Scholarly article on 4-(3-(3-methoxyphenethoxy)prop-1-ynyl)phenol 1602768-67-1 from ChemMedChem p. 129

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DOI: 10.1002/cmdc.201300378

The Fight against the Influenza A Virus H1N1: Synthesis,

Molecular Modeling, and Biological Evaluation of

Benzofurazan Derivatives as Viral RNA Polymerase

Inhibitors

Mafalda Pagano,^[a]Daniele Castagnolo,^[a]Martina Bernardini,^[a]Anna Lucia Fallacara,^{[a, b]}

Ilaria Laurenzana,^[c]Davide Deodato,^[a]Ulrich Kessler,^[d]Beatrice Pilger,^[d]Lilli Stergiou,^[d]

Stephan Strunze,^[d]Cristina Tintori,^[a]and Maurizio Botta*^{[a, e]}

The influenza RNA polymerase complex, which consists of the

three subunits PA, PB1, and PB2, is a promising target for the

development of new antiviral drugs. A large library of benzo-

furazan compounds was synthesized and assayed against influ-

enza virus A/WSN/33 (H1N1). Most of the new derivatives were

found to act by inhibiting the viral RNA polymerase complex

through disruption of the complex formed between subunits

PA and PB1. Docking studies were also performed to elucidate

the binding mode of benzofurazans within the PB1 binding

site in PA and to identify amino acids involved in their mecha-

nism of action. The predicted binding pose is fully consistent

with the biological data and lays the foundation for the ration-

al development of more effective PA–PB1 inhibitors.

Introduction

Influenza is an infectious disease of birds and mammals caused

by ribonucleic acid (RNA) viruses of the family Orthomyxoviri-

dae. It is a contagious disease that occurs seasonally in epi-

demic and sometimes pandemic proportions. On the basis of

serological subtyping, three distinct types of influenza—A, B,

and C—are present, of which types A and B are of great con-

cern as human pathogens.^[1,2]The virus has two major antigen-

ic glycoproteins: hemagglutinin (HA) and neuraminidase (NA).

Three HA subtypes (H1, H2, and H3) and two NA subtypes (N1

and N2) have frequently been found in human influenza

virus,^[1,2]resulting in the possibility for distinct viral strains such

as H1N1, H1N2, H2N1, H2N2, etc. Influenza epidemics and pan-

demics in the past century have had a serious impact on

global morbidity, mortality, and economy.^[3–5]Recent outbreaks

of swine influenza (H1N1) in Mexico and other parts of the

world have led to issuances of pandemic alerts by the World

Health Organization (WHO).^[6]Influenza virus continuously

evolves to avoid host detection systems by changing its anti-

genicity through mutation of its surface glycoprotein (NA, HA)

genes; in this way it maintains the capacity to reinfect the

same individual in subsequent flu seasons. Another major

factor for the emergence of extremely aggressive influenza vi-

ruses is the reassortment of genetic materials between differ-

ent strains of the virus circulating in humans and avians.^[7]

Given this elevated rate of mutation, every season WHO ex-

perts identify the proper combination of antigenic strains for

which vaccines are developed. However, the 9–12-month time

gap between WHO recommendation and actual application of

vaccine could result in a mismatch between the virus and vac-

cine, decreasing its effectiveness against the strains in circula-

tion.^[8]The development of an effective flu vaccine is ham-

pered by these and other complicating factors. Therefore, addi-

tional weapons in the form of efficacious anti-influenza agents

are a must in the fight against future seasonal or pandemic in-

fluenza outbreaks.^[9,10]

[a] Dr. M. Pagano, Dr. D. Castagnolo, Dr. M. Bernardini, A. L. Fallacara,

D. Deodato, Dr. C. Tintori, Prof. M. Botta

Dipartimento Biotecnologie, Chimica e Farmacia

Universitꢀ degli Studi di Siena, Via A. De Gasperi 2, 53100 Siena (Italy)

[b] A. L. Fallacara

Dipartimento di Chimica e Tecnologie del Farmaco

Universitꢀ La Sapienza, Piazzale Aldo Moro 5, 00185 Roma (Italy)

[c] I. Laurenzana

A few antiviral drugs are effective in defending the body

against infection, particularly in patients for whom it is not

possible to administer an influenza vaccine or when a new

type of virus enters circulation (i.e., swine H1N1 virus, 2009).^[11]

NA inhibitors, such as oseltamivir^[12]and zanamivir, which in-

hibit virus budding,^[13]are available for clinical use. Further-

more, M2 protein inhibitors, amantadine and rimantadine, his-

torically the first drugs available for the treatment of influenza,

have been used for over 50 years. Although both drugs can be

effective against influenza virus A infection, they have been re-

IRCCS-Centro di Riferimento Oncologico Basilicata (CROB)

Laboratory of Preclinical and Translational Research

Via Padre Pio 1, Rionero in Vulture, 85028 Potenza (Italy)

[d] Dr. U. Kessler, Dr. B. Pilger, Dr. L. Stergiou, Dr. S. Strunze

PiKe Pharma GmbH, Institute of Pharmaceutical Sciences

Swiss Federal Institute for Technology, 8093 Zꢁrich (Switzerland)

[e] Prof. M. Botta

Biotechnology College of Science and Technology

Temple University, Biolife Science Building, Suite 333

1900 North 12th Street, Philadelphia, PA 19122 (USA)

E-mail: botta.maurizio@gmail.com

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roguanosine (FdG)^[21,22]and T-705^[23–25](Figure 1b). In addition,

flutimide, a 2,6-diketopiperazine identified in extracts of the

fungus Delitschia confertaspora, has been shown to specifically

inhibit the cap-dependent endonuclease activity associated

with influenza viral RNA polymerase and to inhibit the replica-

tion of influenza A and B virus in cell culture.^[26]The discovery

of molecules able to interact with the PA subunit is an ex-

tremely important goal in the synthesis of broad-spectrum an-

tiviral drugs. In fact, because the N-terminal PA interaction

domain of PB1 is highly conserved, a compound that can

block the interaction between these two proteins can be ex-

pected to inhibit most, if not all, influenza strains. We recently

reported the discovery of a novel class of influenza A (H1N1)

inhibitors by taking advantage of a biochemical ELISA-based

screening approach specifically designed to identify com-

pounds that efficiently block the PA–PB1 interaction.^[27,28]How-

ever, because only a limited amount of structure–activity rela-

tionship (SAR) information arose from our previous work, we

decided to synthesize a larger library of benzofurazan deriva-

tives to explore the chemical space around the heterocyclic

nucleus. Both cell-based and enzyme bioassays were planned

in order to test the antiviral activity and the ability of the ben-

zofurazan compounds to interact with and inhibit the viral

RNA polymerase complex. Furthermore, docking studies were

performed to explore the structural features responsible for

the biological activity of benzofurazan derivatives.

Figure 1. a) Structure of influenza RNA polymerase; b) influenza A RNA poly-

merase inhibitors.

ported to cause CNS side effects and have given rise

to the rapid emergence of drug-resistant viral

strains.^[14–19]

In the last few years, influenza RNA polymerase

has been identified as a new target for inhibition of

the virus. This enzyme consists of a complex of three

virus-encoded polypeptides (PB1, PB2, and PA; Fig-

ure 1a). In addition to RNA replicative activity, this

polymerase also contains an endonuclease activity to

ensure “cap snatching” to initiate the transcription

and subsequent translation process. The polymerase

complex genes contribute to the high virulence of

the human H5N1 influenza virus isolate A/Vietnam/

1203/04. This observation highlights the importance

of novel antivirals that target the polymerase for fur-

ther development of therapy and prophylaxis of

human and avian influenza virus infections.^[20]Few

compounds have been reported to operate at the

RNA polymerase. Like the inhibitors that have been

found to act against reverse transcriptase (RNA-de-

pendent DNA polymerase) of HIV or RNA replicase

(RNA-dependent RNA polymerase) of HCV, influenza

RNA polymerase inhibitors can be divided into two

classes: nucleosides and non-nucleosides. Examples

of the nucleoside-type inhibitors are 2’-deoxy-2’-fluo- Figure 2. Second-generation benzofurazan derivatives.

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ence of triethylamine, compounds 9a and 9b were also isolat-

ed. The formation of these latter compounds was due to an in-

tramolecular Smiles rearrangement of the cysteamine chain

catalyzed by triethylamine, as previously reported by our

group.^[29]Finally, reaction of 3 with 12 and 13, in turn obtained

through guanylation and amidation with cysteamine, and acid-

mediated removal of the Boc protecting group led to the for-

mation of derivatives 10a,b. Compounds belonging to the B

series of derivatives, bearing a thiopropionic acid side chain at

C4, were then synthesized according to Scheme 2. Benzofura-

zan 3 was first allowed to react with amides 18a,b, in turn ob-

tained through the reaction of thiopropionic acid 14 with mor-

pholine and thiomorpholine. Reaction of 3 with ethyl 3-thio-

propionate or with N-Boc-cysteine ethyl ester led to derivatives

15a,b. Finally, the allyl derivative 16 was obtained under the

same reaction conditions.

Results and Discussion

Chemistry

To identify novel benzofurazans as RNA polymerase inhibitors

and to study the SARs, five series of analogues (A–E, Figure 2)

of previously discovered compounds 1 and 2^[27]were designed

and synthesized. Both series A and B bear an alkylthio sub-

stituent at C4 of the benzofurazan ring. In the first series of

compounds with general structure A we planned to introduce

a >1-carbon alkyl spacer between the sulfur and the aromatic

ring. Moreover, enhancing the solubility of the compounds by

replacing the aromatic moiety with hydrophilic groups was

also investigated. Similarly, the second series of derivatives B

was planned with the aim to introduce a hydrophilic/hydro-

gen-donor moiety at C4 and to evaluate their importance for

activity. The C series is composed of benzofurazans bearing an

S- or O-aryl moiety at C4. In particular, the introduction of het-

eroaryl moieties bound to the phenyl ring at C4 was planned

in order to improve the solubility of the new compounds and

thus to improve their activity in cellular assays. To determine

the importance of the nitro

Synthesis of derivatives with general structure C

A new series of benzofurazans bearing a thioaryl moiety at C4

was synthesized according to Scheme 3. The starting material

group, the fourth series of com-

pounds D, bearing various

groups at C7 was also planned.

Finally, the synthesis of com-

pounds E with a non-benzofura-

zan core was accomplished. The

benzofurazan core was replaced

by (bio)isosteric (hetero)aromatic

cores in order to investigate the

importance of the oxadiazole

ring for activity against influenza

viruses.

Synthesis of derivatives with

general structures A and B

The synthesis of the first series

of benzofurazan derivatives con-

taining a cysteamine or thioethyl

chain at C4 was carried out ac-

cording to Scheme 1. Commer-

cially available benzofurazan 3

was first treated with protected

thioethanol to afford 4, which,

after deprotection with TBAF, led

to intermediate 5. Acylation of

the latter afforded derivatives

6a–c in excellent yields. Reaction

of benzofurazan 3 with cystea-

mine gave intermediate 7. Acyla-

tion of 7 with various acyl chlor-

Scheme 1. Reagents and conditions: a) HS(CH₂)₂OTBDPS, K₂CO₃, CH₂Cl₂, reflux, 4 h; b) TBAF, THF, RT, 1 h; c) RCOCl,

pyridine, DMAP, RT, 5 h; d) cysteamine, pyridine, KOAc, EtOH, reflux, 1 h; e) RCOCl, pyridine, DMAP, CH₂Cl₂, 6 h;

f) RCOCl, Et₃N, CH₂Cl₂, DMAP, 30 min; g) 12 or 13, pyridine, KOAc, EtOH, reflux, 1 h, then HCl, Et₂O, RT, 24 h;

h) N,N’-di-Boc-N’’-trifluoromethanesulfonylguanidine, Et₃N, CH₂Cl₂, RT, 6 h; i) N-Boc-serine, EDC, HOBt, DIPEA, DMF,

ides in the presence of pyridine

as base selectively afforded com-

pounds 8a–c. If the same reac-

tion was carried out in the pres- RT, 10 h.

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perazine at the para position of the phenyl ring was planned

(Scheme 5). Compound 34 was alkylated, acylated, and guany-

lated to give compounds 35–37, which were in turn coupled

with 4-chloro-7-nitrobenzofurazan 3 to give the desired prod-

ucts 38a–c. Hydrochloride salts 39a,b were also synthesized,

and Boc deprotection of compound 38c led to compound

40c.

Finally, we planned to introduce a heteroaromatic moiety

into the C4 chain. In particular, we focused our attention to

the triazole ring, which is known to be a bioisostere of the

amide bond. Azides 42a,b were allowed to react with various

alkynes through Huisgen-1,3-dipolar cycloaddition to afford tri-

azoles 44a–f, 45a,b, and, after deprotection of 44d, com-

pound 46. These were coupled with compound 3 to furnish

the desired compounds 47–48a–h (Scheme 6, Table 1). Both

Table 1. Triazole derivatives synthesized (series C).

Scheme 2. Reagents and conditions: a) 18a or 18b, pyridine, KOAc, EtOH,

reflux, 1 h; b) ethyl 3-thiopropionate or N-Boc-cysteine ethyl ester, pyridine,

KOAc, EtOH, reflux, 1 h; c) allylthiol, pyridine, KOAc, EtOH, reflux, 1 h; d) mor-

pholine or thiomorpholine, EDC, HOBt, DIPEA, DMF, RT, 10 h.

Entry Azide Alkyne

R

Triazole Benzofurazan

19, obtained from compound 3 by reaction with m-aminothio-

phenol in ethanol at reflux,^[29]was then derivatized at the

amino function. Imidazole derivative 20 was obtained through

a microwave-accelerated one-pot reaction previously reported

by our group,^[30]leading to the formation of the heterocyclic

ring in 10 min. Reaction of 19 with p-toluenesulfonyl isocya-

nate and cyanuric chloride led to derivatives 21 and 22. This

latter compound was also converted into its hydrochloride salt

23 in order to improve aqueous solubility. Finally, reductive

amination of 19 with various aldehydes led to compounds

24a–d. A second series of derivatives bearing an O-aryl moiety

at C4 was then synthesized. With the purpose to avoid poten-

tial oxidation at the sulfur atom, which could reduce antiviral

activity, several derivatives containing an ether moiety were

synthesized. Moreover, the oxy-derivatives 27a–d and 29

(Scheme 4) were synthesized with the aim to introduce an

alkyl spacer between the two aromatic moieties and to obtain

further SARs. Alkynes 26a–d were obtained through Sonoga-

shira coupling from 4-iodophenol 25 and then reacted with

compound 3. Hydrogenation of 26c led to saturated phenol

28, which was in turn converted into derivative 29. Finally, de-

rivative 33 was easily synthesized by starting from commercial-

ly available aldehyde 30, which was converted into diene 31 in

three steps. Ring-closing metathesis and deprotection led to

phenol 32, which, after reaction with 4-chloro-7-nitrobenzofur-

azan 3, led to the desired compound 33. We then turned our

attention to the introduction of a heteroaliphatic group in

order to improve the solubility of target compounds. There-

fore, the synthesis of a series of phenol derivatives bearing a pi-

1

2

3

4

42a

43a

43b

43d

43e

Ph

p-MeOPh

cHex

44a

44b

44d

44 e

47a

47b

47d

47e

CH₂OH

5

42a

43 f

44 f

47 f

6

42a

43g

44g

–

7

8

9

42b

43a

43b

43c

Ph

p-MeOPh

p-MeSPh

45a

45b

45c

46

48a

48b

48c

47h

10

meta- and para-substituted phenols were synthesized. The syn-

theses of benzofurazans 47 f and 47h, respectively bearing an

amino acid and a carbohydrate group, were planned in order

to enhance the aqueous solubility of these derivatives.

Synthesis of derivatives with general structure D

A further series of compounds with general structure D was

then planned with the aim of investigating the role of the sub-

stituents at C7. Compounds in which the nitro group was re-

placed by a halogen or an amino moiety were then designed

and synthesized. Compounds 1, 14a, and 49, which in our pre-

vious studies^[27]showed high antiviral activity in cellular assays,

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tion of 62 with the appropriate

nucleophile led to the desired

products 63a,b. The oxadiazole

ring of the bezofurazans was

then replaced with a pyridine

ring. Hence, a series of quinoline

and isoquinoline derivatives was

synthesized. Skraup reaction be-

tween aniline 64 and acrolein 65

led to quinoline nucleus 66,

which, after coupling with the

appropriate thiol or phenol, led

to desired compounds 67a–d.

The synthesis of isoquinoline de-

rivatives was accomplished by

starting from 68, which was con-

verted into derivative 69 by bro-

mination with N-bromosuccini-

mide (NBS) and nitration with

potassium nitrate. Coupling with

the appropriate thiol led to iso-

quinolines 70a,b (Scheme 9). Fi-

nally, the oxadiazole ring was re-

placed with a 2-methylthiazole

ring. Aniline 71 was acylated

into 72 and cyclized into benzo-

thiazole 73 by reaction with

Lawesson’s reagent. Nitration of

73 led to 74, which was convert-

ed into desired compounds

75a–d after reaction with the

appropriate thiol/phenol. Finally,

derivative 78 was obtained in

Scheme 3. Reagents and conditions: a) H₃PO₄, paraformaldehyde, glyoxal, NH₄Cl, H₂O/dioxane, MW (1208C),

10 min; b) p-toluenesulfonyl isocyanate, CH₂Cl₂, RT, 24 h; c) cyanuric chloride, DME, À308C, 1 h; d) acetyl chloride,

MeOH, CH₂Cl₂, RT, 2 min; e) RCHO, NaBH₄–PTSA, DCE, RT, 16 h.

a

two-step sequence starting

from aniline 76 in order to inves-

were chosen as substrates. Reduction of the nitro group was

carried out with Fe⁰in acidic medium, to afford amines 50a–c.

Chlorination and iodination were performed with CuCl₂–tert-

butylnitrile and NaNO₂–KI, respectively, to allow formation of

51b and 52c. Amines 53a and 54c were also synthesized by

hydrochlorination and acylation reactions. Finally, sulfonamides

58a–d were synthesized from amide 56, in turn obtained from

sulfonyl chloride 55 (Scheme 7).

tigate the importance of the heterocyclic core for antiviral ac-

tivity (Scheme 10).

Biological assays

The synthesized compounds were assayed for their inhibitory

activity toward influenza virus strain A/WSN/33 (H1N1), and

the results are listed in Table 2. With regard to derivatives with

general structure A, compound 6a, containing a thioethanol

chain, showed good activity, with an IC₅₀value of 2.5 mm. How-

ever, 6a proved to be slightly cytotoxic. On the other hand, re-

lated compounds 6b,c were found to be inactive. The best ac-

tivity data were obtained with cysteamine derivative 8. The un-

substituted derivative 7 showed a good biological profile, with

an IC₅₀value of 10 mm and low cytotoxicity. Acylated deriva-

tives 8a–c showed excellent antiviral activity (1–2.5 mm), but

also proved to be highly cytotoxic. It seems clear that the pres-

ence of an aromatic moiety is fundamental for improvement

of activity, yet it is also detrimental in terms of cytotoxicity.

Compound 8c proved to be a perfect compromise, bearing an

aromatic moiety endowed with partial hydrophilic character,

namely a pyridine. Compound 8d was found to be active

Synthesis of the series of derivatives with general structure E:

modifications of the heterocyclic core

The synthesis of a series of non-benzofurazan derivatives relat-

ed to previous compounds was then planned in order to inves-

tigate the role and importance of the benzooxadiazole nucleus

to anti-influenza activity. A series of tricyclic compounds strictly

related to benzofurazans was obtained by Diels–Alder reaction

of 49a–f with the Danishefsky diene, leading to the formation

of diastereomers 59a–f and 60a–f (Scheme 8). The benzo-

2,1,3-thiadiazoles 63a,b were then synthesized from commer-

cially available 61, which was converted into the bromo deriva-

tive 62 by holding it at reflux in concentrated nitric acid. Reac-

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that efficiently block the PA–PB1

interaction. Most of the new an-

tiviral benzofurazan compounds

(8c, 20, 23, 39b, 40, 47a, 47e,

48b, 48c) acted by disrupting

the interaction between the PA

and PB1 subunits of the viral

RNA polymerase at the micro-

molar level, thus blocking an es-

sential protein in the replication

process of the virus. Notably, all

the compounds observed to be

active in the ELISA bear a hetero-

cyclic or heteroaromatic ring in

the benzofurazan chain. Imida-

zole derivative 20 showed

a close correlation between the

results of cell-based assays and

ELISAs, confirming that its bio-

logical activity is due to interac-

tion with the RNA polymerase

complex. Close correlations be-

tween cellular and ELISAs were

also observed for piperazine de-

rivatives 39b and 40, and for tri-

azoles 47a,e and 48b,c. On the

other hand, the high ELISA IC₅₀

values determined for some

compounds (16, 21, 24a, and

33), which were found active in

cells at low micromolar levels,

suggest that their mechanism of

action could be different. Further

studies are in progress to better

elucidate this aspect.

Scheme 4. Reagents and conditions: a) alkyne, (PPh₃)₂PdCl₂, CuI, DMF/Et₃N, MW (1208C), 5 min; b) 3, cat. pyridine

and KOAc, EtOH, reflux, 20 min; c) H₂, Pd/C, EtOAc, RT, 8 h; d) 3, cat. pyridine and KOAc, EtOH, reflux, 20 min;

e) TIPSCl, imidazole, DMF, 608C, 1 h; f) vinylmagnesium bromide, THF, RT, 1 h; g) allyl bromide, NaH, THF, RT, 5 h;

h) 2nd-gen. Grubbs catalyst, CH₂Cl₂, RT, 1 h; i) TBAF, THF, RT, 1 h; j) 3, cat. pyridine and KOAc, EtOH, reflux, 2 h.

against influenza virus H1N1 and showed low cytotoxicity.

Compounds with general structure B (namely 14, 15, and 16)

Molecular modeling

were all inactive against influenza H1N1 virus in cellular assays,

with compound 16 as the only exception: IC₅₀10 mm. In con-

trast, good results were obtained from compounds with gener-

al structure C (compounds 20–48); all were active against in-

fluenza H1N1 virus, with IC₅₀values ranging from 1 to 60 mm.

Overall, the introduction of aryl/benzyl ethers or thioethers or

longer aliphatic/aromatic chains at C4 is favorable for activity

against influenza H1N1. Notably, compound 47e proved to be

active against H1N1 virus, with an IC₅₀value of 1 mm and low

cytotoxicity, thus proving to be the best benzofurazan deriva-

tive identified in this series.

Docking studies were performed on the novel benzofurazans

to explore the structural features responsible for their inhibito-

ry activity toward influenza H1N1 virus. Because most of the

derivatives were shown to act as PA–PB1 inhibitors, they were

subjected to focused docking experiments within the PB1

binding site of PA.

Computational analysis was performed by using a protein

assembly built with the aid of two recently deposited crystallo-

graphic structures of the PA–PB1 complex (PDB IDs 3CM8 and

2ZNL). The complete library of benzofurazans was docked by

using both Glide and Gold programs. Docking results on deriv-

atives reported herein did not converge to a single solution,

but several binding modes were identified. The inability to rec-

ognize a unique pose was likely due to the large size of the

pocket, adapted to the co-crystallized PB1 peptide. However,

the most reliable binding mode identified for active inhibitors

shows the benzofurazan scaffold involved in a p-stacking inter-

action with Trp706. Furthermore, a hydrogen bond between

the oxygen atom of the furazan group and Gln408 and an

Focusing on compounds of series C and D (compounds 49–

78), the biological data indicate that replacement of the nitro

group or change of the benzofurazan core cause a loss of ac-

tivity (data not shown). Similarly, the introduction of substitu-

ents at C6 is detrimental for antiviral activity. On the other

hand, to study the mechanism of action of the new antiviral

benzofurazan compounds, active derivatives were also tested

in the novel biochemical ELISA previously described for influ-

enza peptides and specifically designed to identify compounds

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electrostatic interaction recur-

+

rent between the NH₃

of

Lys643 and the nitro group were

found. As an example, the bind-

ing mode of compound 39b is

shown in Figure 3. Similar inter-

actions were previously de-

scribed for other recently identi-

fied PA–PB1 inhibitors.^[31,32]Ac-

cordingly, the lack of activity of

compounds in which the nitro

group is replaced by another

group may depend on the loss

of the electrostatic interaction

with Lys643. Additionally, re-

placement of the benzofurazan

core is detrimental to activity,

probably from the loss of inter-

actions with Trp706 and Gln408.

The ligand efficiency (LE)^[33]of

benzofurazan derivatives was

calculated as the free energy of

binding divided by the number

of heavy atoms (non-hydrogen

atoms, NHA) of the molecule

(Table 2). Values of LE span from

Scheme 5. Reagents and conditions: a) CH₂O, formic acid, MeOH, reflux, 12 h; b) Ac₂O, pyridine, RT, 1 h; c) N,N’-di-

Boc-N’’-trifluoromethanesulfonylguanidine, DIPEA, DMF, RT, 16 h; d) 35, 36, or 37, cat. pyridine and/or KOAc, EtOH,

reflux, 20 min; e) 38a or 38b, acetyl chloride, MeOH, CH₂Cl₂, RT, 2 min; f) 38c, TFA, CH₂Cl₂, RT, 24 h.

0.16 to 0.34 kcal(mol·NHA)^À1

,

putting compounds 7, 16, and

20 in a promising range for fur-

ther optimization, as an LE value

of 0.24 kcal(mol·NHA)^À1has been described as a limit

over which small molecules are able to disrupt pro-

tein–protein interfaces.^[34]

Conclusions

In summary, the present study demonstrates the po-

tential of benzofurazan compounds as a new family

of influenza virus inhibitors that act by targeting the

interaction between the PA and PB1 subunits of the

viral RNA polymerase. A large library of compounds

was synthesized and biologically evaluated, leading

to the identification of lead compounds (8c and

47e) with high anti-H1N1 activity (IC₅₀=1 mm). ELISA

results confirmed the mechanism of action of benzo-

furazan derivatives being through inhibition of the

viral RNA polymerase. These compounds are a start-

ing point in the identification and development of

novel therapeutic antiviral agents. Results of docking

studies suggest that the residues Trp706, Gln408, and

Lys643 are crucial for the binding of the benzofura-

zan derivatives to the PB1 binding pocket on PA, and

therefore could be targeted for the design of novel

PA–PB1 interaction inhibitors.

Scheme 6. Reagents and conditions: a) NaNO₂, NaN₃, AcOH/H₂O, 15 min; b) appropriate

alkyne, sodium ascorbate/Cu(SO)₄, H₂O/tBuOH, MW (1258C), 10 min; c) 44g, MeOH/

NH₄OH (4:1), RT, 18 h; d) 44–45a–f or 46, cat. pyridine and/or KOAc, EtOH, reflux, 3 h.

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sured on a Bruker Avance DPX400. Chemical shifts are

reported relative to CDCl₃at d=7.24 ppm and tetrame-

thylsilane at d=0.00 ppm. Mass spectrometry (MS) data

were obtained on an Agilent 1100 LC/MSD VL system

(G1946C) at a flow rate of 0.4 mLmin^À1using a binary

solvent system of 95:5 MeOH/H₂O. UV detection was

monitored at l 254 nm. MS data were acquired in both

positive and negative modes, scanning over the mass

range of 50–1500. The following ion source parameters

were used: drying gas flow: 9 mLmin^À1; nebulizer pres-

sure: 40 psig; drying gas temperature: 3508C.

4-(2-(tert-Butyldiphenylsilyloxy)ethylthio)-7-

nitrobenzo[c][1,2,5]oxadiazole (4): To a solution of 4-

chloro-7-nitrobenzofurazan 3 (1 mmol) in 5 mL CH₂Cl₂,

K₂CO₃(1 mmol) and OTBDPS-thioethanol (1.1 mmol)

were added and the reaction mixture was heated at

reflux for 4 h. H₂O was added, and the organic layer was

washed with brine, dried over Na₂SO₄, filtered off and

the solvent was removed. The crude mixture was puri-

fied by flash chromatography (Hex/EtOAc 9:1) to give

1

compound 5 as a yellow solid (0.71 mmol, 71%). H NMR

(400 MHz, CD₃OD): d=8.13–8.11 (d, J=8, 1H), 7.58–7.56

(m, 4H), 7.35–7.32 (m, 2H), 7.30–7.26 (m, 4H), 6.89–6.87

(d, J=8, 1H), 4.00–3.96 (d, J=8, 2H), 3.40–3.36 (d, J=8,

2H), 0.97 ppm (s, 9H); ¹³C NMR (100 MHz, CDCl₃): d=

143.78, 143.42, 135.66, 134.05, 130.07, 129.56, 126.23,

124.48, 119.08, 67.26, 43.39, 31.75, 26.88 ppm; MS (ESI)

m/z: 502 [M+Na]⁺.

2-(7-Nitrobenzo[c][1,2,5]oxadiazol-4-ylthio)ethanol (5):

To a solution of 4 (134 mg, 0.28 mmol), in dry THF (5 mL)

previously cooled to 08C, a 1m TBAF solution in THF

(280 mL, 0.28 mmol) was slowly added. The mixture was

stirred at room temperature for 1 h, then the solvent

was evaporated. The residue was purified by flash chro-

matography (EtOAc/Hex 4:1) giving the desired com-

pound 5 as a colorless oil (0.28 mmol, quant). ¹H NMR

(400 MHz, CDCl₃): d=8.38–8.36 (d, J=8, 1H), 7.37–7.35

(d, J=8, 1H), 3.88–3.85 (t, J=4, 2H), 3.42–3.38 ppm (t,

J=4, 2H); ¹³C NMR (100 MHz, CDCl₃): d=148.98, 140.38,

135.78, 127.22, 124.53, 119.42, 57.13, 38.46 ppm; MS (ESI)

m/z: 264 [M+Na]⁺.

Scheme 7. Reagents and conditions: a) Fe⁰, HCl, CH₂Cl₂/MeOH, RT, 25 min; b) CuCl₂, tert-

butyl nitrite, CH₃CN, 658C, 3 h; c) 1. NaNO₂, H₂O, HCl/H₂SO₄, 08C, 40 min, 2. KI, H₂O, RT,

2 h; d) acetyl chloride, MeOH, CH₂Cl₂, RT, 2 min; e) Ac₂O, pyridine, cat. DMAP, RT, 4 h;

f) Me₂NH, cat. Et₃N, CH₃CN, RT, 3 h; g) appropriate nucleophile, cat. pyridine and/or KOAc,

EtOH, reflux, 2 h.

General procedure for esters 6a–c

To a suspension of 5 (35 mg, 0.14 mmol) in dry CH₂Cl₂

(5 mL), the appropriate acyl chloride (0.43 mmol), pyri-

dine (50 mL, 0.58 mmol), and a catalytic amount of DMAP

were added. The reaction mixture was stirred at room

temperature for 6 h, after which time it was washed with

NaHCO₃and then with 1n HCl. The combined organic

layers were dried over Na₂SO₄, filtered off and the sol-

vent removed under reduced pressure. The crude was

purified by flash chromatography.

Scheme 8. Reagents and conditions: a) Danishefsky diene, CH₂Cl₂, RT, 6 h; b) conc HNO₃,

reflux, 1 h; c) appropriate nucleophile, cat. pyridine and/or KOAc, EtOH, reflux, 3 h.

2-(7-Nitrobenzo[c][1,2,5]oxadiazol-4-ylthio)ethyl 4-bro-

mobenzoate (6a): Yellow solid (0.13 mmol, 94%).

¹H NMR (400 MHz, CDCl₃): d=8.35–8.33 (d, J=7.8, 1H),

7.77–7.75 (d, J=8.3, 2H), 7.52–7.50 (d, J=8.3, 2H), 7.40–

Experimental Section

7.38 (d, J=7.8, 1H), 4.62–4.59 (t, J=6.7, 2H), 3.65–3.62 ppm (t, J=

6.7, 2H); ¹³C NMR (100 MHz, CDCl₃): d=165.46, 149.22, 142.44,

139.31, 133.31, 131.87, 131.04, 130.40, 128.78, 127.96, 121.92,

62.00, 30.25 ppm; MS (ESI) m/z: 447 [M+Na]⁺.

Chemistry

Reagents were obtained from commercial suppliers and were used

without further purification. ¹H and ¹³C NMR spectra were mea-

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Table 2. In vitro inhibition of the PA–PB1 interaction (ELISA) and ligand

efficiency (LE), along with activity (Act) and cytotoxicity (Cytox) of com-

pounds 6–48 toward influenza virus strain A/WSN/33 (H1N1).

Compd

ELISA

IC₅₀[mm]^[a]

LE

Act

IC₅₀[mm]^[a]

Cytox [mm]^[a]

[kcal(mol·NHA)^À1

]

6a

6b

6c

7

8a

8b

8c

16

20

100

inactive

100

0.22

–

2.5

–

10

1

2.5

1

10

20

10

15

–

15

10

5

15

–

0.34

0.23

0.26

0.25

0.34

0.27

0.19

0.21

–

40

2.5

10

20

80

40

–

90

50

45

110

15

200

60

45

21

22

23

24a

24b

24c

24d

33

39a

39b

40

47a

47b

47d

47e

47 f

47h

48a

48b

48c

75

Scheme 9. Reagents and conditions: a) toluene, 1008C, 2 h; b) appropriate

nucleophile, K₂CO₃, DMF, MW (1308C), 10 min; c) 1. NBS, conc H₂SO₄,

À258C/À188C, 3 h, 2. conc H₂SO₄, KNO₃, À108C, 1 h; d) appropriate nucleo-

phile, K₂CO₃, DMF, MW (1308C), 10 min.

inactive

100

–

0.23

0.22

0.21

0.16

0.23

–

0.17

–

0.19

0.20

80

20

40

100

60

40

–

90

–

40

25

30

25

5

200

250

35

inactive

40

50

60

1

–

40

–

inactive

35

5

20

[a] All compounds were tested twice; values are the average of two ex-

periments.

2-(7-Nitrobenzo[c][1,2,5]oxadiazol-4-ylthio)ethyl cinnamate (6b):

1

Yellow solid (0.12 mmol, 87%). H NMR (400 MHz, CDCl₃): d=8.45–

8.43 (d, J=7.8, 1H), 7.71–7.65 (d, J=16, 1H), 7.52–7.39 (m, 6H),

6.41–6.36 (d, J=16, 1H), 4.58–4.54 (t, J=7.2, 2H), 3.66–3.62 ppm (t,

J=7.2, 2H); ¹³C NMR (100 MHz, CDCl₃): d=166.83, 149.52, 146.46,

142.75, 139.97, 134.13, 131.03, 130.88, 129.25, 128.42, 121.92,

116.97, 61.64, 30.44 ppm; MS (ESI) m/z: 394 [M+Na]⁺.

2-(7-Nitrobenzo[c][1,2,5]oxadiazol-4-ylthio)ethyl

thiophene-2-

carboxylate (6c): Yellow solid (0.116 mmol, 83%). ¹H NMR

(400 MHz, CDCl₃): d=8.34–8.32 (d, J=8, 1H), 7.69–7.68 (m, 1H),

7.52–7.51 (m, 1H), 7.43–7.41 (d, J=8, 1H), 7.04–7.02 (m, 1H), 4.60–

4.58 (t, J=7.8, 2H), 3.65–3.62 ppm (t, J=7.8, 2H); ¹³C NMR

(100 MHz, CDCl₃): d=162.95, 143.86, 143.48, 136.72, 134.66, 133.82,

128.98, 126.78, 124.52, 119.13, 57.33, 36.67 ppm; MS (ESI) m/z: 260

[M+Na]⁺.

Scheme 10. Reagents and conditions: a) Ac₂O, reflux, 2 h; b) 1. Lawesson’s re-

agent, xylene, 1108C, 2 h. 2. Cs₂CO₃, reflux, 16 h; c) conc H₂SO₄, KNO₃, 1 h,

À108C, then RT, 2 h; d) appropriate nucleophile, cat. pyridine and/or KOAc,

EtOH, reflux, 3 h; e) 4-OMePhSH, K₂CO₃, EtOH, reflux, 3 h; f) Ac₂O, reflux, 2 h.

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1H), 7.62–7.59 (m, 2H), 7.05–7.03 (m, 1H), 3.76–3.73 (m, 2H), 3.61–

3.57 ppm (m, 2H); ¹³C NMR (100 MHz, CDCl₃): d=162.47, 143.78,

141.07, 136.53, 134.59, 133.44, 132.71, 131.71, 130.49, 128.17,

99.53, 44.62, 27.78 ppm; MS (ESI) m/z: 373 [M+Na]⁺.

N-(2-(7-Nitrobenzo[c][1,2,5]oxadiazol-4-ylthio)ethyl)nicotinamide

1

(8c): Yellow solid (0.072 mmol, 58%). H NMR (400 MHz, CDCl₃): d=

9.18 (s, 1H), 8.76 (brs, 1H), 8.75 (brs, 1H), 8.57 (brs, 1H), 8.51 (d,

1H, J=8 Hz), 7.78 (d, 1H, J=8 Hz), 3.90–3.85 (m, 2H), 3.68–

3.64 ppm (m, 2H); ¹³C NMR (100 MHz, CDCl₃): d=165.92, 147.64,

146.73, 143.74, 143.05, 135.52, 131.46, 126.29, 122.27, 37.43,

30.17 ppm; MS: (ESI): m/z 345 [MÀH]^À, 381 [M+Cl]^À

S-2-(7-Nitrobenzo[c][1,2,5]oxadiazol-4-ylamino)ethylbenzo-

thioate (9a): Red solid (0.018 mmol, 15%). ¹H NMR (400 MHz,

[D₆]DMSO): d=8.46 (d, 1H, J=8.3 Hz), 7.84 (d, 2H, J=8.5 Hz), 7.62

(t, 1H, J=8.5 Hz), 7.48 (t, 2H, J=8.5 Hz), 6.50 (d, 1H, J=8.3 Hz),

3.68 (brs, 1H), 3.35 (t, 2H, J=6.1 Hz), 3.23 ppm (t, 2H, J=6.1 Hz);

¹³C NMR (100 MHz, [D₆]DMSO): d=199.4, 149, 143.4, 141.4, 137.9,

134.5, 132.6, 129.7, 127, 123.8, 95.1, 68.5, 33.3 ppm.

S-2-(7-Nitrobenzo[c][1,2,5]oxadiazol-4-ylamino)ethylthiophene-

2-carbothioate (9b): Red solid (0.027 mmol, 22%). ¹H NMR

(400 MHz, CDCl₃): d=8.47–8.45 (d, J=8, 1H), 7.79–7.77 (m, 1H),

7.65–7.63 (m, 1H), 7.10–7.08 (m, 1H), 6.34–6.32 (d, J=8, 1H), 3.77–

3.75(m, 2H), 3.39–3.36 ppm (m, 2H); ¹³C NMR (100 MHz, CDCl₃): d=

192.33, 143.40, 140.87, 136.17, 133.95, 133.27, 132.11, 131.69,

129.99, 128.17, 99.02, 44.13, 27.49 ppm; MS (ESI) m/z: 373 [M+

Na]⁺.

Figure 3. Binding mode of the benzofurazan derivative 39b.

General procedure (A) for aromatic nucleophilic substitution

To a solution of 4-chloro-7-nitrobenzofuzan (50 mg, 0.25 mmol) in

5 mL EtOH, the appropriate nucleophile was added (0.27 mmol).

The reaction mixture was heated at reflux for 5 min and, if the

color had not changed, a catalytic amount of pyridine (0.05 mL) or

of KOAc (0.01 mmol) was added, and reflux was continued for a fur-

ther 20 min. If a strong red coloration did develop within 5 min, no

catalyst was added, and reflux was continued for a further 15 min.

The mixture was cooled to room temperature, and the precipitate

formed was filtered off to give the desired product pure.

General procedure for compounds 10a,b

Benzofurazans 10a,b were prepared through the reaction of 3

with 12 or 13 according to general procedure (B).

7-(2-Aminoethylthio)-4-nitrobenzofurazan (7): Compound 7 was

synthesized according the general procedure for aromatic nucleo-

philic substitution. Yellow solid (0.217 mmol, 87%). ¹H NMR

(400 MHz, CD₃OD): d=8.48–8.46 (d, J=7.9, 1H), 7.55–7.53 (d, J=

7.9, 1H), 3.65–3.62, (m, 2H), 3.35–3.31 ppm (m, 2H); ¹³C NMR

(100 MHz, CDCl₃): d=149.43, 142.68, 137.06, 133.52, 130.99, 122.86,

37.57, 27.78 ppm; MS (ESI) m/z: 241 [M+H]⁺.

N-Boc-(N-(2-(7-nitrobenzo[c][1,2,5]oxadiazol-4-ylthio)ethyl)guani-

dine (10a): Yellow solid (0.088 mmol, 71%). ¹H NMR (400 MHz,

CDCl₃): d=8.60–8.58 (d, J=8, 1H), 7.24–7.22 (d, J=8, 1H), 3.84–

3.82 (m, 2H), 3.73–3.70 (m, 2H), 1.35 ppm (s, 9H); ¹³C NMR

(100 MHz, CDCl₃): d=163.12, 156.30, 153.17, 144.23, 143.73, 136.19,

124.12, 83.53, 79.47, 39.22, 36.69, 28.13 ppm; MS (ESI) m/z: 483

[M+H]⁺.

(S)-2-Amino-3-hydroxy-N-(2-(7-nitrobenzo[c][1,2,5]oxadiazol-4-

ylthio)ethyl)propanamide hydrochloride (10b): Yellow solid

(0.125 mmol, quant). [a]_D: À2.928 (c=5.7, MeOH); ¹H NMR

(400 MHz, CDCl₃): d=8.45 (d, 1H, J=8 Hz, benzofurazan), 7.55 (d,

1H, J=8 Hz), 3.88–3.85 (m, 2H), 3.78–3.73 (m, 1H), 3.64–3.59 (m,

4H), 3.47–3.44 ppm (m, 2H); ¹³C NMR (100 MHz, CDCl₃): d=167.20,

149.38, 143.53, 142.60, 139.23, 130.99, 121,81, 60.14, 54.79, 37.40,

29.90 ppm; MS (ESI) m/z: 483 [M+H]⁺.

General procedure (B) for compounds 8a–c and 9a–c

To a solution of 7 (30 mg, 0.125 mmol) in dry CH₂Cl₂(2 mL), pyri-

dine (0.15 mmol), catalytic amount of DMAP (0.1) and the appropri-

ate acyl chloride (0.15 mmol) were added. The reaction was stirred

at room temperature for 6 h and then was quenched with

NaHCO₃. The mixture was extracted with EtOAc and washed with

1n HCl and brine. The organic layer was dried over Na₂SO₄, filtered

off and the solvent was removed. The crude residue was purified

by flash chromatography (Hex/EtOAc 1:1).

tert-Butyl-(tert-butoxycarbonylamino) (2- thioethylamino)methy-

lenecarbamate (12): Cysteamine was reacted with protected gua-

nidine according to the general procedure (B). The residue was pu-

rified by flash chromatography (Hex/EtOAc 4:1). White solid

(0.098 mmol, 79%). ¹H NMR (400 MHz, CDCl₃): d=8.53 (brs, 1H),

3.67–3.69 (m, 2H), 2.80–2.77 (m, 2H), 1.40 ppm (s, 18H); ¹³C NMR

(100 MHz, CDCl₃): d=158.78, 157.83, 161.03, 84.92, 80.05, 40.23,

27.87 ppm; MS (ESI) m/z: 320 [M+H]⁺.

N-(2-(7-Nitrobenzo[c][1,2,5]oxadiazol-4-ylthio)ethyl)benzamide

(8a): Yellow solid (0.09 mmol, 62%). ¹H NMR (400 MHz,

[D₆]acetone): d=8.50–8.48 (d, J=8, 1H), 8.19 (brs, 1H), 7.83–7.78

(m, 3H), 7.48–7.44, (m, 1H), 7.40–7.36 (m, 2H), 3.79–3.77 (m, 2H),

3.62–3.59 ppm (m, 2H); ¹³C NMR (100 MHz, [D₆]acetone): d=

167.07, 153.08, 149.97, 140.35, 134.76, 131.62, 131.40, 128.36,

127.12, 122.23, 38.24, 30.32 ppm; MS (ESI) m/z: 367 [M+Na]⁺.

(S)-tert-Butyl-3-hydroxy-1-(2-thioethylamino)-1-oxo-propan-2-yl-

carbamate (13): Compound 13 was obtained using the same pro-

cedure as for 12, but with cysteamine and Boc-serine as the start-

ing materials (0.052 mmol, 42%). [a]_D: À1.768 (c=10, MeOH);

¹H NMR (400 MHz, [D₆]acetone): d=87.47 (brs, 1H), 5.96 (brs, 1H),

N-(2-(7-Nitrobenzo[c][1,2,5]oxadiazol-4-ylthio)ethyl)thiophene-2-

carboxamide (8b): Yellow solid (0.072 mmol, 58%). ¹H NMR

(400 MHz, CDCl₃): d=8.51–8.49 (d, J=8, 1H), 7.77–7.75 (d, J=8,

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4.11–4.08 (m, 1H), 4.03 (brs, 1H), 3.76–3.72 (m, 1H), 3.68–3.63 (m,

1H), 3.36–3.29 (m, 2H), 2.59–2.53 (m, 2H), 1.78 (t, J=8 Hz, 1H),

1.34 ppm (s, 9H); ¹³C NMR (100 MHz, [D₆]acetone): d=170.6, 155.9,

78.6, 62.4, 56.3, 42.3, 27.5, 23.6 ppm; MS (ESI) m/z: 287 [M+H]⁺.

The organic layer was dried over Na₂SO₄, filtered off and the sol-

vent removed under reduced pressure. The crude was purified by

flash chromatography (Hex/EtOAc 20:80) to give the desired amide

as a yellow oil (1.28 mmol, 75%). ¹H NMR (400 MHz, CDCl₃): d=

3.69–3.67 (m, 4H), 3.55–3.53 (m, 4H), 2.64–2.59 (m, 2H), 2.47–2.44

(m, 2H), 1.60–1.56 ppm (m, 1H); ¹³C NMR (100 MHz, CDCl₃): d=

169.00, 47.91, 44.19, 37.10, 27.68, 27.24, 19.94 ppm; MS (ESI) m/z:

214 [M+Na]⁺.

1-Morpholino-3-(7-nitrobenzo[c][1,2,5]oxadiazol-4-ylthio)propan-

1-one (14a): Yellow solid (0.091 mmol, 73%). ¹H NMR (400 MHz,

CDCl₃): d=8.33–8.31 (d, J=8, 1H), 7.21–7.19 (d, J=8, 1H), 3.62–

3.52 (m, 8H), 3.41–3.38 (m, 2H), 2.81–2.77 ppm (m, 2H); ¹³C NMR

(100 MHz, CDCl₃): d=168.17, 149.06, 142.42, 141.00, 132.64, 130.67,

120.74, 66.65, 66.33, 45.62, 42.14, 31.56, 26.86 ppm; MS (ESI) m/z:

361 [M+Na]⁺.

4-(3-(1H-Imidazol-1-yl)phenylthio)-7-nitrobenzo[c]-

[1,2,5]oxadiazole (20): Compound 19 (0.38 mmol) was dissolved

into a solution of H₂O/dioxane (3:1 mL) and H₃PO₄was added until

pH 2 was reached. Solid paraformaldehyde (15 mg), glyoxal sol.

40% in H₂O (0.1 mL), and NH₄Cl saturated solution (0.5 mL) were

then added and the resulting mixture was irradiated by microwave

for 10 min at 1208C. The mixture was then cooled to 08C, and

NaOH was added to pH 12. The alkaline solution was extracted

with EtOAc (2ꢁ10 mL). The combined organic layers were washed

with brine, dried (Na₂SO₄), filtered and evaporated to give the

crude 11. The crude product was then purified by flash chromatog-

raphy (EtOAc/MeOH 9:1) (0.17 mmol, 45%). ¹H NMR (400 MHz,

CDCl₃): d=8.21 (d, J=8 Hz, 1H), 7.96, (s, 1H), 7.70 (s, 1H), 7.67–

7.58 (m, 3H), 7.28, (s, 1H), 7.19 (s, 1H), 6.75 ppm (d, J=8 Hz, 1H);

¹³C NMR (100 MHz, CDCl₃): d=149.03, 142.89, 138.84, 136.77,

136.28, 133.97, 132.52, 131.93, 130.03, 129.89, 125.74, 125.25,

122.51, 121.37, 116.71 ppm; MS (ESI) m/z: 340 [M+H]⁺.

3-(7-Nitrobenzo[c][1,2,5]oxadiazol-4-ylthio)-1-thiomorpholino-

propan-1-one (14b): Yellow solid (0.096 mmol, 77%). ¹H NMR

(400 MHz, CDCl₃): d=8.28–8.26 (d, J=8, 1H), 7.18–7.16 (d, J=8,

1H), 3.78–3.64 (m, 4H), 3.49–3.46 (m, 2H), 2.79–2.76 (m, 2H), 2.54–

2.51 ppm (m, 4H); ¹³C NMR (100 MHz, CDCl₃): d=167.96, 149.00,

142.37, 141.17, 132.37, 130.94, 120.71, 47.96, 44.47, 31.76, 27.71,

27.22, 26.92 ppm; MS (ESI) m/z: 377 [M+Na]⁺.

Ethyl

3-(7-nitrobenzo[c][1,2,5]oxadiazol-4-ylthio)propanoate

(15a): Yellow solid (0.095 mmol, 76%). ¹H NMR (400 MHz, CDCl₃):

d=8.34–8.32 (d, J=8, 1H), 7.20–7.18 (d, J=8, 1H), 4.11–4.06 (q,

J=8, 2H), 3.50–3.46 (t, J=8, 2H), 2.78–2.75 (t, J=8, 2H), 1.19–

1.16 ppm (t, J=8, 4H); ¹³C NMR (100 MHz, CDCl₃): d=170.56,

149.08, 142.42, 140.41, 132.64, 120.91, 61.28, 32.88, 26.57,

14.12 ppm; MS (ESI) m/z: 320 [M+Na]⁺.

4-Methyl-N-(3-(7-nitrobenzo[c][1,2,5]oxadiazol-4-ylthio)phenyl-

carbamoyl)benzenesulfonamide (21): To a solution of compound

19 (0.2 mmol) in dry CH₂Cl₂(4 mL) was added p-toluenesulfonyl

isocyanate (1.5 equiv) and the solution was stirred at room temper-

ature for 24 h. The mixture was then concentrated in vacuo, and

the crude product was purified by flash chromatography (EtOAc/

MeOH 9:1) (0.116 mmol, 58%). ¹H NMR (400 MHz, [D₆]DMSO): d=

8.75 (brs, 1H), 8.41 (d, J=8 Hz, 1H), 7.86 (brs, 1H) 7.65–7.59 (m,

3H), 7.47 (d, J=8 Hz, 1H), 7.33–7.29 (m, 1H) 7.12 (d, J=8 Hz, 1H),

7.55 (d, 1H, J=8 Hz), 6.71 (d, J=8 Hz, 1H), 2.23 ppm (s, 1H);

¹³C NMR (100 MHz, [D₆]DMSO): d=157.95, 151.23, 148.82, 144.44,

143.53, 143.19, 142.23, 141.12, 140.07, 133.09, 130.91, 129.19,

128.60, 126.94, 125.49, 123.64, 120.35 ppm; MS (ESI) m/z: 484

[MÀH]^À.

(R)-Ethyl-2-(tert-butoxycarbonylamino)-3-(7-nitrobenzo[c]-

[1,2,5]oxadiazol-4yl-thio)propanoate

(15b):

Yellow

solid

(0.086 mmol, 69%). ¹H NMR (400 MHz, CD₃OD): d=8.53–8.51 (d,

J=8, 1H), 7.55–7.53 (d, J=8, 1H), 4.55–4.51 (m, 1H), 4.26–4.18 (m,

2H), 3.92–3.87 (m, 1H), 3.64–3.59 (m, 1H), 1.41 (s, 9H), 1.31–

1.28 ppm (m, 3H); ¹³C NMR (100 MHz, CD₃OD): d=169.49, 155.01,

149.23, 142.38, 139.67, 133.20, 130.55, 121.29, 80.83, 62.52, 52.85,

33.88, 28.15 14.06 ppm; MS (ESI) m/z: 413 [M+H]⁺.

4-(Allylthio)-7-nitrobenzo[c][1,2,5]oxadiazole (16): Yellow solid

1

(0.113 mmol, 91%). H NMR (400 MHz, CDCl₃): d=8.33–8.31 (d, J=

7.9, 1H), 7.15–7.13 (d, J=7.9, 2H), 5.94–5.84 (m, 1H), 5.40–5.25 (m,

2H), 3.90–3.88 ppm (m, 2H); ¹³C NMR (100 MHz, CDCl₃): d=148.92,

142.45, 137.41, 135.82, 133.25, 131.02, 130.76, 123.25, 122.68,

119.43, 37.66 ppm; MS (ESI) m/z: 260 [M+Na]⁺.

4,6-Dichloro-N-(3-(7-nitrobenzo[c][1,2,5]oxadiazol-4-ylthio)phen-

yl)-1,3,5-triazin-2-amine (22): To a stirred solution of cyanuric

chloride (51 mg, 0.8 equiv) in DME (10 mL) at À308C, compound

19 (100 mg, 1 equiv) was added and the resulting mixture was

stirred at À308C for 1 h. The reaction mixture was then warmed to

room temperature, diluted with EtOAc, washed with saturated

aqueous solution of NaHCO₃(10 mL), dried and concentrated in

vacuo. The crude product was purified by flash chromatography

3-Thio-1-morpholinopropan-1-one (18a): Morpholine (100 mL,

1.147 mmol) was dissolved in dry CH₂Cl₂(10 mL) and cooled to

08C. Then 3-thiopropionic acid (100 mL, 1.147 mmol), EDC (200 mg,

1.043 mmol), HOBt (141 mg, 1.043 mmol) and DIPEA (218 mL,

1.251 mmol) were added and the mixture was stirred at room tem-

perature for 16 h. The reaction mixture was quenched with saturat-

ed aqueous solution of NaHCO₃(10 mL) and extracted with EtOAc

(3ꢁ10 mL). The combined extract was dried over anhydrous

Na₂SO₄and evaporated to dryness. The crude product was purified

by flash chromatography (EtOAc/Hex 9:1) to provide desired com-

1

(Hex/EtOAc 3:1) (0.263 mmol, 76%). H NMR (400 MHz, CDCl₃): d=

8.22 (d, J=8 Hz, 1H), 7.91 (s, 1H) 7.75 (brs, 1H) 7.67 (d, J=8 Hz,

1H), 7.57–7.53 (t, J=8 Hz, 1H), 7.46 (d, J=8 Hz, 1H), 6.79 ppm (d,

J=8 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃): d=178.48, 165.48, 156.26,

155.05, 150.62, 144.85, 141.60, 141.35, 135.07, 132.22, 128.88,

125.69, 118.07, 117.65 ppm; MS (ESI) m/z: 434–438 [MÀH]^À.

1

pound (0.48 mmol, 42%). H NMR (400 MHz, CDCl₃): d=3.45 (brs,

4H), 3.39 (brs, 2H), 3.26 (brs, 2H), 2.61–2.56 (m, 2H), 2.44–2.41 (m,

2H), 1.55 ppm (t, J=8 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃): d=

169.2, 66.6, 66.3, 45.5, 41.7, 36.7, 18.8 ppm.

4,6-Dichloro-N-(3-(7-nitrobenzo[c][1,2,5]oxadiazol-4-ylthio)phen-

yl)-1,3,5-triazin-2-aminium chloride (23): To a solution of com-

pound 22 (1 equiv) in CH₂Cl₂, a 1m solution of HCl in MeOH

(2 equiv) was added dropwise. The mixture was stirred at room

temperature for 2 min, then the solvent was removed in vacuo to

give the desired pure product (0.31 mmol, quant). ¹H NMR

(400 MHz, [D₆]acetone): d=10.09 (s, 1H), 8.43 (d, J=8 Hz, 1H), 8.11

(s, 1H) 7.90 (d, J=8 Hz, 1H), 7.64–7.60 (t, J=8 Hz, 1H), 7.52 (d, J=

3-Thio-1-thiomorpholinopropan-1-one (18b): To a solution of thi-

omorpholine (172 mL, 1.71 mmol) in dry DMF (5 mL) previously

cooled to 08C, 3-thiopropionic acid 125 (164 mL, 1.88 mmol), EDC

(328 mg, 1.71 mmol), HOBt (231 mg, 1.71 mmol), and DIPEA

(338 mL, 2.05 mmol) were added. The reaction mixture was al-

lowed to stir at room temperature for 10 h. After that time

NaHCO₃was added and the mixture was extracted with EtOAc.

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8 Hz, 1H), 7.03 (d, J=8 Hz, 1H), 2.88 ppm (brs, 1H); ¹³C NMR

(100 MHz, [D₆]acetone): d=179.21, 165.83, 156.31, 155.02, 150.82,

145.15, 141.77, 141.46, 135.12, 132.32, 129.02, 126.08, 118.23,

118.12 ppm; MS (ESI) m/z: 434–438 [MÀH]^À.

tered off and the solvent was removed. The crude residue was pu-

rified by flash chromatography (Hex/EtOAc 4:1) to give the desired

product.

4-(p-Phenylethynyl)phenol (26a): (0.35 mmol, 79%). ¹H NMR

(400 MHz, CD₃OD): d=7.61–7.59 (m, 2H), 7.48–7.36 (m, 5H), 6.91–

6.89 (m, 2H), 5.78 ppm (brs, 1H); ¹³C NMR (100 MHz, CDCl₃): d=

157.35, 133.82, 132.65, 128.47, 128.43, 128.37, 127.32, 115.78,

115.46, 89.88 ppm; MS (ESI) m/z: 195 [M+H]⁺.

General procedure (C) for reductive amination

To a solution of compound 19 (1.0 equiv) in DCE the appropriate

aldehyde (1.0 equiv) was added and the mixture was stirred at

room temperature for 10 min. Then a 1:1 mixture of NaBH₄/PTSA

(1 equiv) was added and the suspension was stirred at room tem-

perature for 16 h. The reaction mixture was quenched with saturat-

ed aqueous solution of NaHCO₃(10 mL) and extracted with CH₂Cl₂

(3ꢁ10 mL). The combined extract was dried over anhydrous

Na₂SO₄, filtered and concentrated. The crude products obtained

were further purified by a flash column chromatography on silica

gel (Hex/EtOAc 3:1).

4-(p-Tolylethynyl)phenol (26b): (0.33 mmol, 75%). ¹H NMR

(400 MHz, CD₃OD): d=7.38–7.36 (d, J=8, 4H), 7.10–7.08 (d, J=8,

2H), 6.75–6.73 (d, J=8, 2H), 2.31 ppm (s, 3H); ¹³C NMR (100 MHz,

CDCl₃): d=157.98, 137.96, 133.56, 132.15, 130.89, 128.32, 119.83,

116.01, 115.65, 89.65, 22.87 ppm; MS (ESI) m/z: 209 [M+H]⁺.

4-((6-Methoxynaphthalen)ethynyl)phenol

(26c):

(0.34 mmol,

78%). ¹H NMR (400 MHz, CD₃OD): d=7.88 (s, 1H), 7.69–7.61 (m,

2H), 7.47–7.45 (m, 1H), 7.10–7.08 (m, 1H), 7.05 (s, 1H), 6.77–

6.75 ppm (m, 2H); ¹³C NMR (100 MHz, CDCl₃): d=158.72, 157.33,

135.02, 134.25, 133.78, 132.32, 129.97, 129.78, 128.92, 127.34,

119.01, 117.98, 115.67, 115.89, 94.75, 89.82, 55.98 ppm; MS (ESI) m/

z: 275 [M+H]+.

N-Benzyl-3-(7-nitrobenzo[c][1,2,5]oxadiazol-4-ylthio)benzena-

1

mine (24a): (0.28 mmol, 91%). H NMR (400 MHz, CDCl₃): d=8.10

(d, J=8 Hz, 1H), 7.28–7.23 (m, 5H), 6.91 (d, J=8 Hz, 1H), 6.78 (s,

1H), 6.59 (d, J=8 Hz, 1H), 4.30 ppm (s, 2H); ¹³C NMR (100 MHz,

CDCl₃): d=152.82, 146.40, 141.57, 141.44, 135.74, 134.64, 134.31,

134.14, 132.0, 130.79, 130.62, 130.03, 127.06, 124.46, 121.71, 119.07,

51.11 ppm; MS (ESI) m/z: 377 [MÀH]^À, 413 [M+Cl]^À.

N-(Biphenyl-4-ylmethyl)-3-(7-nitrobenzo[c][1,2,5]oxa-diazol-4-

ylthio)benzenamine (24b): (0.27 mmol, 87%). ¹H NMR (400 MHz,

CDCl₃): d=8.09 (d, J=8 Hz, 1H), 7.52–7.48 (m, 4H), 7.39–7.34 (m,

4H), 7.31–7.26 (m, 2H), 6.91 (d, J=8 Hz, 1H), 6.81 (s, 1H), 6.61 (d,

J=8 Hz, 1H), 4.35 ppm (s, 2H); ¹³C NMR (100 MHz, CDCl₃): d=

152.62, 151.45, 146.17, 145.50, 143.58, 143.41, 140.19, 134.31,

133.79, 132.40, 132.27, 132.19, 131.87, 130.83, 130.47, 129.93,

126.88, 124.18, 121.50, 118.82, 50.66 ppm; MS (ESI) m/z: 453

[MÀH]^À.

N-(Naphthalen-2-ylmethyl)-3-(7-nitrobenzo[c][1,2,5]oxadiazol-4-

ylthio)benzenamine (24c): (0.26 mmol, 85%). ¹H NMR (400 MHz,

CDCl₃): d=7.74 (d, J=8 Hz, 1H), 7.69–7.64 (m, 4H), 7.43–7.36 (m,

3H), 7.28–7.24 (m, 1H), 6.88 (d, J=8 Hz, 1H), 6.82 (d, J=8 Hz, 1H),

6.75 (s, 1H), 6.35 (d, J=8 Hz, 1H), 4.48 ppm (s, 2H); ¹³C NMR

(100 MHz, CDCl₃): d=144.30, 143.92, 134.97, 134.81, 134.28, 133.89,

132.86, 132.16, 130.65, 130.17, 129.24, 129.01, 128.16, 127.72,

127.44, 126.81, 125.22, 122.53, 119.93, 117.82, 49.49 ppm; MS (ESI)

m/z: 427 [MÀH]^À, 463 [M+Cl]^À.

3-(7-Nitrobenzo[c][1,2,5]oxadiazol-4-ylthio)-N-(3-phenoxyben-

zyl)benzenamine (24d): (0.266 mmol, 86%). ¹H NMR (400 MHz,

CDCl₃): d=8.12 (d, J=8 Hz, 1H), 7.28–7.21 (m, 4H), 7.05–7.00 (m,

2H), 6.94–6.88 (m, 4H), 6.84 (d, J=8 Hz, 1H), 6.75 (s, 1H), 6.59 (d,

J=8 Hz, 1H), 4.29 ppm (s, 2H); ¹³C NMR (100 MHz, CDCl₃): d=

158.69, 158.61, 152.30, 150.49, 144.77, 143.97, 142.67, 134.53,

133.03, 131.97, 127.88, 125.33, 124.90, 124.29, 123.69, 123.29,

121.27, 120.64, 120.40, 119.64, 119.43, 118.95, 118.81, 117.09,

47.90 ppm; MS (ESI) m/z: 469 [MÀH]^À.

4-(3-(4-Methoxybenzyloxy)prop-1-ynyl)phenol (26d): (0.27 mmol,

1

61%). H NMR (400 MHz, CD₃OD): d=7.27–7.25 (d, J=8, 4H), 6.84–

6.82 (d, J=8, 2H), 6.69–6.67 (d, J=8, 2H), 4.55 (s, 2H), 4.29 (s, 2H),

3.74 ppm (s, 3H); ¹³C NMR (100 MHz, CDCl₃): d=158.35, 157.98,

134.22, 130.01, 129.42, 127.89, 116.02, 115.56, 114.77, 97.01, 87.02,

57.53, 54.98 ppm; MS (ESI) m/z: 269 [M+H]⁺.

4-Nitro-7-(4-(phenylethynyl)phenoxy)benzo[c][1,2,5]oxadiazole

(27a): According to general procedure (A) for aromatic nucleophil-

ic substitution. Compound was obtained as

a yellow solid

(0.21 mmol, 65%) ¹H NMR (400 MHz, CDCl₃): d=8.37–8.35 (d, J=

7.8, 1H), 7.57–7.55 (d, J=8, 2H), 7.26–7.24 (d, J=8, 2H), 7.15–7.13

(d, J=8, 2H), 6.97–6.95 (d, J=8, 2H), 6.51–6.49 ppm (d, J=7.8,

1H); ¹³C NMR (100 MHz, CDCl₃): d=159.67, 148.77, 144.86, 141.03,

137.89, 135.84, 133.31, 133.07, 131.48, 120.53, 118.91, 118.73,

117.85, 117.58, 90.43, 90.41 ppm; MS (ESI) m/z: 380 [M+Na]⁺.

4-Nitro-7-(4-(p-tolylethynyl)phenoxy)benzo[c][1,2,5]oxadiazole

(27b): According to general procedure (A) for aromatic nucleophil-

ic substitution. Compound was obtained as

a yellow solid

(0.2 mmol, 66%). ¹H NMR (400 MHz, CD₃OD): d=8.36–8.34 (d, J=

7.8, 1H), 7.55–7.53 (d, J=8, 2H), 7.27–7.25 (d, J=8, 2H), 7.16–7.14

(d, J=8, 2H), 6.94–6.92 (d, J=8, 2H), 6.52–6.50 ppm (d, J=7.8,

1H); ¹³C NMR (100 MHz, CDCl₃): d=158.43, 147.61, 144.56, 140.28,

137.45, 135.21, 133.87, 133.52, 130.07, 120.35, 118.62, 118.03,

117.51, 117.36, 90.45, 90.41, 23.67 ppm; MS (ESI) m/z: 394 [M+

Na]⁺.

4-(4-((6-Methoxynaphthalen-2-yl)ethynyl)phenoxy)-7-

nitrobenzo[c][1,2,5]oxadiazole (27c): According to general proce-

dure (A) for aromatic nucleophilic substitution. Compound was ob-

tained as a yellow solid (0.21 mmol, 68%). ¹H NMR (400 MHz,

CD₃OD): d=8.37–8.35 (d, J=8, 1H), 7.93 (s, 1H), 7.68–7.64 (m, 4H),

7.50–7.48 (m, 1H), 7.20–7.18 (m, 2H), 7.13–7.11 (m, 1H), 7.07 (s,

1H), 6.55–6.53 (d, J=8, 1H), 3.88 ppm (s, 3H); ¹³C NMR (100 MHz,

CDCl₃): d=159.82, 158.87, 145.02, 147.71, 136.75, 135.66, 134.82,

133.98, 132.75, 132.24, 131.69, 130.45, 129.99, 128.77, 121.37,

119.05, 118.27, 118.02, 117.48, 116.73, 116.52, 108.46, 106.81, 94.03,

88.72, 57.42 ppm; MS (ESI) m/z: 460 [M+Na]⁺.

General procedure for Sonogashira reaction

An oven-dried screw-cap tube was charged with 4-iodophenol

(100 mg, 0.45 mmol), the appropriate alkyne (0.49 mmol), CuI

(0.005 mmol), and PdCl₂(PPh₃)₂(0.005 mmol). The reaction mixture

was suspended in a DMF/Et₃N mixture (0.5:1 mL) and heated

under microwave for 5 min at 1208C. After that time the mixture

was poured into 0.1n HCl and extracted with Et₂O. The combined

organic layers were washed with NaHCO₃, dried over Na₂SO₄fil-

4-(4-(3-(3-Methoxyphenethoxy)prop-1-ynyl)phenoxy)-7-

nitrobenzo[c][1,2,5]oxadiazole (27d): According to general proce-

dure (A) for aromatic nucleophilic substitution. Compound was ob-

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tained as a yellow solid (0.15 mmol, 61%). ¹H NMR (400 MHz,

CD₃OD): d=8.37–8.35 (d, J=8, 1H), 7.56–7.54 (d, J=8, 2H), 7.27–

7.25 (d, J=8, 2H), 7.16–7.14 (d, J=8, 2H), 6.52–6.50 (d, J=8, 1H),

4.55 (s, 2H), 4.32 (s, 2H), 3.75 ppm (s, 3H); ¹³C NMR (100 MHz,

CDCl₃): d=159.51, 153.75, 152.62, 145.12, 134.33, 134.13, 133.16,

129.82, 122.05, 120.99, 113.99, 113.86, 108.15, 107.96, 86.76, 84.73,

71.57, 56.94 ppm; MS (ESI) m/z: 454 [M+Na]⁺.

mide (2 mmol) was added dropwise. The reaction mixture was al-

lowed to stir at room temperature for a further 10 h. After that

time H₂O and EtOAc were added. The mixture was extracted with

EtOAc, the organic layer was dried over Na₂SO₄, filtered off and the

solvent removed under reduced pressure. The crude was purified

by flash chromatography (Hex/EtOAc 98:2) to give 31 as a colorless

oil (0.71 mmol, 53%). ¹H NMR (400 MHz, CDCl₃): d=7.15–7.13 (d,

J=8, 2H), 6.77–6.75 (d, J=8, 2H), 5.94–5.84 (m, 2H), 5.25–5.21 (m,

2H), 5.18–5.10 (m, 2H), 4.72–4.69 (m, 1H), 3.92–3.90 (m, 2H), 1.25–

1.18 (m, 3H), 1.07 ppm (s, 18H); ¹³C NMR (100 MHz, CDCl₃): d=

155.52, 139.01, 134.89, 133.25, 128.13, 127.91, 127.66, 119.98,

119.70, 119.40, 117.46, 116.61, 115.83, 114.60, 81.55, 68.97, 17.85,

12.86 ppm; MS (ESI) m/z: 369 [M+Na]⁺.

4-(2-(6-Methoxynaphthalen-2-yl)ethyl)phenol (28): To a solution

of 26c (84 mg, 0.30 mmol) in EtOAc (4 mL) a catalytic amount of

Pd/C (5%) was added. The reaction mixture was stirred under hy-

drogen atmosphere for 10 h. After that time the mixture was fil-

tered on a Celite pad, the solvent was removed and the crude was

purified by flash chromatography (Hex/EtOAc 4:1) to give the de-

1

sired compound (0.24 mmol, 81%). H NMR (400 MHz, CD₃OD): d=

(4-(2,5-Dihydrofuran-2-yl)phenol (32): a) Compound 31 (103 mg,

0.29 mmol) was dissolved in CH₂Cl₂dry (20 mL) under argon at-

mosphere, then second-generation Grubbs catalyst (2%) was

added. The reaction mixture was allowed to stir at room tempera-

ture for 1 h, then the solvent was removed and the crude was puri-

fied by flash chromatography (Hex/Et₂O 96:4) to give the desired

7.64–7.60 (m, 2H), 7.47 (s, 1H), 7.27–7.22 (m, 3H), 7.11–7.07 (m,

4H), 3.89 (s, 3H), 3.03–2.98 ppm (m, 4H); ¹³C NMR (100 MHz,

CDCl₃): d=157.73, 156.34, 135.83, 134.28, 133.42, 131.72, 130.81,

129.78, 129.41, 128.64, 127.19, 119.93, 117.17, 115.85, 106.87, 57.26,

39.41, 38.93 ppm; MS (ESI) m/z: 279 [M+H]⁺.

1

compound as a colorless oil (0.226 mmol, 78%). H NMR (400 MHz,

4-(4-(2-(6-Methoxynaphthalen-2-yl)ethyl)phenoxy)-7-

CDCl₃): d=7.10–7.08 (d, J=8, 2H), 6.80–6.78 (d, J=8, 2H), 5.97–

5.95 (m, 1H), 5.81–5.79 (m, 1H), 5.67–5.66 (m, 1H), 4.81–4.76 (m,

1H), 4.69–4.65 (m, 1H), 1.23–1.14 (m, 3H), 1.05 ppm (s, 18H);

¹³C NMR (100 MHz, CDCl₃): d=155.74, 134.26, 130.08, 127.71,

126.45, 119.67, 87.53, 75.41, 17.85, 12.61 ppm; MS (ESI) m/z: 341

[M+Na]+.

nitrobenzo[c][1,2,5]oxadiazole (29): Compound 29 was obtained

according to general procedure (B) for aromatic nucleophilic sub-

stitution. Compound was obtained as a yellow solid (0.16 mmol,

1

71%). H NMR (400 MHz, CD₃OD): d=8.33–8.31 (d, J=8, 1H), 7.62–

7.58 (m, 2H), 7.44 (s, 1H), 7.25–7.20 (m, 3H), 7.09–7.05 (m, 4H),

6.40–6.38 (d, J=8, 1H), 3.85 (s, 3H), 3.02 ppm (s, 4H); ¹³C NMR

(100 MHz, CDCl₃): d=157.35, 150.92, 141.01, 136.13, 133.38, 133.13,

130.77, 128.88, 127.68, 126.85, 126.49, 120.67, 118.84, 107.42,

105.71, 55.33, 37.70, 37.23 ppm; MS (ESI) m/z: 464 [M+Na]⁺.

b) To a solution of the previous compound (67 mg, 0.21 mmol), in

dry THF (3 mL) previously cooled to 08C was slowly added a solu-

tion 1m of TBAF in THF (250 mL, 0.25 mmol). The mixture was

stirred at room temperature for 1 h and then 1n HCl and EtOAc

were added. The mixture was extracted with EtOAc, the organic

layer was dried over Na₂SO₄, filtered and the solvent evaporated.

The residue was purified by flash chromatography Hex/Et₂O 3:2 to

give desired compound as a colorless oil (0.17 mmol, 82%).

¹H NMR (400 MHz, CDCl₃): d=7.06–7.04 (d, J=8, 2H), 6.64–6.62 (d,

J=8, 2H), 6.40 (brs, 1H), 5.99–5.97 (m, 1H), 5.79–5.78 (m, 1H),

5.70–5.69 (m, 1H), 4.81–4.77 (m, 1H), 4.69–4.66 ppm (m, 1H);

¹³C NMR (100 MHz, CDCl₃): d=155.85, 133.01, 129.70, 128.17,

126.45, 115.41, 87.70, 75.27 ppm; MS (ESI) m/z: 163 [M+H]⁺.

(4-(1-(Allyloxy)allyl)phenoxy)triisopropylsilane (31): a) To a solu-

tion of 4-hydroxybenzaldeyde (200 mg, 1.64 mmol) in dry DMF

(4 mL), imidazole (267 mg, 3.93 mmol) was added. The mixture was

cooled to 08C and TIPS-Cl (416 mL, 1.96 mmol) was slowly added.

The mixture was stirred at 608C for 2 h. After that time H₂O and

EtOAc were added. The mixture was extracted with EtOAc, the or-

ganic layer was dried over Na₂SO₄, filtered off and the solvent re-

moved under reduced pressure. The crude was purified by flash

chromatography (Hex/EtOAc 9:1) to give 4-(triisopropylsilyloxy)-

benzaldehyde as

a

colorless oil (1.64 mmol, quant). ¹H NMR

(400 MHz, CDCl₃): d=9.72 (s, 1H), 7.64–7.62 (d, J=8, 2H), 6.83–

6.81 (d, J=8, 2H), 1.17–1.09 (m, 3H), 0.96 ppm (m, 18H); ¹³C NMR

(100 MHz, CDCl₃): d=190.26, 161.62, 131.67, 130.16, 120.10, 17.64,

12.51 ppm; MS (ESI) m/z: 301 [M+Na]⁺.

4-(4-(2,5-Dihydrofuran-2-yl)phenoxy)-7-nitrobenzo[c]-

[1,2,5]oxadiazole (33): Compound 33 was obtained according to

general procedure (B) for aromatic nucleophilic substitution. Yellow

solid (0.09 mmol, 59%). ¹H NMR (400 MHz, CD₃OD): d=8.35–8.33

(d, J=8, 1H), 7.45–7.43 (d, J=8, 2H), 7.18–7.16 (d, J=8, 2H), 6.47–

6.45 (d, J=8, 1H), 6.05–6.04 (m, 1H), 5.87–5.86 (m, 1H), 5.79–5.78

(m, 1H), 4.87–4.82 (m, 1H), 4.76–4.73 ppm (m, 1H); ¹³C NMR

(100 MHz, CDCl₃): d=154.35, 152.20, 145.09, 144.09, 141.34, 133.22,

129.34, 128.80, 127.32, 120.91, 107.53, 87.03, 75.03, 75.88,

29.58 ppm; MS (ESI) m/z: 348 [M+Na]⁺.

b) To a solution of 4-(triisopropylsilyloxy)benzaldehyde (459.2 mg,

1.64 mmol) in THF dry (20 mL) cooled to 08C was slowly added

a solution 1m of vinylmagnesium bromide in THF (1.96 mL,

1.96 mmol). The mixture was vigorously stirred for 1.5 h; NH₄Cl was

then added and the mixture was extracted with EtOAc (2ꢁ5 mL).

The organic layers were dried over Na₂SO₄, filtered and the solvent

evaporated. The residue was purified by flash chromatography

(Hex/EtOAc 4:1) to give 1-(4-(triisopropylsilyloxy)phenyl)prop-2-en-

4-(4-(4-Methylpiperazin-1-yl)phenoxy)-7-nitrobenzo[c]-

[1,2,5]oxadiazole (38a): A mixture of 1-(4-hydroxyphenyl)pipera-

zine (100 mg, 0.56 mmol), formic acid (16 equiv) and formaldehyde

(1.1 equiv) in MeOH (6 mL) was held at reflux for 12 h. After evapo-

ration of solvents in vacuo the residue was partitioned between

CH₂Cl₂and a saturated aqueous solution of NaHCO₃. The extract

was dried over anhydrous Na₂SO₄, filtered and concentrated to

give intermediate 35. This was dissolved in EtOH and 4-chloro-7-ni-

trobenzofurazan (1 equiv) and a catalytic amount of KOAc and pyr-

idine were added. The resulting mixture was heated at reflux for

3 h, then was concentrated in vacuo and the crude product was

purified by flash chromatography (EtOAc/MeOH 7:3) to give de-

1

1-ol as a colorless oil (1.34 mmol, 82%). H NMR (400 MHz, CDCl₃):

d=7.13–7.11 (d, J=8, 2H), 6.80–6.78 (d, J=8, 2H), 5.97–5.89 (m,

1H), 5.22–5.17 (m, 1H), 5.07–5.04 (m, 1H), 5.00–4.98 (m, 1H), 1.25–

1.15 (m, 3H), 1.05 ppm (s, 18H); ¹³C NMR (100 MHz, CDCl₃): d=

155.47, 140.49, 135.21, 127.56, 119.67, 114.41, 74.62, 17.85,

12.62 ppm; MS (ESI) m/z: 305 [MÀH]^À.

c) To a solution of 1-(4-(triisopropylsilyloxy)phenyl)prop-2-en-1-ol

(411 mg, 1.34 mmol) in dry THF (10 mL) under argon atmosphere

previously cooled to 08C, NaH (32 mg, 1.34 mmol) was added. The

mixture was stirred at room temperature for 15 min and allyl bro-

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1

sired compound (0.44 mmol, 85%). H NMR (400 MHz, CDCl₃): d=

8.32 (d, J=7.9 Hz, 1H), 7.05 (d, J=8.2 Hz, 2H), 6.93 (d, J=8.2 Hz,

2H), 6.45 (d, J=7.9 Hz, 1H), 3.18 (4H, m), 2.54 (4H, m), 2.30 ppm

(3H, s); ¹³C NMR (100 MHz, CDCl₃): d=155.18, 150.05, 146.51,

145.22, 145.09, 133.46, 130.21, 121.36, 117.25, 107.09, 54.86, 48.85,

45.94 ppm; MS (ESI) m/z: 356 [M+H]⁺, 378 [M+Na]⁺.

vacuo and the crude product was purified by flash chromatogra-

phy (EtOAc/Hex 3:2) (0.23 mmol, 94%). H NMR (400 MHz, CD₃OD):

1

d=8.47 (d, J=7.8 Hz, 1H), 7.16 (d, J=8.2 Hz, 2H), 7.07 (d, J=

8.2 Hz, 2H), 6.55 (d, J=7.8 Hz, 1H), 3.65–3.63 (4H, m), 3.23–3.20

(4H, m), 1.42 ppm (18H, s); ¹³C NMR (100 MHz, CD₃OD): d=159.72,

154.14, 151.11, 149.15, 145.24, 144.24, 135.58, 129.74, 121.29,

118.24, 116.99, 115.43, 108.70, 80.09, 77.15, 47.82, 45.09, 27.87,

27.70 ppm; MS (ESI) m/z: 582 [MÀH]^À.

1-(4-(4-(7-Nitrobenzo[c][1,2,5]oxadiazol-4-yloxy)phenyl)pipera-

zin-1-yl)ethanone (38b): To a solution of 1-(4-hydroxyphenyl)pi-

perazine (100 mg, 0.56 mmol) in pyridine (3 mL), Ac₂O (58 mL,

0.61 equiv) were added and the mixture was stirred at room tem-

perature for 1 h. The reaction mixture was quenched with saturat-

ed aqueous solution of NaHCO₃(10 mL) and extracted with EtOAc

(3ꢁ10 mL). The combined extract was dried over anhydrous

Na₂SO₄, filtered and concentrated to give intermediate 36. This

was dissolved in EtOH and 4-chloro-7-nitrobenzofurazan (1 equiv)

and a catalytic amount of KOAc and pyridine were added. The re-

sulting mixture was heated at reflux for 3 h. This was concentrated

in vacuo and the crude product was purified by flash chromatogra-

phy (EtOAc/Hex 3:1) to give desired compound (0.45 mmol, 87%).

¹H NMR (400 MHz, CD₃CN): d=8.41 (d, J=7.9 Hz, 1H), 7.14 (d, J=

8.2 Hz, 2H), 7.03 (d, J=8.2 Hz, 2H), 6.51 (d, J=7.9 Hz, 1H), 3.61

(2H, m), 3.56 (2H, m), 3.16 (2H, m), 3.10 (2H, m), 2.00 ppm (3H, s);

¹³C NMR (100 MHz, CD₃CN): d=170.29, 156.22, 151.42, 147.35,

147.14, 146.34, 137.68, 131.85, 123.36, 119.19, 110.78, 50.56, 50.19,

47.41, 42.64, 23.14 ppm; MS (ESI) m/z: 384 [M+H]⁺, 406 [M+Na]⁺.

1-Acetyl-4-(4-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yloxy)phenyl)pi-

perazin-1-ium chloride (38a) and 1-methyl-4-(4-(7-nitrobenzo[c]-

[1,2,5]oxadiazol-4-yloxy)phenyl)piperazin-1-ium chloride (38b):

To a solution of compounds 38a or 38b (1 equiv) in CH₂Cl₂a solu-

tion of 1m HCl in MeOH (2 equiv) was added dropwise. The mix-

ture was stirred at room temperature for 2 min then the solvent

was removed in vacuo to give the desired product pure. 38a:

(0.44 mmol, quant). ¹H NMR (400 MHz, CD₃OD): d=8.50 (d, J=

7.8 Hz, 1H), 7.69 (d, J=8.3 Hz, 2H), 7.45 (d, J=8.3 Hz, 2H), 6.74 (d,

J=7.8 Hz, 1H), 3.95 (4H, m), 3.63 (2H, m), 3.57 (2H, m), 2.15 ppm

(3H, s); ¹³C NMR (100 MHz, CD₃OD): d=170.63, 153.59, 149.62,

145.59, 145.35, 144.33, 133.76, 130.81, 121.65, 120.15, 108.77,

51.65, 51.35, 44.88, 40.13, 19.55 ppm; MS (ESI) m/z: 418 [MÀH]^À.

1

38b:(0.45 mmol, quant). H NMR (400 MHz, CD₃OD): d=8.47 (d, J=

7.8 Hz, 1H), 7.21 (d, J=8.1 Hz, 2H), 7.14 (d, J=8.1 Hz, 2H), 6.56 (d,

J=7.8 Hz, 1H), 3.85–3.82 (2H, m), 3.60–3.57 (2H, m), 3.28–3.24 (2H,

m), 3.12–3.09 (2H, m), 2.92 ppm (3H, s); ¹³C NMR (100 MHz,

CD₃OD): d=155.99, 150.09, 148.50, 146.94, 145.88, 135.61, 131.94,

122.97, 119.86, 109.39, 54.79, 48.30, 43.76 ppm; MS (ESI) m/z: 390

[MÀH]^À.

tert-Butyl-(4-(4-hydroxyphenyl)piperazin-1-yl)methanediylidene-

dicarbamate (37): Preparation of N,N’-di-Boc-N’’-trifluorometha-

nesulfonylguanidine: To a solution of N,N’-di-Boc-guanidine (7.5 g,

29 mmol) in CH₂Cl₂(100 mL) Et₃N (5.0 mL, 36 mmol) was added

and the temperature of the mixture was equilibrated to À788C

using a dry ice/iPrOH bath. Trifluoromethanesulfanyl anhydride

(5.9 mL, 35 mmol) was added dropwise through the dropping

funnel over a period of 20 min, and the resulting mixture was al-

lowed to warm to À208C over 4 h. A 2m aqueous NaHSO₄solution

was added to the mixture at À208C, such that the reaction tem-

perature did not rise above À108C, and the resulting layers were

stirred vigorously for 5 min. The layers were immediately separat-

ed, and the aqueous phase was extracted with CH₂Cl₂(3ꢁ50 mL).

The combined organic layers were washed with 2m aqueous

NaHSO₄(80 mL), brine (50 mL), dried (Na₂SO₄), filtered, and concen-

trated under reduced pressure. The crude material was purified by

flash column chromatography (CH₂Cl₂/Hex 4:1) to provide desired

compound (24.6 mmol, 85%). ¹H NMR (400 MHz, [D₆]DMSO): d=

1.46 (s, 18H), 11.06 ppm (brs, 2H); ¹³C NMR (100 MHz, [D₆]DMSO):

d=27.5, 83.4, 119.1, 150.0, 152.3 ppm.

4-(4-(7-Nitrobenzo[c][1,2,5]oxadiazol-4-yloxy)phenyl)piperazine-

1-carboximidamide (40c): Compound 38c (0.23 mmol) were dis-

solved in 5 mL dry CH₂Cl₂and added with 0.5 mL of freshly distilled

TFA. The mixtures were stirred at room temperature for 24 h. The

solvent was removed under reduced pressure and the crude com-

pound was washed several times with toluene to yield the desired

1

product (0.23 mmol, quant). H NMR (400 MHz, CD₃OD): d=8.47 (d,

J=7.8 Hz, 1H), 7.19 (d, J=8.2 Hz, 2H), 7.09 (d, J=8.2 Hz, 2H), 6.55

(d, J=7.8 Hz, 1H), 3.81–3.79 (1H, dd), 3.63–3.60 (4H, m), 3.29–3.26

(4H, m), 3.16–3.14 ppm (1H, dd); ¹³C NMR (100 MHz, CD₃OD): d=

159.72, 149.15, 145.24, 144.24, 135.58, 129.74, 121.29, 118.24,

116.99, 115.43, 108.70, 47.82, 45.09 ppm; MS (ESI) m/z: 479

[MÀH]^À.

4-Azidophenol (42a): To a stirred ice cooled solution of 4-hydroxy-

laniline (1.0 g, 18.3 mmol) dissolved in a (1:1) mixture of AcOH/H₂O

(50 mL) was added NaNO₂(2.1 g, 30.4 mmol) slowly. After 5 min

were added NaN₃(2 g, 30.76 mmol) and Et₂O (50 mL) and the reac-

tion was stirred for further 10 min. The Et₂O layer was then separat-

ed, washed with H₂O, dried on Na₂SO₄, evaporated and the resul-

tant black oil triturated with hexane (12.26 mmol, 67%). ¹H NMR

(400 MHz, CDCl₃): d=6.8 ppm (s, 4H); ¹³C NMR (100 MHz, CDCl₃):

d=116, 120, 132, 152.5 ppm.

Preparation of 37: A mixture of N,N’-di-Boc-N’’-trifluoromethane-

sulfonylguanidine (218 mg, 0.56 equiv), 1-(4-hydroxyphenyl)pipera-

zine (100 mg, 0.56 mmol), EDC (1.2 equiv) and DIPEA (1.2 equiv) in

DMF (10 mL) was stirred for 16 h at room temperature. The mixture

was partitioned between NH₄Cl_(aq)(5 mL, 1n) and EtOAc (25 mL).

The organic layer was separated, dried (Na₂SO₄), filtered, and con-

centrated in vacuo. The residue was purified by column chroma-

tography (EtOAc/Hex 3:2) to give desired compound (0.42 mmol,

3-azidophenol (42b): 3-Azidophenol was obtained with the same

procedure as for 42a but starting from 3-hydroxylaniline. After

being stirred for 20 min, the reaction mixture was filtered to

remove black insoluble polymeric residues, extracted with CH₂Cl₂,

dried on Na₂SO₄, and the solvent was removed in vacuo to give

1

76%). H NMR (400 MHz, CD₃OD): d=6.80 (d, J=8.3 Hz, 2H), 6.64

(d, J=8.3 Hz, 2H), 3.65–3.63 (4H, m), 3.23–3.20 (4H, m), 1.42 ppm

(18H, s); MS (ESI) m/z: 419 [MÀH]^À.

1

a yellow/brown oil (6.4 mmol, 35%). H NMR (400 MHz, CDCl₃): d=

7.15 (t, J=8.0 Hz, 1H), 6.6 (d, J=11.7 Hz, 2H), 6.5 ppm (s, 1H);

¹³C NMR (100 MHz, CDCl₃): d=106, 111, 112, 113, 130, 157 ppm.

di-tert-Butyl(4-(4-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yloxy)phe-

nyl)piperazin-1-yl)methanediylidenedicarbamate (38c): To a solu-

tion of 4-chloro-7-nitrobenzofurazan (1 equiv) in EtOH compound

20, a catalytic amount of KOAc and pyridine were added. The re-

sulting mixture was heated at reflux for 3 h, then concentrated in

(4-Ethynylphenyl)(methyl)sulfane (43c): To a solution of triiso-

propyl((4-(methylthio)phenyl)ethynyl)silane (0.46 mmol) in THF dry

(4 mL) TBAF was added (0.56 mmol) and the mixture was stirred at

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room temperature for 2 h. Then a NH₄Cl saturated solution (4 mL)

was added and the aqueous phase was extracted with Et₂O (2ꢁ

10 mL). The organic layers were then collected, washed with satu-

rated NaCl solution, dried with Na₂CO₃, and concentrated in vacuo

to provide desired compound (0.45 mmol, 97%). ¹H NMR

(400 MHz, CDCl₃): d=7.32 (d, 2H, J=7.8 Hz), 7.10 (d, 2H, J=

7.8 Hz), 3.0 (s, 1H), 2.40 ppm (s, 3H); ¹³C NMR (100 MHz, CDCl₃):

d=139.98, 132.29, 125.68, 118.26, 83.37, 77.31, 14.04 ppm; MS

(ESI): m/z: 282 [MÀH]^À.

6.89 ppm (d, J=8.1 Hz, 2H); ¹³C NMR (100 MHz, CD₃OD): d=

158.13, 147.82, 130.05, 129.23, 128.50, 127.99, 125.28, 121.86,

118.90, 115.67 ppm; MS (ESI) m/z: 236 [MÀH]^À.

4-(4-(4-Methoxyphenyl)-1H-1,2,3-triazol-1-yl)phenol

(44b):

1

(2.72 mmol, 85%). H NMR (400 MHz, CD₃OD): d=8.44 (s, 1H), 7.68

(d, J=7.9 Hz, 2H), 7.55 (d, J=7.9 Hz, 2H), 6.89–6.85 (m, 4H),

3.72 ppm (s, 3H); ¹³C NMR (100 MHz, CD₃OD): d=159.90, 158.02,

147.74, 129.26, 126.62, 122.51, 121.78, 117.97, 115.65, 113.88,

54.28 ppm; MS (ESI) m/z: 268 [M+H]⁺, 290 [M+Na]⁺.

N-tert-Butyloxycarbonyl-O-propargyl-l-serine methyl ester (43 f):

To a stirred solution of N-tertbutyloxycarbonyl-l-serine (5.13 g,

25 mmol) in DMF (150 mL) at 08C was carefully added NaH (60%

in mineral oil, 4.0 g, 100 mmol). The reaction mixture was stirred

for 30 min until hydrogen evolution stopped. Propargyl bromide

(4.46 mL, 50 mmol) was added, and the mixture was stirred at 08C

for 30 min and then room temperature for 12 h; MeI (2.40 mL) was

added. Four hours later the reaction mixture was poured into

brine, and extracted with Et₂O (3ꢁ150 mL). The combined organic

layers were washed with brine, dried (Na₂SO₄), and concentrated.

The residue was purified by flash chromatography (Hex/Et₂O 4:1)

to provide desired compound (9.75 mmol, 39%). [a]_D: +17.8 (c=

2.7, CHCl₃); ¹H NMR (400 MHz, CDCl₃): d=5.38 (d, J=8.4 Hz, 1H),

4.46 (dt, J=8.4, 3.3 Hz, 1H), 4.15 (d, J=2.4 Hz, 2H), 3.96 (dd, J=

9.3, 3.3 Hz, 1H), 3.77 (s, 3H), 3.76 (dd, J=9.3, 3.3 Hz, 1H), 2.46 (t,

J=2.4 Hz, 1H), 1.46 ppm (s, 9H); ¹³C NMR (100 MHz, CDCl₃): d=

170.86, 155.40, 79.98, 78.77, 75.03, 69.61, 58.49, 53.72, 52.49,

28.24 ppm.

4-(4-Cyclohexenyl-1H-1,2,3-triazol-1-yl)phenol (44d): (1.46 mmol,

73%). ¹H NMR (400 MHz, CD₃OD): d=8.19 (s, 1H), 7.53 (d, J=

8.1 Hz, 2H), 6.86 (d, J=8.1 Hz, 2H), 6.45 (s, 1H), 2.37 (brs, 2H), 2.15

(brs, 2H), 1.73 (brs, 2H), 1.64 ppm (brs, 2H); ¹³C NMR (100 MHz,

CD₃OD): d=157.95, 149.41, 129.30, 126.83, 124.94, 121.71, 117.53,

115.61, 25.82, 24.80, 22.09, 21.79 ppm; MS (ESI) m/z: 240 [MÀH]^À.

4-(4-(Hydroxymethyl)-1H-1,2,3-triazol-1-yl)phenol

(44e):

1

(1.14 mmol, 57%). H NMR (400 MHz, CD₃OD): d=8.21 (s, 1H), 7.53

(d, J=8.2 Hz, 2H), 6.87 (d, J=8 Hz, 2H), 4.67 ppm (s, 2H); ¹³C NMR

(100 MHz, CD₃OD): d=158.09, 148.11, 129.25, 121.92, 120.91,

115.64, 55 ppm; MS (ESI) m/z: 190 [MÀH]^À.

(S)-Methyl-2-(tert-butoxycarbonylamino)-3-((1-(4-hydroxyphen-

yl)-1H-1,2,3-triazol-4-yl)methoxy)propanoate (44 f): (6.5 mmol,

67%). [a]_D: À1.478 (c=10, CHCl₃); ¹H NMR (400 MHz, CDCl₃): d=

7.91 (s, 1H), 7.85 (brs, 1H), 7.40 (d, J=7.8 Hz, 1H), 6.89 (d, J=8 Hz,

1H), 5.87 (brs, 1H), 5.65 (brs, 1H), 4.78 (brs, 2H), 3.70 (brs, 3H),

2.10 (brs, 2H), 1.36 ppm (s, 9H); ¹³C NMR (100 MHz, CDCl₃): d=

165.30, 157.16, 153.82, 144.74, 139.61, 129.75, 122.16, 121.38,

121.34, 118.48, 116.31, 82.02, 77.26, 76.94, 52.34, 45.16, 28.03 ppm;

MS (ESI) m/z: 393 [M+H]⁺.

1-Propynyl-2,3,4-tri-O-benzoyl-b-d-ribofuranose (43g): To a solu-

tion of b-d-ribofuranose 1-acetate 2,3,5 tribenzoate (200 mg,

0.4 mmol) in CH₂Cl₂(5 mL) was added propargyl alcohol (28 mL,

0.47 mmol) and BF₃·Et₂O (73 mL, 0.6 mmol) at 08C and the reaction

mixture was stirred at room temperature for 1 h. After completion

of the reaction anhydrous K₂CO₃(100 mg) was added and stirring

was continued for further 15 min. Then the reaction mixture was

filtered and washed with CH₂Cl₂. The filtrate was washed with H₂O,

the aqueous phase was separated and extracted with CH₂Cl₂(2ꢁ

10 mL) and the combined organic phases were dried (Na₂SO₄) and

concentrated to yield the desired compound as a crystalline solid

1-(1-(4-Hydroxyphenyl)-1H-1,2,3-triazol-4-yl)-2,3,4-tri-O-benzoyl-

b-d-ribofuranose (44g): (0.256 mmol, 72%). [a]_D: À4.258 (c=10,

1

CHCl₃); H NMR (400 MHz, CHCl₃): d=8.77 (brs, 1H), 7.97–7.91 (m,

4H), 7.80 (d, J=8.2 Hz, 2H), 7.76 (s, 1H), 7.48–7.44 (m, 1H), 7.42–

7.33 (m, 6H), 7.31–7.21 (m, 4H), 6.98 (d, J=8.2 Hz, 2H), 5.88–5.85

(m, 1H, H-2), 5.69 (d, J=4.2 Hz, 1H, H-1), 5.39 (s, 1H, H-4), 4.93–

4.90 (m, 1H), 4.76–4.72 (m, 3H), 4.55–4.51 ppm (m, 1H, H-3);

¹³C NMR (100 MHz, CHCl₃): d=166.34, 165.48, 165.31, 157.66,

144.04, 133.57, 133.45, 133.22, 129.72, 129.67, 129.43, 129.36,

128.93, 128.72, 128.47, 128.34, 122.30, 121.52, 116.48, 104.78, 79.41,

75.59, 72.21, 64.56, 61.00 ppm; MS (ESI) m/z: 634 [MÀH]^À.

3-(4-Phenyl-1H-1,2,3-triazol-1-yl)phenol (45a): (4.72 mmol, 77%).

¹H NMR (400 MHz, CD₃OD): d=8.72 (s, 1H), 7.83 (d, J=8.1 Hz, 2H),

7.40–7.37 (m, 2H), 7.31–7.24 (m, 3H), 6.59 ppm (d, J=8.1 Hz, 2H);

¹³C NMR (100 MHz, CD₃OD): d=158.11, 151.77, 130.31, 129.30,

128.55, 128.10, 125.33, 118.95, 115.54, 112.37 ppm; MS (ESI) m/z:

236 [MÀH]^À.

1

(0.356 mmol, 89%). [a]_D: +12.48 (c=10, CHCl₃); H NMR (400 MHz,

CDCl₃): d=8.02–7.96 (m, 4H), 7.81 (d, J=8.1 Hz, 2H), 7.51–7.41 (m,

3H), 7.38–7.32 (m, 4H), 7.26–7.22 (m, 2H), 5.87–5.84 (m, 1H, H-2),

5.69 (d, J=4.2 Hz, 1H, H-1), 5.44 (s, 1H, H-4), 4.70–4.65 (m, 2H),

4.50–4.46 (m, 1H, H-3), 4.20 (s, 2H), 2.39 ppm (s, 1H); ¹³C NMR

(100 MHz, CDCl₃): d=166.10, 165.26, 165.07, 133.44, 133.33, 133.10,

129.75, 129.67, 129.11, 128.84, 128.43, 128.34, 128.29, 103.30, 79.30,

78.25, 75.48, 75.24, 72.07, 64.42, 54.49 ppm; MS (ESI) m/z: 523 [M+

Na]⁺.

General procedure for the synthesis of triazoles 44–45a–e

3-(4-(4-Methoxyphenyl)-1H-1,2,3-triazol-1-yl)phenol

(45b):

1

(2.24 mmol, 70%). H NMR (400 MHz, CD₃OD): d=8.63 (s, 1H), 7.75

(d, J=7.9 Hz, 1H), 7.31–7.23 (m, 3H), 6.95 (d, J=7.9 Hz, 1H),

6.84 ppm (d, J=8 Hz, 1H); ¹³C NMR (100 MHz, CD₃OD): d=160.04,

158.56, 137.94, 130.27, 126.68, 122.45, 117.89, 115.43, 113.92,

110.63, 107.05, 54.30 ppm; MS (ESI) m/z: 266 [MÀH]^À.

The appropriate alkyne (1 equiv) and the freshly synthesized phe-

nylazide (1 equiv) were suspended in a 1:1 mixture of H₂O and tert-

BuOH (1 mL each). To this, was added sodium ascorbate (0.1 equiv)

and CuSO₄·5H₂O (0.01 equiv). The mixture was then irradiated for

10 min at 1258C. The mixture was then partitioned between a satu-

rated aqueous solution of NH₄Cl (10 mL+2 gtt NH₄OH) and EtOAc

(15 mL) and stirred for 15 min. The organic layer was separated,

dried (Na₂SO₄), and solvent removed in vacuo to furnish the de-

sired triazoles as pure compounds.

3-(4-(4-(Methylthio)phenyl)-1H-1,2,3-triazol-1-yl)phenol

(45c):

1

(0.26 mmol, 58%). H NMR (400 MHz, CD₃OD): d=8.72 (s, 1H), 7.78

(s, 1H), 7.75 (s, 1H), 7.34–7.24 (m, 5H), 6.84 ppm (d, 1H, J=8.2 Hz);

¹³C NMR (100 MHz, CD₃OD): d=157.66, 138.43, 129.82, 126.42,

125.85, 121.90, 118.18, 116, 14.42 ppm; MS (ESI) m/z: 282 [MÀH]^À.

4-(4-Phenyl-1H-1,2,3-triazol-1-yl)phenol (44a): (7.75 mmol, 79%).

¹H NMR (400 MHz, CD₃OD): d=8.65 (s, 1H), 7.82 (d, J=8.1 Hz, 2H),

7.61 (d, J=8.1 Hz, 2H), 7.40–7.36 (m, 2H), 7.31–7.29 (m, 1H),

1-(1-(4-Hydroxyphenyl)-1H-1,2,3-triazol-4-yl)-2,3,4-trihydroxy-b-

d-ribofuranose (46): Compound 44g (61 mg, 0.07 mmol) was dis-

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solved in 4:1 MeOH/conc NH₄OH (5 mL) and stirred at room tem-

perature for 18 h. The reaction mixture was concentrated in vacuo

and azeotroped 3 times with EtOH. The crude product was dis-

solved in H₂O (5 mL), extracted with CH₂Cl₂(3ꢁ50 mL) and the

aqueous layer concentrated in vacuo to provide desired compound

133.06, 131.13, 122.64, 122.25, 117.61, 108.37,81.76, 52.26, 45.19,

27.99 ppm; MS (ESI) m/z: 556 [M+H]⁺.

1-(1-(4-(7-Nitrobenzo[c][1,2,5]oxadiazol-4-yloxy)phenyl)-1H-1,2,3-

triazol-4-yl)-2,3,4-tri-hydroxy-b-d-ribofuranose (47h): (0.08 mmol,

1

32%). [a]_D: À1.098 (c=10, MeOH); H NMR (400 MHz, [D₆]acetone):

1

(0.069 mmol, 99%). [a]_D: À1.288 (c=10, CHCl₃); H NMR (400 MHz,

d=8.60 (d, J=8.0 Hz, 1H), 8.55 (s, 1H), 8.06 (d, J=8.2 Hz, 1H), 7.60

(d, J=8.2 Hz, 1H), 6.91 (d, J=8.0 Hz, 1H), 4.98 (s, 1H, H-4), 4.81 (d,

J=11.8 Hz, 1H), 4.67 (d, J=11.8 Hz, 1H), 4.18–4.16 (m, 1H, H-2),

3.91–3.89 (m, 2H), 5.68 (d, J=11.9 Hz, 1H, H-1), 3.56–3.52 (m, 1H,

H-3), 2.82 ppm (brs, 3H); ¹³C NMR (100 MHz, [D₆]acetone): d=

153.24, 153.08, 145.73, 145.57, 144.56, 135.50, 134.32, 131.23,

122.48, 122.27, 121.71, 109.83, 106.83, 84.56, 75.26, 70.85, 62.92,

60.07 ppm; MS (ESI) m/z: 485 [MÀH]^À.

CD₃OD): d=8.27 (s, 1H), 7.52 (d, J=8.2 Hz, 2H), 6.86 (d, J=8.2 Hz,

2H), 4.95 (s, 1H, H-4), 4.81 (d, J=11.9 Hz, 1H), 4.62 (d, J=11.9 Hz,

1H), 4.08–4.05 (m, 1H, H-2), 3.95–3.88 (m, 2H), 3.72–3.69 (m, 1H,

H-1), 3.56–3.51 ppm (m, 1H, H-3); MS (ESI) m/z: 322 [MÀH]^À.

General procedure for the synthesis of compounds 47–48a–h

To a solution of 4-chloro-7-nitrobenzofurazan (0.25 mmol, 1 equiv)

in EtOH the appropriate triazole (1.1 equiv) and a catalytic amount

of KOAc and pyridine were added. The resulting mixture was

heated at reflux for 3 h. This was concentrated in vacuo and the

crude product was purified by flash chromatography with the ap-

propriate eluent.

4-Nitro-7-(3-(4-phenyl-1H-1,2,3-triazol-1-yl)phenoxy)benzo[c]-

[1,2,5]oxadiazole (48a): (0.225 mmol, 90%). ¹H NMR (400 MHz,

[D₆]acetone): d=8.99 (s, 1H), 8.60 (d, J=7.8 Hz, 1H), 8.02 (s, 1H),

8.00 (d, J=8.2 Hz, 1H), 7.94 (d, J=8.2 Hz, 1H), 7.79 (t, J=8.2 Hz,

1H), 7.51 (d, J=8.1 Hz, 1H), 7.42–7.40 (m, 2H), 7.32–7.29 (m, 1H),

6.99 ppm (d, J=7.8 Hz, 1H); ¹³C NMR (100 MHz, [D₆]acetone): d=

155.94, 154.34, 149.50, 141.41, 146.40, 140.23, 137.26, 134.26,

132.88, 132.03, 131.01, 130.35, 127.36, 122.70, 121.77, 119.98,

114.61, 112.93 ppm; MS (ESI) m/z: 435 [M+Cl]^À.

4-Nitro-7-(4-(4-phenyl-1H-1,2,3-triazol-1-yl)phenoxy)benzo[c]-

[1,2,5]oxadiazole (47a): (0.23 mmol, 91%). ¹H NMR (400 MHz,

[D₆]acetone): d=9.00 (s, 1H), 8.64 (d, 1H, J=7.8 Hz), 8.13 (d, J=

8.2 Hz, 2H) 7.94 (d, J=8.1 Hz, 2H), 7.64 (d, J=8.1 Hz, 2H), 7.45–

7.41 (m, 2H), 7.32 (t, 1H, J=8.2 Hz), 6.94 ppm (d, 1H, J=7.8 Hz);

¹³C NMR (100 MHz, [D₆]acetone): d=153.23, 153.13, 148.03, 135.58,

134.27, 128.77, 128.14, 125.46, 122.39, 122.31, 118.73, 109.82 ppm;

MS (ESI) m/z: 435 [M+Cl]^À.

4-(3-(4-(4-Methoxyphenyl)-1H-1,2,3-triazol-1-yl)phenoxy)-7-

1

nitrobenzo[c][1,2,5]oxadiazole (48b): (0.217 mmol, 87%). H NMR

(400 MHz, [D₆]acetone): d=8.85 (s, 1H), 8.57 (d, J=7.8 Hz, 1H),

7.98 (s, 1H), 7.96 (d, J=8.2 Hz, 1H), 7.80–7.74 (m, 3H), 7.48 (d, J=

8.2 Hz, 1H), 6.97–6.92 (m, 3H), 3.76 ppm (s, 1H); ¹³C NMR

(100 MHz, [D₆]acetone): d=161.43, 155.91, 154.36, 149.46, 147.38,

146.37, 140.29, 137.23, 134.21, 132.84, 128.73, 124.55, 122.56,

120.67, 119.86, 116.45, 114.48, 112.84, 57.20 ppm; MS (ESI) m/z: 429

[MÀH]^À.

4-(3-(4-(4-(Methylthio)phenyl)-1H-1,2,3-triazol-1-yl)phenoxy)-7-

nitrobenzo[c][1,2,5]oxadiazole (48c): (0.14 mmol, 57%). ¹H NMR

(400 MHz, [D₆]acetone): d=8.97 (s, 1H), 8.60 (d, J=7.8 Hz, 1H),

8.02 (s, 1H), 8.00 (d, J=8.2 Hz, 1H), 7.84 (d, J=8.1 Hz, 2H), 7.79 (t,

J=8.2 Hz, 1H), 7.51 (d, J=8.2 Hz, 1H), 7.31 (d, J=8.1 Hz, 2H), 6.99

(d, J=8 Hz, 1H), 2.46 ppm (s, 1H); ¹³C NMR (100 MHz, [D₆]acetone):

d=154.38, 153.02, 147.75, 145.59, 139.13, 138.90, 134.28, 131.37,

127.08, 126.37, 126.00, 125.75, 120.71, 120.48, 118.33, 112.97,

112.81, 110.12, 14.50 ppm; MS (ESI) m/z: 469 [M+Na]⁺, 481 [M+

Cl]^À.

4-(4-(4-(4-Methoxyphenyl)-1H-1,2,3-triazol-1-yl)phenoxy)-7-

nitrobenzo[c][1,2,5]oxadiazole (47b): (0.22 mmol, 88%). ¹H NMR

(400 MHz, [D₆]acetone): d=8.87 (s, 1H), 8.61 (d, J=7.8 Hz, 1H),

8.11 (d, 2H, J=8.2 Hz) 7.86 (d, 2H, J=8.1 Hz), 7.63 (d, J=8.1 Hz,

2H), 6.99 (d, J=8.2 Hz, 2H), 6.93 (d, J=7.8 Hz, 1H), 3.79 ppm (s,

3H); ¹³C NMR (100 MHz, [D₆]acetone): d=161.40, 154.76, 154.62,

149.44, 147.42, 146.41, 137.29, 136.94, 132.75, 128.76, 124.28,

123.79, 120.78, 118.12, 116.47, 112.59, 57.23 ppm; MS (ESI) m/z: 429

[MÀH]^À.

4-(4-(4-Cyclohexenyl-1H-1,2,3-triazol-1-yl)phenoxy)-7-

nitrobenzo[c][1,2,5]oxadiazole (47d): (0.217 mmol, 87%). H NMR

1

(400 MHz, [D₆]acetone): d=8.60 (d, J=7.9 Hz, 1H), 8.48 (s, 1H),

8.06 (d, J=8.0 Hz, 1H) 7.60 (d, J=8.0 Hz, 1H), 6.91 (d, J=7.9 Hz,

1H), 6.57–6.55 (m, 1H), 2.40–2.38 (m, 2H), 2.17–2.14 (m, 2H), 1.74–

1.71 (m, 2H), 1.65–1.61 ppm (m, 2H); ¹³C NMR (100 MHz,

[D₆]acetone): d=152.8, 145.5, 143.2, 135.6, 134.1, 127.3, 124.6,

122.25, 122.13, 117.11, 111.47, 109.73, 25.94, 24.85, 22.23, 22.0 ppm;

MS (ESI) m/z: 403 [MÀH]^À, 439 [M+Cl]^À.

General procedure for reduction of nitro to 7-amino deriva-

tives (50a–c)

(1-(4-(7-Nitrobenzo[c][1,2,5]oxadiazol-4-yloxy)phenyl)-1H-1,2,3-

triazol-4-yl)methanol (47e): 0.21 mmol, 85%. ¹H NMR (400 MHz,

[D₆]acetone): d=8.61 (d, J=7.8 Hz, 1H), 8.43 (s, 1H), 8.06 (d, J=

8.2 Hz, 2H), 7.60 (d, J=8.2 Hz, 2H), 6.91 (d, J=7.8 Hz, 1H),

4.72 ppm (s, 2H); ¹³C NMR (100 MHz, [D₆]acetone): d=154.90,

154.55, 151.20, 147.19, 146.17, 137.27, 135.92, 132.81, 123.94,

123.88, 121.93, 111.36, 57.36 ppm; MS (ESI) m/z: 377 [M+Cl]^À, 389

[M+Na]⁺.

The appropriate nitro compound (5.6 mmol) was dissolved in a mix-

ture of CH₂Cl₂(200 mL), conc HCl (12 mL) and MeOH (90 mL). After

the addition of Fe⁰(2.13 g), the mixture was stirred for 25 min at

room temperature. The reaction mixture was poured into H₂O

(100 mL) and extracted with CH₂Cl₂(3ꢁ150 mL). The organic layer

was dried over anhydrous Na₂SO₄and concentrated in vacuo. The

crude product was then purified by flash chromatography (Hex/

EtOAc 3:2).

(R)-Methyl-2-(tert-butoxycarbonylamino)-3-((1-(4-(7-

7-(4-Bromobenzylthio)benzo[c][1,2,5]oxadiazol-4-amine

(50a):

nitrobenzo[c][1,2,5]oxadiazol-4-yloxy)phenyl)-1H-1,2,3-triazol-4-

yl)methoxy)propanoate (47 f): (0.157 mmol, 63%). [a]_D: +2.658

(c=10, MeOH); ¹H NMR (400 MHz, CDCl₃): d=8.37 (d, J=7.8 Hz,

1H), 8.10 (s, 1H), 7.83 (d, J=8.2 Hz, 1H), 7.37 (d, J=8.2 Hz, 1H),

6.59 (d, J=7.8 Hz, 1H), 5.82 (brs, 1H), 5.61 (brs, 1H), 4.78 (brs,

2H), 3.70 (brs, 4H), 1.34 ppm (s, 9H); ¹³C NMR (100 MHz, CDCl₃):

d=165.24, 153.39, 152.58, 145.82, 144.98, 144.07, 139.82, 135.58,

1

(4.87 mmol, 87%). H NMR (400 MHz, CDCl₃): d=7.25 (d, J=4.5 Hz,

2H), 7.01 (d, J=7.8 Hz, 1H), 6.94 (d, J=4 Hz, 2H), 6.13 (d, J=

7.8 Hz, 1H), 4.57 (brs, 2H), 4.07 ppm (s, 2H); MS (ESI) m/z: 334–336

[MÀH]^À.

7-(4-Bromophenylthio)benzo[c][1,2,5]oxadiazol-4-amine (50b):

(2.91 mmol, 52%). ¹H NMR (400 MHz, CDCl₃): d=7.36–7.34 (d, J=

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7.8, 1H), 7.29–7.27 (d, J=8, 2H), 7.09–7.07 (d, J=8, 2H), 6.29–6.27

(d, J=7.8, 1H), 4.73 ppm (brs, 2H); ¹³C NMR (100 MHz, CDCl₃): d=

147.99, 141.43, 136.89, 134.02, 131.96, 130.65, 129.72, 126.44,

125.05, 121.62 ppm; MS (ESI) m/z: 323 [M+H]⁺.

stirred at room temperature for 1 h. The reaction mixture was

quenched with saturated aqueous solution of NaHCO₃(10 mL) and

extracted with EtOAc (3ꢁ10 mL). The combined extract was dried

over anhydrous Na₂SO₄, filtered and concentrated in vacuo and the

crude product was purified by flash chromatography (EtOAc/Hex

3:1) to provide desired compound (0.509 mmol, 91%). ¹H NMR

(400 MHz, CDCl₃): d=8.12 (d, J=8 Hz, 1H), 8.00 (brs, 1H), 7.25 (d,

J=8 Hz, 1H), 3.59–3.54 (m, 6H), 3.39–3.34 (m, 4H), 2.65 (t, J=8 Hz,

2H), 2.24 ppm (s, 3H); ¹³C NMR (100 MHz, CDCl₃): d=169.13,

168.69, 139.88, 132.31, 124.42, 116.89, 111.18, 90.26, 66.73, 66.41,

45.69, 41.99, 32.99, 28.34, 24.55 ppm.

3-(7-Aminobenzo[c][1,2,5]oxadiazol-4-ylthio)-1-morpholinopro-

1

pan-1-one (50c): (5.15 mmol, 92%). H NMR (400 MHz, CDCl₃): d=

29 (d, J=7.8 Hz, 1H), 6.34 (d, J=7.8 Hz, 1H), 5.99 (brs, 2H), 3.49–

3.44 (m, 4H), 3.38–3.35 (m, 4H), 3.13 (t, J=8 Hz, 2H), 2.59 ppm (t,

J=8 Hz, 2H); ¹³C NMR (100 MHz, CDCl₃): d=169.40, 151.13, 145.56,

139.05, 136.89, 106.84, 105.60, 66.58, 45.83, 41.81, 33.13 ppm; MS

(ESI) m/z: 309 [M+Na]⁺.

7-Chloro-N,N-dimethylbenzo[c][1,2,5]oxadiazole-4-sulfonamide

(56): 4-Chlorosulfonyl-7-chloro-2,1,3-benzoxadiazole 55 (200 mg,

0.92 mmol) was dissolved in CH₃CN (6 mL). After the addition of

Me₂NH (0.9 equiv) and Et₃N (cat.), the mixture was stirred at room

temperature for 3 h. The reaction mixture was evaporated to dry-

ness under reduced pressure, and the residue was separated by

chromatography on silica gel using CH₂Cl₂. To give desired com-

General procedure for 7-chloro derivatives 51a,b

To a solution of CuCl₂(0.15 mmol) and tert-butyl nitrite (0.19 mmol)

in dry CH₃CN at 658C was slowly added the appropriate amine pre-

viously solubilized in CH₃CN. During the addition the reaction solu-

tion turned black from the initial green color. The mixture was

stirred at 658C for further 3 h, then was quenched with 3n HCl

and extracted with Et₂O. The organic layer was dried over Na₂SO₄

filtered off and concentrated and the residue was purified by flash

chromatography (Hex/EtOAc 4:1).

1

pound (0.84 mmol, 92%). H NMR (400 MHz, CDCl₃): d=7.89 (d, J=

7.8 Hz, 1H), 7.49 (d, J=7.8 Hz, 1H), 2.88 ppm (s, 6H); ¹³C NMR

(100 MHz, CDCl₃): d=150.40, 147.20, 135.99, 130.68, 129.21, 127.58,

39.18 ppm; MS (ESI) m/z: 284–286 [M+Na]⁺.

4-Chloro-7-(4-methoxybenzylthio)benzo[c][1,2,5]oxadiazole

(51a): Yellow solid (1.86 mmol, 65%). ¹H NMR (400 MHz, CDCl₃):

d=8.29–8.27 (d, J=8, 1H), 7.29–7.27 (d, J=8, 2H), 7.13–7.11 (d,

J=8, 1H), 6.82–6.80 (d, J=8, 2H), 4.41 (s, 2H), 3.73 ppm (s, 2H);

¹³C NMR (100 MHz, CDCl₃): d=159.76, 146.32, 144.87, 130.48,

128.36, 127.51, 126.83, 117.53, 115.62, 113.56, 57.02, 36.45 ppm; MS

(ESI) m/z: 329 [M+Na]⁺.

General procedure for the synthesis of compounds 58a–d

To a solution of compound 56 (65 mg, 0.25 mmol) in 5 mL of EtOH

was added the appropriate nucleophile (0.27 mmol) and a catalytic

amount of pyridine (0.05 mL) or of KOAc (0.01 mmol). The reaction

mixture was heated at reflux for 2 h. The mixture was then concen-

trated in vacuo and the crude product was purified by flash chro-

matography (Hex/EtOAc 3:2).

4-(4-Bromophenylthio)-7-chlorobenzo[c][1,2,5]oxadiazole (51b):

1

Yellow solid (1.51 mmol, 52%). H NMR (400 MHz, CDCl₃): d=7.51–

7.49 (d, J=8.3, 2H), 7.38–7.36 (d, J=8.3, 2H), 7.19–7.17 (d, J=7.8,

1H), 6.78–6.76 ppm (d, J=7.8, 1H); ¹³C NMR (100 MHz, CDCl₃): d=

136.65, 135.69, 133.20, 130.41, 128.79, 128.28, 126.92, 124.27,

122.41, 119.73 ppm; MS (ESI) m/z: 342 [M+H]⁺.

7-(4-Methoxybenzylthio)-N,N-dimethylbenzo[c][1,2,5]oxadiazole-

4-sulfonamide (58a): (0.23 mmol, 92%). H NMR (400 MHz, CDCl₃):

1

d=7.77 (d, J=7.8 Hz, 1H), 7.26 (d, J=8.1 Hz, 2H), 7.09 (d, J=

7.8 Hz, 1H), 6.80 (d, J=8.1 Hz, 2H), 4.35 (s, 2H), 3.75 (s, 3H),

2.85 ppm (s, 6H); ¹³C NMR (100 MHz, CDCl₃): d=160.96, 150.32,

146.64, 137.24, 136.22, 131.56, 127.71, 124.61, 123.43, 115.90, 56.84,

39.22, 37.61 ppm; MS (ESI) m/z: 402 [M+Na]⁺.

3-(7-Iodobenzo[c][1,2,5]oxadiazol-4-ylthio)-1-morpholinopropan-

1-one (52c): To a cooled solution (08C) of compound 50c

(2.25 mmol) in concentrated HCl (0.56 mL) and 98% H₂SO₄(2 mL)

was added dropwise a solution of NaNO₂(2.23 mmol) in 1 mL H₂O.

The mixture was stirred vigorously at 08C for 40 min. Then, a solu-

tion of KI (4.5 mmol) in 0.9 mL of H₂O was added dropwise, and

the mixture was stirred at room temperature for 2 h. The product

was extracted with EtOAc, washed with a saturated Na₂CO₃solu-

tion, H₂O and brine. After drying on anhydrous Na₂SO₄, the solu-

tion was concentrated in vacuo. The crude product was then puri-

fied by flash chromatography (Hex/EtOAc 3:1) to provide desired

compound (2.07 mmol, 92%). ¹H NMR (400 MHz, CDCl₃): d=7.66

(d, J=8 Hz, 1H), 6.91 (d, J=8 Hz, 1H), 3.59–3.54 (m, 6H), 3.40 (t,

J=8 Hz, 2H), 3.37–3.35 (m, 2H), 2.69 ppm (t, J=8 Hz, 2H); ¹³C NMR

(100 MHz, CDCl₃): d=168.84, 151.25, 147.77, 140.85, 128.12, 127.80,

66.69, 66.39, 45.69, 42.07, 32.42, 27.36 ppm.

7-(4-Bromobenzylthio)-N,N-dimethylbenzo[c][1,2,5]oxadiazole-4-

1

sulfonamide (58b): (0.227 mmol, 91%). H NMR (400 MHz, CDCl₃):

d=7.76 (d, J=7.9 Hz, 1H), 7.39 (d, J=7.7 Hz, 2H), 7.22 (d, J=

7.7 Hz, 2H), 7.08 (d, J=7.9 Hz, 1H), 4.35 (s, 2H), 2.85 ppm (s, 6H);

¹³C NMR (100 MHz, CDCl₃): d=150.32, 146.65, 136.19, 136.08,

135.25, 133.65, 131.96, 125.30, 124.07, 123.64, 39.22, 37.43 ppm;

MS (ESI) m/z: 450–452 [M+Na]⁺.

N,N-Dimethyl-7-(3-nitrobenzylthio)benzo[c][1,2,5]oxadiazole-4-

sulfonamide (58c): (0.23 mmol, 93%). ¹H NMR (400 MHz, CDCl₃):

d=8.21 (s, 1H), 8.08 (d, J=8.2 Hz, 2H), 7.77 (d, J=7.8 Hz, 1H), 7.69

(d, J=8.2 Hz, 2H), 7.49–7.45 (m, 1H), 7.13 (d, J=7.8 Hz, 1H), 4.52

(s, 2H), 2.85 ppm (s, 6H); ¹³C NMR (100 MHz, CDCl₃): d=148.47,

137.19, 134.70, 134.33, 133.47, 129.96, 125.27, 124.71, 123.63,

123.26, 123.07, 117.43, 37.59, 35.73 ppm; MS (ESI) m/z: 393

[MÀH]^À, 429 [M+Cl]^À.

7-(4-Fluorobenzylthio)-N,N-dimethylbenzo[c][1,2,5]oxadiazole-4-

sulfonamide (58d): (0.23 mmol, 92%). ¹H NMR (400 MHz, CDCl₃):

d=7.77 (d, J=7.8 Hz, 1H), 7.34–7.30 (m, 2H), 7.09 (d, J=7.8 Hz,

1H), 6.99–6.94 (m, 2H), 4.38 (s, 2H), 2.85 ppm (s, 6H); ¹³C NMR

(100 MHz, CDCl₃): d=163.58–161.11 (C-F), 148.71, 145.04, 134.94,

134.52, 130.40, 123.48, 122.30, 115.85, 37.61, 35.73 ppm; MS (ESI)

m/z: 366 [MÀH]^À, 402 [M+Cl]^À.

7-(4-Methoxybenzylthio)benzo[c][1,2,5]oxadiazol-4-amine hydro-

chloride (53a): Compound 53a was obtained using the same pro-

cedure as for 39a,b (1.5 mmol, 75%). ¹H NMR (400 MHz, CDCl₃):

d=7.02–7.01 (d, 3H), 6.68 (d, J=8 Hz, 2H), 6.12 (d, J=8 Hz, 1H),

4.55 (brs, 2H), 4.08 (s, 2H), 3.68 ppm (s, 3H); MS (ESI) m/z: 286

[MÀH]^À.

N-(7-(3-Morpholino-3-oxopropylthio)benzo[c][1,2,5]oxadiazol-4-

yl)acetamide (54c): To a solution of compound 50c (0.56 mmol) in

pyridine (3 mL), Ac₂O (0.61 mmol) was added and the mixture was

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(9S,9aR)-4,9-Dimethoxy-9a-nitro-5a,6,9,9a-tetrahydronaphtho-

[2,1-c][1,2,5]oxadiazol-7(8H)-one (60b): White solid (0.142 mmol,

51%). ¹H NMR (400 MHz, CDCl₃): d=5.22–5.21 (m, 1H), 4.52–4.47

(m, 1H), 3.73 (s, 3H), 3.69–3.62 (m, 1H), 3.40 (s, 3H), 3.02–2.97 (m,

1H), 2.66–2.61 (m, 2H), 1.81–1.74 ppm (m, 1H).

General procedure for Diels–Alder with Danishefsky diene

In

a sealed tube the appropriate starting material (49a–f)

(0.28 mmol) was dissolved in freshly distilled CH₂Cl₂(2 mL) then

trans-1-methoxy-3-trimethylsilyloxy-buta-1,3-diene was added. The

reaction mixture was heated at 508C for 6–12 h. After that time 2n

HCl was added and the mixture was stirred for further 1 h then the

mixture was extracted with CH₂Cl₂. The organic layer was dried

over Na₂SO₄, filtered off and the solvent was removed under re-

duced pressure. The crude was then purified by flash chromatogra-

phy to give two diastereomers 59a–f and 60a–f.

(9S,9aR)-9-Methoxy-4-(4-methoxyphenoxy)-9a-nitro-5a,6,9,9a-

tetrahydronaphtho[2,1-c][1,2,5]oxadiazol-7(8H)-one (60c): White

solid (0.134 mmol, 48%). ¹H NMR (400 MHz, CDCl₃): d=7.18–7.16

(d, J=8, 2H), 6.92–6.90 (d, J=8, 2H), 6.52–6.50 (m, 1H), 5.30–5.28

(m, 1H), 4.60–4.56 (m, 1H), 3.87 (s, 3H), 3.66–3.59 129 (m, 1H), 3.49

(s, 3H), 3.10–3.05 (m, 1H), 2.75–2.66 (m, 2H), 2.64–2.62 (m, 1H),

1.91–1.87 ppm (m, 1H).

(9R,9aR)-4-Chloro-9-methoxy-9a-nitro-5a,6,9,9a-

tetrahydronaphtho[2,1-c][1,2,5]oxadiazol-7(8H)-one (59a): White

1

(9S,9aR)-9-Methoxy-4-(4-methoxybenzylthio)-9a-nitro-5a,6,9,9a-

tetrahydronaphtho[2,1-c][1,2,5]oxadiazol-7(8H)-one (60d): White

solid (0.137 mmol, 49%). ¹H NMR (400 MHz, CDCl₃): d=7.07–7.05

(d, J=8, 2H), 6.74–6.72 (d, J=8, 2H), 6.05–6.03 (m, 1H), 4.47–4.43

(m, 1H), 4.03 (s, 2H), 3.69 (s, 3H), 3.61–3.57 (m, 1H), 3.37 (s, 3H),

2.96–2.91 (m, 1H), 2.64–2.57 (m, 1H), 2.46–2.41 (m, 1H), 1.69–

1.61 ppm (m, 1H).

solid (0.07 mmol, 25%). H NMR (400 MHz, CDCl₃): d=6.72–6.70 (m,

1H), 5.11–5.10 (m, 1H), 4.45–4.39 (m, 1H), 3.42 (s, 3H), 2.98–2.94

(m, 1H), 2.83–2.77 (m, 1H), 2.71–2.67 (m, 1H), 1.91–1.84 ppm (m,

1H); ¹³C NMR (100 MHz, CDCl₃): d=200.09, 149.28, 145.75, 132.19,

121.02, 88.98, 79.70, 78.92, 58.73, 42.49, 42.17, 41.04 ppm.

(9R,9aR)-4,9-Dimethoxy-9a-nitro-5a,6,9,9a-tetrahydronaphtho-

[2,1-c][1,2,5]oxadiazol-7(8H)-one (59b): White solid (0.067 mmol,

24%). ¹H NMR (400 MHz, CDCl₃): d=5.48–5.46 (m, 1H), 5.01–5.00

(m, 1H), 4.31–4.24 (m, 1H), 3.74 (s, 3H), 3.35 (s, 3H), 2.89–2.83 (m,

1H), 2.73–2.67 (m, 1H), 2.64–2.59 (m, 1H), 1.80–1.73 ppm (m, 1H).

(9S,9aR)-4-(4-Bromophenylthio)-9-methoxy-9a-nitro-5a,6,9,9a-

tetrahydronaphtho[2,1-c][1,2,5]oxadiazol-7(8H)-one (60e): White

solid (0.154 mmol, 55%). ¹H NMR (400 MHz, CDCl₃): d=7.45–7.43

(d, J=8, 2H), 7.22–7.20 (d, J=8, 2H), 6.02–6.00 (m, 1H), 4.53–4.49

(m, 1H), 3.67–3.61 (m, 1H), 3.40 (s, 3H), 3.02–2.97 (m, 1H), 2.67–

2.61 (m, 1H), 2.58–2.53 (m, 1H), 1.81–1.74 ppm (m, 1H); ¹³C NMR

(100 MHz, CDCl₃): d=200.25, 149.27, 145.51, 134.46, 132.90, 131.43,

128.39, 126.23, 123.71, 89.39, 79.88, 58.71, 42.95, 42.47, 41.02 ppm.

(9R,9aR)-9-Methoxy-4-(4-methoxyphenoxy)-9a-nitro-5a,6,9,9a-

tetrahydronaphtho[2,1-c][1,2,5]oxadiazol-7(8H)-one (59c): White

1

solid (0.07 mmol, 25%). H NMR (400 MHz, CDCl₃): d=7.19–7.17 (d,

J=8, 2H), 6.92–6.90 (d, J=8, 2H), 5.58–5.56 (m, 1H), 5.11–5.09 (m,

1H), 4.28–4.22 (m, 1H), 3.87 (s, 3H), 3.41 (s, 3H), 2.96–2.91 (m, 1H),

2.73–2.68 (m, 2H), 1.90–1.83 ppm (m, 1H).

(9S,9aR)-9-Methoxy-4-(3-morpholino-3-oxopropylthio)-9a-nitro-

5a,6,9,9a-tetrahydronaphtho[2,1-c][1,2,5]oxadiazol-7(8H)-one

(60 f): White solid (0.131 mmol, 47%). ¹H NMR (400 MHz, CDCl₃):

d=6.25–6.23 (m, 1H), 4.51–4.48 (m, 1H), 4.07–4.02 (m, 2H), 3.56–

3.48 (m, 8H), 3.08–2.97 (m, 2H), 2.79–2.75 (m, 2H), 2.69–2.57 (m,

1H), 2.51–2.48 (m, 1H), 1.81–1.75 ppm (m, 1H).

(9R,9aR)-9-Methoxy-4-(4-methoxybenzylthio)-9a-nitro-5a,6,9,9a-

tetrahydronaphtho[2,1-c][1,2,5]oxadiazol-7(8H)-one (59d): White

solid (0.064 mmol, 23%). ¹H NMR (400 MHz, CDCl₃): d=6.98–6.96

(d, J=8, 2H), 6.72–6.69 (d, J=8, 2H), 6.37–6.36 (m, 1H), 4.98–4.97

(m, 1H), 4.18–4.12 (m, 1H), 4.06–3.96 (m, 2H), 3.71 (s, 3H), 3.35 (s,

3H), 2.85–2.80 (m, 1H), 2.59–2.55 (m, 1H), 2.53–2.47 (m, 1H), 1.60–

1.49 ppm (m, 1H); ¹³C NMR (100 MHz, CDCl₃): d=201.11, 159.01,

148.65, 146.00, 138.40, 129.73, 129.20, 127.53, 122.11, 114.08, 87.05,

79.94, 57.77, 55.22, 42.35, 40.20, 37.72, 35.92 ppm.

4-Bromo-7-nitrobenzo[c][1,2,5]thiadiazole (62): A mixture of 4,7-

dibromobenzo[c][1,2,5]thiadiazole (100 mg, 0.34 mmol) in conc

HNO₃(5 mL) was heated at reflux with stirring for 1 h. The resulting

clear solution was poured into 10 mL of ice-H₂O and then extract-

ed with EtOAc (2ꢁ10 mL). The combined organic phases were

washed with brine, dried over Na₂SO₄, filtered and concentrated

under reduced pressure. The crude product was separated by chro-

matography on silica gel (Hex/EtOAc 3:1) to afford the desired

(9R,9aR)-4-(4-Bromophenylthio)-9-methoxy-9a-nitro-5a,6,9,9a-

tetrahydronaphtho[2,1-c][1,2,5]oxadiazol-7(8H)-one (59e): White

solid (0.064 mmol, 23%). ¹H NMR (400 MHz, CDCl₃): d=7.44–7.42

(d, J=8, 2H), 7.23–7.21 (d, J=8, 2H), 6.36–6.34 (m, 1H), 5.04–5.03

(m, 1H), 4.28–4.22 (m, 1H), 3.34 (s, 3H), 2.89–2.83 (m, 1H), 2.64–

2.58 (m, 2H), 1.77–1.70 ppm (m, 1H); ¹³C NMR (100 MHz, CDCl₃):

d=200.81, 146.15, 137.20, 134.43, 132.92, 128.65, 125.98, 123.66,

123.29, 113.73, 87.29, 79.75, 57.79, 42.22, 40.16, 37.96 ppm.

1

product (0.227 mmol, 67%). H NMR (400 MHz, CDCl₃): d=8.41 (d,

J=7.89 Hz, 1H), 7.98 ppm (d, J=7.89 Hz, 1H).

General procedure for the synthesis of compounds 63a,b

(9R,9aR)-9-Methoxy-4-(3-morpholino-3-oxopropylthio)-9a-nitro-

5a,6,9,9a-tetrahydronaphtho[2,1-c][1,2,5]oxadiazol-7(8H)-one

(59 f): White solid (0.07 mmol, 25%). H NMR (400 MHz, CDCl₃): d=

6.47–6.45 (m, 1H), 5.02–5.01 (m, 1H), 4.28–4.22 (m, 1H), 3.57–3.49

(m, 8H), 3.32 (s, 3H), 3.23–3.16 (m, 1H), 3.09–3.02 (m, 1H), 2.86–

2.82 (m, 1H), 2.63–2.59 (m, 2H), 2.53–2.48 (m, 2H), 1.79–1.72 ppm

(m, 1H); ¹³C NMR (100 MHz, CDCl₃): d=201.16, 168.61, 148.50,

146.11, 135.56, 122.65, 87.35, 79.83, 66.69, 66.38, 60.26, 57.76,

45.58, 42.36, 41.99, 40.18, 37.83, 32.38, 26.58, 20.92, 14.09 ppm.

To a solution of compound 62 (1 equiv) in EtOH the appropriate

nucleophile and a catalytic amount of KOAc and pyridine were

added. The resulting mixture was heated at reflux for 3 h. This was

concentrated in vacuo and the crude product was purified by flash

chromatography with the appropriate eluent.

1

4-(4-Methoxybenzylthio)-7-nitrobenzo[c][1,2,5]thiadiazole (63a):

(0.082 mmol, 72%). ¹H NMR (400 MHz, CDCl₃): d=8.42 (d, J=

8.1 Hz, 1H), 7.31–7.29 (m, 3H), 6.81 (d, J=7.8 Hz, 2H), 4.35 (s, 2H),

3.73 ppm(s, 3H); ¹³C NMR (100 MHz, CDCl₃): d=159.39, 153.24,

146.09, 143.64, 135.99, 129.94, 128.06, 125.90, 120.04, 114.34, 55.24,

35.84 ppm.

(9S,9aR)-4-Chloro-9-methoxy-9a-nitro-5a,6,9,9a-

tetrahydronaphtho[2,1-c][1,2,5]oxadiazol-7(8H)-one (60a): White

solid (0.117 mmol, 42%). ¹H NMR (400 MHz, CDCl₃): d=6.37–6.36

(m, 1H), 4.55–4.51 (m, 1H), 3.81–3.75 (m, 1H), 3.39 (s, 3H), 3.02–

2.97 (m, 1H), 2.70–2.64 (m, 2H), 1.94–1.87 ppm (m, 1H).

1-Morpholino-3-(7-nitrobenzo[c][1,2,5]thiadiazol-4-ylthio)pro-

pan-1-one (58b): (0.078 mmol, 69%). ¹H NMR (400 MHz, CDCl₃):

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d=8.46 (d, J=8 Hz, 1H), 7.34 (d, J=8 Hz, 1H), 3.61–3.59 (m, 6H),

3.50 (t, J=8.2 Hz, 2H), 3.40–3.38 (m, 2H), 2.77 ppm (t, J=8 Hz,

2H); ¹³C NMR (100 MHz, CDCl₃): d=168.46, 153.25, 143.18, 135.96,

128.09, 119.52, 66.68, 66.36, 45.66, 42.14, 31.57, 26.46 ppm.

noline (500 mL, 4.21 mmol) was slowly added to the well-stirred

acid at a rate such that the internal temperature was maintained

<308C. The solution was cooled to À258C in a dry ice–acetone

bath and N-bromosuccinimide (976 mg, 5.48 mmol) was added to

the vigorously stirred solution in portions such that the internal

temperature was maintained between À22 and À268C. The sus-

pension was stirred for 2 h at À228C and then for 3 h at À188C.

KNO₃(446 mg, 4.41 mmol) was then added at a rate such as to

maintain the internal temperature below À108C and the mixture

was then stirred at À108C for 1 h. The cooling bath was removed

and the solution was stirred overnight. The resulting homogene-

ous reaction mixture was poured onto crushed ice in a 500 mL

flask and the reaction flask was quickly washed with ice-cold H₂O,

which was added to the 500 mL flask. The resulting mixture was

stirred while the solution was adjusted to pH 8.0 using 25% aque-

ous NH₃with the internal temperature maintained <308C. The re-

sulting suspension was stirred in an ice H₂O bath for 2 h and the

precipitated solids were isolated by filtration. The solids were thor-

oughly washed three times with ice-cold H₂O and then air dried to

constant weight. This material was suspended in 12 mL heptane

and 3 mL toluene and heated at reflux for 1.5 h with stirring. The

hot solution was then filtered through Celite using vacuum suc-

tion. The volume of the filtrate was decreased by distillation and

the resulting orange solution was allowed to slowly cool with stir-

ring overnight. The solids were isolated by filtration, washed with

100 mL ice-cold heptane, and dried to provide desired product

8-Chloro-5-nitroquinoline (66): To a solution of 2-chloro-5-nitroa-

niline (100 mg, 0.579 mmol) in 6n HCl (15 mL), toluene (3 mL) and

acrolein (76 mL, 1.158 mmol) were added and the mixture was

heated at 1008C for 2 h. The aqueous layer was separated and

neutralized with aqueous NaOH to afford crude 66 as a crystalline

solid. The crude product was purified by flash chromatography

(Hex/EtOAc 3:1) to give desired compound (0.55 mmol, 95%).

¹H NMR (400 MHz, [D₆]acetone): d=9.09 (d, J=4.2 Hz, 1H), 8.92 (d,

J=11.9 Hz, 1H), 8.36 (d, J=7.8 Hz, 1H), 8.07 (d, J=7.8 Hz, 1H),

7.83 ppm (dd, J=4.2 Hz and J=11.9 Hz, 1H); ¹³C NMR (100 MHz,

[D₆]acetone): d=152.12, 140.42, 132.12, 128.31, 127.99, 124.80,

124.71, 124.50, 121.97 ppm.

General procedure for aromatic nucleophilic substitution on

quinoline 66

8-Chloro-5-nitroquinoline (0.25 mmol, 1 equiv) and the appropriate

nucleophile (1.1 equiv) were suspended in the minimal amount of

dry DMF (2 mL) in sealed vessels in the presence of K₂CO₃

(1 equiv), and the mixtures were irradiated at 1308C for 10 min The

reaction mixtures were then diluted with H₂O (2 mL) and extracted

with EtOAc (3ꢁ10 mL). Finally, the organic phases were dried over

anhydrous Na₂SO₄and evaporated to dryness. The crude product

was purified by flash chromatography with the appropriate eluent.

1

(2.06 mmol, 49%). H NMR (400 MHz, [D₆]acetone): d=9.77 (s, 1H),

8.77 (d, J=6.2 Hz, 1H), 8.26 (s, 2H), 8.10 ppm (d, J=8.1 Hz, 1H);

¹³C NMR (100 MHz, CDCl₃): d=148.20, 145.81, 145.54, 135.12,

133.16, 127.80, 125.29, 120.49, 118.89 ppm; MS (ESI) m/z: 253–255

[M+H]⁺.

8-(4-Methoxybenzylthio)-5-nitroquinoline (67a): (0.22 mmol,

88%). ¹H NMR (400 MHz, [D₆]acetone): d=9.02 (d, J=4.2 Hz, 1H),

8.92 (d, J=11.9 Hz, 1H), 8.35 (d, J=7.8 Hz, 1H), 7.77 (dd, J=4.2 Hz

and J=11.9 Hz, 1H), 7.71 (d, J=7.8 Hz, 1H), 7.41 (d, J=8.2 Hz, 1H),

6.86 (d, J=8.2 Hz, 1H), 4.33 (s, 2H), 3.72 ppm (s, 3H).

General procedure for aromatic nucleophilic substitution on

isoquinoline 69

1-Morpholino-3-(5-nitroquinolin-8-ylthio)propan-1-one

(67b):

(0.22 mmol, 88%). ¹H NMR (400 MHz, CDCl₃): d=9.04 (d, J=4 Hz,

1H), 8.88 (d, J=11.9 Hz, 1H), 8.31 (d, J=7.8 Hz, 1H), 7.60 (dd, J=

4.2 Hz and J=11.9 Hz, 1H), 7.43 (d, J=7.8 Hz, 1H), 3.58 (brs, 6H),

3.37 (brs, 2H), 3.36–3.34 (m, 2H), 2.77–2.73 ppm (m, 2H); ¹³C NMR

(100 MHz, CDCl₃): d=168.87, 149.94, 149.65, 144.39, 141.11, 132.82,

125.32, 124.61, 120.98, 120.14, 66.70, 66.38, 45.67, 42.09, 31.45,

25.83 ppm; MS (ESI) m/z: 348 [M+H]⁺, 370 [M+Na]⁺.

Compounds 70a,b were obtained by using the same procedure as

for compounds 67a–d but with 5-bromo-8-nitroisoquinoline as the

starting material.

5-(4-Methoxybenzylthio)-8-nitroisoquinoline (70a): (0.207 mmol,

1

83%). H NMR (400 MHz, [D₆]acetone): d=9.87 (s, 1H), 8.65 (d, J=

6.2 Hz, 1H), 8.29 (d, J=8.1 Hz, 1H), 8.05 (d, J=6.2 Hz, 1H), 7.82 (d,

J=8.1 Hz, 1H), 7.36 (d, J=7.8 Hz, 1H), 6.84 (d, J=7.8 Hz, 1H), 4.44

(s, 2H), 3.71 ppm (s, 2H); ¹³C NMR (100 MHz, [D₆]acetone): d=

159.39, 148.52, 148.39, 144.44, 142.98, 133.83, 130.27, 127.00,

125.11, 124.87, 119.72, 116.12, 114.01, 54.60, 35.83 ppm; MS (ESI)

m/z: 327 [M+H]⁺.

8-(4-(4-Methylpiperazin-1-yl)phenoxy)-5-nitroquinoline

(67c):

1

(0.205 mmol, 82%). H NMR (400 MHz, [D₆]acetone): d=9.03 (d, J=

4.2 Hz, 1H), 8.98 (d, J=11.9 Hz, 1H), 8.36 (d, J=7.8 Hz, 1H), 7.78

(dd, J=4.2 Hz and J=11.9 Hz, 1H), 7.08–6.90 (m, 4H), 6.73 (d, J=

8.2 Hz, 2H), 3.15–3.14 (4H, m), 2.47–2.46 (4H, m), 2.21 ppm (3H, s);

¹³C NMR (100 MHz, [D₆]acetone): d=160.81, 150.43, 149.41, 147.26,

139.42, 131.68, 126.78, 124.74, 122.78, 118.03, 115.43, 110.10, 54.86,

48.81, 45.31 ppm; MS (ESI) m/z: 365 [M+H]⁺, 387 [M+Na]⁺.

1-Morpholino-3-(8-nitroisoquinolin-5-ylthio)propan-1-one (70b):

1

(0.217 mmol, 87%). H NMR (400 MHz, CDCl₃): d=9.96 (s, 1H), 8.58

(d, J=6.2 Hz, 1H), 8.18 (d, J=8.1 Hz, 1H), 7.94 (d, J=6.2 Hz, 1H),

7.48 (d, J=8.1 Hz, 1H), 3.58 (brs, 4H), 3.57 (brs, 2H), 3.42–3.49 (m,

2H), 3.36–3.34 (m, 2H), 2.73–2.69 ppm (m, 2H); ¹³C NMR (100 MHz,

CDCl₃): d=168.52, 148.81, 144.44, 142.77, 134.16, 125.04, 123.33,

119.98, 116.26, 66.67, 66.33, 45.61, 42.09, 31.65, 27.17 ppm; MS

(ESI) m/z: 348 [M+H]⁺, 370 [M+Na]⁺.

1-(4-(4-(5-Nitroquinolin-8-yloxy)phenyl)piperazin-1-yl)ethanone

1

(67d): (0.207 mmol, 83%). H NMR (400 MHz, [D₆]acetone): d=9.05

(d, J=4.2 Hz, 1H), 9.00 (d, J=11.9 Hz, 1H), 8.38 (d, J=7.8 Hz, 1H),

7.81 (dd, J=4.2 Hz and J=11.9 Hz, 1H), 7.09–7.06 (m, 4H), 6.95 (d,

J=8.2 Hz, 2H), 3.64–3.61 (4H, m), 3.20–3.18 (2H, m), 3.12–3.10 (2H,

m), 2.01 ppm (3H, s); ¹³C NMR (100 MHz, [D₆]acetone): d=150.48,

149.19, 141.49, 139.13, 132.71, 131.88, 131.69, 126.72, 124.74,

121.28, 118.60, 117.88, 110.33, 49.57, 49.20, 45.76, 40.85,

20.35 ppm; MS (ESI) m/z: 393 [M+H]⁺, 416 [M+Na]⁺.

N-(3-Chloro-2-fluorophenyl)acetamide (72): 3-Chloro-2-fluoroani-

line (200 mL, 2.4 mmol) was mixed with acetic anhydride (10 mL)

and heated at reflux for 2 h. The reaction was quenched with H₂O

(3 mL), neutralized with a saturated solution of Na₂CO₃and extract-

ed with EtOAc (3ꢁ10 mL). The combined extract was dried over

anhydrous Na₂SO₄and evaporated to dryness to provide desired

5-Bromo-8-nitroisoquinoline (69): Concentrated H₂SO₄(96%,

20 mL) was cooled to 08C under nitrogen atmosphere and isoqui-

1

compound (2.4 mmol, quant). H NMR (400 MHz, CDCl₃): d=8.18–

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8.16 (m, 1H), 7.31 (brs, 1H), 7.04–6.99 (m, 2H), 2.17 ppm (s, 3H);

¹³C NMR (100 MHz, CDCl₃): d=170.75, 154.36, 124.82, 124.65,

120.15, 119.90, 29.61 ppm; MS (ESI) m/z: 188 [M+H]⁺.

139.19, 138.45, 137.95, 123.37, 120.47, 66.65, 66.34, 45.59, 42.04,

32.32, 28.16, 20.75 ppm; MS (ESI) m/z: 368 [M+H]⁺.

2-Methyl-7-(4-(4-methylpiperazin-1-yl)phenoxy)-4-nitrobenzo[d]-

thiazole (75c): (0.14 mmol, 56%). ¹H NMR (400 MHz, CDCl₃): d=

8.07 (d, J=8.2 Hz, 1H), 6.97 (d, J=7.9 Hz, 2H), 6.89 (d, J=7.9 Hz,

2H), 6.59 (d, J=8.2 Hz, 1H), 3.16–3.13 (4H, m), 2.88 (s, 3H), 2.53–

2.51 (4H, m), 2.28 ppm (3H, s); ¹³C NMR (100 MHz, CDCl₃): d=

172.35, 157.59, 149.43, 148.09, 146.42, 136.36, 125.33, 121.48,

117.22, 108.20, 55.04, 54.95, 49.18, 46.04, 20.70 ppm; MS (ESI) m/z:

385 [M+H]⁺.

7-Chloro-2-methylbenzo[d]thiazole (73): In a 50 mL flask were

placed compound 72 (74 mg, 0.39 mmol), Lawesson’s reagent

(96 mg, 0.6 equiv), xylene (2 mL) and a magnetic stir bar. The mix-

ture was stirred under argon at 105–1158C for 2.5 h. Then Cs₂CO₃

(302.5 mg, 2 equiv) was added and the reaction mixture was stirred

at reflux for 16 h. The solvent was evaporated to dryness and the

crude product was separated by chromatography on silica gel

(Hex/EtOAc 3:1) to afford the desired product (0.3 mmol, 77%).

¹H NMR (400 MHz, CDCl₃): d=7.76 (d, J=8 Hz, 1H), 7.33–7.24 (m,

2H), 2.76 ppm (s, 3H); ¹³C NMR (100 MHz, CDCl₃): d=167.66,

153.97, 135.77, 126.77, 126.54, 124.33, 120.63, 20.09 ppm; MS (ESI)

m/z: 185 [M+H]⁺.

1-(4-(4-(2-Methyl-4-nitrobenzo[d]thiazol-7-yloxy)phenyl)pipera-

zin-1-yl)ethanone (75d): (0.135 mmol, 54%). ¹H NMR (400 MHz,

CDCl₃): d=8.09 (d, J=8.1 Hz, 1H), 7.01 (d, J=7.8 Hz, 2H), 6.91 (d,

J=7.8 Hz, 2H), 6.61 (d, J=8.1 Hz, 1H), 3.74–3.73 (m, 2H), 3.59–3.58

(m, 2H), 3.15–3.10 (m, 4H), 2.90 (s, 3H), 2.08 ppm (s, 3H); ¹³C NMR

(100 MHz, CDCl₃): d=172.43, 157.38, 149.06, 147.47, 147.18, 136.52,

125.30, 121.62, 117.99, 108.29, 49.80, 49.50, 46.08, 41.21, 21.23,

20.70 ppm; MS (ESI) m/z: 413 [M+H]⁺, 435 [M+Na]⁺.

7-Chloro-2-methyl-4-nitrobenzo[d]thiazole (74): Concentrated

H₂SO₄(96%, 20 mL) was cooled to À108C under nitrogen atmos-

phere and compound 73 (370 mg, 2.02 mmol) was slowly added

to the well-stirred acid at a rate such that the internal temperature

was maintained <308C. KNO₃(213 mg, 2.1 mmol) was then added

at a rate such as to maintain the internal temperature below

À108C and the mixture was then stirred at À108C for 1 h. The

cooling bath was removed and the solution was stirred at room

temperature for 2 h. The resulting homogeneous reaction mixture

was poured onto crushed ice and stirred while adjusting the solu-

tion to pH 8.0 using 25% aqueous NH₃with the internal tempera-

ture maintained <308C. This was extracted with EtOAc (3ꢁ25 mL)

dried over anhydrous Na₂SO₄and solvent removed in vacuo. The

crude product was separated by chromatography on silica gel

(Hex/EtOAc 3:1) to afford the desired product (1.35 mmol, 67%).

¹H NMR (400 MHz, CDCl₃): d=8.09 (d, J=8 Hz, 1H), 7.40 (d, J=

8 Hz, 1H), 2.90 ppm (s, 3H); ¹³C NMR (100 MHz, CDCl₃): d=172.63,

145.67, 140.38, 138.96, 132.35, 123.81, 123.64, 20.75 ppm; MS (ESI)

m/z: 230 [M+H]⁺.

5-(4-Methoxybenzylthio)-2-nitroaniline (77): To a solution of 5-

chloro-2-nitroaniline (100 mg, 0.58 mmol) in 10 mL of EtOH, 4-me-

thoxytoluenethiol (80 mL, 0.58 mmol) and K₂CO₃(1 equiv) were

added. The reaction mixture was heated at reflux for 2 h. The mix-

ture was then cooled to room temperature, the solvent was re-

moved in vacuo. This was extracted with EtOAc (3ꢁ10 mL),

washed with brine, dried over anhydrous Na₂SO₄and the solvent

was removed in vacuo. The crude product was separated by chro-

matography on silica gel (Hex/EtOAc 3:1) to afford the desired

1

product (0.388 mmol, 67%). H NMR (400 MHz, CDCl₃): d=8.06 (d,

J=8 Hz, 1H), 7.47 (s, 1H), 7.25 (d, J=8 Hz, 1H), 6.82 (m, 4H), 4.30

(s, 2H), 3.70 ppm (s, 3H); MS (ESI) m/z: 291 [M+H]⁺.

N-(5-(4-Methoxybenzylthio)-2-nitrophenyl)acetamide (78): To

a solution of compound 77 (50 mg, 0.17 mmol) in CH₂Cl₂, Et₃N

(45 mL, 0.34 mmol) and Ac₂O (24 mL, 0.26 mmol) were added, and

the mixture was stirred at room temperature for 1 h. The reaction

mixture was quenched with saturated aqueous NaHCO₃(10 mL)

and extracted with EtOAc (3ꢁ10 mL). The combined extracts were

washed with brine, dried over anhydrous Na₂SO₄and the solvent

removed in vacuo. The crude product was separated by chroma-

tography on silica gel (Hex/EtOAc 4:1) to afford the desired prod-

General procedure for the synthesis of compounds 75a–d

Compound 74 (0.25 mmol, 1 equiv) and the appropriate nucleo-

phile (1 equiv) were suspended in anhydrous DMF (3 mL) in

a 10 mL glass vial equipped with a small magnetic stir bar. K₂CO₃

(1 equiv) was then added to this solution and the mixture was irra-

diated under microwave for 10 min at 1308C, using an irradiation

power of 300 W. The mixture was then poured into H₂O (10 mL)

and then extracted with EtOAc (2ꢁ10 mL). The combined organic

phases were washed with brine, dried over Na₂SO₄, filtered and

concentrated under reduced pressure. The crude products were

purified by flash column chromatography using the appropriate

eluent.

1

uct (0.147 mmol, 87%). H NMR (400 MHz, CDCl₃): d=8.47 (s, 1H),

8.22 (d, J=8 Hz, 1H), 7.32 (d, J=8 Hz, 1H), 6.82 (m, 4H), 4.30 (s,

2H), 3.70 (s, 3H), 2.07 ppm (s, 3H); ¹³C NMR (100 MHz, CDCl₃): d=

160.2, 143.1, 138.8, 133.7, 129.4, 127.5, 120.1, 114.3, 55.4, 23.7 ppm;

MS (ESI) m/z: 333 [M+H]⁺.

Biology

Virus strains: Infection experiments were carried out using influen-

za A/WSN/33 (H1N1).^[35]

7-(4-Methoxybenzylthio)-2-methyl-4-nitrobenzo[d]thiazole (75a):

(0.187 mmol, 75%). ¹H NMR (400 MHz, CDCl₃): d=8.00 (d, J=

8.2 Hz, 1H), 7.16 (m, 3H), 6.74 (d, J=7.8 Hz, 2H), 4.21 (s, 2H), 3.69

(s, 3H), 2.83 ppm (s, 3H); ¹³C NMR (100 MHz, CDCl₃): d=171.79,

159.23, 144.86, 141.43, 139.40, 138.51, 129.92, 126.91, 123.22,

121.78, 114.16, 55.21, 37.52, 20.73 ppm; MS (ESI) m/z: 347 [M+H]⁺,

369 [M+Na]⁺.

Plasmid constructs: Plasmids pCA-Flag-GFP and pCA-PB_11–25A-GFP,

pCAPB1-HA, the FluA minireplicon plasmids and the expression

plasmids for the FluB minireplicon are described elsewhere.^[36]The

FluB minigenome expression plasmid, pPoll-lucRT_B, was obtained

by cloning the firefly luciferase ORF (inverse orientation) flanked by

the non-coding region of segment 8 of the B/Yamagata/73 into

the SapI-digested plasmid pPoll-SapIRib according to Pleschka

et al.^[28]For the construction of pCA-PB_11–25B-GFP, a linker contain-

ing the first 25 codons of PB1 (B/Yamagata/73) was cloned into the

EcoRI/NotI sites of pCA-Flag-GFP plasmid, replacing the Flag-

coding sequence with PB_11–25B. Site-directed mutagenesis was car-

ried out with pCA-PB_11–25A-GFP to create the plasmid pCAPB_11–

3-(2-Methyl-4-nitrobenzo[d]thiazol-7-ylthio)-1-morpholinopro-

pan-1-one (75b): (0.155 mmol, 62%). ¹H NMR (400 MHz, CDCl₃):

d=8.03 (d, J=8.2 Hz, 1H), 7.21 (d, J=8 Hz, 1H), 3.54–3.52 (m, 6H),

3.38 (t, J=8 Hz, 2H), 3.32–3.30 (m, 2H), 2.83 (s, 3H), 2.65 ppm (t,

J=8 Hz, 2H); ¹³C NMR (100 MHz, CDCl₃): d=171.80, 168.61, 144.95,

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₂₅A_T6Y-GFP. The ORFs of PB1 (B/Yamagata/73) and PA (A/SC35M, A/

Thailand/1(KAN-1)/04, A/Vietnam/1203/04, B/Yamagata/73, B/Lee/

40) were PCR amplified with sense primers containing a NotI site

(FluA strains) or a EcoRI site (FluB strains) upstream of the initiation

codon and antisense primers with a deleted stop codon followed

by an XmaI site, a coding sequence for an HA tag and a XhoI site.

The PCR products were cloned into a modified pCAGGs-vector di-

gested either with EcoRI/XhoI or NotI/XhoI, resulting in pCA-PB1-

HA or pCA-PA-HA plasmids, coding for C-terminal-tagged versions

of the polymerase subunits. To obtain the pCA-PA_A/SC35M-His plas-

mid, pCA-PA_A/SC35M-HA was digested with XmaI/XhoI, and the HA

coding sequence was replaced by a His₆linker. The A/B-chimeric

expression plasmids were obtained by assembly PCR using the

pCA-PB1-HA plasmids of SC35M and B/Yamagata/73 and by clon-

ing the resulting PCR product in pCA-PB1_{B/Yamagata/73}-HA digested

with EcoRI/EcoRV.

infected with 100 PFU A/WSN/33, B/Yamagata/73, A/KAN-1, or VSV/

Indiana in PBS containing BSA at room temperature. After removal

of the inoculums, cells were overlaid with medium (DMEM with

20 mm HEPES, 0.01% DEAE Dextran, 0.001% NaHCO₃) containing

1% Oxoidagar and the benzofurazan compounds at the indicated

concentrations. After incubation for 24 h (VSV), 48 h (A/WSN/33, A/

KAN-1) at 378C with 5% CO₂, or 72 h at 338C with 5% CO₂(B/Ya-

magata/73), cells were fixed with formaldehyde and stained with

crystal violet. Plaques were counted, and mean plaque number of

the water control was set at 100%.

Molecular modeling

Library preparation: All ligands were built with the Schrçdinger

Maestro 9.1 graphical interface.^[38]Compounds were then pro-

cessed with the Schrçdinger LigPrep tool to generate separate files

for all possible enantiomers and protonation states at physiological

pH. OPLS_2005 was used as force field.

Reconstitution of viral polymerase activity: HEK293T cells were tran-

siently transfected with a plasmid mixture containing either FluA-

or FluB-derived PB1-, PB2-, PA- and NP-expression plasmids, poly-

merase I (Pol I)-driven plasmid transcribing an influenza A or influ-

enza B virus-like RNA coding for the reporter protein firefly lucifer-

ase to monitor viral polymerase activity and with expression plas-

mids coding for the indicated GFP fusion proteins. Both minige-

nome RNAs were flanked by noncoding sequences of segment 8

of FluA and FluB, respectively. The transfection mixture also con-

tained a plasmid constitutively expressing Renilla luciferase, which

served to normalize variation in transfection efficiency. The reporter

activity was determined 24 h post-transfection and normalized

using the Dual-Glu Luciferase Assay System (Promega). The activity

observed with transfection reactions containing Flag-GFP were set

to 100%.

Protein preparation: The three-dimensional coordinates of H5N1

influenza A virus PA (C-terminal region, PAC) in complex with the

PA binding region of PB1 (N-terminal region, PB1N, residues 1–25)

were retrieved from the Protein Data Bank (PDB IDs 3CM8^[39]and

2ZNL,^[40]with resolutions of 2.90 and 2.30 ꢂ, respectively). Because

both structures contain gaps, a more complete assembly for PAC

was obtained by applying a homology modeling procedure (Prime,

Schrçdinger). In detail, the FASTA sequence of the protein (influen-

za A virus, strain A/Wilson–Smith/1933 H1N1) was uploaded into

the software, and the model was built using PDB IDs 2ZNL as the

first template and 3CM8 as the second. Indeed, the gaps in the

2ZNL structure were partially filled with the corresponding residues

solved in the 3CM8 structure, while the missing residues in both X-

ray structures were completed by the program according to the

FASTA sequence. The protein was then energy minimized to

remove unfavorable contacts through the all-atom OPLS force field

and Polak–Ribiere conjugate gradient method. A continuum solva-

tion method, with water as the solvent, was also applied. Extended

Immunoprecipitation experiments: HEK293T cells were transfected

with the indicated plasmids in six-well plates using Metafectene

(Biontex, Martinsried, Germany). Cells were incubated 24 h post-

transfection with lysis buffer (20 mm Tris pH 7.5, 100 mm NaCl,

0.5 mm EDTA, 0.5% NP-40, 1% protease inhibitor Mix G, (Serva,

Heidelberg, Germany), 1 mm DTT) for 15 min on ice. After centrifu-

gation at 13000 rpm at 48C, the supernatant was incubated with

anti-HA-specific antibodies coupled to agarose beads (Sigma) for

1 h at 48C. After three washes with 1 mL washing buffer (lysis

buffer without protease inhibitor mix), bound material was eluted

under denaturing conditions and separated by SDS-PAGE and

transferred into PVDF membranes. Viral polymerase subunits and

GFP fusion proteins were detected with antibodies directed against

the HA tag (Covance, Berkeley, CA, USA) or His tag (Qiagen) or GFP

tag (Santa Cruz Biotechnology).

cutoffs were used, and convergence was set to 0.3 kJmol^À1

.

Docking studies: A consensus docking approach that takes ad-

vantage of two widely used docking programs (Glide^[41]and

Gold)^[42]was used in this study. In detail, compounds were first

docked using the Glide standard precision (SP) mode. A grid box

of default size was centered on PB1-derived peptide of the X-ray

crystal structure. No constraints were included during grid genera-

tion, while rotation of hydroxy groups was allowed. Default param-

eters were used for the docking runs. The top-five poses based on

the Glide docking score were saved for each ligand. With regard to

the docking with Gold, GoldScore and ChemScore were used as fit-

ness functions. The GA parameter settings of Gold were used, with

search efficiency set at 200%. Finally, results differing by <1.5 ꢂ in

ligand all-atom RMSD were clustered together. For each inhibitor,

the first-ranked solution as well as the lowest-energy conformation

of the most populated cluster were analyzed by comparing them

with the best poses previously obtained with the Glide software.

Poses were then selected by taking into account the overall match

between the binding modes proposed by the two programs.

Enzyme-linked immunosorbent assay (ELISA): Microwell plates

(Pierce) were incubated at room temperature with saturating con-

centrations of small-molecule competitor compounds, washed, and

subsequently incubated at room temperature with HA-tagged PA.

To obtain PA-HA, HEK293T cells were seeded into 94-mm dishes,

transfected with the respective plasmid, and treated with lysis

buffer 24 h post-transfection as previously described.^[36,37]After

washing the microwell plates, the wells were incubated with an

HA-specific primary antibody (Covance), followed by three washes

and an incubation with a peroxidase-coupled secondary antibody

(Jackson Immuno Research, Newmarket, UK) for a further 30 min.

After the final wash step, ABTS substrate (Sigma, ready-to-use solu-

tion) was added, and the optical density was determined at

l 405 nm.

Ligand efficiency: Ligand efficiency (LE) was calculated by dividing

the free energy of binding by the number of heavy (i.e., non-hy-

drogen) atoms. The activity value of each compound was convert-

ed into the free energy of binding at 300 K using the Gibbs equa-

tion (DG=ÀRTlnK_d), in which K_dis substituted by IC₅₀for the pur-

pose of relative comparison, as previously reported.^[33]

Plaque reduction assay: The experiments were carried out as previ-

ously described^[37]with modifications. Confluent MDCK cells were

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Published online on November 27, 2013

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Article Doi

The fight against the influenza A virus H1N1: Synthesis, molecular modeling, and biological evaluation of benzofurazan derivatives as viral RNA polymerase inhibitors

DOI: 10.1002/cmdc.201300378

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Full text of DOI:10.1002/cmdc.201300378

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