Synthesis and structure investigation of stabilized aromatic oligoamides and their interaction with g quadruplex structures 4

To testify this speculation, ab initio calculation of the designed circular aromatic pentamer 1 was performed with Gaussian 98 at the B3LYP/6-31G level.. The 1H NMR spectrum of 1 reveal

Chapter 4

Synthesis of Folded Circular Aromatic Pentamers with Tunable

Interior Structure

4.1 Introduction

Macrocyclic structures with persistent shape have unique structural features, special physical properties, and chemical behavior that differ from their acyclic counterparts.1,2 In particular, persistent-shaped aromatic macrocycles have attracted wide attention due to their defined structures and functions Usually, the shape persistency and rigidity of these macrocyclic motifs are induced by covalent force,3 intrinsic conformational bias of the backbone4 and built-in hydrogen-bonds.5,6,7 Particularly, multiple-center H-bonding has become a top strategy for designing tailor-made macrocyclic aromatic foldmers due to its robustness and predictablity.6,7 Owning to their specific structural features, these aromatic macrocycle have enabled extensive applications in chemistry and biology For example, their well-defined cavities may serve as species binder8 or ion transporter across cell membrane.9 Aligning rigid macrocycles into columnar assemblies should lead to organic nanotube.10 However, until now, few synthetic macrocyclic systems allow systematic fine-tuning of interior properties while maintain overall topographic feature Herein, a series of 5-fold symmetric aromatic circular oligoamides with tunable interior functional groups were designed and synthesized

4.2 Results and Discussion

4.2.1 Design and Computational Molecular Modeling of Circular Pentamer 1

In chapters 2 and 3, we showed that the oligoamide backbone requires five repeating units per helical turn in forming a helical structure Accordingly, the end-to-tail cyclization of a crescent acyclic pentamer into a circular structure might not impose much angle strain on the backbone and may result in a planar conformation To testify this speculation, ab initio calculation of the designed circular

aromatic pentamer 1 was performed with Gaussian 98 at the B3LYP/6-31G level

a) b)

Figure 4.1 Structure predicted by ab intio calculation of circular pentamer 1 (a) top view and (b) side

view.

As shown in Figure 4.1, circular pentamers adopts almost planar structure with an appreciable cavity size Similar to helical oligoamides, the most stable circular pentamer

prefers the up-down-up-down-up fashion in terms of orientation of five interior methyl

side chains

4.2.2 Synthesis of Circular Pentamer 1

A highly rigid and structurally well defined circular pentamer 1 was synthesized by

Dr Bo Qin.11 This pentamer was efficiently obtained by circlizing the acyclic

pentamer 5a using BOP as the amide coupling reagent

Scheme 4.1 Synthesis of circular pentamer 1a

N H O

O N N

O H O

O O

O

O N H O

O2N O

O N

O N

O N

O N

N O O

O O O O

H H H

1

a, b, c

5a

a

a) 10% Pd/C, 40˚C, THF; b) 1M KOH, MeOH; c) BOP, DIEA, CH 2 Cl 2, 61% (in total)

4.2.3 One-Dimensional 1 H NMR Study of Circular Pentamer 1

1

H NMR was firstly examined to confirm the identity of circular pentamer 1

(Firgure 4.2) The 1H NMR spectrum of 1 revealed five sets of proton signals

corresponding to the methoxy groups (4.09 ppm), aromatic (9.00 ppm, 8.02 ppm & 7.45 ppm), and amide protons (10.89 ppm) that are in excellent agreement with the

symmetrical structure of 1 In particular, the amide protons of 1 resonating at the very

low field (10.88-10.89 ppm) are a diagnostic indicator of the presence of strong H-bonding interactions, leading to the rigidification of the amide linkages and a crescent aromatic backbone

Figure 4.2 1H NMR spectrum (500 MHz, 298 K, CDCl 3) of pentamer 1 at 5 mM

4.2.4 Solid State Structure of Pentamer 1

Single crystal of 1, grown by Dr Qin Bo, was obtained by slow evaporation of 1 in

mixed solvents.11 Consistent to ab initio caculation, the molecules adopted an almost

flat disc arrangement (Firgure 4.3) All the five methoxy oxygen atoms and amide protons point inward and contribute to the formation of a continuous intramolecularly Hydrogen-bonded network (NH•••OMe = 1.9-2.4 Å) The size of the cavity is 2.85 Å

in radius After deducting a covalent radius of 1.4 Å for oxygen atom, the actual radius is 1.45 Å, which is almost the same as K+ (~1.4 Å) The geometrical feature of

circular pentamer 1 made it a potential candidate as cation-binding medium

a) b) c)

Figure 4.3 Crystal structure of pentamer 1 (a) top view with interior methoxy methyl groups omitted

for clarity of view, (b) top view and (c) side view both with methoxy methyl groups in CPK representations

4.2.5 Design of Circular Pentamer 8

According to the crystal structure of 1, the methyl groups form two hydrophobic caps above and below the pentameric plane These hydrophobic caps might prevent 1

from binding to metal cations, such as Na+ and K+ We hypothesised that the replacement of methoxy groups with hydroxyl groups should greatly reduce the steric

hindrance and hydrophobicity imparted by the interior methyl groups in 1

Furthermore, it has been well established that the fluoride atom can act as a good proton acceptor Considering the similarity of the F•••H-N to O•••H-N motif, replacing hydroxyl groups with fluorine may form a new rigid, well-established

conformation Finally, considering the bad solubility of oligoamides, the introduction

of hydrophobic side chains should enhance the solubility of the pentamer

4.2.6 Synthesis of Circular Pentamer 8

The synthetic details for oligoamides have been discussed in Chapter 2 Herein, we focus on discussing two types of new reactions 1) Protection of hydroxyl group with Benzyl group To introduce hydroxyl groups into pentamers’ interior, benzyl protecting group (Bn) was used for protecting hydroxyl groups in case the coupling between amine and phenol group Similar to alkylation, Benzyl protection was realized by reacting monomer with benzyl bromide and potassium carbonate in acetone Since benzyl bromide was hard to remove, no more than 1.1 equiv of the benzyl bromide was used Removal of benzyl protecting groups readily proceeded by catalytic hydrogenation, using Pd-C as the catalyst in MeOH/THF under one atmosphere of hydrogen gas This allows us to introduce three OH groups into the resulting pentamer molecule 2) Reduction of nitro group into amine by iron powder Since benzyl group could be easily moved by catalytic hydrogenation, method to reduce NO2 group using Pd/C could not be used Instead, reduction was carried out by using iron powder and glacial acetic acid, a highly efficient method for reduction of

NO2 into NH2

Scheme 4.2 Synthesis of circular pentamer 8

R

OH

COOCH3

O2N

R

OBn COOCH3

O2N

R

OBn COOH

O2N

1f, 8a, 8b: R=OC8H 17

1o, 8c, 8d: R=CH3

1f, 1o

H O

O

Bn NO2

O Bn

8e

N H O

F

NO2 N

O H O Bn

O Bn

8g

N H O

F N N

O H O Bn

O Bn

F

O2N

8h

N H O

F N N

O H O Bn

O Bn

F N H O

O 2 N O Bn

OC 8 H 17

OBn

COOCH3

O2N

8a

OC8H17

OC8H17

C8H17O

OC8H17

c, e

C8H17O

OC 8 H 17

C 8 H 17 O

OC 8 H 17

8i

c, f

c, g, h

O

N

O N

O N

O N

NH O O

O O

F F Bn Bn H H

H

H Bn

OC8H17

C8H17O

O N

O N

O N

O N

N O O

O O

F F H H H H

H

H H H

OC8H17

C8H17O

a

a) K 2 CO 3 / BnBr, DMF, 60 oC, 4 h, 88~90%; b) NaOH, MeOH/H 2 O, reflux, 2 h, 84~91%; c) Fe, AcOH/EtOH, reflux, 2h, 78%; d) ethyl carbonochloridate, 4-methylmorpholine, CH 2 Cl 2, 8b, overnight, 74%; e) 8f, SOCl2 , reflux, Pyridine/CH 2 Cl 2, 56%; f) 8d, SOCl2 , reflux, Pyridine/CH 2 Cl 2 , 72%; g) KOH, Dioxane/H 2 O, RT, overight; h) BOP, DIEA, CH 2 Cl 2 , 2h, 35%; i) H 2 , Pd/C, THF/MeOH, 40oC, 3h, 40%

To study the intramolecular H-bonds in 8, 1H NMR spectra was first examined As mentioned, amide protons typically exhibit a substantial downfield shift due to the formation of intramolecular H-bonds Surprisingly, only three amide protons resonate

at chemical shifts larger than 10 ppm in CDCl3 According to previous study, these signals should be the amides adjacent to interior hydroxyl group In comparison, two amide protons adjacent to fluorine element were less downfield (7~9 ppm) This experimental observation suggests that the three-centered F•••H-N hydrogen-bonding motif is weaker than O•••H-N hydrogen-bonding motif This may because the small radius of fluorine make it is hard to be a hydrogen acceptor as the distance of F•••H-N and O•••H-N in our design is fixed However, by addition of DMSO-d6 into CDCl3 (1:1), the two amide protons adjacent to fluorine also demonstrated chemical shift more than 10 ppm as a result of the H-bonding ability of DMSO

4.2.7 Crystal Structure of a Tetramer and Computational Molecular Modeling of

Circular Pentamer 8

To demonstrate the existence of intramolecular hydrogen-bonds that restrict the rotational freedom of the aryl-amide bonds to enforce a crescent structure, X-ray

crystallography of tetramer 8h was obtained by slow evaporation of 8h in mixed

solvents of 1:1 dichloromethane and methanol The X-ray results showed that the presence of the bulky benzyloxy group does not disrupt the crescent-shaped conformation In addition, the H-bond distances of F•••NH and OH•••NH are no more than 2.3 Å, indicating the formation of intramolecular hydrogen bonds

However, numerous attempts to obtain single crystal structure of pentamer 8 were

unsuccessful

Figure 4.4 X-ray crystal structure of tetramer 8h

Since our previous study showed that ab initio molecular calculation has

consistently allowed us to reliably predict the 3D topography of helical and circular

oligomers, modeling with the B3LYP/6-31G basis set of circular pentamer 8 was

performed The modeling result showed that the interior cavity dimension of cyclic

pentamer 8 is about 5.64 Å (Figure 4.5), suggesting that the replacement of interior

methoxy groups with hydroxyl or fluorine groups increasingly opened the interior

cavity Moreover, pentamer 8 still folds into a roughly planar disk arrangement akin to pentamer 1

a) b)

Figure 4.5 Structure predicted by ab intio calculation of circular pentamer 8 (a) top view and (b) side

view

4.3 Conclusion

X-ray diffraction results of 1 illustrated that the pentamer was almost planar with

intramolecular hydrogen-bonding between the amide protons and methoxy oxygen

atoms to rigidify the amide linkage A new generation of circular pentamer 8 was

successfully synthesized that folds based on intromolecular F•••N-H H-bonds The planar structure and the interior cavity of these macrocycles may give arise to their potential applications in chemical and biological settings

4.4 Experimental Section

Compound 1:

Compound 5a (0.442 g, 0.559 mmol) was reduced by catalytic hydrogenation in THF

(50 mL) at 50 oC, using Pd-C (0.75 g, 20%) as the catalyst for 3 hours The reaction

mixture was then filtered and the solvent removed in vacuo to give a brown liquid 1x

Yield: 0.425 g, quantitative Compound 1x (0.425 g, 0.558 mmol) was dissolved in

hot methanol (5 mL) to which 1M KOH (1.20 mL, 1.20 mmol) was added The mixture was heated under reflux for 2 hours and then quenched with water (20 mL) The aqueous layer was neutralized with 1M KHSO4 (1.2 mL) The precipitated crude

product 1y was collected by filtration Compound 1y (0.763 g, 1.0 mmol) and BOP

(0.88 g, 2.0 mmol) were dissolved in CH2Cl2 (3.2 ml) at 0 oC DIEA (0.5 ml, 3.0 mmol) was added and the reaction mixture was stirred continuously for 1 hr at 0 oC,

then stirred at room temperature for 2 hours Removal of solvent in vacuo gave the

crude product, which was purified by flash column chromatography on silica gel

using CH2Cl2/CH3CN (1:10) as the eluent to give a pure white product 1 Yield: 0.465

g, 62%; Decomposition in 340-345 oC; 1H NMR (500 MHz, CDCl3) δ 10.88 (s, 5H), 9.00 (d, 5H, J = 8.2, 1.5), 8.02 (d, 5H, J = 8.0, 1.5), 7.44 (t, 5H, J = 8.1), 4.09 (s, 15H).

13

C NMR (125 MHz, CDCl3) δ 162.3, 146.5, 132.9, 126.6, 126.2, 125.6, 124.3, 63.3

HRMS-EI: exact mass calculated for [M]+ (C40H35N5O10): m/z 745.2384, found: m/z

745.2387

Compound 8a:

Methyl 2-hydroxy-3-nitro-5-(octyloxy)benzoate (8.12 g, 25.0 mmol) was dissolved in anhydrous DMF (100 mL), to which anhydrous K2CO3 (14.00 g, 0.1 mol) and benzene bromide (3.1 mL, 26.0 mmol) was added The mixture was heated under reflux for 60 oC hours CH2Cl2 (200 mL) was then added and the reaction mixture was

filtered The solvent was removed in vacuo and the concentrate was dissolved in

CH2Cl2 (200 mL), washed with water (2 x 100 mL) and dried over anhydrous Na2SO4 Removal of CH2Cl2 gave the pure product 8a as a red liqid Yield: 9.40 g, 90% Yield:

1.76 g, 85% 1H NMR (500 MHz, CDCl3): δ 7.56 (d, 1H, J = 3.2 Hz), 7.47 (d, 2H, J = 6.9 Hz), 7.44 (d, 1H, J = 3.8 Hz), 5.11 (s, 2H), 3.99 (t, 2H, J = 6.3 Hz), 3.88 (s, 3H), 1.29 (m, 12H), 0.91 (t, 3H, J = 13.5 Hz) 13C NMR (75 MHz, CDCl3): δ 190.36,

164.85, 154.52, 145.97, 144.65, 136.04, 128.65, 128.57, 128.48, 128.42, 121.57, 114.06, 78.56, 69.22, 52.72, 50.74, 31.73, 29.19, 29.14, 28.91, 25.85, 22.60, 14.02 HRMS-ESI: calculated for [M+Na] + (C23H29NO6Na): m/z 438.1887 found: m/z

438.1868

Compound 8b:

8a (1.25 g, 3.0 mmol) was dissolved in hot MeOH (20 mL), to which 1N NaOH (6

mL, 6.0 mmol) was added The mixture was heated under reflux for 1 h and then quenched with water (20 mL) The aqueous layer was neutralized by addition of 1M HCl (8 mL) The precipitated crude product was collected by filtration, which was

recrystallized from MeOH to give a yellow solid 8b Yield: 0.90 g, 91% 1H NMR (500 MHz, CDCl3): δ 7.82 (d, 1H, J = 3.2 Hz), 7.60 (d, 1H, J = 3.2 Hz), 7.47 (m, 2H), 7.41 (m, 3H), 5.14 (s, 2H), 4.93 (t, 2H, J = 6.4 Hz), 1.81 (m, 3H), 1.46 (m, 3H), 1.30(m, 6H), 0.90 (t, 3H, J = 6.9 Hz) 13C NMR (75 MHz, DMSO-d6): δ 165.62,

154.01, 145.53, 144.01, 135.89, 129.12, 127.90, 121.11, 113.20, 68.68, 31.25, 28.72, 28.66, 28.45, 25.38, 22.12, 13.62 HRMS-ESI: calculated for [M] + (C23H29NO6): m/z 400.1887 found: m/z 400.1868

Compound 8c:

Methyl 2-hydroxy-3-nitro-5-(octyloxy)benzoate (1.05 g, 5.0 mmol) was dissolved in anhydrous DMF (20 mL), to which anhydrous K2CO3 (3.0 g, 21.7 mol) and benzene bromide (0.7 mL, 5.8 mmol) was added The mixture was heated under reflux for 60 o

C hours CH2Cl2 (50 mL) was then added and the reaction mixture was filtered The

solvent was removed in vacuo and the concentrate was dissolved in CH2Cl2 (50 mL), washed with water (2 x 50 mL) and dried over anhydrous Na2SO4 Removal of

CH2Cl2 gave the pure product 8c as a red liquid Yield: 1.32 g, 88% 1H NMR (300 MHz, CDCl3): δ 7.73 (d, 1H, J = 2.0 Hz), 2.2 (d, 1H, J = 2.2 Hz), 7.36 (m, 2H), 7.23

(m, 3H), 5.00 (s, 2H), 3.75 (s, 3H), 2.27 (s, 3H) 13C NMR (75 MHz, CDCl3): δ

164.86, 149.06, 145.36, 135.94, 135.85, 134.39, 128.57, 128.52, 128.34, 127.49, 78.33, 52.50, 20.35 MS-ESI: calculated for [M+Na]+ (C16H15NO5Na): m/z 324.0950, found: m/z 324.0952

Compound 8d:

8c (1.2 g, 4.0 mmol) was dissolved in hot MeOH (20 mL), to which 1N NaOH (8 mL,

8.0 mmol) was added The mixture was heated under reflux for 1 h and then quenched with water (20 mL) The aqueous layer was neutralized by addition of 1M HCl (10 mL) The precipitated crude product was collected by filtration, which was

recrystallized from MeOH to give a yellow solid 8d Yield: 0.97 g, 84%.1H NMR (500 MHz, CDCl3): δ 8.15 (d, 1H, J = 1.9 Hz), 7.91 (d, 1H, J = 1.9 Hz), 7.49 (m, 2H),

7.43 (m, 3H), 5.19 (s, 2H), 2.49 (s, 3H) 13C NMR (75 MHz, CDCl3): δ 165.88,

148.53, 145.04, 135.89, 135.74, 133.87, 128.37, 127.97, 77.71, 20.06 MS-ESI: calculated for [M-H]+ (C15H13NO5): m/z 286.0794 found: m/z 286.0742

Compound 8e:

8a (1.0 g, 2.4 mmol) was first dissolved in ethanol (20 mL), to which acetic acid (5.0

mL), and iron powder (0.6 g, 10.7 mmol) was added The reaction was stirred and heated under reflux for 2 hours The reaction mixture was then filtered and the filtrate

solvent was removed in vacuo The residue after solvent removal was dissolved in

CH2Cl2 (30 mL), washed with water (2 x 30 mL), and dried over anhydrous Na2SO4 Removal of CH2Cl2 solvent gave pure amine which was immediately used for the

next coupling Acid 8b (1.0 g, 2.5 mmol) was dissolved in CH2Cl2 (20 mL) to which