Design and structural analysis of aromatic inhibitors of type II dehydroquinase from mycobacterium tuberculosis

DOI: 10.1002/cmdc.201402298

Full Papers

Design and Structural Analysis of Aromatic Inhibitors of

Type II Dehydroquinase from Mycobacterium tuberculosis

Nigel I. Howard,^[a]Marcio V. B. Dias,^[b]Fabienne Peyrot,^[a]Liuhong Chen,^[a]Marco F. Schmidt,^[a]

Tom L. Blundell,^[b]and Chris Abell*^[a]

3-Dehydroquinase, the third enzyme in the shikimate pathway,

is a potential target for drugs against tuberculosis. Whilst

a number of potent inhibitors of the Mycobacterium tuberculo-

sis enzyme based on a 3-dehydroquinate core have been iden-

tified, they generally show little or no in vivo activity, and were

synthetically complex to prepare. This report describes studies

to develop tractable and drug-like aromatic analogues of the

most potent inhibitors. A range of carbon–carbon linked biaryl

analogues were prepared to investigate the effect of hydrogen

bond acceptor and donor patterns on inhibition. These exhibit-

ed inhibitory activity in the high-micromolar range. The addi-

tion of flexible linkers in the compounds led to the identifica-

tion of more potent 3-nitrobenzylgallate- and 5-aminoiso-

phthalate-based analogues.

Introduction

The shikimate pathway is the essential sequential seven-step

enzymatic pathway that converts erythrose-4-phosphate and

phosphoenol pyruvate into chorismate, the branch point in

the biosynthesis of the aromatic amino acids and other aro-

matic metabolites in plants, fungi, and bacteria.^[1–3]The lack of

this pathway in mammals makes it an attractive target for the

development of new antimicrobials.^[1,4]Dehydroquinase (3-de-

hydroquinate dehydratase, EC 4.2.1.10), the third enzyme in

the pathway, catalyzes the conversion of 3-dehydroquinate (1)

into 3-dehydroshikimate (2) (Scheme 1).

es are homododecameric enzymes that catalyze the anti-elimi-

nation of water via an enol intermediate (Scheme 1).^[6–9]These

are structurally and mechanistically very different from type I

dehydroquinases, which are dimeric and catalyze the syn-elimi-

nation of water via a Schiff base formed with a conserved

lysine residue.^[10,11]The type II enzyme has been identified in

several pathogenic bacteria, including Mycobacterium tubercu-

losis^[12,13]and Helicobacter pylori (gastritis, stomach ulcers),^[14]

that are responsible for enormous mortality and economic loss

across the world, especially in developing countries. Conse-

quently, this enzyme has become an important target for ra-

tional inhibitor design in the past few years.^[15–22]

There are two forms of the enzyme that catalyze the same

reaction, but by differing mechanisms.^[5]Type II dehydroquinas-

Early studies with substrate analogues 3 and 4

(Figure 1), that have K_ivalues of 600 and 30 mm, re-

spectively, against the Streptomyces coelicolor

enzyme,^[15]demonstrated that the flattening of the

six-membered ring in going from quinate to anhy-

droquinate was beneficial to binding. Elaboration of

the anhydroquinate core from the 3-position resulted

in potent (nanomolar) and selective inhibitors of

type II dehydroquinase, with K_ivalues against the

M. tuberculosis form of the enzyme of 940 nm for the

dicarboxylate 5,^[18]54 nm for the 3-nitrophenyl ana-

logue 6,^[17]250 nm for the 2-thienyl analogue 7,^[22]

490 nm for the biaryl analogue 8,^[20]11 mm for the

benzophenone analogue 9,^[20]and 140 nm for the al-

kenyl analogue 10,^[21](Figure 1). However, the use of these

compounds as antimicrobials is not straightforward. Their hy-

drophilicity and charge hinder bioavailability, making them un-

likely to pass though the M. tuberculosis cell wall efficiently.

Attempts to improve the bioavailability of such compounds

by use of propyl ester-based prodrugs shows promise.^[23]How-

ever, the most potent compounds involve multistep syntheses

from quinic acid, requiring several protection and deprotection

steps. The cost of goods suggests this may not be a way for-

Scheme 1. Catalytic mechanism of the type II 3-dehydroquinase from Mycobacterium

tuberculosis.

[a] Dr. N. I. Howard, Dr. F. Peyrot, Dr. L. Chen, Dr. M. F. Schmidt, Prof. C. Abell

Department of Chemistry, University of Cambridge

Lensfield Road, Cambridge CB2 1EW (UK)

E-mail: ca26@cam.ac.uk

[b] Dr. M. V. B. Dias,⁺Prof. T. L. Blundell

Department of Biochemistry, University of Cambridge

80 Tennis Court Road, Cambridge CB2 1GA (UK)

[⁺] Present address: Universidade de S¼o Paulo, Departamento de Microbiolo-

gia, Av. Prof. Lineu Prestes, 1374 – Ed. Biomꢀdicas II, Cidade Universitꢁria,

CEP 05508-900, S¼o Paulo (Brazil)

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greater freedom to adopt favora-

ble hydrogen bond interactions

rather than requiring the core

unit to be conformationally

ideal. The isophthalates differ in

that the C5 oxygen atom of 14

will act as a hydrogen bond ac-

ceptor upon alkylation, whilst

Figure 1. Examples of previously published 3-dehydroquinase inhibitors.^{[15,17,18,20–22]}

the amine of 17 will still act as

a hydrogen bond donor.

Citrazinate 15 and 6-hydroxy-

ward for antibiotics intended for use in developing countries.

In this study, we explored new avenues of inhibitor design by

investigating the impact of flattening the core anhydroquinate

ring further into aromatic structures. The use of simple aromat-

ic structures as a substitute for the complex shikimate ring in

the synthesis of potent inhibitors of 5-enolpyruvoylshikimate-

3-phosphate synthase, the sixth enzyme on the shikimate

pathway, has been previously demonstrated by Miller et al.^[24]

Studies on 5-deoxy- and 4,5-dideoxydehydroquinate have

suggested that the C5 hydroxy group of the natural substrate

forms key hydrogen bond interactions to the enzyme upon

binding, whereas attempts to define the role played by the C4

hydroxy were inconclusive.^[25]As a result, replacement of the

quinate core moiety with analogues based on gallate 11, pro-

tocatechuate 12, 3,5-dihydroxybenzoate 13, 5-hydroxyisoph-

thalate 14, citrazinate 15, 6-hydroxynicotinate 16, and 5-ami-

noisophthalate 17 were investigated in order to further study

the role that the patterns of hydrogen bond donors and ac-

ceptors have on binding and inhibition (Figure 2).

nicotinate 16 are structurally related compounds that can exist

as lactam/lactim tautomers which have reversible hydrogen

bond donor and acceptor arrangements at the 1- and 6-posi-

tions (Figure 3). They differ from the relative position of the

Figure 3. Tautomers of citrazinate 15 and 6-hydroxynicotinate 16. Lactim (I)

and lactam (II) (based on atom arrangement at the 1- and 6-positions).

carboxylate to the ring nitrogen, para in the case of citrazinate

15 and meta for 6-hydroxynicotinate 16. The lactam tautomer

(II) is expected to be the predominant species in aqueous solu-

tion, but it is possible that the compound can bind

as the lactim (I) if that hydrogen bond arrangement

were more favorable.

Results and Discussion

Synthesis

The synthesis of analogues incorporating a citrazinate

core moiety is shown in Scheme 2. Methyl 2-chloro-6-

methoxyisonicotinate 19 was prepared from citrazinic

acid 15 by heating with phosphorous oxychloride

and tetramethylammonium chloride,^[26]followed by

Figure 2. 3-Dehydroshikimate 2 and potential aromatic core mimetics 11–17.

methanol and sulfuric acid to give methyl 2,6-dichlor-

oisonicotinate 18 in 50% yield for the two steps. Dis-

The rationale for each series is as follows: analogues of gal-

late 11 with side chains attached at the C3-hydroxy position

have hydrogen bond donors at both the C4 and C5 positions,

the oxygen atom at C3 becomes a hydrogen bond acceptor,

and replacement of the C3 oxygen with an amine changes

that position to a hydrogen bond donor. Procatechuate 12

and 3,5-dihydroxybenzoate 13 analogues were chosen to

remove a hydrogen bond donor at the C4 and C5 positions, re-

spectively. Isophthalates 14 and 17 were chosen to introduce

a functional group at the C5 position, where its hydrogen

bonding oxygen atoms can move away from the planarity of

the core moiety, potentially allowing the carboxylate group

placement of one of the chlorines was achieved by heating

with sodium methoxide at 808C to give 19 in 52% yield. The

copper-catalyzed Ullmann reaction,^[27]between methyl 2-

chloro-6-methoxyisonicotinate 19 and 3-nitrobenzyl alcohol

generated the free acid 20d rather than the corresponding

methyl ester in a modest 7% yield. Presumably the ester was

cleaved during the aqueous workup, which may also explain

the low yield. The methyl ether of 20d was deprotected by

treatment with iodotrimethylsilane, formed in situ with chloro-

trimethylsilane and sodium iodide, to give the 3-nitrobenoxy

analogue 22d in 82% yield. The 3-nitrophenyl side chain was

attached by palladium-catalyzed Suzuki coupling^[28]between

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Scheme 2. Reagents and conditions: a) POCl₃, Me₄NCl, 1308C; b) MeOH, H₂SO₄, reflux; c) NaOMe, MeOH, reflux; d) 3-nitrobenzyl alcohol, Cu, CuI, K₂CO₃, pyri-

dine, 1008C; e) TMSCl, NaI, MeCN, 658C; f) 3-nitrophenylboronic acid, Pd(Ph₃)₄, Et₃N, DMF, 1008C; g) H₂, Pd/C, EtOAc, RT; h) HBr, AcOH, reflux. See Experimental

Section for reaction times and yields.

methyl 2-chloro-6-methoxyisonicotinate 19 and 3-nitrophenyl-

boronate to give compound 21a in 33% yield. Palladium-cata-

lyzed hydrogenation of 21a gave the 3-amino analogue 21b

in quantitative yield. Both 3-nitro and 3-amino analogues 21a

and 21b were deprotected in one step by heating at reflux in

hydrobromic acid and acetic acid to give 22a and 22b as the

hydrobromide salts in quantitative and 71% yields, respective-

ly.

hydrobromic acid in acetic acid at reflux to give 26a and 26b

in 94 and 71% yields, respectively.

Analogues based on a gallate core were synthesized as out-

lined in Scheme 4. Two of the three aromatic hydroxy groups

of the commercially available methyl gallate 27 were protected

as the diphenyl acetonide 28 by treatment with a,a-dichlorodi-

phenylmethane in 46% yield. Treatment of protected gallate

28 with 1-fluoro-3-nitrobenzene, trimethylsilyl diethylamine

and the strong phosphazene base tBu-P4 gave the diaryl ether

29c in 26% yield using the method of Ebisawa et al.^[30]The

benzylic side chains were at-

The synthesis of analogues incorporating a 6-hydroxynicoti-

nate core was carried out as shown in Scheme 3. Synthetic

tached by reaction of the pro-

tected gallate 28 with 3-nitro-

benzyl- or benzyl bromide to

give the intermediates 29d and

29g in 41 and 51% yields, re-

spectively. The methyl esters

29c, 29d, and 29g were depro-

Scheme 3. Reagents and conditions: a) Br₂, AcOH, 508C; b) PCl₅, POCl₃, reflux; c) MeOH, CH₂Cl₂, reflux; d) NaOMe,

MeOH, 1,4-dioxane, reflux; e) 3-nitrophenyl boronic acid, Pd(OAc)₂, PPh₃, Et₃N, DMF, 1008C; f) H₂, Pd/C, EtOAc, RT;

g) HBr, AcOH, reflux. See Experimental Section for reaction times and yields.

tected by treatment with aque-

ous base and worked up to give

the free acids 30c, 30d, and

30g in yields between 30 and

routes to the 2-pyridone derivatives were based on published

procedures.^[29]Starting from 6-hydroxynicotinic acid 16, methyl

5-bromo-6-chloronicotinate 23 was synthesized in 66% yield

over three steps. In contrast to the published procedure,^[28]it

was found that the introduction of the methoxy group by aro-

matic nucleophilic substitution of 23 gave low yields at room

temperature if methanol was used as the solvent (27% of the

desired product 24 and 54% of recovered starting material). A

slightly better yield of 24 (38%) was obtained by heating 23

with two equivalents of sodium methoxide in 1,4-dioxane at

reflux. Suzuki coupling of the bromide 24 with 3-nitrophenyl-

boronic acid formed the desired product 25a. This was difficult

to separate from decomposition products of the boronic acid

and was consequently obtained in a rather low yield of 32%,

despite the same reaction with phenylboronic acid having

been described in 86% yield.^[29]Hydrogenation with palladium

on charcoal of the nitro group in 25a afforded the amine de-

rivative 25b. Deprotection of both the methyl ether and

methyl esters of 25a and 25b was achieved by heating with

77%. Subsequent deprotection of the diphenyl acetonide with

trifluoroacetic acid gave final compounds 35c, 35d, and 35g

in 73–96% yields. It was necessary to conduct the deprotec-

tion in this order, as removal of the diphenyl acetonide results

in methyl esters that are stabilized by electron donation from

the gallate para-hydroxy group, and could not be cleaved by

any standard chemistry that would not also cleave the ether-

linked side chains.^[31]

The 5-amino analogue 35e was prepared from methyl vanil-

late 31 in four steps. Nitration at the 5-position of 31 with

a mixture of nitric acid and acetic acid afforded 32 in 58%

yield. Both the methyl ester and methyl ether of 32 were de-

protected by heating in 48% aqueous hydrogen bromide solu-

tion to give free acid 33 in 45% yield. The nitro group of 33

was reduced by palladium-catalyzed hydrogenation to give

amine 34 in 98% yield. Reductive amination of 34 with 3-nitro-

benzaldehyde afforded product 35e in 91% yield.

The synthesis of 3-hydroxy-5-substituted benzoic acids is

given in Scheme 5. Compounds 41a and 41b were prepared

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Scheme 4. Reagents and conditions: a) Cl₂CPh₂, Cs₂CO₃, MeCN, RT; b) 1-fluoro-3-nitrobenzene, tBu-P4, TMSNEt₂, DMF, 1008C; c) 3-nitrobenzyl bromide, Cs₂CO₃,

DMF; d) benzyl bromide, Cs₂CO₃, DMF, RT; e) NaOH, H₂O, THF, RT; f) TFA, CH₂Cl₂, RT; g) AcOH, HNO₃, 08C!RT; h) 48% HBr_(aq), reflux; i) H₂, Pd/C, MeOH, RT;

j) 1. 3-nitrobenzaldehyde, AcOH, DMF, 3 ꢁ molecular sieves, 2. NaBH₃CN, RT. See Experimental Section for reaction times and yields.

Scheme 5. Reagents and conditions: a) Ac₂O, RT; b) Tf₂O, pyridine, 08C; c) 3-(nitrophenyl)boronic acid, Pd(PPh₃)₄, Na₂CO_3(aq), DME, reflux; d) KOH, H₂O, THF, RT;

e) H₂, Pd/C, EtOH; f) 1-fluoro-3-nitrobenzene, tBu-P4, TMS-NEt₂, DMF, 1008C; g) 3-nitrobenzyl bromide, NaH, THF, 08C!RT; h) 1. 3-nitrobenzaldehyde, AcOH,

DMF, 3 ꢁ molecular sieves, 2. NaBH₃CN, RT. See Experimental Section for reaction times and yields.

from methyl 3,5-dihydroxybenzoic acid 36, which was convert-

ed into the triflate 38, to be used as a substrate in Suzuki cou-

plings. Treatment of 36 with one equivalent of acetyl chloride

was non-selective, and gave an approximate 1:1:1 mixture of

the mono- and di-acylated products and the starting material,

which were separable by column chromatography. Trifluoro-

methanesulfonic anhydride was used to convert the mono-

protected compound 37 into triflate 38, with pyridine acting

as both base and nucleophilic catalyst. Suzuki coupling was

then used to couple 38 with 3-nitrophenylboronic acid. The

acyl-protecting group was removed under the mildly basic

conditions of the coupling reaction to give 39a. A further hy-

drolysis step gave the free acid 41a. The 3-nitro compound

41a was then converted by catalytic hydrogenation into the

corresponding amine 41b.

mide to give intermediate 39d, followed by hydrolysis to give

41d. The 5-amino analogue 41e was prepared by reductive

amination between 3-amino-5-hydroxybenzoic acid 40 and 3-

nitrobenzaldehyde in 93% yield without the need for protec-

tion/deprotection steps.

Scheme 6 shows the synthesis of analogues incorporating

a 3-aryl-4-hydroxybenzoate core. It was originally proposed

that 44a and 44b could be synthesized by following the same

scheme used for the meta-hydroxy compound 41a, using 3,4-

dihydroxybenzoic acid as a starting point. This strategy was

The 5-(3-nitrophenoxy)-3-hydroxy analogue 41c was synthe-

sized from methyl 3,5-dihydroxybenzoate 36 in 45% yield by

similar methodology used for 31c (Scheme 4). The 5-(3-nitro-

benzoxy)-3-hydroxybenzoic acid analogue 41d was synthe-

sized from methyl 3,5-dihydroxybenzoic acid 36 by treatment

with one equivalent of sodium hydride then 3-nitrobenzyl bro-

Scheme 6. Reagents and conditions: a) 3-nitrophenylboronic acid, Pd(PPh₃)₄,

Na₂CO_3(aq), DME, reflux; b) KOH, H₂O, THF, RT; c) H₂, Pd/C, EtOH, RT. See Exper-

imental Section for reaction times and yields.

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abandoned, however, as the acylation protection step was con-

sidered insufficiently selective. Consequently the commercially

available 4-hydroxy-3-iodobenzoic acid methyl ester 42 was

used as a Suzuki coupling partner with 3-nitrophenylboronic

acid to give product 43a in 46% yield. Hydrolysis of the

methyl ester in 43a gave the free acid 44a. The 3-nitro group

of 44a was reduced by hydrogenation to give the aromatic

amine 44b.

mophore of the product 3-dehydroshikimate 2, or by a coupled

assay with E. coli shikimate dehydrogenase, monitoring the de-

crease in absorption of NADPH at l 340 nm (see the Experi-

mental Section below for details). All compounds were initially

tested at a concentration of 200 mm with a substrate concen-

tration at K_M(30 mm), and the percentage inhibition measured

(Table 1). Further testing was performed to determine the K_i

values of those compounds that exhibited inhibition above

40% under these conditions (Table 1). All experiments were

performed in duplicate.

Analogues incorporating an isophthalate core were synthe-

sized as illustrated in Scheme 7. The ether-linked isophthalate

Comparison of the percentage

inhibition (Table 1), by the corre-

sponding pairs of 3-nitro and 3-

amino compounds 22a (15%)

and 22b (13%), 26a (18%) and

26b (32%), 41a (32%) and 41b

(13%), and 44a (25%) and 44b

(12%) showed that they have

Scheme 7. Reagents and conditions: a) 3-nitrobenzyl bromide, Cs₂CO₃, DMF, RT; b) 1. 3-nitrobenzaldehyde, toluene,

1308C (Dean–Stark), 2. NaBH₃CN, AcOH, MeOH, RT; c) KOH, H₂O, THF, 608C; d) 1. 4-nitrobenzaldehyde, AcOH,

DMF, 3 ꢁ molecular sieves, 2. NaBH₃CN, RT. See Experimental Section for reaction times and yields.

similar potencies, although the

3-nitro compounds were often

marginally more potent than

their 3-amino analogues. A com-

parison of the effect of C3 and

49d was prepared from dimethyl 5-hydroxyisophthlate 45 by

alkylation with 3-nitrobenzyl bromide to give 47d in 79%

yield, followed by saponification of the methyl ester with aque-

ous base to give 49d in 64% yield. The 3-nitrobenzyl amino-

linked isophthalic acid 49e was prepared from dimethyl 5-ami-

noisophthalate 46. Amine 46 and 3-nitrobenzaldehyde were

heated under Dean–Stark conditions, and the resultant imine

was reduced with sodium cyanoborohydride to give the secon-

dary amine 47e in 65% yield. Saponification of 47e with aque-

ous base gave 49e in quantitative yield. A shorter synthetic

route avoiding the need for protecting groups was investigat-

ed for the synthesis of the 4-nitrobenzyl amino analogue 49 f.

Reductive amination between the free di-acid 48 and 4-nitro-

benzaldehyde afforded the 49 f in one step and 95% yield.

C4 hydroxy substitution on inhibition between corresponding

pairs of compounds, 41a (32%) and 44a (25%), 41b (13%)

and 44b (12%) (Table 1) showed little difference between the

respective pairs. This suggests that binding from both the C3

and C4 hydroxy groups of the inhibitors is important.

Analyses of the compounds designed using different flat

quinate analogues show that both the citrazinate analogue

22a and 6-hydroxynicotinate analogue 26a are less potent in-

hibitors than their corresponding 3-hydroxy 41a and 4-hy-

droxy 44a benzoate analogues, and all four compounds less

than citrazinate 15 itself (K_i=300 mm). Citrazinate 15 has three

possible binding motifs in the active site of the enzyme

(Figure 4): in the first, the compound has the O2 oxygen as

a carbonyl directed toward the “active site” pocket and both

the ring nitrogen N1 and the O6 oxygen are protonated (Fig-

ure 4a); secondly, both oxygen atoms are protonated, and the

ring nitrogen is deprotonated (Figure 4b); and thirdly, the O6

oxygen is a carbonyl and both ring nitrogen N1 and O2

oxygen are protonated (Figure 4c).

Protein production, crystallization, and data collection

Type II dehydroquinase from M. tuberculosis (MtDHQase) was

expressed, purified and crystallized according to Dias et al.^[32]

X-ray crystallographic data were collected using various syn-

chrotron X-ray sources. MtDHQase structures were solved by

molecular replacement using MOLREP^[33]or Phaser^[34]imple-

mented in CCP4.^[35]The MtDHQase dodecamers (PDB: 3N7A)

and (PDB: 3N76) were used as probes for the P2₁and F23 crys-

tals, respectively. Refinement was carried out using the

REFMAC 5.2^[36]also implemented in CCP4.^[35]Coot 0.3.1 was

used for visual analysis, manual inspection and to add water

molecules.^[37]The figures were prepared using PyMOL.^[38]

In compound 22a, the attachment of a six-membered ring

side chain to the C6 position and the subsequent binding of

that side chain in the “active site” pocket means that the bind-

ing motif adopted can only be that shown in either Figure 4b

or 4c. It is proposed that citrazinate 15 binds in the manner

shown in Figure 4a, where the compound is a hydrogen bond

donor at the 3- and 4-positions to both Asp88 and His81. The

loss of inhibition observed in compound 22a would result

from the inability to adopt this two-hydrogen-bond-donor ar-

rangement, as compound 22a is forced to adopt either of the

hydrogen bond donor/acceptor arrangements (Figure 4b and

4c).

MtDHQase assays

M. tuberculosis type II dehydroquinase was assayed either by

direct enzyme assay, monitoring the increase in absorbance at

l 234 nm due to the formation of the enone–carboxylate chro-

For compound 26a, this can adopt one of the two one-

donor/one-acceptor

hydrogen

bonding

arrangements

(Figure 5), albeit with the functional groups at the 1- and 6-po-

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Table 1. Inhibition of type II 3-dehydroquinase from M. tuberculosis.

Table 1. (Continued)

Compd

Inhib. [%]^[a]

K_i[mm]^[b]

Inhib. [%]^[a]

K_i[mm]^[b]

130ꢀ10

22a

22b

22d

26a

15

ND

41d

41e

44a

44b

49d

49e

66

13

51

18

ND

92

25

12

62

99

20ꢀ3

190ꢀ10

ND

26b

35c

35d

32

90

94

ND

140ꢀ10

7.5ꢀ1

50ꢀ4

10ꢀ1

49 f

92

20ꢀ5

[a] Percent inhibition caused by test compound at 200 mm, with a sub-

strate concentration of 30 mm (K_M). [b] K_ivalues were determined for com-

pounds with inhibition >40% (ND: not determined).

35e

35g

41a

91

47

32

20ꢀ4

240ꢀ20

ND

sitions reversed relative to the carboxylate and side chain of

citrazinate-based compound 22a. The similarity in levels of in-

hibition between 22a and 26a suggest neither configuration

gives an advantage over the other, and if both compounds

bind with the generally favorable lactam tautomer, there is

little difference in whether the compounds form a hydrogen

bond to Asp88 (Figure 4c) or His81 (Figure 5a). In addition,

the observation that these compounds result in less potent in-

hibitors than a single hydrogen bond donor alone, the corre-

sponding 3-hydroxybenzoate 41a and 4-hydroxybenzoate 44a

suggest that the presence of hydrogen bond donors at both

the C3 and C4 positions (benzoate numbering) is advanta-

geous for binding and inhibition, and hydrogen bond accept-

ors at these positions are unfavorable. This is backed up by

the observation that the 3,4-dihydroxy-5-(3-nitrophenoxy)ben-

zoate 35c (K_i=49 mm) is more potent than the 3-hydroxy-5-(3-

nitrophenoxy)benzoate 41c (K_i=165 mm), and that 3,4-dihy-

41b

41c

13

61

ND

170ꢀ10

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Whilst the carboxylate and 3-

hydroxy groups of the two com-

pounds overlap, and can form

the same hydrogen bond inter-

actions, the vector at which the

CꢁC bond linking the aromatic

moiety of 9 and the quinate

core leaves the puckered six-

membered ring is ~308 away

from the vector to which the Cꢁ

O bond of the citrazinate points.

In the case of the former, the

result is that the aromatic

Figure 4. a) Binding of citrazinate 15-lactam tautomer with carbonyl directed toward the “active site” pocket.

b) Binding of citrazinate 15 and inhibitor 22a-lactim tautomers. c) Binding of citrazinate 15 and inhibitor 22a-

lactam tautomers with carbonyl directed toward His81.

Figure 5. a) Binding of inhibitor 26a-lactam tautomer. b) Binding of inhibitor

26a-lactim tautomer.

Figure 6. Superposition of MtDHQase–35c (ribbon diagram of protein in

orange and molecular structure of compound in grey) and MtDHQase–41c

(ribbon diagram of protein in green and molecular structure of compound

in yellow). The molecular structures of the active site residues correspond to

the MtDHQase–41c complex. The key hydrogen bond/electrostatic interac-

tions between the central gallate core of compound 35c and protein are

shown as black lines, and dashed lines indicate the interactions lost if an in-

hibitor lacks a hydroxy group at the 4-position.

droxy-5-(3-nitrobenzoxy)benzoate 35d (K_i=10 mm) is more

potent than both the 3-hydroxy-5-(3-nitrobenzoxy)benzoate

41d (K_i=130 mm) and 3-hydroxy-5-(3-nitrophenoxy)citrazinate

22d (K_i=186 mm).

To get a better understanding of the roles of the 4- and 5-

hydroxy groups, crystals of MtDHQase in complex with com-

pounds 35c, 35d, and 41c were obtained and their structures

solved (PDB codes 4KIJ, 4KIM, and 4KI7, respectively). An over-

lay of the crystal structures of MtDHQase in complex with 41c

(PDB: 4KI7) and 35c (PDB: 4KIJ) (Figure 6) reveals that three of

the key hydrogen bond/electrostatic interactions to the C4 hy-

droxy group of the gallate moiety of 35c, and presumably to

the C4 hydroxy group of the quinate core of inhibitors 4–10 as

well, are lost when 3-hydroxy-5-benzoate is used as a scaffold.

As a result, compound 41c, lacking the C4 hydroxy group, is

displaced ~0.5 ꢁ in comparison with 35c (PDB: 4KIJ) (Figure 6)

and also 35d (PDB: 4KIM); consequently the orientation of the

nitrophenyl group is slightly changed.

moiety sits in a pocket formed by the active site loop, whilst

for the latter, the vector is directed into the main body of the

enzyme. Therefore, it is postulated that the CꢁC linked citrazi-

nate analogues cannot bind with the pyridinyl moiety bound

in the same configuration as citrazinate itself, which in turn re-

sults in the loss of key hydrogen bond interactions and lower

potency. The addition of ether linkages improved potency:

41c (K_i=165 mm) and 35c (K_i=49 mm), whilst extension of the

linker with a methylene unit increased potency even further:

41d (K_i=130 mm) and 35d (K_i=10 mm), respectively. Further-

more, superposition of the MtDHQase–35c and MtDHQase–

35d (Figure 8) shows that the different linkages affect the rela-

tive orientation of the rings of these compounds and conse-

quently different interactions are observed. Despite the gallate

ring occupying the same position as the quinate, conserving

most of the interactions, the nitrophenyl rings are displaced by

~0.7 ꢁ, and some of the interactions observed in the

MtDHQase–35d complex are absent in the MtDHQase–35c

complex. Due to the extra methylene carbon in the linkage,

35d is more flexible, and the rings may be able to optimize

their interactions with the active site of the enzyme. Thus the

The CꢁC linked biaryl compounds 22a, 22b, 26a, 26b, 41a,

41b, 44a, and 44b proved to be relatively poor inhibitors, the

best 26b and 41a resulting in only 32% inhibition at 200 mm.

During the course of this investigation, the crystal structures of

citrazinate 15 and inhibitor 9 bound in the active site of

MtDHQase were obtained (PDB codes 3N8K and 3N87).^[33]An

overlay of compounds 9 and 15 (Figure 7) gives a possible ex-

planation for the poor inhibition for the biaryl compounds

(N.B.: the protein structure used is from the complex with 9,

wherein the side chain of residue Arg18 is displaced away

from the active site by the biaryl side chain of the inhibitor).

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gallate 35d (K_i=10 mm). The crystal structures of amino-linked

isophthalate 49e (Figure 7, PDB: 4KIW) and ether-linked iso-

phthalate 49d (PDB: 4KIU) bound in the active site of

MtDHQase were solved. In these structures, both the 5-amino

and 5-ether groups from the linkage form a hydrogen bond

network with N^d-Asn12 and the b-carboxylate side chain from

Asp88 of the neighboring protomer via a water molecule

(Figure 9). This water molecule has been observed in other

MtDHQase–inhibitor complexes.^[21]

Figure 7. Overlay of citrazinate 15 (orange) and inhibitor 9 (yellow) bound

to 3-dehydroquinase with residues Arg18, Tyr24, and His101 highlighted.

The angle shown is defined by atoms O6 (15)–C3 (9)–C1’ (9), and is an esti-

mate of the difference in vector trajectory into the side chain pocket of the

C6ꢁC1’ bond of 9 and C6ꢁO6 bond of 15.

Figure 9. Crystal structure of 49e bound in the 3-dehydroquinase active

site, showing key electrostatic interactions. Residues Asn12 and Tyr24 from

chain 1 (green), and Asp88 from chain 2 (cyan) are highlighted, with water

shown as a red sphere.

The affinity of compound 49e (K_i=7.5 mm) is significantly

higher than 49d (K_i=136 mm). The oxygen of the ether linkage

in 49e forms an angle of 1098, compared with 1188 for the ni-

trogen of amino linkage in 49d. This difference affects the po-

sitions of the nitrophenyl and isophthalate rings (Figure 10).

Figure 8. Superposition of MtDHQase–35c (protein is green and compound

in yellow) and MtDHQase–35d (protein in white and compound in grey),

showing the effect the additional methylene group in 35d has on the loca-

tion of the 3-nitrophenyl ring and active site residue Tyr24.

linkage of 35c is more distended than 35d, and it conserves

the key interactions of the quinate binding site and the p in-

teraction with Tyr24.

The presence of an amino group at the 5-position has

a marked effect on potency in compounds lacking a non-hy-

drogen substituent at the 4-position. Both 5-amino-3-hydroxy-

benzoate 41e (K_i=17 mm) and 5-amino-isophthalate 49e (K_i=

7.5 mm) are an order of magnitude more potent than their cor-

responding ether-linked analogues 41d (K_i=130 mm) and 49d

(K_i=136 mm). However, with a hydroxy group at the 4-position

the amino analogue 35e (K_i=19 mm) has similar potency to

Figure 10. Superposition of MtDHQase–49d (protein in salmon and com-

pound in white) and MtDHQase–49e (protein in green and compound in

yellow). The image shows the different angles of the two compounds 49d

and 49e and their effects on the orientation of the nitrophenyl groups.

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are quoted with respect to the solvent system used to develop the

plate. Column chromatography was carried out using 230–

400 mesh silica gel 60 or on a Biotage Isolera 1 (using KP-SIL car-

tridges: size and gradient used as described). Where quoted, high-

performance liquid chromatography (HPLC) was performed on

a Gilson HPLC system fitted with a Gilson 118 UV/Vis detector set

at l 220 nm. HPLC purification was performed with either: A) a

semi-preparative Phenomenex Luna 5 mm C₁₈(2) 10ꢂ250 mm

column (flow rate: 4.6 mLmin^ꢁ1), B) a semi-preparative Hi-Chrom

Kromasil 100 5 mm C₁₈10ꢂ250 mm column (4.0 mLmin^ꢁ1), C) a

preparative Hi-Chrom Kromasil 100 5 mm C₁₈21.2ꢂ250 mm column

(14 mLmin^ꢁ1), or D) a preparative Macherey–Nagel Nucleosil 100

5 mm C₁₈21.2ꢂ250 mm column (21.2 mLmin^ꢁ1). The columns were

eluted with MeCN in H₂O (containing 0.1% trifluoroacetic acid

(TFA)) with gradients as described. Amberlite IR-120 (H⁺) (cation

exchanger) was washed alternately with Milli-Q H₂O, 10% HCl, and

finally Milli-Q H₂O before use. Infrared spectra were recorded on

a PerkinElmer Spectrum One FTIR spectrometer using attenuated

transmittance reflectance (ATR); n_maxvalues are quoted in wave-

The nitrophenyl ring of 49e is displaced ~0.7 ꢁ in relation to

49d forward to the flexible loop region, and a network of five

hydrogen bond interactions with the protein is formed. In

comparison, 49d has only one hydrogen bond interaction in

the same region. The isophthalate is identically located in both

structures.

The significance of the 3-nitro group was investigated by

first preparing the benzyl analogue 35g of the most potent

gallate-based inhibitor 35d (K_i=10 mm). This analogue was

found to be considerably less potent (K_i=240 mm). Second, the

4-nitro analogue 49 f of the most potent isophthalate inhibitor

49e (K_i=7.5 mm) was prepared. This analogue was found to

have slightly lower potency (K_i=19 mm), which suggests that

substitution at the 3-position is preferable. It is known that in

general, the presence of electron-withdrawing groups on the

aromatic side chain improves potency of the anhydroquinate-

core-based inhibitors,^[17]and it appears that it is the same for

inhibitors with an aromatic core.

1

numbers (cm^ꢁ1). H NMR spectra were recorded on a Bruker DPX-

400 spectrometer or a Bruker Avance 500 spectrometer in deuter-

ated solvents, as indicated. ¹³C NMR spectra were recorded on

a Bruker DPX-400 spectrometer or a Bruker Avance 500 spectrome-

ter linked to a Bruker 5 mm dual Cryoprobe (operating at 100 and

125 MHz, respectively). ¹⁹F NMR spectra were recorded on a Bruker

Avance 400 QNP Ultrashield spectrometer (operating at 376 MHz).

All chemical shifts (d) are quoted in parts per million (ppm) refer-

enced with residual solvent peaks.^[39]Coupling constants (J) are as-

signed where possible and are given in Hz. Yields of water-soluble

Conclusions

The simple flattening of the anhydroquinate core of the

known highly potent inhibitors of 3-dehydroiquinase to

a number of aromatic systems (the CꢁC linked range of inhibi-

tors) was found to lead to a loss of potency. The crystal struc-

tures suggest that the rigidity of, and the conformations

adopted by, such compounds are such that they cannot

occupy the “side chain” pocket and the substrate binding

pocket at the same time in a manner that maximizes hydrogen

bond interactions in the same way as the potent quinate-

based inhibitors. Attempts to increase flexibility between the

aromatic rings in the inhibitors to allow greater conformational

freedom generally resulted in increased potency due to the ar-

omatic “side chains” being able to maximize p-stacking inter-

actions with Tyr24 of the flexible active site loop, whilst the

central aromatic core maintains favorable hydrogen bond in-

teractions. It was observed that electron-withdrawing side

chains were favored, and that hydrogen bond donors in the 3-,

4-, and 5-positions are all favorable. The present studies identi-

fied a number of ways in which the potency of these syntheti-

cally tractable series can be improved.

1

compounds were determined by H NMR spectroscopy and 2,2,3,4-

tetradeutero-3-(trimethylsilyl)propionic acid sodium salt (TSP) as an

internal standard. Liquid chromatography–mass spectrometry (LC–

MS) was carried out using an Alliance HT Waters 2795 Separations

Module fitted with a Waters Atlantis dC₁₈4.6ꢂ30 mm, 3 mm

column and coupled to a photodiode array detector and a Waters

Micromass ZQ Quadrapole Mass Analyzer (using electrospray ioni-

zation, ES). Samples were run with an isocratic elution of 10 mm

NH₄OAc containing 0.1% formic acid for 0.7 min, followed by a gra-

dient over a period of 3.5 min to reach 95% MeCN with 0.05%

formic acid, which was then run under isocratic conditions for

3.5 min before going back to the initial eluent over 0.3 min (flow

rate: 1 mLmin^ꢁ1). Low- and high-resolution mass spectrometry

(LRMS and HRMS, respectively) were performed at the EPSRC Na-

tional Mass Spectrometry Service Centre (Swansea, UK). Electron

ionization (EI) LRMS data were collected on a Micromass Quattro

quadrupole instrument. Accurate masses were recorded using

either a Finnigan MAT 900 XLT or a Finnigan MAT 95 XP instrument.

Perfluorotributylamine was used as the reference compound for EI,

and polyethylenamine for electrospray ionization (ES).

Experimental Section

Methyl 2,6-dichloroisonicotinate (18): Citrazinic acid 15 (2.50 g,

16.1 mmol) and Me₄NCl (1.84 g, 16.8 mmol) were suspended in

POCl₃(7.4 g, 4.5 mL, 47.7 mmol) and heated at 1308C. When the in-

ternal temperature reached 758C, the solids dissolved to yield

a clear brown solution. After 18 h, the temperature was increased

to 1458C for 2 h. After cooling to 258C, the mixture was poured

onto ice (30 g) and stirred for 2 h. The resulting solids were filtered,

dried, and then stirred with 25 mL EtOAc. Insoluble material

(mostly citrazinic acid) was removed by filtration. The organic solu-

tion was dried over Na₂SO₄, and the solvent was removed to yield

2,6-dichloroisonicotinic acid as a light-brown solid (1.77 g, 57%).

¹H NMR (400 MHz, [D₆]DMSO): d=7.81 ppm (s, 2H); ¹³C NMR

(100 MHz, [D₆]DMSO): d=163.7, 150.1, 144.6, 122.9 ppm; IR (ATR):

n˜_max=3078, 3000–2400, 1722 cm^ꢁ1; LCMS-ES m/z 214 [M+Na]⁺,

Chemistry

All non-aqueous reactions were carried out in pre-dried glassware

under an inert atmosphere (N₂or Ar). All starting materials and re-

agents were commercially available and used without further pu-

rification. Organic solvents were freshly distilled prior to use, and

Milli-Q deionized H₂O was used for all biochemical work. Unless

otherwise stated, petroleum ether (PE) refers to the fraction collect-

ed between 40 and 608C. Analytical thin-layer chromatography

(TLC) was carried out on commercial silica gel 60 0.25 mm plates

using UV absorption, potassium permanganate stain (3 g KMnO₄,

20 g K₂CO₃, 5 mL 5% NaOH, 300 mL H₂O), or cerium molybdate

stain (2 g ammonium cerium(IV) sulfate, 5 g ammonium heptamo-

lybdate, 12 mL 98% H₂SO₄, 188 mL H₂O) for visualization. R_fvalues

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192 [M+H]⁺, 190 [MꢁH]^ꢁ. These data are in agreement with

sure. The residue was purified by column chromatography, eluting

with 3:17 v/v EtOAc/PE, to afford the title compound 21a as

a white solid (65 mg, 33%) and 48% of the starting chloride 19.

those previously reported by Henegar et al.^[26]

2,6-Dichloroisonicotinic acid (1.0 g, 5.2 mmol) was dissolved in

freshly distilled MeOH (25 mL), and concentrated H₂SO₄(1 mL) was

added dropwise. The mixture was heated at reflux for 2 h. After

cooling to 258C, the solvent was removed under reduced pressure,

and the residue was partitioned between H₂O (30 mL) and CH₂Cl₂

(30 mL). The organic phase was washed with saturated aqueous

NaHCO₃(2ꢂ30 mL) and brine (30 mL), dried (Na₂SO₄), and the sol-

vent was removed under reduced pressure. The residue was puri-

fied by silica gel column chromatography (eluent: 3:17 v/v EtOAc/

PE) to afford the desired methyl ester 18 as white needles (0.94 g,

1

R_f=0.31 (3:17 v/v EtOAc/PE); H NMR (400 MHz, CDCl₃): d=8.92 (t,

J=2.0 Hz, 1H), 8.40 (ddd, J=1.1, 2.0, 8.0 Hz, 1H), 8.25 (ddd, J=1.1,

2.0, 8.0 Hz, 1H), 7.93 (d, J=1.1 Hz, 1H), 7.64 (t, J=8.0 Hz, 1H), 7.32

(d, J=1.1 Hz, 1H), 4.09 (s, 3H), 3.98 ppm (s, 3H); ¹³C NMR

(100 MHz, CDCl₃): d=165.4, 164.8, 153.0, 149.0, 141.5, 140.2, 132.6,

129.8, 124.0, 121.8, 112.4, 111.3, 54.1, 52.9 ppm; IR (ATR): n˜_max

=

3094, 2949, 1723 cm^ꢁ1; LCMS-ES m/z 289 [M+H]⁺; HRMS-ES m/z

[M+H]⁺calcd for C₁₄H₁₃N₂O₅289.0819, found 289.0820.

Methyl 6-(3’-aminophenyl)-2-methoxyisonicotinate (21b): Methyl

2-methoxy-6-(3’-nitrophenyl)isonicotinate 21a (24 mg, 83 mmol)

and Pd/C (2.5 mg, 10 wt% Pd) were stirred in EtOAc (1.5 mL) under

a balloon of H₂for 30 min. The reaction mixture was then filtered

through Celite, and the solvent removed under reduced pressure.

The residue was purified by silica gel chromatography, eluting with

2:3 v/v EtOAc/PE to afford the title compound 21b as a yellow oil

(21 mg, quant). ¹H NMR (400 MHz, CDCl₃): d=7.85 (d, J=1.0 Hz,

1H), 7.44–7.48 (m, 2H), 7.23–7.28 (m, 2H), 6.75 (ddd, J=1.0, 2.2,

7.5 Hz, 1H), 4.06 (s, 3H), 3.96 ppm (s, 3H); ¹³C NMR (100 MHz,

CDCl₃): d=165.9, 164.4, 156.0, 146.9, 140.9, 139.5, 129.8, 117.4,

116.3, 113.6, 112.3, 109.4, 53.9, 52.7 ppm; IR (ATR): n˜_max=3461,

3376, 3225, 2949, 1723 cm^ꢁ1; LCMS-ES m/z 259 [M+H]⁺; HRMS-ES

m/z [M+H]⁺calcd for C₁₄H₁₅N₂O₃259.1077, found 259.1081.

1

88%). H NMR (400 MHz, CDCl₃): d=7.80 (s, 2H), 3.97 ppm (s, 3H);

¹³C NMR (100 MHz, CDCl₃): d=163.4, 151.7, 142.5, 122.8, 53.4 ppm;

IR (ATR): n˜_max=3090, 2960, 1730 cm^ꢁ1; LCMS-ES m/z: 206 [M+H]⁺;

HRMS-ES m/z [M⁺] calcd for C₇H₅Cl₂NO₂204.9692, found 204.9690.

Methyl 2-chloro-6-methoxyisonicotinate (19): Methyl 2,6-dichlor-

oisonicotinate 18 (0.50 g, 2.4 mmol) was dissolved in freshly dis-

tilled MeOH (15 mL) and NaOMe (0.45 g, 8.4 mmol) was added

slowly. The mixture was heated at reflux for 5 h with a CaCl₂guard.

After cooling to 258C, the mixture was neutralized with glacial

AcOH (~0.5 mL) and concentrated to dryness. The residue was par-

titioned between CH₂Cl₂(50 mL) and saturated NaHCO₃solution

(50 mL). The organic layer was dried over MgSO₄, and the solvent

was removed under reduced pressure to afford the title compound

19 as a white solid (250 mg, 52%); mp: 101–1028C; ¹H NMR

(400 MHz, CDCl₃): d=7.43 (d, J=1.1 Hz, 1H), 7.21 (d, J=1.1 Hz,

1H), 3.97 (s, 3H), 3.93 ppm (s, 3H); ¹³C NMR (100 MHz, CDCl₃): d=

2-Hydroxy-6-(3-nitrophenyl)isonicotinic acid (22a): Methyl 2-me-

thoxy-6-(3’-nitrophenyl)isonicotinate 21a (25 mg, 87 mmol) was dis-

solved in 48% HBr solution (0.6 mL) and glacial AcOH (0.6 mL), and

the mixture was heated at reflux for 1 h. After cooling to 258C, the

solvent was removed under reduced pressure. The resulting solid

was collected by filtration, washed with Et₂O (1 mL) to extract an

orange impurity, and dried to give the desired product 22a as

164.4, 149.2, 142.3, 115.7, 109.7, 54.5, 52.8 ppm; IR (ATR): n˜_max

=

3110, 3090, 2960, 1736 cm^ꢁ1; LCMS-ES m/z 202 [M+H]⁺; HRMS-ES

m/z [M+H]⁺calcd for C₈H₉ClNO₃202.0265, found 202.0264.

2-Methoxy-6-((3-nitrobenzyl)oxy)isonicotinate (20d): 3-Nitroben-

zyl alcohol (456 mg, 2.98 mmol), methyl 2-chloro-6-methoxyisoni-

cotinate 19 (300 mg, 1.49 mmol), K₂CO₃(414 mg, 3.00 mmol),

copper (7.2 mg, 0.11 mmol), and copper(I) iodide (6 mg,

0.03 mmol) were suspended in anhydrous pyridine (15 mL) under

N₂and heated at 1008C for 24 h. The reaction was allowed to cool

to RT, and partitioned between water (150 mL)and EtOAc (2ꢂ

150 mL). The combined organic layers were washed with saturated

brine (150 mL), dried over Na₂SO₄, and the solvent was removed

under reduced pressure. The residue was purified by silica gel

column chromatography (eluent 1:9 v/v EtOAc/hexane then 1:3 v/v

EtOAc/hexane) to give the title product 20d as a white solid

(23 mg, 7%). ¹H NMR (400 MHz, [D₆]DMSO): d=11.26 (brs, 1H),

8.30 (t, J=2.3 Hz, 1H), 8.18 (ddd, J=0.9, 2.3, 8.2 Hz, 1H), 7.92 (ddd,

J=0.8, 2.3, 8.1 Hz, 1H), 7.69 (t, J=8.0 Hz, 1H), 6.75 (brs, 1H), 6.67

(d, J=1.1 Hz, 1H), 5.46 (s, 2H), 3.85 ppm (s, 3H); ¹³C NMR

(100 MHz, [D₆]DMSO): d=169.7, 168.1, 167.2, 152.7, 147.7, 144.2,

139.1, 134.8, 127.6, 127.0, 105.8, 104.6, 70.9, 57.5 ppm; IR (ATR):

n˜_max=2957, 1722, 1660, 1618, 1527 cm^ꢁ1; LCMS-ES m/z 305 [M+

H]⁺, 303 [MꢁH]^ꢁ; HRMS-ES m/z [M+H]⁺calcd for C₁₄H₁₃N₂O₆

305.0774, found 305.0784.

1

a yellow solid (23 mg, quant). H NMR (500 MHz, D₂O+NaOD): d=

8.54 (t, J=2.0 Hz, 1H), 8.09–8.15 (m, 2H), 7.56 (t, J=8.0 Hz, 1H),

7.12 (d, J=1.0 Hz, 1H), 6.62 ppm (d, J=1.0 Hz, 1H); ¹³C NMR

(125 MHz, D₂O+NaOD): d=174.8, 172.1, 153.7, 148.0, 141.2, 133.4,

129.6, 123.0, 121.7, 112.5, 107.4 ppm; IR (ATR): n˜_max=3094, 3000,

2450, 1681, 1621 cm^ꢁ1; LCMS-ES m/z 261 [M+H]⁺, 259 [MꢁH]^ꢁ;

HRMS-ES m/z [MꢁH]^ꢁcalcd for C₁₂H₇N₂O₅: 259.0360, found

259.0358.

3-(4-Carboxy-6-hydroxypyridin-2-yl)benzenaminium

bromide

(22b): Methyl 6-(3’-aminophenyl)-2-methoxyisonicotinate 21b

(14 mg, 54 mmol) was dissolved in 48% HBr solution (0.6 mL) and

glacial AcOH (0.6 mL), and the mixture was heated at reflux for

30 min. After cooling to 258C, the solvent was removed under re-

duced pressure. The resulting solid was collected by filtration,

washed with Et₂O (1 mL) to extract an orange impurity, and dried

to give the desired product 22b as a pale-yellow solid (12 mg,

1

71%). H NMR (400 MHz, D₂O+NaOD): d=7.28–7.34 (m, 3H), 7.15

(d, J=2.0 Hz, 1H), 6.88 (m,1H), 6.67 ppm (d, J=2.0 Hz, 1H);

¹³C NMR (125 MHz, D₂O+NaOD): d=175.1, 172.0, 156.1, 147.8,

146.3, 141.0, 129.6, 118.1, 116.5, 114.7, 111.4, 107.0 ppm; IR (ATR):

n˜_max=3195, 3144, 3089, 2849, 2617, 2544, 1711 cm^ꢁ1; LCMS-ES m/z

231 [M+H]⁺, 229 [MꢁH]^ꢁ; HRMS-ES m/z [M+H]⁺calcd for

C₁₂H₁₁N₂O₃231.0764, found 231.0761.

Methyl 2-methoxy-6-(3’-nitrophenyl)isonicotinate (21a): Methyl

2-chloro-6-methoxyisonicotinate 19 (140 mg, 0.69 mmol), 3-nitro-

phenylboronic acid (173 mg, 1.04 mmol), Et₃N (0.3 mL, 2.07 mmol),

and tetrakis(triphenylphosphine)palladium (40 mg, 35 mmol) were

heated at 1008C in dry DMF (2.8 mL) under argon for 21 h. After

cooling to 258C, the solvent was removed under reduced pressure.

The residue was dissolved in CH₂Cl₂(30 mL) and washed with satu-

rated NaHCO₃solution (3ꢂ30 mL), saturated brine (30 mL), dried

over Na₂SO₄, and the solvent was removed under reduced pres-

2-Hydroxy-6-((3-nitrobenzyl)oxy)isonicotinic acid (22d): 2-Me-

thoxy-6-((3-nitrobenzyl)oxy)isonicotinate 20d (23 mg, 0.072 mmol)

and NaI (17 mg, 0.113 mmol) were suspended in anhydrous MeCN

(4 mL) in the dark under N₂. Chlorotrimethylsilane (14 mL, 12 mg,

0.11 mmol) was added, and the mixture heated at 658C for 7 h.

The reaction was allowed to cool to RT, 1:1 v/v 5% sodium sulfite/

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saturated brine (5 mL) was added and extracted with EtOAc (3ꢂ

5 mL). The combined organics were dried over Na₂SO₄, and solvent

concentrated under reduced pressure. The residue was purified by

silica gel column chromatography (eluent 1:9 v/v MeOH/CH₂Cl₂) to

rated NaHCO₃solution (4ꢂ50 mL), saturated brine (50 mL), dried

(Na₂SO₄), and the solvent was removed under reduced pressure.

The residue was purified by column chromatography, eluting with

3:17 v/v EtOAc/PE followed by 1:1 v/v EtOAc/PE, and recrystallized

from CHCl₃/EtOAc to afford the title compound 25a as a yellow

solid (137 mg, 32%). ¹H NMR (400 MHz, CDCl₃): d=8.86 (d, J=

2.1 Hz, 1H), 8.43 (t, J=2.0 Hz, 1H), 8.24 (d, J=2.1 Hz, 1H), 8.21

(ddd, J=1.1, 2.0, 7.9 Hz, 1H), 7.89 (ddd, J=1.1, 2.0, 7.9 Hz, 1H),

7.60 (t, J=7.9 Hz, 1H), 4.05 (s, 3H), 3.93 ppm (s, 3H); ¹³C NMR

(100 MHz, CDCl₃): d=165.6, 163.3, 149.6, 148.3, 139.4, 137.3, 135.1,

1

give the title product 22d as a white solid (15 mg, 82%). H NMR

(400 MHz, [D₆]DMSO): d=8.30 (t, J=1.9 Hz, 1H), 8.18 (dd, J=2.3,

8.4 Hz, 1H), 7.92 (td, J=1.3, 7.7 Hz, 1H), 7.69 (t, J=7.9 Hz, 1H), 6.72

(d, J=1.0 Hz, 1H), 6.66 (d, J=1.1 Hz, 1H), 5.46 ppm (s, 2H);

¹³C NMR (100 MHz, [D₆]DMSO): d=165.9, 163.3, 162.3, 147.9, 144.4,

139.5, 134.4, 130.1, 122.8, 122.3, 101.2, 99.8, 66.1 ppm; IR (ATR):

n˜_max=3210, 2919, 1675, 1628, 1529, 1464, 1346 cm^ꢁ1; LCMS-ES m/z

291 [M+H]⁺, 289 [MꢁH]^ꢁ; HRMS-ES m/z [M+H]⁺calcd for

C₁₃H₁₁N₂O₆291.0617, found 291.0636.

129.3, 124.2, 122.8, 121.8, 120.3, 54.6, 52.2 ppm; IR ATR): n˜_max

=

3029, 2961, 2917, 2849, 1720 cm^ꢁ1; LCMS-ES m/z 289 [M+H]⁺;

HRMS-ES m/z [M+H]⁺calcd for C₁₄H₁₃N₂O₅289.0819, found

289.0820.

Methyl 5-bromo-6-chloronicotinate (23):^[29,40]Bromine (1.5 mL,

30 mmol) was added to a suspension of 6-hydroxynicotinic acid 15

(2.78 g, 20 mmol) in glacial AcOH (5 mL) with stirring. The mixture

was heated at 508C for 18 h then concentrated to dryness under

reduced pressure. POCl₃(5 mL) and PCl₅(8.4 g, 40 mmol) were

added to the crude residue, and the mixture was heated at reflux

for 12 h. Excess POCl₃was distilled off under reduced pressure (the

bath temperature was kept <508C). The resulting crude brown oil

was dissolved in CH₂Cl₂(25 mL) and MeOH (10 mL) and heated at

reflux for 2 h. The solvents were removed under reduced pressure,

and the residue was dissolved in Et₂O (40 mL) and washed with sa-

turated aqueous NaHCO₃solution (3ꢂ15 mL). The Et₂O layer was

dried over MgSO₄, and the solvent was removed under reduced

pressure. The residue was purified by column chromatography,

eluting with 3:17 v/v EtOAc/PE, to afford the title compound 23 as

a white crystalline solid (3.32 g, 66%); mp: 76–778C (lit.:^[41]76–

778C); ¹H NMR (400 MHz, CDCl₃): d=8.90 (d, J=2.0 Hz, 1H), 8.50

(d, J=2.0 Hz, 1H), 3.95 ppm (s, 3H); ¹³C NMR (100 MHz, CDCl₃): d=

Methyl 6-methoxy-5-(3-aminophenyl)nicotinate (25b): Methyl 6-

methoxy-5-(3-nitrophenyl)nicotinate 25a (10 mg, 35 mmol) and Pd/

C (2.5 mg, 10 wt% Pd) were stirred in EtOAc (1.5 mL) under a bal-

loon of H₂for 1 h. The reaction mixture was then filtered through

Celite and the solvent removed under reduced pressure. The resi-

due was purified by column chromatography, eluting with 6:4 v/v

EtOAc/PE, to afford the desired amine 25b as a yellow solid (9 mg,

1

quant). H NMR (400 MHz, CD₃OD): d=8.74 (d, J=2.3 Hz, 1H), 8.15

(d, J=2.3 Hz, 1H), 7.17 (t, J=7.8 Hz, 1H), 6.93 (t, J=2.0 Hz, 1H),

6.86 (ddd, J=0.9, 2.0, 7.8 Hz, 1H), 6.76 (ddd, J=0.9, 2.0, 7.8 Hz,

1H), 4.01 (s, 3H), 3.93 ppm (s, 3H); ¹³C NMR (100 MHz, CD₃OD): d=

167.4, 165.1, 149.0, 148.6, 140.0, 137.7, 130.0, 126.4, 121.3, 120.1,

117.3, 116.3, 54.6, 52.6 ppm; IR (ATR): n˜_max=3427, 3321, 3220, 3013,

2954, 1718 cm^ꢁ1; LCMS-ES m/z 259 [M+H]⁺; HRMS-ES m/z [M+

H]⁺calcd for C₁₄H₁₅N₂O₃259.1077, found 259.1075.

5-(3-Nitrophenyl)-6-oxo-1,6-dihydropyridine-3-carboxylic

acid

(26a): Methyl 6-methoxy-5-(3-nitrophenyl)nicotinate 25a (31 mg,

0.11 mmol) was dissolved in 48% HBr solution (0.6 mL) and glacial

AcOH (0.6 mL) and the mixture was heated at reflux for 1 h. After

cooling to 258C, the solvent was removed under reduced pressure.

The resulting solid was collected by filtration, washed with Et₂O

(1 mL) to extract an orange impurity and dried to afford the de-

sired pyridone 26a as an off-white solid (27 mg, 94%). ¹H NMR

(500 MHz, [D₆]DMSO): d=8.66 (t, J=2.0 Hz, 1H), 8.21 (ddd, J=1.0,

2.0, 8.0 Hz, 1H), 8.17 (ddd, J=1.0, 2.0, 8.0 Hz, 1H), 8.12 (d, J=

2.5 Hz, 1H), 8.09 (br, 1H), 7.71 ppm (t, J=8.0 Hz, 1H); ¹³C NMR

(125 MHz, [D₆]DMSO): d=165.3, 161.3, 147.6, 140.4, 138.3, 137.3,

134.6, 129.8, 126.7, 122.9, 122.6, 109.7 ppm; IR (ATR): n˜_max=3088,

2511, 1685, 1638 cm^ꢁ1; LCMS-ES m/z 261 [M+H]⁺, 259 [MꢁH]^ꢁ;

HRMS-ES m/z [M+H]⁺calcd for C₁₂H₉N₂O₅261.0506, found

261.0505.

163.9, 155.1, 149.0, 143.0, 126.2, 120.4, 53.0 ppm; IR (ATR): n˜_max

=

3067, 2954, 1722 cm^ꢁ1; LCMS-ES m/z 250, 252 [M+H]⁺. These

data are in agreement with those previously reported.^[29,40]

Methyl 5-bromo-6-methoxynicotinate (24): NaOMe (288 mg,

5.28 mmol) in MeOH (5 mL), was added to a solution of methyl 5-

bromo-6-chloronicotinate 23 (0.66 g, 2.65 mmol) in 1,4-dioxane

(7 mL). The mixture was heated at reflux for 16 h under a CaCl₂

guard. The reaction mixture was neutralized with glacial AcOH

(0.3 mL). After removal of the solvent under reduced pressure, the

residue was dissolved in CH₂Cl₂(15 mL) and washed with saturated

NaHCO₃solution (5 mL). The organic extract was dried over MgSO₄,

and the solvent was removed under reduced pressure. The residual

solid was recrystallized from Et₂O/hexane to afford the title com-

pound 24 as a white crystalline solid (0.25 g, 37%); mp: 104–

1

1058C (Et₂O/hexane; lit.:^[29]104–1068C); H NMR (400 MHz, CDCl₃):

3-(5-carboxy-2-oxo-1,2-dihydropyridin-3-yl)benzenaminium bro-

mide (26b): Methyl 6-methoxy-5-(3-aminophenyl)nicotinate 25b

(7.0 mg, 27 mmol) was dissolved in 48% HBr solution (0.6 mL) and

glacial AcOH (0.6 mL) and the mixture was heated at reflux for 1 h.

After cooling to 258C, the solvent was removed under reduced

pressure. The resulting solid was collected by filtration, washed

with Et₂O (1 mL) to extract an orange impurity, and dried to give

the desired pyridone 26b was obtained as a light-brown solid

(6.0 mg, 71%). ¹H NMR (400 MHz, D₂O+NaOD): d=8.35 (d, J=

2.5 Hz, 1H), 7.86 (d, J=2.5 Hz, 1H), 7.26 (t, J=7.8 Hz, 1H), 6.96–

7.01 (m, 2H), 6.79 ppm (brd, J=7.8 Hz, 1H); ¹³C NMR (125 MHz,

D₂O+NaOD): d=175.1, 170.6, 149.1, 145.8, 140.6, 138.8, 129.2,

125.2, 120.2, 117.8, 116.8, 114.9 ppm; IR (ATR): n˜_max=3421, 3368,

3200, 2500, 1721, 1636 cm^ꢁ1; LCMS-ES m/z 231 [M+H]⁺, 229

[MꢁH]^ꢁ; HRMS-ES m/z [M+H]⁺calcd for C₁₂H₁₀N₂O₃231.0764,

found 231.0761.

d=8.74 (d, J=2.1 Hz, 1H), 8.39 (d, J=2.1 Hz, 1H), 4.08 (s, 3H),

3.92 ppm (s, 3H); ¹³C NMR (100 MHz, CDCl₃): d=164.9, 162.9, 148.2,

142.4, 121.1, 106.8, 55.3, 52.4 ppm; IR (ATR): n˜_max=3066, 3022,

2954, 1719 cm^ꢁ1; LCMS-ES m/z 246, 248 [M+H]⁺. These data are in

agreement with those previously reported.^[29]

Methyl 6-methoxy-5-(3-nitrophenyl)nicotinate (25a): Methyl 5-

bromo-6-methoxynicotinate 24 (369 mg, 1.5 mmol), 3-nitrophenyl-

boronic acid (376 mg, 2.25 mmol), Et₃N (0.63 mL, 4.5 mmol),

Pd(OAc)₂(10 mg, 45 mmol), and PPh₃(24 mg, 93 mmol) in DMF

(6 mL) were heated at 1008C under argon for 24 h. Additional 3-ni-

trophenylboronic acid (84 mg, 0.5 mmol), Pd(OAc)₂(10 mg,

45 mmol) and PPh₃(24 mg, 93 mmol) were added and the heating

was continued for 4 h. After cooling to 258C, the solvent was re-

moved under reduced pressure (over a water bath at 608C). The

residue was partitioned between CH₂Cl₂(50 mL) and saturated

NaHCO₃solution (50 mL). The organic layer was washed with satu-

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Methyl 7-hydroxy-2,2-diphenylbenzo[d][1,3]dioxole-5-carboxyl-

ate (28): Methyl 3,4,5-trihydroxybenzoate 27 (2.00 g, 10.86 mmol),

a,a-dichlorodiphenylmethane (3.35 g, 14.12 mmol), and K₂CO₃

(4.50 g, 32.58 mmol) in anhydrous MeCN (30 mL) were stirred at

room temperature under N₂for 24 h. The mixture was diluted with

saturated NH₄Cl (75 mL) and H₂O (75 mL), then extracted with

EtOAc (2ꢂ100 mL). The combined organics were washed with sa-

turated brine (50 mL), dried over Na₂SO₄, filtered, and concentrated

under reduced pressure. The residue was purified by flash silica

chromatography (Biotage Isolera). Column 1, 50 g column,

40 mLmin^ꢁ1, gradient: 5% EtOAc/hexane 1 cv; 5!25% EtOAc

5 cv; 25% EtOAc/hexane 10 cv; Column 2, 50 g column,

40 mLmin^ꢁ1, gradient: 1% MeOH/CH₂Cl₂1 cv; 1!10% MeOH

10 cv, 10% MeOH/CH₂Cl₂2 cv, to give product 28, a white solid

MeOH/CH₂Cl₂1 cv, 2!20% MeOH 10 cv, 20% MeOH/CH₂Cl₂2 cv,

to give product 29c as a white solid (355 mg, 51%). ¹H NMR

(400 MHz, CDCl₃): d=8.32 (s, 1H), 8.16 (m, 1H), 7.75 (dd, J=0.4,

7.6 Hz, dd), 7.54–7.59 (m, 5H), 7.36–7.42 (m, 7H), 7.30 (d, J=1.4 Hz,

1H), 5.32 (s, 2H), 3.86 ppm (s, 3H); ¹³C NMR (100 MHz, CDCl₃): d=

166.6, 149.1, 148.8, 141.8, 140.1, 139.8, 133.7, 130.0, 129.9, 128.7,

126.7, 124.9, 123.5, 122.8, 119.2, 113.3, 105.1, 70.9 ppm; IR (ATR):

n˜_max=3675, 2988, 2901, 1716, 1632 cm^ꢁ1; LCMS-ES m/z 484 [M+

H]⁺; HRMS-ES m/z [M+H]⁺calcd for C₂₈H₂₂NO₇484.1391, found

484.1382.

Methyl 7-(benzyloxy)-2,2-diphenylbenzo[d][1,3]dioxole-5-carbox-

ylate (29g): Methyl 7-hydroxy-2,2-diphenylbenzo[d][1,3]dioxole-5-

carboxylate 28 (107 mg, 0.307 mmol), benzyl bromide (37 mL,

0.311 mmol), and Cs₂CO₃(200 mg, 0.614 mmol) in anhydrous DMF

(3 mL) were stirred at room temperature under N₂for 18 h. The

mixture was diluted with saturated NH₄Cl (10 mL) and H₂O (10 mL),

and extracted with EtOAc (2ꢂ15 mL). The combined organics were

dried over Na₂SO₄, filtered, and concentrated under reduced pres-

sure to ~1.5 mL. The sample was purified by flash silica chroma-

tography (Biotage Isolera). Column, 10 g, 12 mLmin^ꢁ1, gradient:

5% EtOAc/hexane 1 cv, 5!80% EtOAc 10 cv, 80% EtOAc/hexane

2 cv, to give the product 29g as a white solid (56 mg, 41%).

¹H NMR (400 MHz, CDCl₃): d=7.57–7.61 (m, 4H), 7.45 (m, 2H),

7.37–7.41 (m, 8H), 7.33–7.36 (m, 2H), 7.29 (d, J=1.5 Hz, 1H), 5.25

(s, 2H), 3.86 ppm (s, 3H); ¹³C NMR (100 MHz, CDCl₃): d=166.8,

148.9, 142.5, 140.1, 140.0, 136.9, 129.7, 129.0, 128.7, 128.6, 128.1,

1

(1.74 g, 46%). H NMR (400 MHz, CDCl₃): d=7.50–7.55 (m, 4H), 7.41

(d, J=1.5 Hz, 1H), 7.26–7.31 (m, 6H), 7.18 (d, J=1.5 Hz, 1H),

3.77 ppm (s, 3H); ¹³C NMR (100 MHz, CDCl₃): d=167.4, 148.8, 139.9,

139.6, 138.7, 129.8, 128.7, 126.7, 124.5, 119.1, 114.6, 103.7,

52.7 ppm; IR (ATR): n˜_max=3670, 3387, 2988, 1697, 1643 cm^ꢁ1

;

LCMS-ES m/z 349 [M+H]⁺, 347 [MꢁH]^ꢁ; HRMS-ES m/z [M+H]⁺

calcd for C₂₁H₁₇O₅349.1076, found 349.1083.

Methyl 7-(3-nitrophenoxy)-2,2-diphenylbenzo[d][1,3]dioxole-5-

carboxylate (29c): Methyl 7-hydroxy-2,2-diphenylbenzo[d]-

[1,3]dioxole-5-carboxylate 28 (125 mg, 0.359 mmol), and 1-fluoro-3-

nitrobenzene (46 mL, 0.432 mmol) were dissolved in anhydrous

DMF (1 mL) under argon. Trimethylsilyl diethylamine (165 mL,

0.871 mmol) was added, followed by tBu-P4 phosphazene base

(1m in hexane; 45 mL, 0.045 mmol) dropwise over a period of

1 min (N.B. phosphazene solution warmed to 458C prior to use).

The reaction was heated at 1008C for 17 h, then allowed to cool to

room temperature. The reaction was diluted with saturated NH₄Cl

(10 mL) and H₂O (10 mL), and extracted with EtOAc (2ꢂ10 mL).

The combined organics were washed with H₂O (20 mL), saturated

brine (20 mL), dried over Na₂SO₄, filtered, and concentrated under

reduced pressure. The residue was purified by flash silica chroma-

tography (Biotage Isolera), 10 g column, 12 mLmin^ꢁ1, gradient: 5%

EtOAc/hexane 1 cv; 5!40% EtOAc 10 cv, 40% EtOAc/hexane 3 cv,

to give the product 29c as a yellow solid (13 mg, 26% based on

126.8, 124.7, 118.9, 113.2, 104.6, 72.1, 52.5 ppm; IR (ATR): n˜_max

=

3670, 2989, 2901, 1710, 1634 cm^ꢁ1;LCMS-ES m/z 439 [M+H]⁺;

HRMS-ES m/z [M+H]⁺calcd for C₂₈H₂₃O₅439.1545, found

439.1555.

7-(3-Nitrophenoxy)-2,2-diphenylbenzo[d][1,3]dioxole-5-carboxyl-

ic acid (30c): Methyl 7-(3-nitrophenoxy)-2,2-diphenylbenzo[d]-

[1,3]dioxole-5-carboxylate 29c (22 mg, 0.047 mmol) was dissolved

in 2.5m NaOH solution (1 mL) and THF (5 mL), and heated at 408C

for 24 h. The reaction was allowed to cool to room temperature,

and THF removed under reduced pressure. The remaining solution

was diluted with H₂O (10 mL), and extracted with EtOAc (2ꢂ

10 mL). The combined organics were dried over Na₂SO₄, filtered

and concentrated under reduced pressure. The residue was puri-

fied by flash silica chromatography (Biotage Isolera), 10 g column,

15 mLmin^ꢁ1, gradient: 1% MeOH/CH₂Cl₂1 cv, 1!15% MeOH

10 cv, 15% MeOH/CH₂Cl₂2 cv, to give the product 30c as a white

solid (13 mg, 75% based on recovery of 4 mg starting material).

¹H NMR (400 MHz, CDCl₃): d=7.98 (ddd, J=0.8, 2.0, 8.1 Hz, 1H),

7.77 (t, J=2.3 Hz, 1H), 7.58 (m, 1H), 7.43–7.47 (m, 5H), 7.35–7.40

(m, 7H), 7.22 ppm (d, J=1.5 Hz, 1H); ¹³C NMR (100 MHz,

[D₆]DMSO): d=167.3, 157.5, 149.5, 148.2, 143.6, 139.9, 138.5, 137.6,

132.1, 130.5, 130.2, 129.3, 126.5, 119.6, 119.2, 118.4, 117.9, 112.4,

110.5, 107.5, 104.0 ppm; IR (ATR): n˜_max=2932, 1684, 1631, 1608 cm

^ꢁ1; LCMS-ES m/z 456 [M+H]⁺, 454 [MꢁH]^ꢁ; HRMS-ES m/z [M+H]⁺

calcd for C₂₆H₁₈NO₇456.1083, found 456.1086.

1

recovery of 88 mg starting material 28). H NMR (400 MHz, CDCl₃):

d=8.11 (t, J=1.0 Hz, 1H), 7.97 (ddd, J=0.9, 2.1, 8.1 Hz, 1H), 7.65 (t,

J=8.4 Hz, 1H), 7.55–7.60 (m, 5H), 7.35–7.42 (m, 7H), 7.22 (d, J=

1.5 Hz, 1H), 3.87 ppm (s, 3H); ¹³C NMR (100 MHz, CDCl₃): d=168.0,

165.8, 157.7, 149.6, 139.0, 137.0, 130.4, 129.7, 129.4, 128.4, 126.4,

126.3, 125.3, 123.3, 118.1, 112.1, 110.0, 107.3, 52.4 ppm; IR (ATR):

n˜_max=3385, 1696, 1643, 1610 cm^ꢁ1; LCMS-ES m/z 470 [M+H]⁺;

HRMS-ES m/z [M+H]⁺calcd for C₂₇H₂₀NO₇470.1240, found

470.1254.

Methyl 7-(3-nitrobenzyloxy)-2,2-diphenylbenzo[d][1,3]dioxole-5-

carboxylate (29d): NaH (60% in mineral oil; 60 mg, 1.5 mmol) was

washed with hexane (4 mL) under N₂. A solution of methyl 7-hy-

droxy-2,2-diphenylbenzo[d][1,3]dioxole-5-carboxylate 28 (400 mg,

1.448 mmol) in anhydrous THF (7.5 mL) was added and stirred

under N₂at room temperature for 5 min. A solution of 3-nitroben-

zyl bromide (312 mg, 1.444 mmol) in anhydrous THF (7.5 mL) was

added dropwise over a period of 3 min. The reaction was stirred at

room temperature for 7 h, then 508C for 16 h. The reaction was

cooled to room temperature, diluted with saturated brine (100 mL)

and H₂O (100 mL), and extracted with EtOAc (2ꢂ100 mL). The com-

bined organics were washed with saturated brine (150 mL), dried

over Na₂SO₄, filtered through Celite, and concentrated under re-

duced pressure. The residue was purified by flash silica chromatog-

raphy (Biotage Isolera), 25 g column, 25 mLmin^ꢁ1, gradient: 2%

7-(3-Nitrobenzyloxy)-2,2-diphenylbenzo[d][1,3]dioxole-5-carbox-

ylate (30d): Methyl 7-(3-nitrobenzyloxy)-2,2-diphenylbenzo[d]-

[1,3]dioxole-5-carboxylate 29d (77 mg, 0.159 mmol) was dissolved

in 2.5m NaOH solution (1 mL) and THF (7 mL), and heated at 408C

for 16 h. The reaction was allowed to cool to room temperature,

acidified with 1n HCl (9 mL), and extracted with EtOAc (2ꢂ25 mL).

The combined organics were washed with saturated brine (25 mL),

dried over Na₂SO₄, filtered, and concentrated under reduced pres-

sure. The residue was purified by flash silica chromatography (Biot-

age Isolera), 10 g column, 12 mLmin^ꢁ1, gradient: 5% EtOAc/hexane

1 cv, 5!40% EtOAc 10 cv, 40% EtOAc/hexane 3 cv, then 5!20%

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MeOH/CH₂Cl₂5 cv, to give product 30d as a white solid (8 mg,

30% based on recovery of 50 mg starting material). ¹H NMR

(400 MHz, CDCl₃): d=8.33 (s, 1H), 8.18 (d, J=8.1 Hz, 1H), 7.76 (d,

J=7.5 Hz, 1H), 7.53–7.59 (m, 5H), 7.33–7.46 (m, 8H), 5.33 ppm (s,

2H); ¹³C NMR (100 MHz, CDCl₃): d=167.0, 148.4, 148.1, 141.2,

139.4, 139.2, 138.6, 133.2, 129.4, 129.1, 128.1, 126.0, 124.3, 122.7,

122.0, 118.4, 112.9, 104.6, 70.2 ppm; IR (ATR): n˜_max=3677, 2919,

1683, 1636 cm^ꢁ1;LCMS-ES m/z 470 [M+H]⁺, 468 [MꢁH]^ꢁ; HRMS-ES

m/z [MꢁH]^ꢁcalcd for C₂₇H₁₈NO₇468.1089, found 468.1104.

116.3, 116.0 ppm; IR (ATR): n˜_max=3020, 1778, 1694, 1614, 1573,

1534, 1489, 1450, 1392 cm^ꢁ1; LCMS-ES m/z 168 [MꢁH]^ꢁ; HRMS-ES

m/z [M+H]⁺calcd for C₇H₈NO₄170.0453, found 170.0462.

3,4-Dihydroxy-5-(3-nitrophenoxy)benzoic acid (35c): 7-(3-Nitro-

phenoxy)-2,2-diphenylbenzo[d][1,3]dioxole-5-carboxylic acid 30c

(13 mg, 0.028 mmol) was dissolved in CH₂Cl₂(1 mL) and TFA

(1 mL), and stirred for 3 h. The reaction was concentrated under re-

duced pressure, and the residue was purified by HPLC, column D,

solvent A: H₂O, solvent B: MeCN, gradient: 5% B 1 cv, 5!95% B

6 cv, 95% B 1 cv, 95!5% B 1 cv, 5% B 2 cv. The sample containing

fractions were pooled, flash-frozen in liquid N₂, and lyophilized to

give the product 31c as a white solid (6 mg, 73%). ¹H NMR

(400 MHz, [D₆]DMSO): d=10.04 (brs, 1H), 9.72 (brs, 1H), 7.91 (ddd,

J=0.8, 2.2, 8.2 Hz, 1H), 7.62 (t, J=8.2 Hz, 1H), 7.56 (t, J=2.3 Hz,

1H), 7.38 (ddd, J=0.8, 2.2, 8.2 Hz, 1H), 7.36 (d, J=2.0 Hz, 1H),

7.13 ppm (d, J=2.3 Hz, 1H); ¹³C NMR (125 MHz, [D₆]DMSO): d=

166.7, 158.4, 148.8, 147.0, 142.6, 141.6, 131.2, 123.0, 121.5, 117.1,

114.5, 113.9, 110.3 ppm; IR (ATR): n˜_max=3280, 1665, 1605,

1521 cm^ꢁ1; LCMS-ES m/z 290 [MꢁH]^ꢁ; HRMS-ES m/z [MꢁH]^ꢁcalcd

for C₁₃H₈NO₇290.0295, found 290.0300.

7-(Benzyloxy)-2,2-diphenylbenzo[d][1,3]dioxole-5-carboxylate

(30g): Methyl 7-(benzyloxy)-2,2-diphenylbenzo[d][1,3]dioxole-5-car-

boxylate 29g (56 mg, 0.128 mmol) was suspended in 100 mm KOH

(5.12 mL, 0.512 mmol) and THF (3 mL), and heated at 608C for 6 h.

The reaction was allowed to cool to room temperature, acidified

with 3n HCl (10 mL), and extracted with EtOAc (2ꢂ20 mL). The

combined organics were dried over Na₂SO₄, filtered, and concen-

trated under reduced pressure. The residue was purified by flash

silica chromatography (Biotage Isolera), 10 g column, 12 mLmin^ꢁ1

,

gradient: 1% MeOH/CH₂Cl₂1 cv; 1!15% MeOH 10 cv, 15%

MeOH/CH₂Cl₂2 cv, to give the product 30g (15 mg, 65% based on

1

recovery of 32 mg starting material). H NMR (400 MHz, CDCl₃): d=

3,4-Dihydroxy-5-((3-nitrobenzyl)oxy)benzoic acid (35d): 7-(3-Ni-

trobenzyloxy)-2,2-diphenylbenzo[d][1,3]dioxole-5-carboxylate 30d

(8 mg, 0.017 mmol) was dissolved in CH₂Cl₂(1 mL) and TFA (1 mL)

and stirred at room temperature for 3 h. The solvents were re-

moved under reduced pressure, and the residue was purified by

HPLC, column D, solvent A: H₂O, solvent B: MeCN, gradient: 5% B

1 cv, 5!95% B 6 cv, 95% B 1 cv, 95!5% B 1 cv, 5% B 2 cv, to give

product 31d as a white solid (5 mg, 96%). ¹H NMR (400 MHz,

CD₃OD): d=8.42 (s, 1H), 8.21 (d, J=8.1 Hz, 1H), 7.91 (d, J=7.6 Hz,

1H), 7.66 (d, J=8.0 Hz, 1H), 7.24 (d, J=1.9 Hz, 1H), 7.23 (d, J=

1.8 Hz, 1H), 5.32 ppm (s, 2H); ¹³C NMR (100 MHz, [D₆]DMSO): d=

167.6, 148.2, 146.7, 146.0, 140.1, 140.0, 134.3, 130.2, 122.9, 122.2,

120.9, 111.5, 107.3, 69.3 ppm; IR (ATR): n˜_max=3205, 1685,

1606 cm^ꢁ1; LCMS-ES m/z 304 [MꢁH]^ꢁ; HRMS-ES m/z [MꢁH]^ꢁcalcd

for C₁₄H₁₀NO₇304.0463, found 304.0474.

7.54–7.62 (m, 4H), 7.42–7.48 (m, 3H), 7.38 (m, 8H), 7.33 (m, 2H),

5.25 ppm (s, 2H); ¹³C NMR (100 MHz, CDCl₃): d=166.3, 158.0, 157.4,

148.8, 142.7, 140.2, 139.8, 139.5, 130.4, 130.3, 129.8, 128.8, 126.5,

125.4, 123.8, 122.8, 119.3, 118.7, 118.5, 118.1, 113.3, 104.3,

71.2 ppm; IR (ATR): n˜_max=3488, 2524, 2159, 2030, 1679, 1628 cm^ꢁ1

;

LCMS-ES m/z 425 [M+H]⁺, 423 [MꢁH]^ꢁ; HRMS-ES m/z [M+H]⁺

calcd for C₂₇H₂₁O₅425.1389, found 425.1401.

Methyl 4-hydroxy-3-methoxy-5-nitrobenzoate (32): Methyl vanil-

late 31 (1.0 g, 5.5 mmol) was dissolved in AcOH (5 mL) at 08C. A

mixture of fuming nitric acid (0.4 mL) and AcOH (2 mL) was added

slowly. The reaction mixture was allowed to warm to room temper-

ature and stirred for 3 h. The resulted precipitate was filtered and

recrystallized from EtOAc to give the product 32 as a yellow solid

(725 mg, 58%). ¹H NMR (400 MHz, CDCl₃): d=8.39 (s, 1H) 7.71 (s,

1H), 3.95 (s, 3H), 3.81 ppm (s, 3H); ¹³C NMR (100 MHz, CDCl₃): d=

165.4, 150.4, 150.0, 133.8, 121.7, 118.8, 117.7, 57.0, 53.2 ppm; IR

(ATR): n˜_max=3263, 3079, 3007, 2952, 2928, 1715, 1617, 1539, 1439,

1375 cm^ꢁ1; LCMS-ES m/z 228 [M+H]⁺; HRMS-ES m/z [M+H]⁺

calcd for C₉H₁₀NO₆228.0503, found 228.0518.

3,4-Dihydroxy-5-((3-nitrobenzyl)amino)benzoic acid (35e): 3-Ni-

trobenzaldehyde (67 mg, 0.44 mmol) and crude 3-amino-4,5-dihy-

droxybenzoic acid 34 (100 mg, 0.59 mmol) were stirred in anhy-

drous DMF (1 mL) containing 1% AcOH and molecular sieves (3 ꢁ)

under nitrogen for 18 h. NaBH₃CN (80 mg, 1.27 mmol) was added,

and reaction stirred for 1 h then quenched by the addition of H₂O

(100 mL). The molecular sieves were removed by filtration, and the

filtrate evaporated in vacuo to dryness. The crude product was pu-

rified by flash silica chromatography (CHCl₃/MeOH/AcOH/H₂O,

120:15:3:2 v/v/v/v) to give the product 35e as a brown solid

(120 mg, 91%). ¹H NMR (400 MHz, [D₆]DMSO): d=11.98 (brs, 1H)

8.21 (s, 1H), 8.16 (d, J=1.2 Hz, 1H), 7.81 (d, J=1.1 Hz, 1H), 7.61 (t,

J=2.3 Hz, 1H), 6.83 (s, 1H), 6.52 (s, 1H), 5.78 (brs, 2H), 4.48 (s, 2H),

4.12 ppm (brs, 1H); ¹³C NMR (100 MHz, [D₆]DMSO): d=172.3,

168.7, 128.2, 145.6, 144.2, 136.1, 134.0, 130.1, 122.0, 107.2, 103.8,

46.1 ppm; IR (ATR): n˜_max=3455, 3441, 3273, 3097, 1675, 1621, 1595,

1523, 1458, 1427, 1382 cm^ꢁ1; LCMS-ES m/z 305 [M+H]⁺; HRMS-ES

m/z [M+H]⁺calcd for C₁₄H₁₃N₂O₆305.0774, found 305.0798.

3,4-Dihydroxy-5-nitrobenzoic acid (33): Methyl 4-hydroxy-3-me-

thoxy-5-nitrobenzoate 32 (500 mg, 2.2 mmol) was dissolved in

48% HBr aqueous solution (20 mL) and heated at reflux until

a clear solution was observed. After complete demethylation (TLC

monitoring), the solution was cooled on ice, and the resulted pre-

cipitate was filtered, washed with H₂O and dried to give the crude

product 33 as a yellow solid (195 mg, 45%). ¹H NMR (400 MHz,

[D₆]DMSO): d=8.01 (s, 1H), 7.83 (s, 1H), 5.31 ppm (s, 2H); ¹³C NMR

(100 MHz, [D₆]DMSO): d=168.7, 149.6, 146.0, 139.5, 125.1, 119.3,

118.3 ppm; IR (ATR): n˜_max=3537, 3088, 2822, 2648, 2523, 1791,

1682, 1616, 1588, 1542, 1455, 1407 cm^ꢁ1; LCMS-ES m/z 200 [M+

H]⁺; HRMS-ES m/z [M+H]⁺calcd for C₇H₆NO₆200.0190, found

200.0203.

3-Amino-4,5-dihydroxybenzoic acid (34): Crude 3,4-dihydroxy-5-

nitrobenzoic acid 33 (100 mg, 0.5 mmol) was dissolved in MeOH

(1 mL) and Pd/C (20 mg, 10 wt% Pd) was added. The mixture was

stirred under hydrogen for 18 h. The mixture was filtered, and the

filtrate was dried in vacuo to give the crude product 34 as a grey

solid (83 mg, 98%). ¹H NMR (400 MHz, [D₆]DMSO): d=10.56 (br,

1H), 7.49 (s, 1H), 7.45 (s, 1H), 3.99 (s, 2H), 3.76 ppm (s, 2H);

¹³C NMR (100 MHz, [D₆]DMSO): d=166.7, 146.1, 144.0, 121.7, 119.2,

3-(Benzyloxy)-4,5-dihydroxybenzoic acid (35g): 7-(Benzyloxy)-2,2-

diphenylbenzo[d][1,3]dioxole-5-carboxylate

30g

(15 mg,

0.035 mmol) was dissolved in CH₂Cl₂(1.5 mL), and TFA (50 mL) was

added. The reaction was stirred at room temperature for 2 h. Addi-

tional TFA (50 mL) was added, and the reaction was stirred for a fur-

ther 2 h. The solvents were removed under reduced pressure, and

the residue purified by flash silica chromatography (Biotage Iso-

lera), 10 g column, 12 mLmin^ꢁ1, gradient: 2% MeOH/CH₂Cl₂1 cv;

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2!20% MeOH 10 cv, 20% MeOH/CH₂Cl₂2 cv, to give product 31g

tracted with EtOAc (4ꢂ20 mL) and the combined organic phases

were washed with saturated NaHCO₃solution (80 mL), saturated

brine (80 mL), and dried over MgSO₄. The product 39a was puri-

fied by column chromatography, eluting with 2:1 v/v PE/EtOAc,

and collected as an off-white solid (160 mg, 88%). ¹H NMR

(400 MHz, [D₆]DMSO): d=8.39 (t, J=2.0 Hz, 1H), 8.25 (ddd, J=1.0,

2.0, 8.0 Hz, 1H), 8.12 (ddd, J=1.0, 2.0, 8.0 Hz, 1H), 7.77 (t, J=

8.0 Hz, 1H), 7.71 (t, J=1.6 Hz, 1H), 7.43 (dd, J=1.6, 2.4 Hz, 1H),

7.39 (dd, J=1.6, 2.4 Hz, 1H), 3.88 ppm (s, 3H); ¹³C NMR (100 MHz,

[D₆]DMSO): d=165.8, 158.2, 148.3, 140.7, 139.7, 133.3, 131.7, 130.5,

122.5, 121.1, 118.5, 118.2, 115.7, 52.2 ppm; IR (ATR): n˜_max=3358,

2953, 2855, 1686, 1600, 1526, 1485, 1455, 1433, 1413, 1332,

1314 cm^ꢁ1; LCMS-ES m/z 274 [M+H]⁺, 272 [MꢁH]^ꢁ; HRMS-ES m/z

[M]⁺calcd for C₁₄H₁₁NO₅273.0632, found 273.0629.

1

as a white solid (8 mg, 88%). H NMR (400 MHz, CD₃OD): d=7.46–

7.53 (m, 2H), 7.35–7.42 (m, 2H), 7.32 (d, J=7.2 Hz, 1H), 7.24 (d, J=

1.9 Hz, 1H), 7.20 (d, J=1.9 Hz, 1H), 5.19 ppm (s, 2H); ¹³C NMR

(100 MHz, [D₆]DMSO): d=167.6, 147.1, 145.8, 139.9, 137.6, 128.7,

128.1, 127.8, 120.9, 111.1, 107.2, 70.4 ppm; IR (ATR): n˜_max=3398,

2926, 1648, 1614 cm^ꢁ1; LCMS-ES m/z 259 [MꢁH]^ꢁ; HRMS-ES m/z

[MꢁH]^ꢁcalcd for C₁₄H₁₁O₅259.0612, found 259.0619.

Methyl 3-acetoxy-5-hydroxybenzoate (37):^[24]Acetyl chloride

(1.10 mL, 15.2 mmol) was added dropwise to a cooled solution of

methyl 3,5-dihydroxybenzoate 36 (2.56 g, 15.2 mmol) in dry pyri-

dine (10 mL) at 08C. The reaction mixture was stirred for 1 h at 08C

and then at 258C for a further 1 h. The reaction mixture was dilut-

ed with EtOAc (30 mL) before being washed with saturated NH₄Cl

solution (2ꢂ20 mL), brine (20 mL), and dried (MgSO₄). The solvent

was removed under reduced pressure to give the crude mixture of

the mono- and di-acylated methyl ester as a white solid. The

mono-acylated product was purified by flash column chromatogra-

phy (initially eluting with 4:1 v/v PE/EtOAc; switching to progres-

sively more polar solvent systems as each product was collected).

The mono-acylated product 37 was collected as a white solid

(0.86 g, 27%). Some di-acylated product was collected as a white

Methyl 3-hydroxy-5-(3-nitrophenoxy)benzoate (39c): Methyl 3,5-

dihydroxybenzoate 36 (250 mg, 1.49 mmol) was dissolved in anhy-

drous DMF (2 mL) under argon. 1-Fluoro-3-nitrobenzene (160 mL,

1.50 mmol) was added, followed by trimethylsilyl diethylamine

(570 mL, 3.01 mmol), and tBu-P4 phosphazene base (1m in hexane;

150 mL, 0.15 mmol) dropwise over a period of 1 min (N.B. phospha-

zene solution warmed to 458C prior to use). The reaction was

heated at 1008C for 17 h, then allowed to cool to room tempera-

ture. The reaction was diluted with saturated NH₄Cl (10 mL) and

H₂O (10 mL), and extracted with EtOAc (2ꢂ10 mL). The combined

organics were washed with H₂O (20 mL), saturated brine (20 mL),

dried over Na₂SO₄, filtered, and concentrated under reduced pres-

sure. The residue was purified by flash silica chromatography (Biot-

age Isolera), 25 g column, 25 mLmin^ꢁ1, gradient: 2% MeOH/CH₂Cl₂

1 cv, 2!20% MeOH 10 cv, 20% MeOH/CH₂Cl₂2 cv, to give the

product 39c as a yellow solid (53 mg, 12%). ¹H NMR (400 MHz,

CDCl₃): d=7.99 (ddd, J=0.9, 2.1, 8.2 Hz, 1H), 7.82 (t, J=2.3 Hz,

1H), 7.52 (t, J=8.2 Hz, 1H), 7.36 (m, 2H), 7.26 (m, 1H), 6.77 (t, J=

2.3 Hz, 1H), 3.90 ppm (s, 3H); ¹³C NMR (100 MHz, CDCl₃): d=163.5,

159.5, 158.3, 157.0, 149.6, 132.0, 130.8, 124.8, 118.3, 113.6, 112.3,

1

solid (1.00 g, 24%). H NMR (400 MHz, CD₃OD): d=7.30 (dd, J=1.5,

2.3 Hz, 1H), 7.19 (dd, J=1.5, 2.3 Hz, 1H), 6.77 (t, J=2.3 Hz, 1H),

3.87 (s, 3H), 2.26 ppm (s, 3H); ¹³C NMR (100 MHz, CD₃OD): d=

170.9, 167.7, 159.8, 153.1, 133.2, 114.8, 114.7, 52.8, 20.9 ppm; IR

(ATR): n˜_max=3283, 3128, 3087, 2962, 1757, 1695, 1601, 1547,

1500 cm^ꢁ1; LCMS-ES m/z 209 [MꢁH]^ꢁ; HRMS-ES m/z [M+NH₄]⁺

calcd for C₁₀H₁₄NO₅228.0866, found 228.0866.

Methyl 3-acetoxy-5-trifluromethanesulfonyloxybenzoate (38):

Methyl 3-acetoxy-5-hydroxybenzoate 37 (0.65 g, 3.0 mmol) and

pyridine (0.74 mL, 9.3 mmol) were dissolved in THF (15 mL) under

argon, and the colorless solution cooled to 08C. Trifluoromethane-

sulfonic anhydride (0.66 mL, 3.9 mmol) was added dropwise over

1 min and the reaction mixture stirred for a further 10 min which

gave a yellow solution and a white precipitate. The precipitate was

filtered off under reduced pressure, washed on the filter with THF

(15 mL) and discarded. THF was removed from the filtrate under

reduced pressure and the crude product was re-dissolved in EtOAc

(40 mL) and washed consecutively with saturated NaHCO₃solution

(40 mL), 1n HCl solution (40 mL), saturated NaHCO₃solution

(40 mL), saturated brine solution (40 mL), and dried over MgSO₄.

The solvent was removed under reduced pressure to give the

crude product as an off-white solid, which was then purified by

column chromatography, eluting with 3:1 then 1:1 v/v PE/EtOAc.

108.7, 108.0, 52.4 ppm; IR (ATR): n˜_max=3098, 1718, 1585 cm^ꢁ1

LCMS-ES m/z 288 [MꢁH]^ꢁ

;

5-Methyl

3-hydroxy-5-((3-nitrobenzyl)oxy)benzoate

(39d):

Methyl 3,5-dihydroxybenzoate 36 (250 mg, 1.49 mmol), and NaH

(60% in mineral oil; 60 mg, 1.49 mmol) were cooled to 08C under

N₂. Anhydrous THF (5 mL) was added and mixture stirred for

10 min. A solution of 3-nitrobenzyl bromide (322 mg, 1.49 mmol)

in anhydrous THF (5 mL) was added dropwise over a period of

5 min. The mixture was stirred at 08C for 15 min, then allowed to

warm to room temperature and stirred for 17 h. Very little reaction

was observed by TLC. An additional 60 mg of NaH (60% in mineral

oil) was added, and reaction stirred for a further 8 h. The reaction

was acidified with 1n HCl (15 mL), and extracted with EtOAc (2ꢂ

15 mL). The combined organics were washed with saturated brine

(15 mL), dried over Na₂SO₄, filtered, and concentrated under re-

duced pressure. The residue was purified by flash silica chromatog-

raphy (Biotage Isolera), 25 g column, 25 mLmin^ꢁ1, gradient: 1%

MeOH/CH₂Cl₂1 cv; 1!20% MeOH 10 cv; 20% MeOH/CH₂Cl₂3 cv,

to give the product 39d as a yellow solid (21 mg, 6% based on re-

covery of 56 mg starting material). ¹H NMR (400 MHz, CDCl₃): d=

8.32 (s, 1H), 8.20 (dd, J=1.3, 8.1 Hz, 1H), 7.76 (d, J=7.6 Hz, 1H),

7.58 (t, J=7.9 Hz, 1H), 7.24 (dd, J=1.3, 2.3 Hz, 1H), 7.17 (dd, J=

1.3, 2.3 Hz, 1H), 6.70 (t, J=2.3 Hz, 1H), 5.17 (s, 2H), 3.91 ppm (s,

3H); ¹³C NMR (100 MHz, CDCl₃): d=166.2, 158.4, 156.9, 147.5,

137.9, 132.3, 131.2, 128.7, 122.1, 121.2, 109.4, 106.7, 106.4, 67.9,

51.5 ppm; IR (ATR): n˜_max=3678, 3075, 2924, 1722 cm^ꢁ1; LCMS-ES m/

z 304 [M+H]⁺, 302 [MꢁH]^ꢁ; HRMS-ES m/z [MꢁH]^ꢁcalcd for

C₁₅H₁₂NO₆302.0670, found 302.0679.

1

The triflate 38 was collected as a yellow oil (0.40 g, 64%). H NMR

(400 MHz, CDCl₃): d=7.84 (dd, J=1.3, 2.3 Hz, 1H), 7.81 (dd, J=1.3,

2.3 Hz, 1H), 7.31 (t, J=2.3 Hz, 1H), 3.95 (s, 3H), 2.34 ppm (s, 3H);

¹³C NMR (100 MHz, CDCl₃): d=168.3, 164.3, 151.4, 149.2, 133.2,

123.0, 119.8, 118.7 (q, ¹J_CF=323 Hz, CF₃), 52.9, 20.9 ppm; ¹⁹F NMR

(376 MHz, CDCl₃): d=ꢁ72.9 ppm; IR (ATR): n˜_max=3093, 3026, 2958,

2845, 1777, 1730, 1615, 1595, 1464, 1424, 1370, 1185 cm^ꢁ1; LCMS-

ES m/z 341 [MꢁH]^ꢁ; HRMS-ES m/z [M+NH₄]⁺calcd for

C₁₁H₁₃F₃NO₇S 360.0359, found 360.0359.

Methyl 5-hydroxy-3’-nitro-[1,1’-biphenyl]-3-carboxylate (39a):

Methyl

3-acetoxy-5-trifluromethanesulfonyloxybenzoate

35

(200 mg, 0.58 mmol) and 3-nitrophenylboronic acid (167 mg,

1.0 mmol), 2m Na₂CO₃solution (0.45 mL, 0.90 mmol) and tetrakis(-

triphenylphosphine)palladium (19 mg, 16 mmol) were dissolved se-

quentially in 1,2-dimethoxyethane (2.2 mL) under argon. The reac-

tion mixture was heated under reflux for 19 h before cooling to

258C and H₂O (20 mL) was added. The reaction mixture was ex-

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Hydroxy-3’-nitro-[1,1’-biphenyl]-3-carboxylic acid (41a): KOH

(38 mg, 0.68 mmol) in H₂O (1 mL) was added dropwise to a solution

of methyl 5-hydroxy-3’-nitro-[1,1’-biphenyl]-3-carboxylate 39a

(75 mg, 0.27 mmol) in THF (1 mL). The reaction mixture was stirred

for 19 h at 258C. The reaction mixture was diluted with 1m NaOH

(10 mL) and washed with EtOAc (3ꢂ10 mL) before the aqueous

phase was acidified by careful addition of 1n HCl, and then ex-

tracted with EtOAc (3ꢂ70 mL). The organic phase was then

washed with brine (200 mL), and dried over MgSO₄, before the sol-

vent was removed under reduced pressure. Product 41a was ob-

20 mL). The combined organics were washed with saturated brine

(20 mL), dried over Na₂SO₄, filtered, and concentrated under re-

duced pressure. The residue was purified by flash silica chromatog-

raphy (Biotage Isolera), 10 g column, 12 mLmin^ꢁ1, gradient: 10%

MeOH/CH₂Cl₂1 cv; 1!20% MeOH 10 cv; 20% MeOH/CH₂Cl₂1 cv;

20!30% MeOH, 1 cv; 30% MeOH/CH₂Cl₂3 cv, to give the product

1

41d as a white solid (11 mg, 55%). H NMR (400 MHz, CD₃OD): d=

8.33 (s, 1H), 8.19 (dd, J=1.4, 8.2 Hz, 1H), 7.85 (d, J=7.6 Hz, 1H),

7.63 (t, J=7.9 Hz, 1H), 7.15 (d, J=2.1 Hz, 1H), 7.09 (d, J=1.9 Hz,

1H), 6.66 (t, J=2.2 Hz, 1H), 5.21 ppm (s, 2H); ¹³C NMR (100 MHz,

[D₆]acetone): d=167.3, 160.0, 159.0, 148.8, 140.0, 133.9, 133.2,

tained as

a

white solid (61 mg, 89%). ¹H NMR (400 MHz,

[D₆]DMSO): d=8.38 (t, J=2.0 Hz, 1H), 8.24 (ddd, J=1.0, 2.0, 8.1 Hz,

1H), 8.13 (ddd, J=1.0, 2.0, 8.1 Hz, 1H), 7.77 (t, J=8.1 Hz, 1H), 7.70

(t, J=1.6 Hz, 1H), 7.42 (dd, J=1.6, 2.4 Hz, 1H), 7.36 ppm (dd, J=

1.6, 2.4 Hz, 1H); ¹³C NMR (100 MHz, [D₆]DMSO): d=166.9, 158.1,

148.3, 140.9, 139.4, 133.2, 133.0, 130.5, 122.4, 121.1, 118.4, 118.0,

115.9 ppm; IR (ATR): n˜_max=3169, 3078, 2848, 2651, 1715, 1616,

1602, 1532, 1505, 1476, 1451, 1431, 1341 cm^ꢁ1; LCMS-ES m/z 277

[M+NH₄]⁺, 258 [MꢁH]^ꢁ; HRMS-ES m/z [MꢁH]^ꢁcalcd for C₁₃H₈NO₅

258.0408, found 258.0412.

130.2, 123.0, 122.2, 110.1, 107.2, 107.1, 68.8 ppm; IR (ATR): n˜_max=

3162, 1719, 1616 cm^ꢁ1; LCMS-ES m/z 288 [MꢁH]^ꢁ; HRMS-ES m/z

calcd for C₁₄H₁₀NO₆[MꢁH]^ꢁ288.0514, found 288.0527.

3-Hydroxy-5-((3-nitrobenzyl)amino)benzoic acid (41e): 3-Nitro-

benzaldehyde (79 mg, 0.52 mmol) and 3-amino-5-hydroxybenzoic

acid 40 (90 mg, 0.59 mmol) were stirred in anhydrous DMF (1 mL)

containing 1% AcOH and molecular sieves (3 ꢁ) under nitrogen for

17 h. NaBH₃CN (80 mg, 1.27 mmol) was added and reaction stirred

for 1 h, then quenched by addition of H₂O (100 mL). The molecular

sieves were removed by filtration, and the filtrate evaporated in

vacuo to dryness. The crude product was purified by flash silica

chromatography (CHCl₃/MeOH/AcOH/H₂O, 120:15:3:2 v/v/v/v) to

give the product (41e) as a yellow solid, (138 mg, 93%). ¹H NMR

(400 MHz, CD₃OD): d=8.39 (s, 1H), 8.18 (d, J=2.4 Hz, 1H), 7.86 (1d,

J=2.4 Hz, 1H), 7.54 (t, J=3.1 Hz, 1H), 6.87 (s, 1H), 6.72 (s, 1H), 6.28

(s, 1H), 4.86 ppm (s, 2H); ¹³C NMR (100 MHz, CD₃OD): d=169.4,

158.5, 151.1, 147.7, 143.5, 134.6, 133.4, 129.6, 121.8 (ꢂ2), 106.2,

105.6, 104.0, 46.7 ppm; IR (ATR): n˜_max=3303, 3289, 3121, 3085,

1696, 1669, 1616, 1595, 1531, 1504, 1440, 1372 cm^ꢁ1; HRMS-ES m/z

[M+H]⁺calcd for C₁₄H₁₃N₂O₅289.0819, found 289.0835.

3’-Amino-5-hydroxy-[1,1’-biphenyl]-3-carboxylic acid (41b): 5-Hy-

droxy-3’-nitro-[1,1’-biphenyl]-3-carboxylic acid 41a (5.0 mg,

19 mmol) and Pd/C (2.5 mg, 10 wt% Pd) were stirred in EtOH

(1.5 mL) under a balloon of H₂for 1 h. The reaction mixture was

then filtered through Celite and the solvent removed under re-

duced pressure. The residue was purified by HPLC, column B, gra-

dient: elution with 5% MeCN for 2.5 min then linear increase to

40% MeCN over 15 min, t_R=13.4 min; to afford the title com-

pound 41b as a white solid (3.7 mg, 85%). ¹H NMR (500 MHz,

CD₃OD): d=7.74 (t, J=1.4 Hz, 1H), 7.60 (dt, J=1.4, 7.8 Hz, 1H),

7.55 (t, J=7.8 Hz, 1H), 7.49 (t J=2.0 Hz, 1H), 7.47 (dd, J=1.4,

2.0 Hz, 1H), 7.27–7.29 ppm (m, 2H); ¹³C NMR (125 MHz, CD₃OD):

d=169.6, 159.5, 143.7, 142.6, 135.9, 134.0, 131.7, 126.7, 121.9,

121.6, 120.4, 119.4, 117.0 ppm; IR (ATR): n˜_max=3049, 2886, 2707,

Methyl 6-hydroxy-3’-nitro-[1,1’-biphenyl]-3-carboxylate (43a):

Methyl 4-hydroxy-3-iodobenzoate 42 (200 mg, 0.72 mmol) and 3-

nitrophenylboronic acid (170 mg, 1.0 mmol), 2m Na₂CO₃solution

(0.45 mL, 0.90 mmol) and tetrakis(triphenylphosphine) palladium

(19 mg, 16 mmol) were dissolved sequentially in 1,2-dimethoxy-

ethane (2.2 mL) under argon. The reaction mixture was heated

under reflux for 17 h before cooling to 258C and H₂O (20 mL) was

added. The reaction mixture was extracted with EtOAc (4ꢂ20 mL)

and the combined organic phases were washed with saturated

NaHCO₃solution (80 mL), brine (80 mL) and dried over MgSO₄. The

residue was triturated and sonicated in PE (6ꢂ20 mL) to remove

residual starting material before purification by column chromatog-

raphy, eluting with 2:1 v/v PE/EtOAc. The product 43a was collect-

ed as an off-white solid (97 mg, 46%). ¹H NMR (400 MHz,

[D₆]DMSO): d=10.90 (brs, 1H), 8.40 (brt, J=1.9 Hz, 1H), 8.21 (ddd,

J=1.0, 2.4, 8.0 Hz, 1H), 8.03 (ddd, J=1.0, 1.8, 8.0 Hz, 1H), 7.94 (d,

J=2.2 Hz, 1H), 7.89 (dd, J=2.2, 8.5 Hz, 1H), 7.73 (t, J=8.0 Hz, 1H),

7.10 (d, J=8.5 Hz, 1H), 3.82 ppm (s, 3H); ¹³C NMR (100 MHz,

[D₆]DMSO): d=165.7, 158.9, 147.6, 138.7, 135.5, 131.7, 131.1, 129.7,

125.3, 123.4, 121.8, 120.9, 116.3, 51.7 ppm; IR (ATR): n˜_max=3390,

3079, 3048, 3007, 2961, 1688, 1603, 1517, 1508, 1477, 1449, 1428,

1409, 1345, 1312 cm^ꢁ1; LCMS-ES m/z 291 [M+NH₄]⁺, 272 [MꢁH]^ꢁ;

HRMS-EI m/z [M]^ꢁcalcd for C₁₄H₁₁NO₅273.0643, found 273.0646.

2608, 1691, 1655, 1595, 1513, 1490, 1422, 1383, 1318, 1307 cm^ꢁ1

;

LCMS-ES m/z 230 [M+H]⁺, 228 [MꢁH]^ꢁ; HRMS-ES m/z [M+H]⁺

calcd for C₁₃H₁₂NO₃230.0812, found 230.0809.

3-Hydroxy-5-(3-nitrophenoxy)benzoic acid (41c): Methyl 3-hy-

droxy-5-(3-nitrophenoxy)benzoate 39c (53 mg, 0.183 mmol) was

dissolved in THF (4 mL) and 100 mm KOH (3 mL), and heated at

708C for 8 h. The reaction was allowed to cool to room tempera-

ture, acidified with 1n HCl (15 mL), and extracted with EtOAc (2ꢂ

15 mL). The combined organics were washed with saturated brine

(15 mL), dried over Na₂SO₄, filtered, and concentrated under re-

duced pressure. The residue was purified by flash silica chromatog-

raphy (Biotage Isolera), 10 g column, 12 mLmin^ꢁ1, gradient: 4%

MeOH/CH₂Cl₂1 cv; 4!20% MeOH 9 cv; 20!30% MeOH, 2 cv;

30% MeOH/CH₂Cl₂6 cv, to give the product 41c as an off-white

solid (15 mg, 45% based on recovery of 18 mg starting material).

¹H NMR (400 MHz, [D₆]DMSO): d=7.98 (dd, J=2.1, 8.0 Hz, 1H), 7.73

(t, J=2.3 Hz, 1H), 7.67 (t, J=8.2 Hz, 1H), 7.50 (dd, J=2.4, 8.0 Hz,

1H), 7.26 (t, J=1.7 Hz, 1H), 7.00 (t, J=1.8 Hz, 1H), 6.68 ppm (t, J=

2.3 Hz, 1H); ¹³C NMR (100 MHz, [D₆]DMSO): d=159.4, 157.8, 156.5,

149.2, 131.7, 125.4, 118.5, 114.5, 113.2, 113.1, 110.6, 110.0,

104.5 ppm; IR (ATR): n˜_max=3255, 1716, 1605, 1585 cm^ꢁ1; LCMS-ES

m/z 274 [MꢁH]^ꢁ; HRMS-ES m/z [MꢁH]^ꢁcalcd for C₁₃H₈NO₆

274.0357, found 274.0367.

6-Hydroxy-3’-nitro-[1,1’-biphenyl]-3-carboxylic acid (44a): NaOH

(17.6 mg, 0.40 mmol) in H₂O (0.5 mL) was added dropwise to a solu-

tion of methyl 6-hydroxy-3’-nitro-[1,1’-biphenyl]-3-carboxylate 43a

(40 mg, 0.15 mmol) in THF (0.5 mL). The reaction mixture was

stirred for 3 h at 708C. The reaction mixture was diluted with 1m

NaOH (10 mL) and washed with EtOAc (3ꢂ10 mL) before the aque-

ous phase was acidified by careful addition of 1n HCl and then ex-

tracted with EtOAc (3ꢂ70 mL). The organic phase was then

3-Hydroxy-5-((3-nitrobenzyl)oxy)benzoic acid (41d): Methyl 3-hy-

droxy-5-((3-nitrobenzyl)oxy)benzoate 39d (21 mg, 0.07 mmol) was

dissolved in 100 mm KOH (2 mL) and THF (2 mL), and heated at

608C for 6 h. The reaction was allowed to cool to room tempera-

ture, acidified with 3n HCl (20 mL), and extracted with EtOAc (2ꢂ

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washed with brine (220 mL) and dried (MgSO₄) before the solvent

1H), 8.02 (t, J=1.4 Hz, 1H), 7.69 (d, J=7.7 Hz, 1H), 7.51 (d, J=

7.9 Hz, 1H), 7.45 (d, J=1.4 Hz, 2H), 4.62 (br, 1H), 4.53 (s, 2H),

3.89 ppm (s, 6H); ¹³C NMR (100 MHz, CDCl₃): d=166.9, 149.0, 147.9,

141.3, 133.6, 132.0, 130.2, 123.0, 122.5, 120.6, 118.1, 52.7, 47.7 ppm;

IR (ATR): n˜_max=3403, 2952, 1705, 1608 cm^ꢁ1; LCMS-ES m/z 345 [M+

H]⁺; HRMS-ES m/z [M+H]⁺calcd for C₁₇H₁₇N₂O₆345.1087, found

345.1086.

was removed under reduced pressure. The product 44a was ob-

tained as

a

white solid (37 mg, 98%). ¹H NMR (400 MHz,

[D₆]DMSO): d=12.62 (brs, 1H), 10.79 (brs, 1H), 8.40 (t, J=2.0 Hz,

1H), 8.20 (ddd, J=1.5, 2.0, 8.0 Hz, 1H), 8.03 (ddd, J=1.5, 2.0,

8.0 Hz, 1H), 7.93 (d, J=2.0 Hz, 1H), 7.85 (dd, J=2.0, 8.5 Hz, 1H),

7.73 (t, J=8.0 Hz, 1H), 7.08 ppm (d, J=8.5 Hz, 1H); ¹³C NMR

(100 MHz, [D₆]DMSO): d=166.8, 158.5, 147.6, 139.0, 135.5, 131.9,

5-((3-Nitrobenzyl)oxy)isophthalic acid (49d): Dimethyl 5-((3-nitro-

benzyl)oxy)isophthalate 47d (51 mg, 0.148 mmol) was dissolved in

THF (4 mL) and 100 mm KOH solution (4 mL), and heated at 608C

for 18 h. The reaction was allowed to cool to room temperature,

diluted with saturated NH₄Cl solution (10 mL) and H₂O (10 mL),

and extracted with EtOAc (2ꢂ20 mL). The combined organics were

dried over Na₂SO₄, filtered, and concentrated under reduced pres-

sure to give the product 49d as a white solid (30 mg, 64%).

¹H NMR (400 MHz, [D₆]DMSO): d=8.36 (s, 1H), 8.21 (d, J=8.2 Hz,

1H), 8.11 (t, J=1.4 Hz, 1H), 7.96 (d, J=7.9 Hz, 1H), 7.78 (d, J=

1.4 Hz, 2H), 7.72 (t, J=8.0 Hz, 1H), 5.41 ppm (s, 2H); ¹³C NMR

(100 MHz, [D₆]DMSO): d=166.7, 158.4, 148.2, 139.2, 134.2, 133.0,

130.3, 123.1, 123.1, 122.3, 119.8, 68.8 ppm; IR (ATR): n˜_max=2829,

2549, 1692, 1596 cm^ꢁ1; LCMS-ES m/z 316 [MꢁH]^ꢁ; HRMS-ES m/z

[MꢁH]^ꢁcalcd for C₁₅H₁₀NO₇316.0463, found 316.0472.

131.3, 129.7, 125.0, 123.4, 122.1, 121.8, 116.1 ppm; IR (ATR): n˜_max

=

3397, 2824, 2548, 1683, 1609, 1592, 1572, 1527, 1509, 1479, 1454,

1426, 1401, 1349, 1314 cm^ꢁ1; LCMS-ES m/z 258 [MꢁH]^ꢁ; HRMS-EI

m/z [M]^ꢁcalcd for C₁₃H₉NO₅259.0486, found 259.0480.

3’-Amino-6-hydroxy-[1,1’-biphenyl]-3-carboxylic acid (44b): 6-Hy-

droxy-3’-nitro-[1,1’-biphenyl]-3-carboxylic acid 44a (3.0 mg,

12 mmol) and Pd/C (2.5 mg, 10 wt% Pd) were stirred in EtOH

(1.5 mL) under a balloon of H₂for 1 h. The reaction mixture was

then filtered through Celite, and the solvent removed under re-

duced pressure. The residue was purified by HPLC, column B, gra-

dient: elution with 10% MeCN for 2.5 min then linear increase to

40% MeCN over 8 min, t_R=9.8 min, to afford the desired product

1

44b as a white solid (2.4 mg, 79%). H NMR (500 MHz, CD₃OD): d=

7.99 (d, J=2.2 Hz, 1H), 7.90 (dd, J=2.2, 8.5 Hz, 1H), 7.59–7.61 (m,

2H), 7.54 (t, J=8.2 Hz, 1H), 7.27 (ddd, J=1.0, 2.2, 8.2 Hz, 1H),

6.98 ppm (d, J=8.5 Hz, 1H); ¹³C NMR (125 MHz, CD₃OD): d=169.8,

160.3, 141.5, 134.0, 133.7, 132.5, 130.9, 129.6, 128.1, 124.0, 123.4,

121.6, 116.9 ppm; LCMS-ES m/z 230 [M+H]⁺, 228 [MꢁH]^ꢁ; HRMS-

ES m/z [M+H]⁺calcd for C₁₃H₁₂NO₃230.0812, found 230.0813.

5-((3-Nitrobenzyl)amino)isophthalic acid (49e): Dimethyl 5-((3-ni-

trobenzyl)amino)isophthalate 47e (210 mg, 0.610 mmol) was sus-

pended in 2.5m NaOH (5 mL), THF (5 mL) and MeOH (5 mL), and

stirred at room temperature for 3 h. The reaction was acidified

with 3n HCl (20 mL) and extracted with EtOAc (2ꢂ50 mL). The

combined organics were dried over Na₂SO₄, filtered, and concen-

trated under reduced pressure to give the product 49e as a yellow

Dimethyl 5-((3-nitrobenzyl)oxy)isophthalate (47d): 3-Nitrobenzyl

bromide (103 mg, 0.477 mmol), dimethyl 5-hydroxyisophthalate 45

(100 mg, 0.476 mmol), and Cs₂CO₃(308 mg, 0.945 mmol) were sus-

pended in anhydrous DMF (5 mL) under N₂, and stirred at room

temperature for 18 h. The reaction was diluted with saturated

NH₄Cl (20 mL) and H₂O (20 mL), and extracted with EtOAc (2ꢂ

20 mL). The combined organics were washed with saturated brine

(20 mL), dried over Na₂SO₄, filtered, and concentrated under re-

duced pressure. The residue was purified by flash silica chromatog-

raphy (Biotage Isolera), 10 g column, 12 mLmin^ꢁ1, gradient: 20%

EtOAc/hexane 1 cv, 20!80% EtOAc10 cv, 80% EtOAc/hexane 2 cv,

1

solid (193 mg, quant). H NMR (400 MHz, [D₆]DMSO): d=12.87 (br,

2H), 8.23 (s, 1H), 8.11 (d, J=8.2 Hz, 1H), 7.83 (d, J=7.8 Hz, 1H),

7.70 (t, J=1.4 Hz, 1H), 7.65 (t, J=7.9 Hz, 1H), 7.37 (d, J=1.4 Hz,

1H), 6.99 (t, J=6.0 Hz, 1H), 4.52 ppm (d, J=5.7 Hz, 2H); IR (ATR):

n˜_max=3395, 3369, 2917, 2528, 1716, 1688 cm^ꢁ1; LCMS-ES m/z 317

[M+H]⁺, 315 [MꢁH]^ꢁ; HRM-ES m/z [M+H]⁺calcd for C₁₅H₁₃N₂O₆

317.0774, found 317.0776.

5-((4-Nitrobenzyl)amino)isophthalic acid (49 f): 4-Nitrobenzalde-

hyde (347 mg, 2.30 mmol) and 5-aminoisophthalic acid 48 (500 mg,

2.76 mmol) were stirred in anhydrous DMF (3 mL) containing 1%

AcOH and molecular sieves (3 ꢁ) under nitrogen for 17 h. NaBH₃CN

(580 mg, 9.2 mmol) was added and reaction stirred for 1 h, then

quenched by addition of H₂O (500 mL). The molecular sieves were

removed by filtration and the filtrate evaporated in vacuo to dry-

ness. The crude product was purified by flash silica chromatography

(CHCl₃/MeOH/AcOH/H₂O, 120:15:3:2 v/v/v/v) to give the product

1

to give the product 47d as a white solid (130 mg, 79%). H NMR

(400 MHz, CDCl₃): d=8.31 (s, 1H), 8.29 (s, 1H), 8.18 (d, J=8.1 Hz,

1H), 7.81 (s, 2H), 7.77 (d, J=7.6 Hz, 1H), 7.57 (t, J=7.9 Hz, 1H),

5.22 (s, 2H), 3.92 ppm (s, 6H); ¹³C NMR (100 MHz, CDCl₃): d=166.3,

158.6, 148.9, 138.7, 133.5, 132.4, 130.1, 124.1, 123.6, 122.6, 120.4,

69.4, 52.9 ppm; IR (ATR): n˜_max=3094, 2959, 1719, 1594 cm^ꢁ1; LCMS-

ES m/z 363 [M+NH₄]⁺; HRMS-ES m/z [MꢁH]^ꢁcalcd for

C₁₇H₁₄NO₇344.0776, found 344.0787.

1

49 f as a brown solid (695 mg, 95%); H NMR (400 MHz, [D₆]DMSO):

Dimethyl 5-((3-nitrobenzyl)amino)isophthalate (47e): 3-Nitroben-

zaldehyde (166 mg, 1.10 mmol) and dimethyl 5-aminoisophthalate

46 (209 mg, 1.00 mmol) in anhydrous toluene (8 mL) were heated

at 1408C under N₂in a flask with a Dean–Stark apparatus attached

for 3 h. The solvent was removed under reduced pressure,

NaBH₃CN (69 mg, 1.10 mmol), AcOH (200 mL), and anhydrous THF

(8 mL) were added, and the mixture was stirred under N₂at room

temperature for 15 h. The reaction was diluted with saturated

NaHCO₃solution (12.5 mL) and H₂O (12.5 mmol), and extracted

with EtOAc (2ꢂ25 mL). The combined organics were dried over

Na₂SO₄, filtered, and concentrated under reduced pressure. The

residue was purified by flash silica chromatography (Biotage Iso-

lera), 25 g column, 25 mLmin^ꢁ1, gradient: 1% MeOH/CH₂Cl₂1 cv,

1!10% MeOH 10 cv, 10!20% MeOH 2 cv, 20% MeOH/CH₂Cl₂

5.7 cv, to give the product 47e as an off-white solid (245 mg,

d=10.98 (brs, 2H), 8.23 (d, J=2.0 Hz, 2H), 7.72 (s, 1H), 7.61 (d, J=

2.3 Hz, 2H), 7.36 (s, 2H), 4.52 (s, 1H), 4.51 ppm (s, 2H); ¹³C NMR

(100 MHz, [D₆]DMSO): d=169.3, 168.5, 148.8, 147.5, 145.3, 133.6,

131.6, 129.3, 128.7, 125.8, 124.6, 121.7, 119.7, 118.8, 48.8 ppm; IR

(ATR): n˜_max=3431, 3076, 2836, 2620, 1688, 1607, 1513, 1438,

1358 cm^ꢁ1; LCMS-ES m/z 317 [M+H]⁺; HRMS-ES m/z [M+H]⁺calcd

for C₁₅H₁₃N₂O₆317.0774, found 317.0770.

Synthesis of 3-dehydroquinate (1): 3-Dehydroquinate 1 was syn-

thesized (as the potassium salt) from (ꢁ)-quinic acid using the

method described by Le Sann et al.^[41]Calibration of 3-dehydroqui-

nate solutions (in H₂O) were determined from the absorbance dif-

ference at l 234 nm resulting from the total conversion of an ali-

quot of 3-dehydroquinate to 3-dehydroshikimate by 1 mL of S. coe-

licolor type II dehydroquinase (5.1 mgmL^ꢁ1

) using the kinetic

assay A described below.

1

65%). H NMR (400 MHz, CDCl₃): d=8.22 (s, 1H), 8.12 (d, J=8.2 Hz,

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dehydrogenase reaction was not limiting, and that the K_Mand k_cat

of the enzymes were in agreement with published data.^[44]There

was no change in the measured rate when using twice as much

shikimate dehydrogenase (2 mm) and the measured velocity was

proportional to the amount of dehydroquinase (when doubled or

trebled). Using a concentration of 0.5 mm NADPH allowed a linear

consumption of NADPH vs. time over 5–10 min. Some of the as-

sayed compounds were not soluble in water at high concentra-

tions, hence stock solutions in DMSO had to be prepared previous

to further dilution in buffer or water. DMSO (5%) was used in the

assay in this case.

Enzyme purification

M. tuberculosis type II dehydroquinase was purified as described

previously.^[42]

E. coli shikimate dehydrogenase: The aroE gene (819 bp) from

E. coli K12 was amplified by PCR and cloned into the NdeI/XhoI-di-

gested pET-28a vector for expression in order to place the gene in-

frame with an N-terminal (His)₆-tag sequence cleavable by throm-

bin. An overnight culture (100 mL) of E. coli C41(DE3) competent

cells transformed with plasmid pET-28a/aroE was added to 1 L of

fresh 2YT media supplemented with 30 mgmL^ꢁ1kanamycin and di-

vided into 2ꢂ500 mL cultures. Overexpression of (His)₆–AroE was

induced at an OD₆₀₀of 0.6 by the addition of isopropyl-b-d-thioga-

lactopyranoside (IPTG) to a final concentration of 1 mm. After 4 h

shaking at 378C, the IPTG-induced cells were harvested by centri-

fuging at 6084ꢂg for 15 min at 48C. Cell pellets were stored at

ꢁ208C. The harvested cells were suspended in 0.02m potassium

phosphate, 0.5m NaCl, 0.02m imidazole, pH 7.4 (buffer A) contain-

ing lysozyme (0.35 mgmL^ꢁ1) and stirred at room temperature for

30 min. The suspension was homogenized for 20 min by sonication

on ice. The crude extract was then centrifuged for 30 min at

38724ꢂg. The resulting supernatant was applied at a flow rate of

1.0 mLmin^ꢁ1to a 5 mL HiTrap chelating column (Amersham Biosci-

ences) previously charged with 0.1m NiSO₄and equilibrated with

buffer A at 48C. After washing with buffer A (10 bed volumes;

2.5 mLmin^ꢁ1), the protein was eluted using a linear gradient of 20–

500 mm imidazole in 100 mL buffer A (2.5 mLmin^ꢁ1). The concen-

trated protein sample was then dialyzed at 48C against 100 mm

potassium phosphate, pH 7.0, using a PD-10 column containing Se-

phadex G-25 resin (Amersham Biosciences). The purity of the pro-

tein was determined to be ꢂ95% by SDS-PAGE. Enzyme concen-

trations were evaluated by measuring the absorbance at 280 nm

and using the conversion factor (OD₂₈₀=1.0 corresponds to

1.74 mgmL^ꢁ1for (His)₆-AroE) calculated using the software Vector

NTI (version 6; Invitrogen). Aliquots of pure (His)₆-tag recombinant

proteins were frozen with liquid nitrogen and stored at ꢁ808C

until use.

The inhibition kinetics data were obtained by measuring the initial

rates of reaction over a range of inhibitor concentrations (between

4 and 7 different concentrations) at either 4 or 5 different substrate

concentrations. The inhibition constants and standard deviations

were obtained by least-squares fitting using the software GraFit.^[45]

Acknowledgements

The authors thank the Bill & Melinda Gates Foundation for finan-

cial support, and the European Commission for a Marie Curie

Intra-European Fellowship (M.F.S.).

Keywords: dehydroquinase

· drug design · inhibitors ·

Mycobacterium tuberculosis · protein structures

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Kinetic assays for type II dehydroquinases

Two different assays for type II dehydroquinases were used in this

work: a direct assay (A) and a coupled assay with E. coli shikimate

dehydrogenase (B).

Assay A was previously described for S. coelicolor and M. tuberculo-

sis type II dehydroquinases.^[43]It consists of monitoring the increase

in absorbance at l 234 nm due to the formation of the enone–car-

boxylate chromophore of 3-dehydroshikimate (2). The assay was

performed at 258C in Tris·HCl buffer (0.05m, pH 7.0) using 90 nm

M. tuberculosis dehydroquinase.

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Assay B: Enzymes were assayed by coupling the formation of 3-de-

hydroshikimate to the oxidation of NADPH in the presence of

E. coli shikimate dehydrogenase (EC 1.1.1.25). This assay was devel-

oped to overcome the problems caused by the absorption of

some inhibitors at l 234 nm. Moreover, due to their poor solubility

in water, some of the compounds had to be assayed using DMSO,

which also absorbs at l 234 nm. The initial rate of the reaction was

determined by monitoring the decrease in absorption of NADPH at

l 340 nm using a Bio-tek Powerwave XS multi-well plate spectro-

photometer, thermostatically controlled at 288C. All coupled reac-

tions were performed in 60 mm Tris·HCl buffer (pH 7.0), 0.5 mm

NADPH, 1 mm E. coli shikimate dehydrogenase and included 90 nm

M. tuberculosis dehydroquinase. It was ensured that the shikimate

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Received: July 18, 2014

Published online on September 18, 2014

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Design and structural analysis of aromatic inhibitors of type II dehydroquinase from mycobacterium tuberculosis

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