [4].
All these natural products show very potent antitumor activity due to inhibition of tubulin polymerization combined with a very powerful cytotoxicity up to picomolar activity against various human cancer cell lines [5,6].This exciting biological profile generated a tremendous interest in the scientific community in both total synthesis and biology [7].In addition, their enormous biological potency makes them very attractive in personalized medicine as potesntial payloads for antibody-drug conjugates (ADCs) in targeted cancer therapy [8].
We recently published a flexible and robust new route to synthesize (-)-disorazole C 1 , which involved, at the endgame, a coupling of the building blocks via Yamaguchi esterification and a final Yamaguchi macrolactonization [9].
The advantage of this powerful strategy to construct the disorazole core is that it offers high diversity in each building block before the desired coupling takes place and provides great opportunities to design a variety of disorazole analogues to study SAR (structure-activity relationships) [10].
Based on this strategy, we wish to show our efforts along these lines and report an efficient synthesis to construct a non-symmetrical disorazole C 1 analogue with potent antitumor activity.Most of the published analogues of this highly active natural product family are based on symmetrical compounds, and to the best of our knowledge, only one non-symmetrical disorazole synthesis has been published in the past by K. C. Nicolaou [11].

Results
In a recent study, we focused on the structure-activity relationship of the disorazole C 1 core by exchanging the oxazole ring with thiazole, and we studied the influence of chiral centers within the disorazole framework [10].Employing the same strategy we successfully used in our total synthesis of disorazole C 1 , we have prepared an analogue with the oxazole ring replaced by a thiazole unit.To our surprise, this replacement resulted in an increase in IC 50 values between 200-and 800-fold.Even more dramatic was the inversion of configuration at C(14)/C(14') stereocenters, with a drop in activity of several thousand-fold [10].
Much less influence had the inversion of configuration at C(5)/C(5') stereocenters, with a loss of activity of only between 40-and 160-fold [10].
Based on these findings, it would be of great interest to synthesize a kind of hybrid structure connecting one half of the C2-symmetric macrocycle containing the natural oxazole heterocycle and the other half containing a thiazole unit.
Our published strategy offers the possibility of synthesizing a large variety of disorazole analogues.In order to construct hybrid 16, one just has to construct the two halves 9 and 10.Fragment 10 can be used from the total synthesis of disorazole C 1 , and the thiazole fragment 9 can be used from the synthesis of bis-thiazole 17.
Alcohol 9 was esterified using the Yamaguchi protocol with acid 10 to obtain compound 11 at 81% as a single compound (Scheme 1).Mild deprotection of TES-protected compound 11 with CSA provided the desired alcohol 12, which was directly hydrolyzed with LiOH, providing the starting material 13 for the Yamaguchi macrolactonization.Macrocycle 14 was isolated in 58% yield as a single isomer, which was transferred to the required (E,Z,Z)-triene derivative 15 in 62% using the Boland hydrogenation protocol.After completion of the synthesis of 16, the biological activity of the analogue was evaluated against several immortalized animal and human cancer cell lines and compared to the cytotoxicity of synthetic disorazole C 1 and epothilone B as internal controls (Figure 2) [12,13].Table 1 shows these results and demonstrates again the very high activity of disorazole C 1 in the low sub-nanomolar range (between 0.11 and 0.6 ng/mL).Compound 16 is only one order of magnitude less active than the natural product 4, but several orders of magnitude more active than the bis-thiazole analogue 17.

Discussion
The hybrid disorazole 16 provides a very important piece of information to understand the biological activity of disorazoles because we can compare a series of analogues and can now offer a rationale to understand the activity differences and bring some light into the structure-activity relationship (SAR) of these fascinating molecules.
So far, only for disorazole Z, an X-ray exists to provide competitive data about the interaction of the molecule with the target protein tubulin [14,15].Given the fact that more or less the same interactions take place with other disorazoles, only one half of disorazole C 1 is involved in protein binding to tubulin, and therefore, this can explain the activity differences with our compounds.
Disorazole C 1 is still the most active compound in our series, whereas bis-thiazole 17 showed around 200-800 fold less activity.However, hybrid 16 still retained most of the activity of the natural product 4, with an increased IC 50 value of about a maximum of 10.This clearly demonstrates that the natural oxazole half of 16 preferentially binds to the protein, leaving the thiazole half outside of the close interaction with tubulin.In addition, it explains the quite large drop in activity of the bis-thiazole compound 17.

Materials and Methods
Solvents were dried by standard procedures and redistilled under an N 2 atmosphere prior to use.All reactions were run under nitrogen, unless otherwise stated.For reactions that require heating, an oil bath was used as an external heat source.When the reactions were run at room temperature, a temperature of 22 • C ± 2 • C was implied.The products were purified by flash chromatography on Merck silica gel 60 (40-63 µm).POLYGRAM SIL G/UV254 prefabricated TLC plates with fluorescent indicators from Macherey-Nagel have been used for analytical thin layer chromatography (TLC).The separated substances were detected by irradiation with UV light with a wavelength of 254 nm, staining with vanillin or potassium permanganate reagent, and subsequent warming with a heat gun.Electrospray ionization (ESI) mass spectra were recorded on Waters Xevo G2-TOF spectrometers. 1 H and 13 C-NMR spectra were recorded on Bruker AVIII 400 and Bruker AVI 600 spectrometers (Supplementary Materials).Chemical shifts (δ) are reported in ppm, referencing the resonance signal of the residual undeuterated solvent for 1 H-NMR and the deuterated solvent for 13 C-NMR ( 1 H-NMR = CDCl 3 : δ 7.26, CD 3 OD: δ 3.31; 13 C-NMR = CDCl 3 : δ 77.16, CD 3 OD: δ 49.00).Data are reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, br = broad, m = multiplet, app = apparent), coupling constants (Hz), and integration.Optical rotations were recorded on Perkin-Elmer 341 and Anton Paar MCP150 polarimeters employing the solvent and concentration indicated and are reported in units of 10 −1 (deg cm 2 g −1 ).

Conclusions
The disorazoles are a fascinating group of natural products that attracted both the synthetic chemistry community and biologists.Within the last decade, quite a few research groups have reported new total synthesis efforts and strategies to synthesize these attractive molecules.In addition, several SAR studies have been reported to shed some light on the biology of these interesting compounds.Our results provide some more rationale for these findings for the future because still no in vivo data is reported about the therapeutic usefulness of these highly active molecules.Based on the outstanding cytotoxicity of the compounds, it is still very reasonable that they could be powerful payloads for ADCs.

Figure 1 .
Figure 1.Selected members of the disorazole family.The percentages correspond to the relative abundance from the initial isolation from Sorangium cellulosum, So ce12.

Table 1 .
Biological activities of Disorazole C 1 and thio analogues.Epothilone B was used as internal standard.