Synthesis and structure investigation of stabilized aromatic oligoamides and their interaction with g quadruplex structures 5

The binding selectivity of pentamer 10a is similar to Se2SAP which was reported to prefer the mixed parallel/antiparallel hybrid structure.1 The result suggests that our circular pentame

Intramolecular quadruplexes have been intensively investigated due to their biological relevance For example, intramolecular quadruplex formation in the promoter region of some oncogenes seems to play a vital role in regulating transcription of the corresponding gene.4,5 Among all reported intramolecular quadruplexes, human telomere DNA is of particular interest.6 Telomere DNAs, located at the end of chromosomes, are known to contain repeating single-strand sequences of (TTAGGG)n folding into a quadruplex structure.7 The formation of

G-quadruplexes has been shown to inhibit the activity of telomerase (an enzyme overexpressed in 84-90% of cancer cells), which catalyses the addition of telomeric DNA repeats onto the single-stranded 3’ end, contributing to the immortalization of human tumor cells.8-10

The topologies of telomere G-quadruplex in different salts have been extensively studies In Na+ solution, the 22nt sequence d[AGGG(TTAGGG)3] was proven to form

a basket-type structure which has one diagonal and two lateral TTA loops in Na+solution.11 In the presence of K+, the telomere DNA adopts a propeller-type parallel-stranded G-quadruplex structure that contains no loop structure by a solid state study.9,12 However, the topology of telomere DNA in K+ solution was controversial and many scientists have made efforts in elucidating it.13-16 It was generally suggested that telomere DNA adopts a mixed-parallel/antiparallel qudruplex stucture.17-20

Figure 5.1 Human telomeric DNA of different conformations:18 (A) Antiparallel basket-type quadruplex in a Na + solution determined by NMR, (B) Parallel propeller-type quadruplex in K + crystal determined by X-ray, (C) Mixed-parallel/antiparallel quadruplex in a K + solution proposed based on

CD and NMR

As mentioned, G-quadruplexs are stabilized with either potassium or sodium ions Since Zahler and co-workers demonstrated in 1991 the telomerase inhibitory

behaviors of potassium-stabilized G-quadruplex structures, G-quadruplex DNA has emerged as an attractive target for designing telomerase inhibitors, leading to the development of new anticancer drugs.21-23 Recently, there is a growing interest in developing ligands that stabilize quadruplex DNA structures and so disrupt the interactions between quadruplex DNA and telomerase, causing telomerase to lose its function.41-43

Among all the reported G-quadruplex stabilizers, most of them are based on cyclic

π-delocalized systems owing to the cyclic nature of G-tetrad, a repeating unit composed of four guanines that forms the G-quadruplex structure Telomestain24 is the first natural telomerase inhibitor of high potency due to its ability to stabilize the G-qudruplex structure It appears to interact preferably with basket-type

intramolecular G-quadruplexes rather than intermolecular G-qudruplexes However, this macrocyclic compound tends to occupy the whole G-quartet with little selectivity over different intromolecular structures Heterocyclic compounds such as porphyines,25,26 porphyrazines27 and cyclopyyroles28,29 have also been demonstrated

to display high binding affinities for G-quadruplex Among the most popular macrocyclic ligands are oligoamides which showed unique G-quadruplex selectivity.30,31 For example, oxazole-based peptide32 which contains three stereo amine side chains demonstrated high selectivity toward c-kit over human telomeric

quadruplex Recently, Masayuki Tera, et al, also developed the macrocyclic hexazole

binders selective for telo23 DNA sequence.33

In the study of G-quadruplex structure, effective assays are required for studying the G-quadruplex formation and for determining the interaction between ligands and G-quadruplexes Titration experiments analyzed by spectroscopic methods such as fluorescence,34 circular dichroism35 (CD) and ultraviolet (UV)36 can provide significant information on ligand-quadruplex interactions CD was used mostly to identify G-quadruplex structures, especially to distinguish parallel from antiparallel structures It also serves as an efficient method to measure the melting temperature of oligonucleotides Calorimetric techniques such as isothermal titration calorimetry (ITC) can also provide thermodynamics information on ligand-quadruplex interactions.37 However, those analytical methods always require a large amount of oligonucleotides and are time-consuming Therefore, gel-shift assays have been extensively used, including polyacrylamide gel electrophoresis38 (PAGE) and

polymerase chain reaction (PCR) stop assays.39 PCR stop assays could selectively detect G-quadruplexes ligands by using only trace amounts (pmole) of oligonucleotides

Although the topologies of telomere G-quadruplexs have been recently elucidated

in a good detail, few compounds have been reported as selective binders to telomere G-quadruplexes with different structures In this study, we developed a new generation of aromatic oligoamides for stabilizing telomeric G-quadruplex structures These stabilizers demonstrated better binding affinity to mixed-parallel/antiparallel over anti-parallel basket-type G-quadruplexes PCR stop assay has been extensively adopted herein because of its high sensitivity and selectivity We also confirmed the switching oligoamides from macrocylic to helical structures resulted in loss of G-quadruplex binding affinity and selectivity

5.2 Result and discussion

Table 5.1 Sequences of oligomers (primers) used in this study

ODN1 5’-GGGTTAGGGTTAGGGTTAGGGT-3’ Telomeric DNA forming mixed

-parallel/antiparallel G-quadruplex in K +

Primer1 5’-TCTCTGTCACACCCTAAC-3’ Partial complementary sequence of ODN1,ODN2

and ODN3 in PCR stop assay Primer2 5’-TCTCTGTCACACTCTAAC-3’ Partial complementary sequence of ODN4 in

PCR stop assay

Scheme 5.1 Cyclic pentamers recognizing G-quadruplexes

Scheme5 2 Synthetic route for 9a, 10a, 10b, 11a

HN O HN

O

NH O

HN O HN

O HN

O HN

O H

NH O

O HN

O HN

O H

NH O

O HN

O HN

O NH

NH O

O H

OMe MeO MeO OMe 9a

O N O HN

O HN

O NH

NH O

OH

O Me MeO

HO

OMe 10a

O N O HN

O HN

O NH

NH O

OH

O Me MeO MeO

O NH

NH O

O HN

Scheme 5.3 Synthetic route for 9b

O 2 N OBn COOH H 2 N

O OMe O

OC 8 H 17

5f 6e

OMe O O N O

O

NO 2

O

O OMe

O MeO Bn

MeO

OC8H17

HN O HN O

NO 2

O MeO

NH O

O NH O

NH

O

N HN O

O

O O

O NH O

NH

O N HN O

O

O O O O

5.2.1 Binding Selectivity of Oligoamides toward Telemeric G-quadruplexes

The ligands’ ability to stabilize a quadruplex structure was primarily evaluated by PCR stop assay The detection principle is based on the fact that the binding of ligands with G-quadruplex structures could inhibit the action of the DNA polymerase.41 The single-stranded DNA could be induced into a G-quadruplex structure that blocks hybridization with a complementary strand overlapping the last G repeat in the

presence of aromatic oligoamides As a result, the DNA extension with Taq polymerse

is inhibited, causing a reduction on the final double-stranded PCR product

For all the Oligonucleotides (ODNs), PCR stop assay was performed in the presence of different concentrations of aromatic oligoamides A typical PAGE for

PCR stop assay on circular pentamer 10a is shown in Figure 5.3 The synthesis of

double-stranded PCR product was inhibited by oligoamides in a dose-dependent manner IC50 was used to represent the concentration of oligoamides required to

achieve 50% inhibition of the reaction Oligoamide 10a was investigated at different

concentrations ranging from 0.5 to 16 μM With the increasing concentration, PCR product decreased correspondingly PCR products can be hardly detected when the

concentration of 10a reached 8 μM or higher This micro-molar inhibition capability

of our circular pentamer was almost the same as TMPyP4 (1.9 μM) and telomestatin (3 μM) to Pu22mu with G-quadruplex structure39

c)

Figure 5.3 Effect of 10a on the double-stranded PCR product using ODN-1 as the PCR template (a)

PAGE analysis of the inhibiting effect in 50 mM KCl, (b) PAGE analysis of inhibiting effect in 50 mM

NaCl, (c) Comparison of inhibiting effect by 10a in 50 mM KCl, 50 mM NaCl, and without salts

A control experiment was carried out with an oligonuclotide (ODN-4) that contains one G to A mutation in every guanine tandem repeat (5’-GAGTTAGAGTTAGAGTTAGAGT-3’) and no inhibition was observed even at a very high concentration of 20 μM (Figure 5.4)

Figure 5.4 Effect of 10a on the double-stranded PCR product of ODN-4 in 50 mM KCl

Interestingly, pentamer 10a demonstrated distinct inhibitory behaviors in K+

solution compared to Na+ solution At lower concentrations (< 4 μM), the intensity of the PCR product decreased more slowly in Na+ solution than in K+ solution The IC50

of 10a is found to be 2.68 μM in 50 mM K+ solution and 6μM in 50 mM Na+ solution, respectively The difference may be attributed to the variation of G-quadruplex

structures in different salt solutions It can be deduced that pentamer 10a was a better

stabilizer for hybrid G-quadruplex in K+ solution than the basket structure in Na+

solution The binding selectivity of pentamer 10a is similar to Se2SAP which was reported to prefer the mixed parallel/antiparallel hybrid structure.1 The result suggests

that our circular pentamer 10a may use the same binding mechanism as Se2SAP that requires external loops for ionic interactions with the phosphate contained in these loops.38

5.2.2 Stability of Telemeric G-quadruplex

It was found that, the inhibition still occurred in PCR stop assay without adding any

salts This may be due to the presence of potassium chloride (KCl) in Taq polymerase

buffer Some interesting results could be obtained by comparing the IC50 of 10a with

and without 50 mM KCl or sodium chloride (NaCl) The IC50 of 10a was 2.98 μM

without adding additional salts, slightly higher than 2.68 μM in 50 mM KCl This result suggests that higher KCl concentration causes a decrease in IC50 value Moreover, compared with that in 50 mM NaCl solution, IC50 of 10a without

additional salts decreased from 6 μM to 2.98 μM, indicating that the addition of sodium salt into the buffer produces more PCR product This may be attributed to the fact that the anti-parallel G-qudruplex stabilized by Na+ solution is less stable than the mixed-parallel/antiparallel G-quadruplex in K+ To support this assumption, the variation of the Taq polymerase activity in different salts was tested by performing

PCR stop assay in 50 mM KCl or NaCl solution without 10a It was found that the

quantity of PCR products were equal in 50 mM KCl and 50 mM NaCl, indicating that

the polymerase activity showed no difference in K+ and Na+ solution

Circular dichroism (CD) spectroscopy (Figure 5.4) was used to examine the stability of G-quadruplex with different structures The CD spectrum of the d[AGGG(TTAGGG)3] (ODN-1) in the presence of 100 mM Na+ had a 295 nm positive band and a 265nm negative band (Figure 5.4a, red), indicative of a typical anti-parallel structure In contrast, the CD spectrum of ODN-1 in 100 mM K+ ion showed a stronger positive band at 290nm, with a weak negative peak near 255 and

235 nm (Figure 5.4a, black), demonstrating that the DNA adopts a mixed parallel/antiparallel G-quadruplex structure as reported.2 It was found that, by adding

K+ ion into the solution which already has Na+ ion inside, the CD spectrum changed from the red line into the green line, demonstrating a structural change from the anti-parallel into mixed-parallel/antiparallel G-quadruplex structure This phenomenon suggests that the mixed-parallel/antiparallel G-quadruplex is more stable than the antiparallel structure The thermal stabilities were also examined by CD melting experiments According to the sigmoidal fitting using Origin 7.0, the melting temperature of the anti-parallel G-quadruplex formed by ODN-1 in 100 mM Na+ was about 61 oC and that of the mixed-parallel/antiparallel G-quadruplex was about 71 oC

in 100 mM K+ The CD results, therefore, suggest that the stability of mixed-parallel/antiparallel G-quadruplex is higher than that of anti-parallel structure

a) b)

Figure 5.5 CD spectra and melting points of ODN-1 in different salt solution (a) CD Spectra of

ODN-1 in the presence of 100 mM KCl, 100 mM NaCl and 50 mM KCl/50 mM NaCl mixture, (b) melting points of ODN-1 G-quadruplex in 100 mM KCl and 100 mM NaCl, respectively

Figure 5.6 Melting study of G-quadruplex ODN-5 in the phosphate buffer (pH = 8.0, 10 mM) in the

presence of 100 mM KCl

The ability of 10a to stabilize G-quadruplex was also investigated by CD melting experiment This study demonstrates that 10a stabilizes the DNA quadruplex structure

as evidenced by an increase in T m from 63.03 oC to 65.73 oC (Figure 5.6) This also

indicate that 10a showed stabilization of G-quadruplex, However, compared to other literature33, the change of melting temperature is too slight to be powerful evidence

5.2.3 The Unique Binding Affinity of Macrocylic Oligoamides

Scheme 5.2 Acyclic oligomers for the study of G-quadruplexes ligands

10 20 30 40 50 60 70 80 90 100 -2

-1 0 1 2 3

Temperature ( 0C )

Na K

NO 2 N O H O

O O O

OC 8 H 1 7

N O

O

NO 2 N

O H O

O O O

OCH(CH 3 ) 2

OC 8 H 1 7

N H O

O N N

O H O

O O

O

H OO N O

O 2 N O

OCH(CH 3 ) 2

5d

10 20 30 40 50 60 70 80 90 100 -1.0

-0.5 0.0 0.5 1.0 1.5 2.0 2.5

To determine whether the good planarity in the circular aromatic pentamers is the prerequisite for G-quadruplex stabilization, other acyclic aromatic oligomers were also studied (Figure 5.7) PCR stop assay demonstrated that the acyclic oligomers had little effect on the inhibition of PCR products As reported in our previous studies,

acyclic oligoamides were shown to adopt the crescent (2c and 3f) or helical (5d)

conformations, while cyclic pentamers folded into an almost planar disc arrangement reinforced by strong intramolecular H-bonding forces The PCR stop assay implies that good planarity found in macrocycles is crucial for stabilizing G-qudruplexes

a) b)

c)

Figure 5.7 Effect of oligoamides on the PCR product using ODN-1 as the PCR template in 50 mM

KCl: (a) 2c, (b) 3f and (c) 5d

5.2.4 The Tunable Binding Affinity of Macrocyclic Oligoamides

One remarkable characteristic of this new class of aromatic oligoamides lies in the modular nature that makes them amenable to chemical modification, which allows a fine-tuning of binding affinities and selectivities toward quadruplexes Crystal structure in our previous research revealed that the modification of macrocycle has

little effects on its planarity Based on this, a series of circular pentamers (9b, 9a, 10b, 11a, 1) were designed by either adding the exterior side chains or replacing the

interior methoxyl groups with hydroxyl groups The binding affinities of these aromatic oligomers have also been studied For example, Figure 5.8 showed the effect

of 10b on the formation of double-stranded DNA by PCR

a) b)

Figure 5.8 Effect of 10b and 9a on the double-stranded PCR product by using ODN-1 as the template

in different salt solutions: (a) 10b and (b) 9a

a) b)

Figure 5.9 Comparison of effects of different oligomers 9a, 10a and 10b on the double-stranded PCR

product by using ODN-1 as the template in different salt solutions: (a) 50 mM KCl, (b) 50 mM NaCl

The PCR stop assay using different macrocyclic compounds showed significant differential inhibition extents caused by the subtle difference in structure Figure 5.9a

showed the inhibitory behaviors of 9a, 10a and 10b on ODN-1 in K+ solution where ODN-1 adopts a mixed-parallel/antiparallel G-quadruplex structure Circular

pentamer 10a was found to show best inhibitory ability on PCR product, suggesting it

to be the best stabilizer of mixed-parallel/antiparallel G-quadruplex among the

circular pentamers studied For 9a and 10b, IC50 values were both larger than 4 μM

and 10b demonstrated better inhibitory ability than 9a However, in Na+ solution

where ODN-1 adopts an anti-parallel G-quadruplex structure, 9a showed the best

inhibitory ability (Figure 5.9b) The PCR stop assay on these compounds provided a clear evidence of structure-dependent inhibitory effects on G-quadruplex-forming

DNAs The results on 9a, 10a and 10b further suggest that circular pentamers containing more interior hydroxyl group may have higher binding affinity since 10a has one more interior hydroxyl group than 9a

Figure 5.10 Influence of 1 on the synthesis of double-stranded PCR product by using ODN-1 as the

PCR template in 50 mM KCl

To verify this assumption, PCR stop assays in the presence of 1 and 11a were

carried out in K+ solution From Figure 5.10, the intensity of the PCR product remains

unchanged even when the concentration of 1 was as high as 20 μM, indicating 1 was a

weak stabilizer of G-quadruplex region found in ODN-1 Figure 5.11 illustrated the

inhibitory behavior of 11a, which has three interior hydroxyl groups At a

concentration lower than 1 μM, PCR product disappeared both in K+ and Na+ solution

To determine the IC50 of 11a, further PCR stop assay was carried out at very low

concentrations The IC50 of 11a in K+ solution was estimated to be 0.75 μM The above results suggested that by replacing more interior methoxy groups with hydroxyl groups, the binding affinity toward DNA G-quadruplex structures was greatly

improved On the other hand, the quantity of PCR product of 11a in Na+ solution also

decreased sharply compared to 10a, indicating the decrease of the ligands’ binding

selectivity in company with the increase of affinity Therefore, the binding affinity

toward G-quadruplex region in ODN-1 is 11a >10a >10b >9a >1

Figure 5.11 Influence of 11a on the double-stranded PCR product by using ODN-1 as the PCR

template: (a) 50 mM KCl, (b) 50 mM NaCl and (c) comparison of inhibitory effect of 11a in 50 mM

-ss

The almost planar disc arrangement of nearly perfect 5-fold symmetry of the macrocycle presents a 5-fold attachment point that allows the facile attachment of functionalized pendant arms at its periphery In order to investigate the effect of

exterior side chain, the inhibition activities of 9b and 9a were compared According to Figure 5.12, 9a showed a more smoothly curve than 9b, which indicated that the

presence of hydrophobic side chains on the pentamers’ exterior surface has positive effects on G-quadruplex stabilization This could be explained by the increase of solubility between the macrocycles and DNA with the involving of hydrophobic side chains

a) b)

c)

Figure 5.12 Comparison of the effect of 9b and 9a on the double-stranded PCR product by using

ODN-1 as the PCR template: (a) PAGE analysis of inhibitory effect of 9b in 50 mM KCl, (b) PAGE analysis of inhibitory effect of 9a in 50 mM KCl and (c) comparison of the effect of 9b and 9a on the

double-stranded PCR product

5.2.5 Poor Binding Affinity of Macrocyclic Oligoamides to Other G-qudruplexs

To further validate the selectivity of circular pentamers to certain G-quadruplex

ss

structure, other ODNs with the ability to form G-qudruplex structures were also

studied As shown in Figure 5.13, 10a demonstrated the same inhibitation activities to

ODN-2 as to ODN-1 since they adopted the same G-qudruplex structures in both K+

and Na+ solutions (Figure 5.13d and Figure 5.5a) According to the CD measurement,

the melting temperature of the anti-parallel G-quadruplex is about 61 oC and that of

the mixed-parallel/antiparallel G-quadruplex is about 66 oC

a) b)

c) d)

Figure 5.13 Effect of 10a on the double-stranded PCR product by using ODN-2 as the PCR template:

(a) in 50 mM KCl, (b) in 50 mM NaCl, (c) Comparison of inhibitory effect of 10a in KCl and NaCl, (d)

CD Spectra of ODN-2 in the presence of 100 mM KCl or NaCl

The inhibitory behavior varied when it comes to telemetric G-quadruplex ODN-3,

which adopts a parallel G-quadruplex structure in both K+ and Na+ solutions (Figure

5.14a) The intensity of the PCR product decreased slowly with the increasing

concentration of 10a Even for 11a, the best stabilizer of the

mixed-parallel/antiparallel G-quadruplex, little inhibitory effect can be seen This

indicated the preference of circular pentamers to recognize the

220 240 260 280 300 320 -5

-4 -3 -2 -1 0 1 2 3 4

ss

Lane

ss

-mixed-parallel/antiparallel over parallel G-quadruplex structures

Figure 5.14 Influence of 10a and 11a on PCR product by using ODN-3 as the PCR template: (a) CD

spectra of ODN-1 in the presence of 100 mM KCl and NaCl, (b) PAGE analysis of inhibitory effect of 10a in 50 mM KCl, (c) PAGE analysis of inhibitory effect of 10a in 50 mM NaCl, (d) PAGE analysis

of inhibitory effect of 11a in 50 mM KCl and (e) PAGE analysis of inhibitory effect of 11a in 50 mM NaCl (f) Comparison of inhibitory effect of 10a in KCl and NaCl (g) Comparison of inhibitory effect

of 11a in KCl and NaCl,

+ 8 2 +

ds ss

5.3 Conclusion

The interactions between aromatic oligoamides and G-quadruplex-forming oligonucleotides were studied Circular pentamers demonstrated the inhibition of the double-stranded PCR products while acyclic oligomers showed little effect Furthermore, our circular pentamers showed better binding affinities to mixed-parallel/antiparallel over anti-parallel telomere G-quadruplex By slightly modifying the chemical structure, the binding affinity or selectivity to different G-quadruplexs could be varied However, the solubility of these aromatic oligoamides

is still a limiting factor that constrains the research scope Further research can be focus on introducing positively charged substituents which have been used very often

in quadruplex-targeting ligands to enable the ligand to efficiently interact with the grooves and loops of quadruplex DNA and the negatively charged backbone phosphates In our design, it could also help to increase the ligands solubility in aqueous solution Therefore, modification of some selected pentamers with positively charged substituents such as NH3+ and NR3+ will be our next step

5.4 Experimental Section

Taq Polymerase Stop Assay:

Oligonucleotides and the corresponding complementary sequence 3’-CAATCCCACACTGTCTCT-5’ were used The chain-extension reaction was performed in PCR buffer containing 0.2 mM dNTP, 2.5 U Taq polymerase, 7.5 pmol oligonucleotides, 50 mM KCl or NaCl, and cyclic pentamers of various concentrations The mixture was incubated in a thermocycler with the following

cycling conditions: 94 oC for 30 s, 47 oC for 30 s and 72 oC for 30 s PCR products were resolved on 20% native polyacrylamides gels in 1 x TBE buffer and stained with Sybe Gold The intensity of Sybe Gold was measured and quantified

CD Spectroscopy:

CD spectra were recorded on a Jasco-810 spectropolarimer (Jasco, Easton, MD) using a quartz cell of 1-mm optical path length and an instrument scanning speed of 100nm/min with a response time 1s, and over a wavelength range of 220-320 nm Telomeric DNA (10 mM) was dissolved in Tris-HCl buffer (10 mM, pH 7.5) with KCl/NaCl (100 mM), and the solution was heated to 90 oC for 5 min, then slowly cooled to 25 oC The CD spectra are representative of three averaged scans taken at 25

oC The CD melting curves of the Telemetric G-quadruplex were determined from

measurements of the CD intensity at 295 nm The heating rate was 2.0 oC /min

Materials:

All oligomers/primers used in this study were purchased from Sigma (Singapore), and their sequences were listed in Table 5.1 Taq DNA polymerase kit was purchased from Biolabs, Singapore All the derivatives were synthesized as follows Stock solutions of all the drerivatives (2 mM) were made using DMF

Compound 5h:

Acid 5e (3.00 g, 15.2 mmol) was dissolved in CH2Cl2 (30 mL) to which 4-methylmorpholine, NMM (2.2 mL, 17.9 mmol) and ethyl chloroformate (1.96 mL, 16.4 mmol) was added at 0 oC The reaction mixture was stirred for at least 15 min

then a solution of amine 5g (2.70 g, 14.9 mmol) dissolved in CH2Cl2 (30 mL) was

added The reaction mixture was allowed to stir continuously overnight at room temperature The reaction mixture was washed with 1M KHSO4(100 mL), followed

by saturated NaHCO3 (100 mL) and saturated NaCl (100 mL) Drying over Na2SO4

and removal of solvent in vacuo gave the crude product, which was recrystallized

from methanol to give the pure product 5h as a white solid Yield: 3.49 g, 71%. 1H NMR (300 MHz, CDCl3) δ 10.37 (s, 1H), 8.80 (dd, 1H, J = 8.2, 1.6 Hz), 8.45 (dd, 1H,

J = 7.9, 1.8 Hz), 7.99 (dd, 1H, J = 8.1, 1.8 Hz), 7.63 (dd, 1H, J = 7.9, 1.6 Hz), 7.41 (t,

1H, J = 8.1 Hz), 7.24 (t, 1H, J = 8.1 Hz), 4.10 (s, 3H), 3.95 (s, 6H) 13C NMR (125 MHz, CDCl3) δ 165.9, 161.3, 151.5, 149.4, 136.5, 132.7, 128.8, 126.6, 124.6, 123.5,

64.5, 62.6 MS-ESI: calculated for [M]+ (C17H16N2O7): m/z 360.2, found: m/z 360.1

Compound 5i:

Acid 5f (5.71 g, 20.9 mmol) was dissolved in CH2Cl2 (75 mL) to which 4-methylmorpholine, NMM (2.7 mL, 24.7 mmol) and ethyl chloroformate (2.4 mL, 24.7 mmol) was added at 0 oC The reaction mixture was stirred for at least 15 min

then a solution of amine 5g (3.5 g, 19.0 mmol) dissolved in CH2Cl2 (75 mL) was added The reaction mixture was allowed to stir continuously overnight at room temperature The reaction mixture was washed with 1M KHSO4(200 mL), followed

by saturated NaHCO3 (100 mL) and saturated NaCl (100 mL) Drying over Na2SO4

and removal of solvent in vacuo gave the crude product, which was recrystallized

from methanol to give the pure product 5i as a white solid Yield: 5.35 g, 64%.1H NMR (500 MHz, CDCl3) δ 9.80 (s, 1H), 8.69 (dd, 1H, J = 8.2, 1.6 Hz), 8.29 (dd, 1H,

J = 7.9, 1.8 Hz), 7.98 (dd, 1H, J = 8.0, 1.8 Hz), 7.58 (dd, 1H, J = 7.9, 1.6 Hz), 7.40 (t,

1H, J = 7.9 Hz), 7.27 – 7.24 (m, 3H), 7.19 (dd, 1H , J = 15.3, 7.4 Hz), 5.17 (s, 2H),

3.92 (s, 3H), 3.56 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 166.0, 161.8, 149.3, 149.2, 144.9, 135.9, 133.9, 132.3, 131.1, 129.4, 129.1, 128.5, 128.3, 126.4, 124.9, 124.3, 123.8, 123.3, 79.8, 62.1, 52.2; MS-ESI: calculated for [M+Na]+ (C23H20N2O7+Na):

m/z 459.1, found: m/z 459.1

Compound 5j:

Compound 5h (3.46 g, 10.0 mmol) was reduced by catalytic hydrogenation in THF

(50 mL) at 40 oC, using Pd-C (0.35 g, 10%) as the catalyst for 3 hours The reaction

mixture was then filtered and the solvent removed in vacuo to give the pure amine

product Yield: 3.16 g, qualitative Acid 5e (2.06 g, 10.5 mmol) was dissolved in

CH2Cl2 (50 mL) to which NMM (1.35mL, 12.4mmol) and ethyl chloroformate (1.08

mL, 11.3 mmol) was added at 0 oC The reaction mixture was stirred for at least 15

min after which a solution of amine product (3.1 g, 9.04 mmol) dissolved in CH2Cl2

(50 mL) was added The reaction mixture was allowed to stir continuously overnight

at room temperature The reaction mixture was washed with 1M KHSO4 (100 mL), followed by saturated NaHCO3 (100 mL) and saturated NaCl (100 mL) Drying over

Na2SO4 and removal of solvent in vacuo gave the crude product, which was

recrystallized from methanol to give the pure product 5j as a white solid Yield: 3.64 g,

82% 1H NMR (300 MHz, CDCl3) δ 10.23 (s, 1H), 10.22 (s, 1H), 8.85 (dd, 1H, J = 8.2, 1.5 Hz), 8.78 (dd, 1H, J = 8.2, 1.6 Hz), 8.47 (dd,, 1H, J = 7.9, 1.8 Hz), 8.03 (dd, 1H, J = 8.0, 1.8 Hz), 7.92 (dd, 1H, J = 7.9, 1.7 Hz), 7.62 (dd, 1H, J = 7.9, 1.7 Hz),

7.48-7.34 (m, 2H), 7.23-7.20 (m, 1H), 4.14 (s, 3H), 3.98 (s, 3H), 3.96 (s, H); 13C