3.1.Synthesis of the functionalized DMQA:

  Figure IV-3: Chemical structure of the desired molecules shown in racemic form

As already said, the correct selection of the structural motifs for the “click” reaction is essential and the introduction of the azide and alkyne functionalities could be performed on the periphery of either of the reactants. The azide could thus be attached to the cation with the alkyne on the biotin or vice versa.

Since we had already successfully achieved the first strategy with the two-carbon azido side chain-containing DMQA 1m (chapter II, § II-2.5.), formed from the corresponding alcohol 1c in good yield (85%), we maintained this strategy and undertook the formation of the longer five-carbon side chain. Also, the biotin propargylic ester was already described¹⁴ in the literature which was a definite advantage to our selected route. The details on the preparation of all these new dyes are presented below.

IV-3.1.Synthesis of the functionalized DMQA:

First, we turned our attention to the preparation of the azido containing side chains. They were prepared according to described procedures^15,16,17 from commercially available compounds. In the case of the fully carbonated side chain, we started from the 1,5-dibromopentane 21a which reacted at 80 °C with NaN3 in DMF to give 1,5-diazidopentane 22a in excellent yield (97%). The Staudinger reduction of 22a provided after 24 h the expected 5-azidopentan-1-amine 23a in 96 %       

14 Siegwart, D. J.; Oh, J. K.; Gao, H.; Bencherif, S. A.; Perineau, F.; Bohaty, A. K.; Hollinger, J. O.; Matyjaszewski, K. Macromol. Chem. Phys. 2008, 209, 2179-2193.

88 | P a g e  yield. For the synthesis of 22b where an oxygen atom replaced the central methylene group in the previous case, the same conditions were applied from the corresponding dibromide 21b and the desired motifs were isolated in good yields (89% for 22b and 60% for 23b) (Scheme IV-2).

These reactions were so straightforward and the procedures highly scalable that these amines were obtained in larger quantities (up to 15 g) without any noticeable effect on the yields.

Scheme IV-2: Synthetic procedure to obtain the new primary amines 23a-b

The 1,5-dibromo-3-oxapentane 21b was not commercially available so we needed to synthesize it¹⁵ along with two other precursors following literature procedures, starting from the very cheap diethylene glycol 24. Peripheral substitution of the two OH groups was performed either by mesylation with methanesulfonyl chloride and Et3N in Et2O at 0 °C¹⁶ or by tosylation with 2 equivalents of p-toluenesulfonyl chloride in the presence of powdered KOH at 0 °C (Scheme IV-3).¹⁷To form 21b, the dimesylated derivative 24a was heated to reflux in acetone with excess of LiBr for 20 h. The heterogeneous medium obtained was then filtered on a celite/silica pad (1:3) and washed with hexanes as eluent, dried with Na2SO4, filtered again and evaporated. The desired 1,5-dibromo-3-oxapentane 21b was then isolated as a light yellow oil in 60% yield (Scheme IV-3).

15 Bérubé, G.; Rabouin, D.; Perron, V.; N'Zemba, B.; Gaudreault, R.-C.; Parent, S.; Asselin, É. Steroids 2006, 71, 911-921.

16 Sun, X.; Hai, L.; Wu, Y.; Hu, H.-Y.; Zhang, Z.-R. J. Drug Target. 2005, 13, 121-128.

17 Bonger, K. M.; van den Berg, R. J. B. H. N.; Heitman, L. H.; Ijzerman, A. P.; Oosterom, J.; Timmers, C. M.;

Overkleeft, H. S.; van der Marel, G. A. Bioorg. Med. Chem. 2007, 15, 4841-4856.

Scheme IV-3: Synthesis of three precursors of 22b

However, we realized at that stage that the reactivity of these azido side chains with the cationic precursor 6 was essentially unknown and a successful coupling was probable but not guaranteed.

As a backup solution, we considered that it would be safer to prepare a side chain that ought to react readily with 6 and which could, on the helicene itself, be transformed later to contain the azido group.¹⁸ This was done by mono-functionalizing diethylene glycol 24 to leave a single free OH group (that will be later transformed). The results are summarized in scheme IV-4.

This time, with only one equivalent of MsCl in THF the addition was carefully done at -5 °C after deprotonation with Et3N. Then the medium was left overnight (almost 14 h) at 25 °C before undergoing the nucleophilic substitution reaction with NaN3 at 82 °C. Due to the regioselectivity issue, the reactions were left longer than the ones in scheme IV-3 and required purification by chromatography whereas previously, compounds would be immediately used after extraction.

Except for the first step (45%) where the regioselectivity was an issue, excellent yields were overall obtained (92-99%) as reported in the literature.¹⁹

18 Only the 2-(2-aminoethoxy)ethanol 27b was synthesized since 5-aminopentan-1-ol 27a was commercially available as a 50% m/m water solution.

19 Lebeau, L.; Oudet, P.; Mioskowski, C. Helvetica Chimica Acta 1991, 74, 1697-1706.

90 | P a g e  Scheme IV-4: Synthetic procedure of the 2-(2-aminoethoxy)ethanol 27b

With the amines 23a-b and 27a-b now in hand, several new structures were afforded by reaction with the (DMP)3C⁺BF4- 6, giving at room temperature acridinium salts 3.

Scheme IV-5: Synthesis of acridinium derivatives 3q-t

It is worth mentioning that, having these amines in hand, care was taken to try to introduce the

“expensive” azido side chains first, as the acridinium formation requires 2.5 equivalents of amines, whereas in the second step the quinacridinium synthesis needs 25 equivalents. These derivatives were formed in excellent yields (73-96%) as orange needle-like crystals, following the exact same conditions detailed previously.²⁰ Their purification was done by precipitation in a 0.2 M aqueous KPF6 solution. Increasing the reaction temperature to 90 °C, unsymmetrical DMQA were obtained by reaction of the mentioned acridinium derivatives with a different amine.

Initially, not only n-propyl but also methyl residues were chosen as second side chains of the unsymmetrical helicenes, since the dramatic effect of the methyl group(s) on the “resolution”

procedure was not known at the time (Chapter II, §II-3.6). All the derivatives were obtained in       

20 Laursen, B. W.; Krebs, F. C. Chem. Eur. J. 2001, 7, 1773-1783

excellent purity and yields (80-87%) by selective precipitation in aqueous KPF6 solution followed by several recrystallisations from CH2Cl2/(Et2O or pentane).

  Scheme IV-6: Synthetic procedure of the new unsymmetrical azide derivatives 1u-x

Cation R R1 yield

1u (CH2)5N3 Me 87%

1v (CH2)5N3 Pr 80%

1w (CH2)2O(CH2)2N3 Me 87%

1x (CH2)2O(CH2)2N3 Pr 80%

Table IV-1: Yields of the new unsymmetrical azide derivatives 1u-x

As discussed earlier, we also tried to introduce the side chains twice. For that purpose, we reacted 6 with 25 equivalents of the amines 23a-b and 27a-b at 90 °C for 4 h to form symmetrical derivatives in only one step. All these reactions proceeded smoothly, forming the desired products in good to excellent yields (60-85%).

  Scheme IV-7: Synthetic procedure of the new symmetrical DMQA derivatives 1q-t

92 | P a g e 

Cation R yield

1q (CH2)5N3 80%

1r (CH2)2O(CH2)2N3 85%

1s (CH2)5OH 70%

1t (CH2)2O(CH2)2OH 60%^a

a unoptimised data

Table IV-2: Yields of the new symmetrical DMQA derivatives 1q-t

The purification of 1q, 1s and 1t was performed as previously described, by precipitation in 100 mL of a 0.2M solution of KPF6(aq) followed by a thorough wash with water. All the compounds were then further purified by dissolving in CH2Cl2 and adding an excess of Et2O and/or pentane to afford a dark green precipitate in good yields (60-80%). However, in the case of DMQA 1r, the purification procedure was different. The excess of amine and NMP was distilled from the crude reaction mixture followed by silica gel column chromatography to remove unreacted material with CH2Cl2/MeOH 95:5 as eluent. The isolated fraction was evaporated and dried on the vacuum line but the material appeared viscuous and did not give a solid precipitate as in the previous cases. The ¹H-NMR spectrum showed no presence of solvent responsible for such a behavior and an entirely clean compound in 85% yield. It seems that the presence of the oxygen is as expected, affecting its general aspect and increasing its water solubility. Indeed, this derivative is water soluble even in the presence of the hydrophobic PF6 counterion.

In any case, compound 1t could be transformed into 1r using the traditional sequence of mesylation (MsCl, Et3N, CH2Cl2, 25 °C, 20 h) and nucleophilic substitution (NaN3, DMF, 25°C, 24 h) in 85% overall yield.²¹ Overall, it was found that the direct addition of the amine 23b was easier to perform.