# Arylbiamidines: synthesis and structural studies en route to anticancer applications

Oleksandr Grytsai, Leticia Christina Pires Gonçalves, Rostyslav Bardovskyi, Nedra Hamouda-Tekaya, Stéphane Rocchi, Cyril Ronco, Rachid Benhida

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Oleksandr Grytsai, Leticia Christina Pires Gonçalves, Rostyslav Bardovskyi, Nedra Hamouda-Tekaya, Stéphane Rocchi, et al.. Arylbiamidines: synthesis and structural studies en route to anticancer applications. New Journal of Chemistry, 2021, 45 (27), pp.11893-11897. 10.1039/D1NJ01943K. hal-03870875

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# Arylbiamidines: synthesis and structural studies en route to anticancer applications†

Oleksandr Grytsai,‡ª Leticia Christina Pires Gonçalves, ID‡ª Rostyslav Bardovskyi,‡ª Nedra Hamouda-Tekaya,ᵇ Stéphane Rocchi, IDᵇ Cyril Ronco ID*ª and Rachid Benhida*ac

Biamidines are a unique and poorly studied class of nitrogenous compounds prone to tautomerization and H-bonding. Four series of heteroaryl diarylbiamidines were synthesized and the antimelanoma activity and physicochemical properties of the resulting 37 new compounds were evaluated. The dimethylthiazolyl 3-bromophenyl biamidine derivative B6 inhibits the growth of six different melanoma cell lines, having higher activity than the positive control drug, the B-RAF inhibitor PLX4032. This study introduces diarylbiamidines as promising frameworks for drug discovery.

Biamidine (N'-carbamimidoylimidamide) is a functional group in which two amidine moieties are linked by a C-N bond (Fig. 1). Examination of the structural features of biamidines suggests that these compounds share the strong base character, water solubility, tautomerism capability, and coordination properties of guanidines and biguanides.¹⁻³

Guanidine is one of the most versatile functional groups in chemistry. Compounds containing this function are ubiquitous in bioactive molecules with diverse biological properties.⁴,⁵ Similarly, biguanides are known for their broad biological activity and diverse therapeutic applications, e.g. antidiabetic, antimalaria, antiseptic and anticancer activity.⁶,⁷ Although the features of guanidine and biguanides, which contain three and five nitrogen atoms, are well known, knowledge on the four nitrogen-containing biamidines is lacking. Notably, details on the synthesis and properties of biamidines are scarce in the literature.⁸⁻¹¹ Here, we report a new class of compounds, the diarylbiamidines, a general and efficient synthetic protocol

leading to different derivatives and we present their in vitro antimelanoma activity. Besides, we demonstrate their peculiar tautomerism and H-bonding features, relevant for drug discovery.

According to the Lipinski's rule of five, increasing the lipophilicity of biamidines may enhance their bioactivity. Hence, we designed derivatives in which a thiazolyl substituent is linked to the N-atom of the biamidine group and a second aryl group is attached to the C-atom (Fig. 2a). This functionalization is expected to increase the lipophilicity of the resulting biamidine and to affect its basicity and H-bonding capability. Thiazolyl group is found in a variety of drugs, such as antitumor, antifungal, and anti-inflammatory therapeutics,¹²⁻¹⁵ and its electronic and steric properties can be tuned by adding substituents at positions 4 and 5 of the heterocyclic ring.¹⁴ The aromatic substituent R² enables the fine tuning of the physicochemical properties of the derivatives, and their effect on the biological activity.

No general approach is available towards the synthesis of arylbiamidines, which were mainly obtained as side-products.⁸⁻¹⁰ Therefore, a new synthetic method was developed to obtain the diarylbiamidines. For that, the guanidine intermediates 6–9 were first prepared in >70% yields by thiazole-Hantzsch synthesis from amidinothiourea 5 and α-haloketones 1–4 (Fig. 2a, see ESI†). Next, these 2-thiazolylguanidines were coupled to arylnitriles in DMF using sodium hydride as base. Full conversion was obtained in 2–6 h at 25 °C, resulting in the desired arylbiamidines within

<figure><img src="image_2.png" /><figcaption>**Fig. 1** Comparison of the chemical structures of the biamidine (4× N) function to the amidine (2× N), guanidine (3× N), and biguanide (5× N) functions.</figcaption></figure>

<footnote>ª Institut de Chimie de Nice CRNS UMR7272, Université Côte d'Azur, Nice, France.
E-mail: cyril.ronco@univ-cotedazur.fr, rachid.benhida@univ-cotedazur.fr</footnote>

<footnote>ᵇCentre Méditerranéen de Médecine Moléculaire (C3M), INSERM, U1065, Université Côte d'Azur, Nice, France</footnote>

<footnote>ᶜDepartment of Chemical and Biochemical Sciences, Green Process Engineering, CBS, Mohamed VI Polytechnic University, UM6P, 43150 Ben Guerir, Morocco</footnote>

<footnote>†Electronic supplementary information (ESI) available. CCDC 2042663. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/d1nj01943k</footnote>

<footnote>‡These authors contributed equally.</footnote>
<figure><img src="image_3.png" /><figcaption>Fig. 2 Arylbiamidines: (a) Design and synthetic route for the obtention of arylbiamidines containing the biamidine core and two aryl groups. (b) Scope of arylbiamidines synthesized in this work.</figcaption></figure>

up to 95% isolated yield. The use of other bases, Brønsted and Lewis acids, heating, and other solvents was investigated but did not result in the desired product (see ESI†). By using this approach, we synthesized 37 novel arylbiamidines within four different thiazole-substituted groups, namely groups A (4-Methiazolyl), B (4,5-diMe-thiazolyl), C (4-Ph-thiazolyl) and D (4-(m-NO₂)Ph-thiazolyl) (Fig. 2b). All compounds are air-stable as solids and in solution (DMSO, THF, DCM, PB pH 7, DMEN-PS + FBS cell media, see ESI†).

Experimental evidence on the solubility of the arylbiamidines in apolar solvents such as dichloromethane and diethyl ether indicated higher lipophilicity than expected. To rationalize these observations and to evaluate the effect of the addition of the thiazolyl group on the physicochemical properties of biamidines, we performed predictions of pK<sub>aH</sub>, log D and log S of different biamidines, in comparison to guanidines and biguanides (Table 1, see ESI†). Biguanides are known to be slightly less basic than guanidines (less than 1 log unit); however, biamidines are substantially weaker bases with a difference of ≈2pK<sub>aH</sub> log units. Substitution with a phenyl leads to a decrease in basicity in all classes of compounds, with phenylbiamidine showing lower basicity and higher polarity than phenylguanidine and o-tolylbiguanide. Similarly, the introduction of the thiazolyl group leads to a significant reduction in basicity (Table 1). As expected,

<table><caption>Table 1 Determination of pK<sub>aH</sub>, log D and log S of biamidines in comparison to guanidines and biguanides</caption><thead><tr><th>Compound</th><th>s<sub>s</sub>pK<sub>aH</sub> (exp.)<sup>a</sup></th><th>pK<sub>aH</sub> (calc.)<sup>b</sup></th><th>log D (pH 7)<sup>b</sup></th><th>log S (pH 7)<sup>b</sup></th></tr></thead><tbody><tr><td>Guanidine</td><td>13.09 ± 0.11</td><td>11.40</td><td>-3.65</td><td>2.42</td></tr><tr><td>Phenyl</td><td>n.d.</td><td>9.70</td><td>-1.45</td><td>0.37</td></tr><tr><td>diMe-thiazolyl 7</td><td>7.30 ± 0.02</td><td>7.30</td><td>-0.7</td><td>0.25</td></tr><tr><td>Biamidine</td><td>n.d.</td><td>9.00</td><td>-5.57</td><td>1.63</td></tr><tr><td>R<sup>1</sup>-Phenyl</td><td>n.d.</td><td>8.30</td><td>-3.22</td><td>0.00</td></tr><tr><td>R<sup>1</sup>,R<sup>2</sup>-Diphenyl</td><td>n.d.</td><td>6.85</td><td>0.45</td><td>-1.32</td></tr><tr><td>diMe-thiazolyl B1</td><td>7.00 ± 0.02</td><td>6.40</td><td>0.73</td><td>-1.92</td></tr><tr><td>Biguanide</td><td>n.d.</td><td>11.00</td><td>-4.17</td><td>2.15</td></tr><tr><td>o-Tolyl</td><td>10.94 ± 0.02</td><td>10.20</td><td>-1.58</td><td>0.00</td></tr><tr><td>Diphenyl</td><td>n.d.</td><td>6.77</td><td>0.49</td><td>-1.54</td></tr></tbody></table>

<sup>a</sup> pK<sub>aH</sub> experimental (exp.) values were determined with two different potentiometric procedures (see ESI); pK<sub>aH</sub> (couples amidine/amidine-H<sup>+</sup>); the s<sub>s</sub>pK<sub>aH</sub> value in the pH scale is relative to each acetonitrile-water mixture. <sup>b</sup> Calculated values were predicted with the ChemAxon online calculator (see ESI). log D values refer to log P estimations at pH 7 and log S = log (solubility measured in mol L<sup>-1</sup>). n.d.: not determined.

the substitution of the thiazole ring does not change significantly the pK<sub>aH</sub> (<0.5 log) of the biamidine, but it has a progressive effect on the log D (Me < diMe < m-NO₂Ph < Ph, see ESI†). Finally, we correlated these predictions with the experimental determination of pK<sub>aH</sub> for compounds B1 and 7, with guanidine
and o-tolylbiguanide as reference compounds (Table 1). The experimental values were in accordance with the predictions, with measured $pK_{aH}$ (B1) = 7.00 ± 0.02.

The effect of the thiazolyl moiety on the structure of the biamidine core was studied by crystal structure analysis of derivative B1 (Fig. 3a). Surprisingly, the biamidine core proved to appear as the tautomer with two $NH_2$ groups, contrary to the representation in previous reports.⁸⁻¹¹ This is supported by infrared analysis, which shows a strong absorption band at $1652 cm^{-1}$ (see ESI†), assigned to the out-of-phase deformation planar mode of the $NH_2$ group ($1660 cm^{-1}$ for guanidine $NH_2$),¹⁶ and correlated by ¹H-¹H NOESY NMR experiments (see ESI†). Moreover, the molecular conformation of the studied compounds is stabilized by two strong intramolecular hydrogen bonds N3-H3A···N5 of 205 pm and N1-H1A···N4 of 199 pm (Fig. 3a).

The ¹H NMR shifts corresponding to the $NH_2$ are represented as four characteristic peaks largely split over the range around 5-12 ppm. This pattern involving two sharp and two broad peaks corresponding to individual $NH_2$ groups is found in all thiazolyl biamidine derivatives and represent a clear signature of this family of compounds. Dynamic NMR experiments at different temperatures in DMSO-d6 were performed using B1 as model to compare the strength of the H-bonding in solution (Fig. 3b). The two sharp peaks appearing more downfield correspond to the unshielded $H_{a,b}$ atoms with $H_a$ involved in intramolecular H-bonding (10.5 ppm). ¹H NMR signals of $H_{c,d}$ coalesce at 62 °C while the other $NH_2$ displays split signals, demonstrating the faster rotation speed of C8-N3 bond and lower energy barrier for the corresponding H-bond. At 86 °C, we observe the merge of both N-H peaks into a single one for each $NH_2$ group, implying the fast exchange of both hydrogen atoms in H-bonds. Yet, additional experiments were carried out in $CDCl_3/CD_3OD$ mixtures at 25 °C to show the faster deuterium exchange for the weaker H-bond. At fixed time (10 min), the addition of 5% of protic deuterated solvent promotes a deuterium exchange with the most accessible protons due to faster rotation and exchange

of $NH_2H_d$ group, rather than with the protons involved in stronger hydrogen bond N1-H1A···N4, as demonstrated by the disappearance of the corresponding signals from the spectrum (Fig. 3c).

In summary, the thiazolyl biamidine derivatives show strong double intramolecular H-bonding that freezes their conformation and explains their high solubility in apolar solvents. Besides, 2-pyridyl derivatives (A10, B7, C7, D6) seem to show an additional H-bond between the pyridyl N and $H_{ab}$, as supported by sharper NMR $H_{ab}$ peaks and higher apolarity than the respective 3- and 4-pyridyl analogues (see ESI†). More generally, these features help reducing the polarity and flexibility of the biamidine function and can improve the physicochemical characteristics of biamidines towards druglikeness criteria.

We evaluated the biological activity of the 37 biamidine derivatives on the viability of metastatic melanoma cells, a cancer known for its high resistance to the current targeted therapies.¹⁷ The rationale for this model relies on the fact that antimelanoma activity has been recently described for biguanide drugs.¹⁸⁻²⁰ Nevertheless, the high polarity and flexibility related to these compounds lead to reduced efficacy.²¹ Thus, our arylbiamidines can be considered as biguanide analogues, with reduced polarity and flexibility. Hence, the cell viability of all compounds (at 5 µM and 10 µM) was evaluated on A375 human aggressive melanoma cells after treatment for 24 h and 48 h (see ESI†). Overall, dimethylthiazolyl derivatives from group B showed the highest activity among all groups (<50% at 10 µM after 48 h, Fig. 4a), with compound B6 being the most active at any of the conditions tested (see ESI†), although compound B7 showed similar results but only at 10 µM after 48 h. Comparison to PLX-4032, the current first line treatment for cutaneous melanoma patients carrying B-RAFV600 mutations,²²,²³ demonstrated a clearly higher activity at 2.5 and 5 µM concentrations (Fig. 4b, see ESI†), confirming B6 efficacy. Moreover, we identified apoptosis as type of cell death occurring after B6 treatment, as assessed by the cleavage of PARP protein (Fig. 4c).

<figure><img src="image_4.png" /><figcaption>Fig. 3 Tautomerism and fine structure of B1. (a) View of the structure of the crystals of B1. Hydrogen atoms appear in white, carbon in grey, nitrogen in blue and sulphur in yellow. (b) Effect of the temperature on the ¹H NMR shifts of $NH_2$. (c) Effect of deuterium exchange with increasing amounts of $CD_3OD$.</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>Fig. 4 Biological evaluation of diarylbiamidines. (a) Cell viability of A375 melanoma cells relative to DMSO upon treatment of 10 µM of the arylbiamidine derivatives of groups A, B, C and D at 48 h. Values are shown in the ESI.† Blue trace indicates the EC₅₀. N = 3 ± SD. Mean is the average between all values within each group. (b) Cell viability assessed with crystal violet staining of A375 melanoma cells incubated for 24 h with DMSO (negative control), PLX-4032 or B6. (c) Western blotting analysis of apoptosis related protein PARP from A375 melanoma cells incubated for 24 h with DMSO, B6 or PLX-4032 (actin is used as loading control). (d) Selected *in vitro* tumour growth data (<20%) for compound B6. Data obtained from the NCI's *in vitro* disease-oriented human tumour cells screening against 60-cancer cell lines. Results of the complete study are presented in the ESI.†</figcaption></figure>

Compound B6 was subsequently evaluated on a panel of 60 other cancer cell lines through the DTP program of the National Cancer Institute.²⁴ This screening led to cancer cell growth lower than 50% in 28 cancer cell lines (see ESI†) and lower than 20% in 6 cancer cell lines (Fig. 4d). The best results were obtained against CNS cancer and melanoma, specifically on the SF-295 and MDA-MB-435 cell lines, respectively.

Based on these results, we performed an *in silico* study to assess the druglikeness of compound **B6**.²⁵ As a result, it showed compatibility with the main druglike criteria (Lipinski, Ghose, Veber, Egan, Muegge, see ESI†). Taken together, these results strongly suggest anticancerous effects of compound **B6** on melanoma cells and, overall, confirm the pertinence of our designed molecules of group B as promising anticancer scaffold to be optimized.

In conclusion, we designed a novel family of biamidine derivatives containing one thiazolyl and one aryl moiety. A simple, robust and high-yielding synthetic route was developed for the synthesis of 37 derivatives. The study of the fine molecular structure of the dimethylthiazolyl biamidine compound **B1** by X-ray crystal analysis, IR and NMR experiments revealed the existence of a single tautomer with a strong double intramolecular H-bonding network, which deeply affects the polarity, solubility and stability properties of this family of compounds. Moreover, we determined their weak basicity ($pK_{aH} \approx 7$). The biological evaluation of all compounds on a metastatic melanoma model showed a structure activity relationship favourable for dimethylthiazolyl derivatives (group B). The most active compound **B6** demonstrated superior activity compared to reference standard drug PLX-4032, and activity towards other cancer cell lines. Overall, the peculiar structure of these arylbiamidine derivatives provides attractive physicochemical properties that might influence their biological activity. These results support the development of new arylbiamidine analogues as anticancer compounds and highlight the applicability of this non-exploited functional group in drug discovery.

## Conflicts of interest

There are no conflicts to declare.

## Acknowledgements

This work was supported by the Cancéropôle PACA and the SATT-Sud Est (EmA grant). Université Côte d'Azur (IDEX UCA-JEDI Innovation Program), CNRS, ANR and INCA are also acknowledged for additional funding. The authors thank Dr Michel Giorgi for X-ray crystallography and Prof. Erick L. Bastos for constructive discussions.

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