**5.1 Epothilones**

Epothilones are one of the most promising natural products discovered with paclitaxel-like activity. Their advantages come from the fact that they can be produced in large amounts by fermentation (epothilones are secondary metabolites from the myxobacterium *Sorangiun celulosum*), their higher solubility in water, their simplicity in molecular architecture which makes possible their total synthesis and production of many analogs, and their effectiveness against multi-drug resistant cells due to they are worse substrates for P-glycoprotein.

The structure affinity-relationship of a group of chemically modified epothilones was studied. Epothilones derivatives with several modifications in positions C12 and C13 and the side chain in C15 were used in this work.

Fig. 1. Epothilone atom numbering.

Epothilone binding affinities to microtubules were measured by displacement of Flutax-2, a fluorescent taxoid probe (fluorescein tagged paclitaxel). Both epothilones A and B binding constants were determined by direct sedimentation which further validates Flutax-2 displacement method.

All compounds studied are related by a series of single group modifications. The measurement of the binding affinity of such a series can be a good approximation of the incremental binding energy provided by each group. Binding free energies are easily calculated from binding constants applying equation 29. The incremental free energies (ΔG0) change associated with the modification of ligand L into ligand S is defined as:

Thermodynamics as a Tool for the Optimization of Drug Binding 779

Fig. 2. Dependence of the IC50 of epothilone analogs against 1A9 cells on their Ka to

transmembrane proteins like P-glycoprotein (Shabbits *et al.* 2001).

four different locations of the taxane scaffold (C2, C13, C7 and C10).

Fig. 3. Taxanes head compounds. Atom numbering

discussed above for epothilones (table 2).

Paclitaxel and docetaxel are widely used in the clinics for the treatment of several carcinoma and Kaposi's sarcoma. Nevertheless, their effectiveness is limited due to the development of resistance, beeing its main cause the overexpression and drug efflux activity of

We have studied the thermodynamics of binding of a set of nearly 50 taxanes to crosslinked stabilized microtubules with the aim to quantify the contributions of single modifications at

Once confirmed that all the compounds were paclitaxel-like MSA, their affinities were measured using the same competition method mentioned above (section 5.1. displacement of Flutax-2). Seven of the compounds completely displaced Flutax-2 at equimolar concentrations indicating that they have very high affinities and so they are in the limit of the range to be accurately calculated by this method (Diaz&Buey 2007). The affinities of these compounds were then measured using a direct competition experiment with epothilone-B, a higher-affinity ligand (Ka 75.0 x 107 at 35ºC compared with 3.0 x 107 for Flutax-2). With all the binding constants determined at a given temperature, it is possible to determine the changes in binding free energy caused by every single modification as

microtubules. Data from (Buey *et al.* 2004).

**5.2 Taxanes** 

$$
\Delta\Delta\mathbf{G}^{\circ}(\mathbf{L}\rightarrow\mathbf{S}) = \Delta\mathbf{G}^{\circ}(\mathbf{L}) - \Delta\mathbf{G}^{\circ}(\mathbf{S})\tag{55}
$$

These incremental binding energies were calculated for a collection of 20 different epothilones as reported in (Buey *et al.* 2004).


Table 1. Incremental binding energies of epothilone analogs to microtubules. (ΔΔG in kJ/mol at 35ºC). Data from (Buey *et al.* 2004).

The data in table 1 show that the incremental binding free energy changes of single modifications give a good estimation of the binding energy provided by each group. Moreover, the effect of the modifications is accumulative, resulting the epothilone derivative with the most favourable modifications (a thiomethyl group at C21 of the thiazole side chain, a methyl group at C12 in the *S* configuration, a pyridine side chain with C15 in the *S* configuration and a cyclopropyl moiety between C12 and C13) the one with the highest affinity of all the compounds studied (Ka 2.1±0.4 x 1010 M-1 at 35ºC).

The study of these compounds showed also a correlation between their citotoxic potencial and their affinities to microtubules. The plot of log IC50 in human ovarian carcinoma cells versus log Ka shows a good correlation (figure 2), suggesting binding affinity as an important parameter affecting citotoxicity.

Fig. 2. Dependence of the IC50 of epothilone analogs against 1A9 cells on their Ka to microtubules. Data from (Buey *et al.* 2004).

### **5.2 Taxanes**

778 Thermodynamics – Interaction Studies – Solids, Liquids and Gases

These incremental binding energies were calculated for a collection of 20 different

Site Modification Compounds ΔΔG C15 *S* → *R* 4 → 17 ~ 27

 Thiazole → Pyridine 5 → 7 -2.9 ± 0.2 6 → 8 -2.1 ± 0.3 14 → 4 -0.2 ± 0.4 16 → 17 ~ 9.4 C21 Methyl → Thiomethyl 2 → 3 -2.8 ± 0.8

 Methyl → Hydroxymethyl 8 → 9 1.4 ± 0.3 5-Thiomethyl-pyridine → 6-Thiomethyl-pyridine 12 → 13 4.1 ± 0.5

C12 *S* → *R* 4 → 7 -2.1 ± 0.3 → 5 0.6 ± 0.3 → 18 ~ -2 → 11 9.0 ± 0.6 → 8 1.9 ± 0.4

Cyclopropyl → Cyclobutyl 5 → 15 4.1 ± 0.2

 7 → 8 1.2 ± 0.2 10 → 11 2.7 ± 0.7

The data in table 1 show that the incremental binding free energy changes of single modifications give a good estimation of the binding energy provided by each group. Moreover, the effect of the modifications is accumulative, resulting the epothilone derivative with the most favourable modifications (a thiomethyl group at C21 of the thiazole side chain, a methyl group at C12 in the *S* configuration, a pyridine side chain with C15 in the *S* configuration and a cyclopropyl moiety between C12 and C13) the one with the highest

The study of these compounds showed also a correlation between their citotoxic potencial and their affinities to microtubules. The plot of log IC50 in human ovarian carcinoma cells versus log Ka shows a good correlation (figure 2), suggesting binding affinity as an

Table 1. Incremental binding energies of epothilone analogs to microtubules. (ΔΔG in

affinity of all the compounds studied (Ka 2.1±0.4 x 1010 M-1 at 35ºC).

Epoxide → Cyclopropyl 1 → 14 -4.7 ± 0.4

*S* H → Methyl 1 → 2 -8.1 ± 0.6

*R* H → Methyl 5 → 6 0.4 ± 0.3

epothilones as reported in (Buey *et al.* 2004).

kJ/mol at 35ºC). Data from (Buey *et al.* 2004).

important parameter affecting citotoxicity.

ΔΔG0(L→S) = ΔG0(L) – ΔG0(S) (55)

7 → 18 ~ 27 14 → 16 17.8 ± 0.3

5 → 10 -5.9 ± 0.6 6 → 11 -3.6 ± 0.3 8 → 12 2.6 ± 0.3

3 → 19 -5.4 ± 0.8

4 → 20 -1.8 ± 0.5

Paclitaxel and docetaxel are widely used in the clinics for the treatment of several carcinoma and Kaposi's sarcoma. Nevertheless, their effectiveness is limited due to the development of resistance, beeing its main cause the overexpression and drug efflux activity of transmembrane proteins like P-glycoprotein (Shabbits *et al.* 2001).

We have studied the thermodynamics of binding of a set of nearly 50 taxanes to crosslinked stabilized microtubules with the aim to quantify the contributions of single modifications at four different locations of the taxane scaffold (C2, C13, C7 and C10).

Fig. 3. Taxanes head compounds. Atom numbering

Once confirmed that all the compounds were paclitaxel-like MSA, their affinities were measured using the same competition method mentioned above (section 5.1. displacement of Flutax-2). Seven of the compounds completely displaced Flutax-2 at equimolar concentrations indicating that they have very high affinities and so they are in the limit of the range to be accurately calculated by this method (Diaz&Buey 2007). The affinities of these compounds were then measured using a direct competition experiment with epothilone-B, a higher-affinity ligand (Ka 75.0 x 107 at 35ºC compared with 3.0 x 107 for Flutax-2). With all the binding constants determined at a given temperature, it is possible to determine the changes in binding free energy caused by every single modification as discussed above for epothilones (table 2).

Thermodynamics as a Tool for the Optimization of Drug Binding 781

cephalomannine → docetaxel C → 21 -3.8 -5.6 ± 1.1

C10 acetyl → hydroxyl T → 15 -1.3 -1.7 ± 0.8

Table 2. Incremental binding energies of taxane analogs to microtubules. (ΔΔG in kJ/mol at

In this way, it is possible to select the most favourable substituents at the positions studied and design optimized taxanes. According to the data obtained, the optimal taxane should have the docetaxel side chain at C13, a 3-N3-benzoyl at C2, a propionyl at C10, and a hydroxyl at C7. From compound 1 with a binding energy of -39.4 kJ/mol, the modifications selected would increase the binding affinity in -5.6 kJ/mol from the change of the cephalomannine side chain at C13 to the docetaxel one, -11.2 kJ/mol from the introduction of 3-N3-benzoyl instead of benzoyl at C2, -1.6 kJ/mol from the substitution of a propionyl at C7 with a hydroxyl, and -0.9 kJ/mol from the change of a hydroxyl at C10 to a propionyl. Thus, this optimal taxane would have a predicted ΔG at 35ºC of -58.7 kJ/mol. This molecule was synthesized (compound 40) and its binding affinity measured using the epothilone-B displacement method and the value obtained is in good corespondence with the predicted one: Ka = 6.28±0.15 x 109 M-1; ΔG = -57.7±0.1 kJ/mol (Matesanz *et al.* 2008). This value means

It is also possible to check the influence of the modifications on the cytotoxic activity determining the IC50 of each compound in the human ovarian carcinoma cells A2780 and their MDR counterparts (A2780AD). The plots of log IC50 versus log Ka (figure 4) indicate that, as in the case of epothilones, both magnitudes are related, and the binding affinity acts as a good predictor of citotoxicity. In this type of MDR cells the high-affinity drugs are circa 100-fold more cytotoxic than the clinically used taxanes (paclitaxel and docetaxel) and

The plot of log resistance index against log Ka shows a bell-shaped curve (figure 5). Resistance index present a maximum for taxanes with similar affinities for microtubules and P-glycoprotein, then rapidly decreases when the affinity for microtubules either increases or decreases. To find an explanation for this behaviour we should note that the intracellular free concentration of the high-affinity compounds will be low. To be pumped out by Pglycoprotein ligands must first bind it, so ligand outflow will decrease with lower free ligand concentrations (discussed in (Matesanz *et al.* 2008)). In the case of the low-affinity drugs, the concentrations needed to exert their citotoxicity are so high that the pump gets

saturated and cannot effectively reduced the intracellular free ligand concentration.

acetyl → propionyl C → 18 -1.6 -0.5 ± 0.4

 17 → D -7.7 20 → 40 -5.2

propionyl → hydroxyl 18 → 17 0.9

C7 propionyl → hydroxyl 17 → 1 -1.6

35ºC). Data from (Matesanz *et al.* 2008).

a 500-fold increment over the paclitaxel affinity.

exhibit very low resistance indexes.

27 → 26 -1.3 38 → 39 -2.8 T → 21 -4.2

C → 17 -0.7 21 → D -3.2

13 → 19 0.2 14 → 20 0


Site Modification Compounds ΔΔG Average C2 benzoyl → benzylether T → 25 13.2 +13.0 ± 0.2

benzoyl → benzylsulphur T → 27 13.6 +15.9 ± 2.3

benzoyl → thiobenzoyl T → 23 19.6 +15.9 ± 3.8

benzamide → 3-Cl-benzamide 42 → 44 5.3

benzoyl → 3 methyl- 3 butenoyl 1 → 3 4.9

benzoyl → 3 methyl- butanoyl 1 → 10 6.3

benzoyl → 3-methoxy-benzoyl 1 → 5 -6.2 -7.2 ± 0.6

benzoyl → 3-Cl-benzoyl 1 → 6 -3.1

benzoyl → 3-I-benzoyl 1 → 30 -3.3

benzoyl → 3-methyl-benzoyl 1 → 8 0

benzoyl → 3-hydroxy-benzoyl 18 → 37 9.2

benzoyl → 2,4-di-F-benzoyl 1 → 28 2.7

benzoyl → 6-carboxy-pyran-2-one 1 → 41 8.1 C13 paclitaxel → cephalomannine T → C 1.9 +2.0 ± 0.2

benzoyl → 2-thienoyl 1 → 31 4.1

paclitaxel → docetaxel 23 → 22 -1.7 -3.2 ± 0.9

25 → 24 -6.2

benzoyl → benzamide 21 → 42 19.2

benzoyl → benzylamine T → 38 18.6 +20.1 ± 1.5

benzoyl → 3-azido-benzoyl 1 → 4 -8 -11.2 ± 1.3

21 → 26 18.1

21 → 22 12.1

benzamide → 3-methoxy-benzamide 42 → 43 -3.4

benzoyl → 3 methyl- 2 butenoyl 1 → 2 6.2

benzoyl → 2(E)-butenoyl 1 → 9 7.3

benzoyl → 2-debenzoyl-1,2-carbonate C → 16 5.8

 T → 11 -8.3 C → 13 -8.1 18 → 19 -6.3

benzoyl → 3-Br-benzoyl 1 → 34 -2.3

benzoyl → 3-ciano-benzoyl 1 → 7 0.6

benzoyl → 3-hydroxymethyl-benzoyl 1 → 36 7.2

3-Cl-benzoyl → 2,4-di-Cl-benzoyl 6 → 29 4.8

3-methoxy-benzoyl → 2,5-di-methoxy-benzoyl 5 → 35 4.6

benzoyl → 3-thienoyl 1 → 32 1.8

 11 → 13 1.9 12 → 14 1.6 15 → 17 2.4

21 → 24 12.8

21 → 39 21.6

T → 12 -13.9 C → 14 -12.2 18 → 20 -10.6


Table 2. Incremental binding energies of taxane analogs to microtubules. (ΔΔG in kJ/mol at 35ºC). Data from (Matesanz *et al.* 2008).

In this way, it is possible to select the most favourable substituents at the positions studied and design optimized taxanes. According to the data obtained, the optimal taxane should have the docetaxel side chain at C13, a 3-N3-benzoyl at C2, a propionyl at C10, and a hydroxyl at C7. From compound 1 with a binding energy of -39.4 kJ/mol, the modifications selected would increase the binding affinity in -5.6 kJ/mol from the change of the cephalomannine side chain at C13 to the docetaxel one, -11.2 kJ/mol from the introduction of 3-N3-benzoyl instead of benzoyl at C2, -1.6 kJ/mol from the substitution of a propionyl at C7 with a hydroxyl, and -0.9 kJ/mol from the change of a hydroxyl at C10 to a propionyl. Thus, this optimal taxane would have a predicted ΔG at 35ºC of -58.7 kJ/mol. This molecule was synthesized (compound 40) and its binding affinity measured using the epothilone-B displacement method and the value obtained is in good corespondence with the predicted one: Ka = 6.28±0.15 x 109 M-1; ΔG = -57.7±0.1 kJ/mol (Matesanz *et al.* 2008). This value means a 500-fold increment over the paclitaxel affinity.

It is also possible to check the influence of the modifications on the cytotoxic activity determining the IC50 of each compound in the human ovarian carcinoma cells A2780 and their MDR counterparts (A2780AD). The plots of log IC50 versus log Ka (figure 4) indicate that, as in the case of epothilones, both magnitudes are related, and the binding affinity acts as a good predictor of citotoxicity. In this type of MDR cells the high-affinity drugs are circa 100-fold more cytotoxic than the clinically used taxanes (paclitaxel and docetaxel) and exhibit very low resistance indexes.

The plot of log resistance index against log Ka shows a bell-shaped curve (figure 5). Resistance index present a maximum for taxanes with similar affinities for microtubules and P-glycoprotein, then rapidly decreases when the affinity for microtubules either increases or decreases. To find an explanation for this behaviour we should note that the intracellular free concentration of the high-affinity compounds will be low. To be pumped out by Pglycoprotein ligands must first bind it, so ligand outflow will decrease with lower free ligand concentrations (discussed in (Matesanz *et al.* 2008)). In the case of the low-affinity drugs, the concentrations needed to exert their citotoxicity are so high that the pump gets saturated and cannot effectively reduced the intracellular free ligand concentration.

Thermodynamics as a Tool for the Optimization of Drug Binding 783

We found a correlation between binding affinities of paclitaxel-like MSA to microtubules and their citotoxicities in tumoral cells both MDR and non-resistant. The results with taxanes further validate the binding affinity approach as a tool to be used in drug optimization as it was previously discuss for the case of epothilones. Moreover, from the thermodynamic data we could design novel high-affinity taxanes with the ability to overcome resistance in P-glycoprotein overexpressing cells. Anyway, there is a limit concentration below which MSA are not able to kill cells (discussed in (Matesanz *et al.* 2008)), the highest-affinity compounds studied have no dramatically better citotoxicities than paclitaxel or docetaxel have. Thus, the goal is not to find the drug with the highest cytotoxicity possible but rather to find one able to overcome resistances. The study of taxanes indicates that increased drug affinity could be an improvement in this direction. The extreme example of that come from the covalent binding of cyclostreptin (Buey *et al.* 2007)

(that might be consider as infinite affinity) having a resistance index close to one.

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cytotoxicity." Chem Biol 12(12): 1269-1279.

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methods." Methods Mol Med 137: 245-260.

taxoid binding sites." Nat Chem Biol 3(2): 117-125.

stabilization, and cytotoxicity." Chem Biol 11(2): 225-236.

However, in the case of chemically diverse paclitaxel-like MSA, the inhibition of cell proliferation correlates better with enthalpy change than with binding constants (Buey *et al.* 2005) suggesting that favourable enthalpic contributions to the binding are important to improve drug activity as it has been shown for statins and HIV protease inhibitors (Freire

Alberts, B., D. Bray, J. Lewis, M. Raff, K. Roberts&J. D. Watson, Eds. (1994). Molecular

Benesi, H. A.&J. H. Hildebrand (1949). "A Spectrophotometric Investigation of the

Buey, R. M., I. Barasoain, E. Jackson, A. Meyer, P. Giannakakou, I. Paterson, S. Mooberry, J.

Buey, R. M., E. Calvo, I. Barasoain, O. Pineda, M. C. Edler, R. Matesanz, G. Cerezo, C. D.

Buey, R. M., J. F. Diaz, J. M. Andreu, A. O'Brate, P. Giannakakou, K. C. Nicolaou, P. K.

Connors, K. A., Ed. (1987). Binding Constants: The Measurement of Molecular Complex

Connors, K. A.&S. Mecozzi, Eds. (2010). Thermodynamics of Pharmaceutical Systems. An Introduction to Theory and Applications. new york, wiley-intersciences. Diaz, J. F.&R. M. Buey (2007). "Characterizing ligand-microtubule binding by competition

Interaction of Iodine with Aromatic Hydrocarbons." journal of the american

M. Andreu&J. F. Diaz (2005). "Microtubule interactions with chemically diverse stabilizing agents: thermodynamics of binding to the paclitaxel site predicts

Vanderwal, B. W. Day, E. J. Sorensen, J. A. Lopez, J. M. Andreu, E. Hamel&J. F. Diaz (2007). "Cyclostreptin binds covalently to microtubule pores and lumenal

Sasmal, A. Ritzen&K. Namoto (2004). "Interaction of epothilone analogs with the paclitaxel binding site: relationship between binding affinity, microtubule

**6. Conclusion** 

2008).

**7. References** 

Fig. 4. Dependence of the IC50 of taxane analogs against A2780 non-resistant cells (black circles, solid line) and A2780AD resistant cells (white circles, dashed line) on their Ka to microtubules. Data from (Matesanz *et al.* 2008).

Fig. 5. Dependence of the resistance index of the A2780AD MDR cells on the Ka of the taxanes to microtubules. Data from (Yang et al. 2007; Matesanz et al. 2008).
