Design, synthesis and biological evaluation of imidazole and oxazole fragments as HIV-1 integrase-LEDGF/p75 disruptors and inhibitors of microbial pathogens

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Design, synthesis and biological evaluation of imidazole and oxazole frag-

ments as HIV-1 integrase-LEDGF/p75 disruptors and inhibitors of microbial

pathogens

Thompho J. Rashamuse, Angela T. Harrison, Salerwe Mosebi, Sandy van

Vuuren, E. Mabel Coyanis, Moira L. Bode

PII:

S0968-0896(19)31034-X

https://doi.org/10.1016/j.bmc.2019.115210

BMC 115210

DOI:

Reference:

To appear in:

Bioorganic & Medicinal Chemistry

Received Date:

Revised Date:

Accepted Date:

22 June 2019

23 October 2019

5 November 2019

Please cite this article as: T.J. Rashamuse, A.T. Harrison, S. Mosebi, S. van Vuuren, E. Mabel Coyanis, M.L.

Bode, Design, synthesis and biological evaluation of imidazole and oxazole fragments as HIV-1 integrase-

LEDGF/p75 disruptors and inhibitors of microbial pathogens, Bioorganic & Medicinal Chemistry (2019), doi:

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Design, synthesis and biological evaluation of imidazole and oxazole fragments as

HIV-1 integrase-LEDGF/p75 disruptors and inhibitors of microbial pathogens

Thompho J. Rashamuse, Angela T. Harrison, Salerwe Mosebi, Sandy van Vuuren, E. Mabel Coyanis, and Moira

L. Bode

2

Bioorganic & Medicinal Chemistry

journal homepage: www.elsevier.com

Design, synthesis and biological evaluation of imidazole and oxazole fragments as

HIV-1 integrase-LEDGF/p75 disruptors and inhibitors of microbial pathogens

Thompho J. Rashamuse^a,b, Angela T. Harrison^a, Salerwe Mosebi^a, Sandy van Vuuren^c, E. Mabel Coyanis^a,

and Moira L. Bode^b

^aAdvanced Materials Division, Mintek, 200 Malibongwe Drive, Randburg, 2125, South Africa

^bMolecular Sciences Institute, School of Chemistry, University of the Witwatersrand, Private Bag 3, PO WITS, 2050, South Africa

^cDepartment of Pharmacy and Pharmacology, Faculty of Health Sciences, University of the Witwatersrand, 7 York Road, Parktown, 2193, South Africa

ARTICLE INFO

ABSTRACT

Article history:

Received

Received in revised form

Accepted

We describe here the synthesis of libraries of novel 1-subtituted-5-aryl-1H-imidazole, 5-aryl-4-tosyl-4,5-dihydro-

1,3-oxazole and 5-aryl-1,3-oxazole fragments via microwave (MW)-assisted cycloaddition of para-

toluenesulfonylmethyl isocyanide (TosMIC) to imines and aldehydes. The compounds obtained were biologically

evaluated in an AlphaScreen HIV-1 IN-LEDGF/p75 inhibition assay with six imidazole-based compounds (16c, 16f,

17c, 17f, 20a and 20d) displaying more than 50% inhibition at 10 µM, with IC₅₀values ranging from 7.0 to 30.4 µM.

Additionally the hypothesis model developed predicts all active scaffolds except 20d to occupy similar areas as the N-

heterocyclic (A) moiety and two aromatic rings (B and C) of previously identified inhibitor 5. These results indicate

that the identified compounds represent a viable starting points for their use as templates in the design of a next

generation of inhibitors targeting the HIV-1 IN and LEDGF/p75 protein-protein interaction. In addition, the in vitro

antimicrobial properties of these fragments were tested by minimum inhibitory concentration (MIC) assays showing

that compound 16f exhibited a MIC value of 15.6 μg/ml against S. aureus, while 17f displayed a similar MIC value

against B. cereus, suggesting that these compounds could be further developed to specifically target those microbial

pathogens.

Available online

Keywords:

TosMIC

1-substituted-5-aryl-1H-imidazoles

5-aryl-1,3-oxazoles

HIV-1 integrase

LEDGF/p75

Antibacterial

———

 Corresponding author. E-mail: MabelC@mintek.co.za (E. M Coyanis).

 Corresponding author. E-mail: Moira.Bode@wits.ac.za (M. L. Bode).

1

1. Introduction

screened (usually by biophysical methods) for binding affinity

to the target. One of the advantages of using in-house produced

fragments is that the structure activity relationships (SAR) of a

family of related compounds can be exploited at an early

stage.²⁹FBDD can provide good starting points for binding

fragments to be grown (or linked) into larger drug-like

molecules with greater target affinities.³⁰

Some five-membered nitrogen-containing heterocycles are

found as the central core of compounds inhibiting various

stages of the HIV-1 replication cycle.^31-33Reagents such as

para-toluenesulfonylmethyl isocyanide (TosMIC) (9) can

serve as the starting point for the synthesis of various nitrogen

containing 5-membered rings, by varying the reaction

conditions. Unlike most of the isocyanide family members,

TosMIC is stable, non-volatile and odourless at room

temperature.³⁴This α-acidic isocyanide is a versatile building

block which has been used to access biologically relevant

heterocycles such as imidazoles,^34-36oxazoles^37,38and

pyrroles.^39,40

The small molecules 5, 7 and 8 that interrupt the HIV-1 IN-

LEDGF/p75 interaction can be disconnected into four chemical

features: the oxoacetic acid moiety, the central N-heterocycle

core (A), and the two aromatic rings (B and C). As a starting

point, we set out to design possible compounds with the

minimal structural features (i.e. one or two aromatic moieties

linked to a central N-heterocyclic core) that could be

synthesised and biologically evaluated to identify possible

hit(s) that could later be improved through a structure-activity

relationship (SAR) strategy.

Infectious diseases triggered by viral and bacterial

pathogens continue to be a major threat to global health. The

emergence of multi-drug resistance to current therapeutics due

to the evolution of existing strains has hampered the treatment

of both viral and bacterial pathogens, resulting in higher

morbidity and mortality rates.^1-4Thus, the search for new

effective inhibitors to augment existing treatments remains a

key objective in many drug discovery programs.

The human immunodeficiency virus type 1 integrase

enzyme (HIV-1 IN) has been identified as an attractive target

for the development of the next generation antiretroviral

drugs.^5,6The major role of this catalytic enzyme lies in

facilitating the insertion of the reverse-transcribed viral DNA

into the host DNA.^7-9To date there are four FDA approved

HIV-1 IN drugs (referred to as IN strand transfer inhibitors or

INSTIs) that tightly bind the active site of the enzyme:

Elvitegravir (1), Dolutegravir (2) Raltegravir (3) and

Bictegravir (4) (Fig. 1).^10-14Although these inhibitors manage

to suppress HIV-1 as part of highly active antiretroviral therapy

(HAART), cross resistance and poor tolerability has restricted

the long-term use of these inhibitors.^2-4,15

During the last decade, several small molecules have been

identified as disruptors of the HIV-1 IN - host Lens Epithelium-

Derived Growth Factor (LEDGF/p75) interaction, by binding

to the IN catalytic core domain (CCD) dimer interface in the

LEDGF/p75 binding pocket.^16-19These include BI-1001 (5),

CX05168 (6), BI-224436 (7) and CX14442 (8) (Fig. 1).^18-22

These allosteric compounds inhibit binding of IN with its

cofactor LEDGF/p75 protein and promote aberrant IN

multimerization, ultimately resulting in defective virions.^18,23-25

Although these compounds are not yet clinically approved,

they provide proof-of-concept for the disruption of the HIV-1

IN-LEDGF/p75 interaction as a genuine target for the

development of the next generation of anti-HIV treatment.

Fragment-based drug discovery (FBDD) has emerged as a

reliable approach to identify small compounds that can be

elaborated into good inhibitors.^26-30Collections of these low

molecular weight and low chemical complexity compounds

(commercially obtained or chemically synthesized) are

We identified versatile isocyanide chemistry⁴¹as key to the

synthesis of suitable small fragment-based libraries of 1-

substituted-5-aryl-1H-imidazoles, 5-aryl-1,3-oxazoles and

(4R,5R)/(4S,5S)-5-aryl-4-tosyl-2,4-dihydro-1,3-oxazoles

which were subsequently evaluated for their ability to disrupt

the interactions between HIV-1 IN and the host LEDGF/p75

proteins. The serious problem of opportunistic infections in

HIV patients also led us to evaluate the compounds for their

ability to inhibit infections which are prominent in these

patients. Pathogens such as Pseudomonas aeruginosa and

Staphylococcus aureus, amongst others, have demonstrated

multidrug resistance and have become challenging in the

clinical setting and are of great concern in hospital infections.

H

O

N

F

CH₃

N

O

OH

O

OH

F

Cl

O

F

O

N

F

N

H

HO

H

N

O

N

O

1

O

H

O

H

F

O

4

O

3

HO

O

2

Cl

C

O

C

O

N

B

O

Cl

OH

Br

OH

S

O

N

OH

C

B

A

N

A

N

7

B

A

N

O

A

B

S

O

N

8

6

9

5

Fig. 1: Structures of different classes of HIV-1 IN inhibitors (1-8) and TosMIC (9).

2. Results and Discussion

2.1. Chemistry

As intermediates for the isocyanide-based synthesis of

imidazoles, N-benzylidene alkylamines were prepared by

2

condensation of commercially available aldehydes (10a-i) with

primary amines (11a-d), in DCM at room temperature for 16 h,

as outlined in Scheme 1 (Method A).⁴²Although the reaction

afforded N-benzylidene alkylamines 12-15 in good to excellent

yields (71-96%,), reaction times could be shortened to just 4 min

in excellent yields by neat microwave irradiation at a set

temperature of 60^oC (Method B).

TosMIC 9, K₂CO₃and MeCN (Scheme 1, Method E). However,

when dihalogen-substituted anilines 11f-h were used the desired

products could not be isolated.

We extended the microwave-assisted cycloaddition of

TosMIC to the preparation of a small library of 5-aryl-1,3-

oxazoles 21 from commercially available aldehydes 10. Initially

we selected aldehydes 10e, 10h and 10j as model substrates to

examine the reaction. Thus, a mixture of these aldehydes,

TosMIC 9 and K₂CO₃in MeCN were microwave-irradiated in a

pressurized vessel under inert conditions at a set temperature of

75^oC and 120 Watts (Scheme 1, Method F). After 12 min the

trans-5-aryl-4-tosyl-2,4-dihydro-1,3-oxazole intermediates 22e,

22h, and 22i were isolated instead, as racemic mixtures, in yields

ranging from 65-80% (Table 2).⁴³These dihydro-1,3-oxazole

intermediates were converted to their corresponding 5-aryl-1,3-

oxazoles 21e, 21h and 21i by refluxing in toluene (Method G) for

1 h as per Companyo et al.⁴⁴

In order to investigate the effect of other solvents on this

reaction, aldehydes 10a-j were reacted under the same

microwave conditions using anhydrous MeOH as solvent as

shown in Scheme 1 (Method H). Methanol proved to be a far

superior solvent for the reaction, with the corresponding 5-aryl-

1,3-oxazoles 21a-f, 21h-j being obtained in just 7 min in yields

ranging from 43 % to 84% (Table 2)

The cycloaddition of TosMIC 9 to the imines (12a-i, 13a-f, h-i,

14a, c-d, and 15a, c-e) was then carried out initially using K₂CO₃

in refluxing MeCN for 72 h to produce 1-substituted-5-aryl-1H-

imidazoles (16a-b, 17d-g, 18a-f) (Scheme 1, Method C).

Imidazoles (16a-f, 16h-i; 17a-f, 17h-i; 18a, 18c-d; 19a, 19c-e)

were also obtained by microwave irradiation in significantly

shorter times (from 3 days by conventional heating to just 7 h) in

slightly improved yields (Scheme 1, Method D, Table 1).

Imidazoles 16d-e and 17d-e were also obtained in a two-step-

one-pot reaction in comparable yields to those obtained in the

stepwise manner, by neat microwave irradiation of the aldehydes

10d-e and aliphatic amines 11a-b for 4 min at 60^oC, followed by

the addition of TosMIC 9, K₂CO₃and MeCN, and then subjecting

the reaction mixture to the microwave conditions described in

Method C (Scheme 1, Method E). 1,5-Diaryl-1H-imidazoles 20a,

d-e derived from aryl amines were also prepared in a two-step

one pot reaction by neat irradiation of substituted benzaldehydes

10a, d-e and 4-bromoaniline 11e, followed by addition of

R'

H₂N

11a

R' =n-butyl

11b R' =

11c

11d

cyclohexyl

R' = cyclopropyl

R' = CH₂Ph

R'

O

N

O

3

N

10a

10b

10c

10d

10e

10f

R = 4-F

R= 2-Cl

R= 3-OMe

R= 2,4-diOMe

R= 3,4-OCH₂O-

R= 4-t-butyl

S

4

12

13

14

15

R' = n-butyl

R'= cyclohexy

R'= cyclopropyl

R'= CH₂Ph

Method A

O

2

H

l

1

O

Method F

5

Method B

22e

22h

22j

R

10g

10h

10i

R= 3,4-(OCH₂)₂O-

R= 2-NO₂

R= 4-NO₂

R= H

Method C Method D

Method G

10j

M

e

t

h

o

H

R'

d

E

o

H₂N

3

N

h

t

3

N

e

M

4

16a-i

11a

11b

11e

11f

11g

11h

R' =n-butyl

2

17a-f, h-i

18a, c-d

19a, c-e

20a, d-e

R' = cyclohexyl

R'= 4-BrPh

R'= 2,4-diClPh

R'= 2.4-diFPh

R'= 2,4-diBrPh

1

O

1

5

N

5

R'

R

21a-f, h-j

Scheme 1: Synthesis of 1-substituted-5-aryl-1H-imidazoles 16-20, 5-aryl-1,3-oxazoles 21 and (4R,5R)/(4S,5S)-5-aryl-4-tosyl-2,4-

dihydro-1,3-oxazoles 22: Reaction conditions: Method A: MgSO_4,DCM, r.t., 16 h; Method B: MW, 60^oC, 4 min; Method C: 9,

K₂CO₃, anhydrous MeCN, reflux, 72 h.; Method D: MW: 9_,K₂CO₃, anhydrous MeCN, 120W, 90^oC, 7 h.; Method E: (i) MW:

60^oC, 1 min-1 h; (ii) 9, K₂CO₃, anhydrous MeCN, 120W, 90^oC, 7 h.; Method F: MW: 9, K₂CO₃, anhydrous MeCN, 120W, 90^oC,

12 min.; Method G: toluene, reflux, 1 h; Method H: MW: 9, K₂CO₃, anhydrous MeOH, 120W, 75^oC, 7 min.

3

Table 2: Isolated yields of 5-aryl-1,3-oxazoles 21 and

(4R,5R)/(4S,5S)-5-aryl-4-tosyl-2,4-dihydro-1,3-oxazoles 22

Table 1: Isolated yields of 1-substituted-5-aryl-1H-imidazoles 16-20

Compds

R

R’

Yields

C: 38%

D: 55%

C: 20%

D: 26%

D: 60%

C: 80%

D: 87%

E: 81%

C: 43%

D: 48%

E: 55%

C: 44%

D: 52%

Structure

Compds

21a

21b

21c

21d

R

4-F

2-Cl

Yields

H: 84%

H: 74%

H: 60%

H: 44%

H: 43%

G: 69%

H: 59%

H: 73%

H: 68%

G: 63%

G 68%

Yields

Structure

16a

4-F

n-butyl

3-OMe

2,4-diOMe

16b

16c

2-Cl

n-butyl

N

3-OMe

21e

3,4-OCH₂O-

O

16d

16e

2,4-diOMe

n-butyl

21f

21h

4-t-butyl

2-NO₂

R

21i

4-NO₂

3,4-OCH₂O-

21j

Compds

H

R

Structure

16f

4-t-buty

n-butyl

3,4-

O(CH₂)₂O-

2-NO₂

22e

22h

22j

3,4-OCH₂O-

2-NO₂

H

F: 80%

F: 65%

F: 76%

16g

C: 54%

O

S

16h

16i

n-butyl

D: 15%

D: 13%

C: 46%

D: 51%

C: 23%

D: 29%

C: 47%

D: 53%

C: 64%

D: 74%

E: 73%

C: 35%

D: 54%

E: 50%

C: 29%

D:50%

D: 12%

D: 10%

D: 23%

D: 40%

C: 68%

D: 26%

D: 24%

D: 74%

C: 22%

E: 68%

N

O

4-NO₂

N

17a

17b

17c

4-F

2-Cl

cyclohexyl

R

N

R'

R

3-OMe

which possess a para-bromophenyl group at the 1-postion of

the imidazole moiety, showed 52% inhibition of the IN-

LEDGF/p75 interaction.

17d

2,4-diOMe

cyclohexyl

Table 3: The HIV-1 IN-LEDGF/p75 inhibition activities of the 5-aryl-1H-

imidazoles 16-20, 5-aryl-1,3-oxazoles 21 and 4,5-dihydro-1,3-oxazoles

22.

17e

17f

3,4-OCH₂O-

cyclohexyl

Biochemical

Target assays

Cell-based assay

(MT-4 cells)

4-t-butyl

Entry

17h

17i

2-NO₂

4-NO₂

4-F

3-OMe

2,4-diOMe

4-F

3-OMe

2,4-diOMe

3,4-OCH₂O-

4-F

cyclohexyl

cyclopropyl

Benzyl

4-BrPh

%Inhibition

Cytotoxicity

Antiviral

EC₅₀(µM)

IC₅₀(µM)

(@10 µM)

30

37

51

26

35

56

21

30

27

45

31

55

40

62

31

40

28

25

21

17

43

52

48

23

33

42

35

30

33

31

33

38

34

98

CC₅₀(µM)

29.6±11.00

23.9± 2.36

18a

18c

18d

19a

19c

19d

19e

20a

16a

16b

16c

16d

16e

16f

16g

16h

16i

17a

17b

17c

17d

17e

17f

18a

18c

18d

19a

19c

19d

19e

20a

20d

20e

21a

21b

21c

21d

21e

21f

30.5 ±4.92

22.4 ±1.46

>10

20d

20e

2,4-diOMe

4-BrPh

E: 38%

E: 62%

3,4-OCH₂O-

21.9 ±0.49

14.6± 0.24

27.7 ± 5.44

21.8 ± 2.84

>10

2.2. Biological evaluation

2.2.1. HIV-1 IN - LEDGF AlphaScreen^TMassays

The synthesized libraries of 1-substituted-5-aryl-1H-

imidazoles 16-20, 5-aryl-1,3-oxazoles 21 and (4R,5R)/(4S,5S)-

5-aryl-4-tosyl-2,4-dihydro-1,3-oxazoles 22, of average

molecular weight 359, were assessed in the AlphaScreen^TM

assay⁴⁵to determine the percentage inhibition for the disruption

of HIV-1 IN-LEDGF/p75 interaction at a concentration of 10

µM. Out of thirty seven compounds screened, six (16c, 16f,

17c, 17f, 20a, and 20d) showed more than our pre-defined 50%

inhibition benchmark (Table 3). Amongst them compound 17f,

containing a cyclohexyl moiety, showed the highest percentage

inhibition (62%) and produced an IC₅₀value of 15 µM in a dose

response assay. Compound 16f, with an n-butyl moiety instead

of the cyclohexyl ring gave an IC₅₀value of 22 µM. Both 16f

and 17f contain a tert-butyl substituent at the para-position of

the phenyl ring attached to the imidazole motif at the 5-position.

Compound 17c, an analogue of 17f, containing a methoxy

substituent at the meta-position inhibited the protein-protein

interaction by 55% with an IC₅₀value of 22 µM, while

compound 16c, also with a meta-methoxy substituent, showed

an IC₅₀value of 30 µM. Two compounds, 20a and 20d, both of

25.1±2.47

7.0 ±1.49

48.0±13.00

55.5±16.25

>10

21h

21i

21j

22e

22h

22j

CX05168

* IC₅₀- concentration of compound required to inhibit 50% of the specific

biological process.

4

*CC₅₀– concentration of compounds that causes 50% reduction of cell growth

the antimicrobial activity. Selected antimicrobial activity results

of the synthesized compounds are shown in Table 4.

Compound 20d with methoxy substituents at the ortho- and

para-positions of the 5-aryl ring displayed the best IC₅₀value in

the series (7 µM), while its counterpart with a fluorine atom at

the para-position 20a produced an IC₅₀value of 25 µM. The

oxygen atoms present on the methoxy substituent groups could

provide the possibility of hydrogen-bonding interactions with

some of the amino acid residues within the binding site, leading

to higher binding affinity. The IC₅₀data suggest that the presence

of the para-bromophenyl moiety at the 1-position of the

imidazole motif might be of significance for the binding ability

of these scaffolds. It is interesting to note that two previously

identified allosteric inhibitors, 5 and 6, both also have aryl halide

moieties within their structures.

These best six compounds (16c, 16f, 17c, 17f, 20a and 20d)

were further tested in the AlphaScreen TruHits^TMKit counter

assay⁴⁶to identify false positive results (which may arise from

compound signal interfering with the AlphaScreen^TMassay) and

all six compounds were confirmed as true inhibitors of the

interaction between the HIV-1 integrase and LEDGF/p75

proteins.

Compounds which exhibited an MIC value of 15.6 μg/ml

were considered to have good antibacterial activity, while MIC

values of 31.3 and 62.5 μg/ml were considered to represent

moderate and marginal activities, respectively. Compounds with

antimicrobial activities of less than 100 μg/ml are noted to have

clinical relevance.⁵²The analysis of the antimicrobial results

revealed that compound 16f exhibited good MIC values of 15.6

μg/ml against S. aureus and moderate MIC values of 31.3 μg/ml

against B. cereus and C. tropicalis. Compound 17f, with a

cyclohexyl group instead of the n-butyl group of 16f, but with the

same tert-butyl group at the para-position of the 5-aryl ring, was

shown to be potent against B. cereus by exhibiting an MIC value

of 15.6 μg/ml while against C. tropicalis it demonstrated a

comparable MIC value to that of compound 16f.Both compounds

16f and 17f were less active against the two Gram-negative

bacterial strains. Compound 16e was the only other compound

showing noteworthy activity, with an MIC of 31.3 µg/ml against

B. cereus. It was interesting to note that only the imidazoles

showed activity, while the oxazoles and dihydrooxazoles were

completely inactive. MIC values obtained suggest that the

imidazole-based scaffolds could serve as a framework for further

development through synthetic modification to obtain more

potent biologically active compounds.

2.2.2. Cytotoxicity and antiviral studies

The newly synthesized 1-substituted-5-aryl-1H-imidazoles 16-

20, 5-aryl-1,3-oxazoles 21 and (4R,5R)/(4S,5S)-5-aryl-4-tosyl-

2,4-dihydro-1,3-oxazoles 22 were evaluated for cytotoxicity in

the metallothionein type 4 (MT-4) cell line. At an initial

concentration of 10 µM, none of the compounds tested reduced

the viability of the cells in culture as detected by MTS [(4,5-

dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-

sulfophenyl)-2H-tetrazolium)].^18,47-48The CC₅₀data obtained for

the six compounds found to be most active in the AlphaScreen^TM

assay (16c, 16f, 17c, 17f, 20a and 20d) showed that these

compounds were not overtly toxic and produced CC₅₀values

ranging from 55.5 to 21.8 µM (Table 3). In general, however, the

IC₅₀values of compounds 16c, 16f, 17c, 17f and 20a were close

to their cytotoxicity values. Compound 20d was more potent

(IC₅₀= 7.0 µM) and was not overly toxic (CC₅₀= 55.5 µM),

suggesting a smaller adverse effect on host cells, and thus

representing a good starting point for improvement of the

biological activity. Finally, the six compounds were tested in a

cell-based antiviral assay at 10 µM within the MT-4 cell line.^18,48

Unfortunately, the compounds did not reach potency levels to

allow for antiviral activity at this relatively low compound

concentration for fragment evaluation, which was chosen to avoid

any direct toxic effect on the cells.

Table 4: Selected MIC (μg/ml) results for synthesized compounds

Gram-positive

bacteria

Gram-negative

bacteria

Yeast

S.

aureus

ATCC

2592

> 250

-

15.6

62.5

B.

cereus

ATCC

11779

62.5

31.3

62.5

250

E.

P.

aeruginosa

ATCC

27858

>250

C.

tropicalis

ATCC

750

62.5

-

31.3

62.5

125

31.3

>250

250

coli

ATCC

8739

16c

16e

16f

17c

17d

17f

19a

19d

125

>250

250

>250

250

>250

15.6

125

Cipro-

floxacin

Nysta-

tin

0.156

-

0.313

-

0.313

-

1.25

3. Receptor-ligand pharmacophore model and screening

A pharmacophore hypothesis was developed manually using

the receptor-ligand complex protocol of Maestro (Schrodinger

software) in order to understand the binding similarity between

the six identified hits with compounds known to induce

multimerization of HIV-1 IN. The hypothesis was created based

on the representative structure of ligand 5 binding at the

LEDGF/p75 binding pocket in HIV-1 IN.^54-55A 9-point

hypothesis pharmacophore was generated as illustrated by the

flow chart in Fig. 2 and is comprised of the following chemical

features: A1 and A2 acceptor sites (pink); H3, H4 and H5 donor

sites (green); the N6 negatively charged site (red) and R7, R8 and

R9 hydrophobic sites (orange) with distance matching tolerance

of 2.0 and excluded volume. The 3D hypothesis pharmacophore

model was then used as a query to assess the best six hits 16c,

16f, 17c, 17f, 20a and 20d. The phase screen data showed that

ligand 5 used as a reference mapped well with all features with a

fitness score of 2.713. Compound 20a shows a relatively higher

fitness score of 1.824 than the other five compounds (i.e. 16f, 17f,

2.2.3. Antimicrobial activities

The synthesized fragments (16-20, 21 and 22) were also

evaluated for their in vitro antimicrobial efficacy towards two

Gram-positive bacterial species (Staphylococcus aureus ATCC

25923 and Bacillus cereus ATCC 11779), two Gram-negative

bacterial species (Escherichia coli ATCC 8739 and

Pseudomonas aeruginosa ATCC 27858) and one yeast (Candida

tropicalis ATCC 750) using the Minimum Inhibitory

Concentration (MIC) assay.^49-51Ciprofloxacin and nystatin were

used as positive controls for bacteria and yeast, respectively,

while 10% dimethyl sulfoxide (DMSO) in water and 100%

acetone were used as the negative controls. All negative controls

demonstrated that dissolution solvents on further serial dilution

(resulting in a starting concentrations of 2.5%) had no effect on

5

16c, 17c, and 20d) which displayed fitness scores of 1.776, 1.756,

1.689, 1.646 & 1.392, respectively. Furthermore, compound 20a

mapped well into five essential features on the hypothesis model,

specifically, the two aromatic rings and the imidazole moiety

predicted to occupy the R7, R8 and R9 hydrophobic spheres

(orange) while the halide substituents were projected onto the H4

and H5 donor sites (green) as shown in Fig. 3A. The overlaying

of 20a with 5 displays the similar chemical features exhibited by

the two aromatic rings (B and C) and N-heterocyclic moiety (A)

of ligand 5 (Fig. 3B). Moreover, scaffolds 16c, 16f, 17c and 17f

are predicted to have similar binding models and were projected

to fit onto five chemical features on the hypothesis model. The n-

butyl (for 16c and 16f) and cyclohexyl moieties (for 17c and 17f)

were projected through R7 towards an H5 donor site (green)

while their substituted aromatic rings pointed towards an R9

hydrophobic (orange) and an H4 donor site (green) as shown in

see Fig 3C to 3J. Moreover, the imidazole moiety (for 16c, 16f,

17c and 17f) projected towards an R8 hydrophobic site (orange)

while a nitrogen atom of the same imidazole motif projected

towards the A1 acceptor point (pink). The superimposed models

of 16c, 16f, 17c and 17f with 5 projected that the aliphatic and the

aryl motifs lie in the same area occupied by the two aromatic rings

(B and C) of 5 while the imidazole motif settled in the same area

occupied by the N-heterocycle (A) of 5. In contrast, the model of

scaffold 20d is predicted to have a different binding model in

comparison to the other five hits (see. Fig 3K and 3L). The 2,4-

dimethoxyphenyl group of 20d is projected to occupy the A1 and

A2 acceptor (pink) and the R8 hydrophobic sites (orange) while

the para-bromophenyl group is shown to be pointing towards the

R9 hydrophobic site (orange) and the H4 donor site (green). The

imidazole motif of 20d was predicted to be on the R7

hydrophobic point (orange).

low molecular weight. Evaluation of the synthesized compounds

in a direct HIV1 IN-LEDGF/p75 AlphaScreen^TMassay at a single

dose of 10 µM resulted in identification of six novel 1-

substituted-5-aryl-1H-imidazoles (16c, 16f, 17c, 16f, 20a, and

20d) able to disrupt the HIV-1 IN- LEDGF/p75 protein-protein

interaction. To understand the structural features responsible for

the biological activity of these compounds, a receptor-ligand

pharmacophore hypothesis model was generated, built on

previously identified inhibitor 5 at the LEDGF/p75 binding

pocket in HIV-1 IN. The models superimposing 5 with the active

compounds 16c, 16f, 17c, 17f, 20a and 20d showed that all the

compounds besides 20d settled in the same region occupied by

the two aromatic rings (B and C) and the N-heterocyclic moiety

(A) of compound 5. Based on the hypothesis model, it is likely

that the aromatic rings of these arylimidazoles occupy the

hydrophobic pockets formed by helices at the interface made by

two integrase monomers, as previously proposed for the

quinoline and chlorophenyl rings of ligand 5.^53-54Despite having

inhibitory activity within the biochemical assay, none of the best

six active compounds inhibited viral replication within a cell

based assay.

In an in vitro antimicrobial assay by the MIC method,

compounds 16e, 16f and 17f displayed activity against Gram-

positive bacterial and yeast pathogens. Interestingly, compounds

16f and 17f both exhibited activity in inhibiting the HIV-1 IN-

LEDGF/p75 interaction and antimicrobial activity and represent

promising starting candidates for synthetic modifications to

increase their potency. Once again, the versatility of the

privileged imidazole moiety, which exhibits a variety of

biological activities,^33,57-58has been demonstrated. To the best of

our knowledge, these compounds are novel and have not been

reported previously in the literature as HIV-1 IN disruptors or

inhibitors of bacterial pathogens. The success in the generation of

diverse five membered nitrogen-containing heterocycle

collections in the present study was made possible by the

versatility of the TosMIC synthon. The viable hits identified

provide a good starting point for synthetic variations that could

result in compounds with improved potency, that are currently

under investigation in our laboratory.

4. Conclusion

In the present study, we have established the use of

microwave-assisted cycloaddition of TosMIC to imines and

aldehyde as an efficient approach for directly preparing small

libraries of 1-substituted-5-aryl-1H-imidazoles, 5-aryl-1,3-

oxazoles and trans-5-aryl-4-tosyl-4,5-dihydro-1,3-oxazoles with

6

Fig 2: Overall flowchart of pharmacophore derived from 5 binding at the LEDGF/p75 binding pocket in HIV-1 IN.

Fig. 3: mapping of: [A] 20a; [C] 16f; [E] 17f; [G] 16c; [I] 17c and [K] 20d onto the hypothesis model. Superposition mapping

of 5 (yellow) with: [B] 20a (orange); [D] 16f (red); [F] 17f (green); [H] 16c (purple); [J] 17c (blue) and [L] 20d (red) onto the

hypothesis model.

6

5. Experimental protocols

(N=CHCH₂), 32.8 (N=CHCH₂CH₂), 20.4 (N=CHCH₂CH₂CH₂),

13.8 (N=CHCH₂CH₂CH₂CH₃).

5.1. General methods and materials

5.2.5. (E)-N-(3-Methoxybenzylidene)butan-1-amine (12c).Yield:

97% (Method B) as colourless oil. H NMR: δ 8.26 (1H, s,

N=CH), 7.28-7.37 (3H, m, ArH), 6.98 (1H, d, J = 8.4 Hz, ArH),

3.87 (3H, s, ArOCH₃), 3.62 (2H, t, J = 7.0 Hz, N=CHCH₂), 1.70-

1.75 (2H, m, N=CHCH₂CH₂), 1.39-1.50 (2H, m,

All commercially available reagents were purchased from

Sigma Aldrich. Dry solvents were used from an LC-Tech SP-1

Solvent Purification System stored under argon. Microwave-

assisted reactions were performed in a CEM Discover reactor

(Dynamic Temperature and Power set). Column chromatography

was performed using Merck Silica gel 60 [particle size 0.040-

0.063 mm (230-400 mesh)]. TLC was performed on pre-coated

Merck silica gel F254 plates and viewed under UV light (254 nm)

or following exposure to iodine. NMR spectra were recorded on a

Bruker 400 Avance III Spectrometer at 298 K equipped with a 5

mm BBI probe Chemical shifts (δ) are given in parts per million

and referenced by the solvent residual peak [all spectra were run

in CDCl₃δ: 7.26 ppm for ¹H NMR (400 MHz), and 77.0 ppm for

¹³C NMR (101 MHz)]. High-resolution mass spectra were

recorded on an LC-MS system consisting of a Dionex Ultimate

3000 Rapid Separation LC system equipped with a C-18 pre-

coated column and coupled to a MicrOTOF QII Bruker mass

spectrometer fitted with an electrospray source and operating in

positive mode. FTIR spectra were recorded using a Thermo

Nicolet 5700 spectrometer and samples were prepared as KBr

mixtures. All melting points were obtained using a Stuart SMP10

melting point apparatus and are uncorrected.

1

N=CHCH₂CH₂CH₂), 0.96 (3H, t,

J

=

7.4 Hz,

N=CHCH₂CH₂CH₂CH₃); ¹³C NMR: δ 160.5 (ArCOCH₃), 159.8

(N=CH), 137.8, 129.4, 121.2, 117.1, 111.4 (ArC), 61.3

(N=CHCH₂), 55.2 (ArCOCH₃), 32.9 (N=CHCH₂CH₂), 20.4

(N=CHCH₂CH₂CH₂), 13.8 (N=CHCH₂CH₂CH₂CH₃).

5.2.6. (E)-N-(2,4-Dimethoxybenzylidene)butan-1-amine (12d).

Yields: 84% (Method A), 98% (Method B) as light brown oil; ¹H

NMR: δ 8.48 (1H, s, N=CH ), 7.77 (1H, d, J = 8.8 Hz, ArH), 6.37

(1H, d, J = 8.8 Hz, ArH), 6.28 (1H, s, ArH), 3.66 and 3.68 (6H, 2

x s, 2 x ArOCH₃), 3.44 (2H, t, J = 7.0 Hz, N=CHCH₂), 1.51-1.57

(2H, m, N=CHCH₂CH₂), 1.24-1.30 (2H, m, N=CHCH₂CH₂CH₂),

0.81 (3H, t, J = 7.4 Hz, N=CHCH₂CH₂CH₂CH₃); ¹³C NMR: δ

162.5 and 159.6 (2 x ArCOCH₃), 155.8 (N=CH), 128.1, 117.9,

105.0, 97.6 (ArC), 61.4 (N=CHCH₂), 55.0 and 54.9 (2 x

ArCOCH₃), 33.0 (N=CHCH₂CH₂), 20.2 (N=CHCH₂CH₂CH₂),

13.7 (N=CHCH₂CH₂CH₂CH₃).

5.2.7. (E)-N-(Benzo[d][1,3]dioxol-5-ylmethylene)butan-1-amine

(12e). Yield: 96% (Method A), 100% (Method B) as brown oil;

¹H NMR: δ 8.11 (1H, s, N=CH), 7.32 (1H, s, ArH), 7.05 (1H, d, J

= 7.6 Hz, ArH), 6.77 (1H, d, J = 8.0 Hz, ArH), 5.94 (2H, s,

ArOCH₂O-), 3.52 (2H, t, J = 7.0 Hz, N=CHCH₂), 1.60-1.67 (2H,

m, N=CHCH₂CH₂), 1.33-1.39 (2H, m, N=CHCH₂CH₂CH₂), 0.90

(3H, t, J = 7.4 Hz, N=CHCH₂CH₂CH₂CH₃); ¹³C NMR: δ 159.6

(N=CH), 149.5, 148.1, 131.1, 123.9, 107.8, 106.4 (ArC), 101.2

(ArOCH₂O), 61.0 (N=CHCH₂), 33.0 (N=CHCH₂CH₂), 20.3

(N=CHCH₂CH₂CH₂), 13.8 (N=CHCH₂CH₂CH₂CH₃).

5.2. Synthesis of N-benzylidene alkylamines (12-15)

5.2.1. Synthetic Method A: Conventional Method A

To a solution of aldehyde (2.72 mmol) in DCM (5 ml) was added

primary amine (4.08 mmol) and MgSO₄(5.44mmol). The resulting

reaction mixture was then stirred at room temperature for 16h. The

solution was filtered and the collected solid was washed with

DCM (2 x 5 ml). The combined DCM filtrates were concentrated

in vacuo and dried under high vacuum to give pure products.

5.2.2. Synthetic Method B: Microwave Irradiation

5.2.8. (E)-N-(4-tert-butylbenzylidene)butan-1-amine (12f). Yield:

84% (Method A), 93% (Method B) as pale brown oil; ¹H NMR:

δ 8.30 (1H, s, N=CH), 7.65-7.68 (2H, m, ArH), 7.41-7.43 (2H, m,

ArH), 3.15 (2H, t, J = 7.0 Hz, N=CHCH₂), 1.60-1.77 (2H, m,

N=CHCH₂CH₂), 1.31-1.33 [11H, m, N=CHCH₂CH₂CH₂and

A mixture of aldehyde (4.80 mmol) and primary amine (5.76

mmol) in a 10 ml microwave vessel was irradiated neat at 60°C for

4 min (power applied controlled by the instrument). At the end of

the reaction time, DCM (15 ml) was added and reaction mixture

was transferred into round bottom flask and the vessel was then

rinsed with DCM (2 x 5 ml). The combined DCM fractions were

then concentrated in vacuo to give pure N-arylidene alkylamine.

ArC(CH₃)₃], 0.98 (3H, t, J = 7.4 Hz, N=CHCH₂CH₂CH₂CH₃); ¹³

C

NMR: δ 158.4 (N=CH), 153.7, 134.0, 127.9 , 125.5 (ArC), 70.1

(N=CHCH₂), 34.9 [ArCC(CH₃)], 33.0 (N=CHCH₂CH₂), 20.3

(N=CHCH₂CH₂CH₂), 13.8 (N=CHCH₂CH₂CH₂CH₃), 25.4

[ArCC(CH₃)₃].

5.2.3. (E)-N-(4-Fluorobenzylidene)butan-1-amine (12a). Yield:

87% (Method A), 98% (Method B) as pale brown oil; ¹H NMR:

δ = 8.21 (1H, s, N=CH), 7.12 (2H, m, Hz, ArH), 7.02 (2H, t, J =

8.8 Hz, ArH ), 3.57 (2H, t, J = 7.0 Hz, N=CHCH₂), 1.63-1.70 (2H,

m, N=CHCH₂CH₂), 1.33-1.42 (2H, m, N=CHCH₂CH₂CH₂), 0.92

(3H t, J = 7.4 Hz, N=CHCH₂CH₂CH₂CH₃); ¹³C NMR: δ 164.1 (d,

¹J_CF= 250 Hz, para-CF), 159.1 (6-C) 132.6 (d, ⁴J_CF= 3 Hz, 1’-C),

129.8 (d, ³J_CF= 9 Hz, ortho-C), 115.5 (d, ²J_CF= 22 Hz, meta-C),

5.2.9. (E)-N-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)methylene)-

butan-1-amine (12g). Yield: 84% (Method A) as pale brown oil;

¹H NMR: δ 8.03 (1H, s, N=CH), 7.18 (1H, s, ArH) 7.10 (1H, d, J

= 8.4 Hz, ArH), 6.77 (1H, d, J = 8.0 Hz, ArH), 4.16 (4H, s,

ArOCH₂CH₂O), 3.45-3.49 (2H, t, J = 7.0 Hz, N=CHCH₂), 1.55-

159 (2H, m, N=CHCH₂CH₂), 1.26-1.31 (2H, m,

61.3

(N=CHCH₂),

32.9

(N=CHCH₂CH2),

20.4

N=CHCH₂CH₂CH₂), 0.83 (3H, t,

J

=

7.4 Hz,

N=CHCH₂CH₂CH₂CH₃); ¹³C NMR: δ 159.7 (N=CH), 145.5 and

143.5 (ArCOCH₂CH₂O), 130.1, 121.5, 117.2, 116.5 (ArC), 64.4

and 64.0 (ArCOCH₂CH₂O-), 61.3 (N=CHCH₂), 32.9

(N=CHCH₂CH₂CH₂), 13.8 (N=CHCH₂CH₂CH₂CH₃).

5.2.4. (E)-N-(2-Chlorobenzylidene)butan-1-amine (12b). Yield:

94% (Method A), 99% (Method B) as colourless oil; ¹H NMR: δ

8.61 (1H, s, N=CH), 7.92 (1H, d, J = 8.4 Hz, ArH), 7.17-7.28 (3H,

m, ArH), 3.56 (2H, t, J = 7.0 Hz N=CHCH₂), 1.58-1.65 (2H, m,

N=CHCH₂CH₂), 1.29-1.34 (2H, m, N=CHCH₂CH₂CH₂), 0.85

(3H, t, J = 7.4 Hz, N=CHCH₂CH₂CH₂CH₃); ¹³C NMR δ 157.4

(N=CH), 134.9, 133.3, 131.2, 129.7, 128.2, 126.9 (ArC), 61.5

(N=CHCH₂CH₂),

(N=CHCH₂CH₂CH₂CH₃).

20.3

(N=CHCH₂CH₂CH₂),

13.8

5.2.10. (E)-N-(2-Nitrobenzylidene)butan-1-amine (12h). Yield:

73% (Method B) as yellow oil; ¹H NMR: δ 8.66 (1H, s, N=CH),

7.97-8.03 (2H, m, ArH), 7.51-7.66 (2H, m, ArH), 3.65 (2H, t, J =

7.0 Hz, N=CHCH₂), 1.66-1.73 (2H, m, N=CHCH₂CH₂), 1.35-1.44

(2H, m, N=CHCH₂CH₂CH₂), 0.92 (3H, t, J = 7.4 Hz,

7

N=CHCH₂CH₂CH₂CH₃); ¹³C NMR: δ 156.6 (N=CH), 148.7,

133.4, 131.3, 130.4, 129.6, 124.2 (ArC), 61.4 (N=CHCH₂), 32.6

(NCHCH₂CH₂CH₂CH₂CH₂), 25.6 (NCHCH₂CH₂CH₂CH₂CH₂),

24.8 (NCHCH₂CH₂CH₂CH₂CH₂).

(N=CHCH₂CH₂),

(N=CHCH₂CH₂CH₂CH₃).

20.3(N=CHCH₂CH₂CH₂),

13.8

5.2.17. (E)-N-(4-tert-Butylbenzylidene)cyclohexanamine (13f).

Yield: 93% (Method A), 99% (Method B) as a white solid; Mp.:

45-48^oC; ¹H NMR: δ 8.30 (1H, s, N=CH), 7.65 (2H, d, J = 8.4 Hz,

ArH), 7.41 (2H, d, J =8.4 Hz, ArH), 3.15.-3.20 (1H, m,

5.2.11. (E)-N-(4-Nitrobenzylidene)butan-1-amine (12i). Yield:

91% (Method B) as yellow oil; ¹H NMR: δ 8.34 (1H, s, N=CH),

8.22-8.25 (2H, m, ArH), 7.86-7.90 (2H, m, ArH), 3.64 (2H, t, J =

7.0 Hz, N=CHCH₂), 1.65-1.73 (2H, m N=CHCH₂CH₂), 1.38-1.43

(2H, m, N=CHCH₂CH₂CH₂), 0.92-0.96 (3H, t, J = 7.4 Hz,

N=CHCH₂CH₂CH₂CH₃); ¹³C NMR: δ 158.3 (N=CH), 148.8,

141.8, 128.6, 123.8 (ArC), 61.6 (N=CHCH₂), 32.7

NCHCH₂CH₂CH₂CH₂CH₂),

1.33-1.86

[19H,

m,

NCHCH₂CH₂CH₂CH₂CH₂and ArC(CH₃)]; ¹³C NMR: δ 158.4

(N=CH), 153.7 [ArCC(CH₃)₃], 134.0, 127.9, 125.5 (ArC), 70.1

(NCHCH₂CH₂CH₂CH₂CH₂),

34.9

31.3

[ArCC(CH₃)₃],

34.4

25.7

(N=CHCH₂CH₂),

20.4

(N=CHCH₂CH₂CH₂),

13.8

(NCHCH₂CH₂CH₂CH₂CH₂) 24.9 (NCHCH₂CH₂CH₂CH₂CH₂).

(N=CHCH₂CH₂CH₂CH₃).

5.2.18. (E)-N-(2-Nitrobenzylidene)cyclohexanamine (13h). Yield:

95% (Method B) as a yellow oil; ¹H NMR: δ 8.70 (1H, s, N=CH),

7.97-8.03 (2H, m, ArH), 7.61-7.65 (1H, m, ArH), 7.49 (1H, m,

ArH), 3.28-3.33 (1H, m, NCHCH₂CH₂CH₂CH₂CH₂), 1.23-1.85

(10H, m, NCHCH₂CH₂CH₂CH₂CH₂); ¹³C NMR: δ 154.6 (N=CH),

148.7 (ArCNO₂), 133.4, 131.6, 130.3, 12.7, 124.1 (ArC), 70.0 (

NCHCH₂CH₂CH₂CH₂CH₂), 34.0 (NCHCH₂CH₂CH₂CH₂CH₂),

5.2.12. (E)-N-(4-Fluorobenzylidene)cyclohexanamine (13a).

Yield: 85% (Method A), 88% (Method B) as pale yellow oil; ¹H

NMR: δ 8.26 (1H, s, N=CH), 7.69 (2H, m, ArH), 7.04 (2H, t, J =

8.8 Hz, ArH), 3.14 (1H, m, NCHCH₂CH₂CH₂CH₂CH₂), 1.38-1.85

(10H, m, NCHCH₂CH₂CH₂CH₂CH₂); ¹³C NMR: δ 164.0 (d, J_C,F

1

4

=249.6 Hz, para-CF), 157.0 (8-C), 132.9 (d, J_C,F=3.0 Hz 1’-C),

129.8 (d, ³J_C,F= 8.1 Hz, ortho-C), 115.6 (d, ²J_C,F= 22.0 Hz, meta-

25.5

(NCHCH₂CH₂CH₂CH₂CH₂),

24.5

C),

69.8

(NCHCH₂CH₂CH₂CH₂CH₂),

34.3

(NCHCH₂CH₂CH₂CH₂CH₂).

(NCHCH₂CH₂CH₂CH₂CH₂), 25.6 (NCHCH₂CH₂CH₂CH₂CH₂),

24.7 (NCHCH₂CH₂CH₂CH₂CH₂).

5.2.19. (E)-N-(4-Nitrobenzylidene)cyclohexanamine (13i). Yield:

91% (Method B) as a yellow oil; ¹H NMR: δ 8.38 (1H, s, N=CH),

8.23-8.25 (2H, m, ArH), 7.86-7.89 (2H, m, ArH), 3.25-3.30 (1H,

5.2.13. (E)-N-(2-Chlorobenzylidene)cyclohexanamine (13b).

Yield: 86% (Method A), 89% (Method B) as pale yellow solid;

¹H NMR: δ 8.71 (1H, s, N=CH), 7.99 (1H, d, J = 7.6 Hz, ArH),

m,

NCHCH₂CH₂CH₂CH₂CH₂); ¹³C NMR: δ 156.2 (N=CH), 148.8

(ArCNO₂), 142.1, 128.7, 128.8 (ArC), 70.1

(NCHCH₂CH₂CH₂CH₂CH₂), 34.1 (NCHCH₂CH₂CH₂CH₂CH₂),

25.5 (NCHCH₂CH₂CH₂CH₂CH₂), 24.6

(NCHCH₂CH₂CH₂CH₂CH₂).

NCHCH₂CH₂CH₂CH₂CH₂),

1.25-1.87

(10H,

m,

7.23-7.34

(3H,

m,

ArH),

3.22-3.28

(1H,

(10H,

m,

NCHCH₂CH₂CH₂CH₂CH₂),

1.38-1.83

NCHCH₂CH₂CH₂CH₂CH₂); ¹³C NMR: δ 155.4 (N=CH), 134.9,

133.6,

(NCHCH₂CH₂CH₂CH₂CH₂), 34.3 (NCHCH₂CH₂CH₂CH₂CH₂),

25.6 (NCHCH₂CH₂CH₂CH₂CH₂), 24.7

(NCHCH₂CH₂CH₂CH₂CH₂).

131.1,

129.6,

128.4,

126.9

(ArC),

70.0

5.2.20. (E)-N-(4-Fluorobenzylidene)cyclopropaneamine (14a).

Yield: 77% (Method A) as a brown oil; ¹H NMR: δ 8.40 (1H, s,

N=CH), 7.64 (2H, d, J = 8.6 Hz, ArH), 7.04 (2H, t, J = 8.6 Hz,

ArH), 2.98-3.02 (1H, m, NCHCH₂CH₂), 0.89-0.99 (4H, m, 3- &

5.2.14. (E)-N-(3-Methoxybenzylidene)cyclohexanamine (13c).

Yield: 72% (Method A), 88% (Method B) as white solid; Mp: 58-

1

4-CH₂); ¹³C NMR: δ 163.9 (d, J_F,C= 249.6 Hz, para-CF)_,156.9

1

61^oC; H NMR: δ 8.30 (1H, s, N=CH), 7.28-7.34 (3H, m, ArH),

4

3

(N=CH), 132.8 (d, J_F,C= 3.0 Hz, 1’-C), 129.8 (d, J_F,C= 8.1 Hz,

ortho-C), 115.5 (d, ²J_F,C= 21.1 Hz, meta-C), 41.8 (NCHCH₂CH₂),

8.70 (NCHCH₂CH₂).

6.95 (1H, dd, J = 8.0 Hz, 1.2 Hz, ArH), 3.86 (3H, s, ArOCH₃),

3.17-3.25 (1H, m, NCHCH₂CH₂CH₂CH₂CH₂), 1.38-1.88 (10H, m,

NCHCH₂CH₂CH₂CH₂CH₂); ¹³C NMR: δ 159.8 (ArCOCH₃), 158.4

(N=CH), 138.1, 129.4, 121.2, 116.9, 111.7 (ArC), 69.9

5.2.21. (E)-N-(3-Methoxybenzylidene)cyclopropanamine (14c).

Yield: 92% (Method A) as a brown oil; ¹H NMR: δ 8.47 (1H, s,

N=CH), 7.28-7.37 (3H, m, ArH), 6.98 (1H, dd, J = 3.5 Hz, ArH),

3.89 (3H, s, ArOCH₃), 3.07-3.10 (1H, m, NCHCH₂CH₂), 0.98-

1.05 (4H, m, NCHCH₂CH₂); ¹³C NMR: δ159.8 (ArCOCH₃), 158.2

(N=CH), 137.9, 129.5, 120.7, 116.6, 111.2 (ArC), 55.3

(ArCOCH₃), 41.8 (NCHCH₂CH₂), 8.77 (NCHCH₂CH₂).

(NCHCH₂CH₂CH₂CH₂CH₂),

55.3

(ArCOCH₃),

34.2

(NCHCH₂CH₂CH₂CH₂CH₂), 25.6 (4 NCHCH₂CH₂CH₂CH₂CH₂),

24.8 (NCHCH₂CH₂CH₂CH₂CH₂).

5.2.15.

(E)-N-(2,4-Dimethoxybenzylidene)cyclohexanamine

(13d). Yield: 81% (Method A), 99% (Method B) as a pale yellow

solid; Mp: 55-58^oC; ¹H NMR: δ 8.63 (1H, s, N=CH), 7.87 (1H, d,

J= 8.8 Hz, ArH), 6.47 (1H, d, J = 6.8 Hz, ArH), 6.40 (1H, s, ArH),

5.2.22.

(E)-N-(2,4-Dimethoxybenzylidene)cyclopropanamine

(14d). Yield: 87% (Method A) as a pale yellow solid; ¹H NMR: δ

8.77 (1H, s, N=CH), 7.79 (1H, d, J = 8.4 Hz, ArH), 6.47 (1H, d, J

= 8.4 Hz, ArH), 6.42 (1H, s, ArH), 3.81 and 3.84 (6H 2 x s, 2 x

ArOCH₃), 2.99-3.02 (1H, m, NCHCH₂CH₂), 0.85-0.93 (4H, m,

NCHCH₂CH₂); ¹³C NMR: δ 162.7 and 159.8 (2 x ArCOCH₃),

158.6 (N=CH), 129.5, 116.6, 111.2, 98.0 (ArC), 55.4 and 55.3 (2

x ArCOCH₃), 41.8 (NCHCH₂CH₂), 8.77 (NCHCH₂CH₂).

3.81 (6H

NCHCH₂CH₂CH₂CH₂CH₂),

2

x

s,

2

x

ArOCH₃), 3.11-3.80 (1H, m,

1.20-1.82 (10H, m,

NCHCH₂CH₂CH₂CH₂CH₂); ¹³C NMR: δ 162.7 and 159.8 (2 x

ArCOCH₃), 154.1 (N=CH), 127.5, 118.4, 105.2, 98.0 (ArC), 70.2

(NCHCH₂CH₂CH₂CH₂CH₂), 55.4 and 55.3 (2 x ArCOCH₃), 34.6

(NCHCH₂CH₂CH₂CH₂CH₂), 25.7 (NCHCH₂CH₂CH₂CH₂CH₂),

25.0 (NCHCH₂CH₂CH₂CH₂CH₂).

5.2.23.

(E)-N-(4-Fluorobenzylidene)-1-phenylmethanamine

5.2.16. (E)-N-(Benzo[d][1,3]dioxol-5-ylmethylene)cyclohexan-

amine (13e). Yield: 90% (Method A), 97% (Method B) as a pale

yellow solid; Mp: 64-67^oC (lit. ⁵⁸); ¹H NMR: δ8.18 (1H, s, N=CH),

7.34 (1H, s, ArH) 7.07 (1H, d, J = 6.8 Hz, ArH), 6.79 (1H, d, J =

8.0 Hz, ArH), 5.96 (2H, s, ArOCH₂O), 3.10-3.16 (1H, m,

(15a). Yield: 92% (Method B) as an orange solid; ¹H NMR: δ8.37

(1H, s, CH=N), 7.78-7.82 (2H, m, ArH) 7.05 (7H, m, ArH), 4.83

(2H, s, CH=NCH₂); ¹³C NMR: δ 165.5 (d, J_C,F= 250 Hz, para-

1

CF), 160.4 (CH=N), 139.1 (ArC), 132.4 (d, J_C,F= 3 Hz, 1’-C ),

130.1 , 130.0, 128.5 (d, ³J_C,F= 8 Hz, ortho-C) 127.9 (ArC), 115.6

(d, ¹J_C,F= 22 Hz, meta-C), 64.9 (CH=NCH₂).

NCHCH₂CH₂CH₂CH₂CH₂),

1.36-1.84

(10H,

m,

NCHCH₂CH₂CH₂CH₂CH₂); ¹³C NMR: δ 157.6 (N=CH), 149.4

and 148.1 (ArCOCH₂O), 131.4, 123.9, 107.9 (ArC), 101.3

5.2.24.

(E)-N-(2-Chlorobenzylidene)-1-phenylmethanamine

1

(ArCOCH₂O),

61.0

(NCHCH₂CH₂CH₂CH₂CH₂),

34.3

(15b). Yield: 93% (Method B) as a pale yellow oil; H NMR: δ

8

8.86 (1H, s, CH=N), 8.01 (1H, dd, J = 6 Hz, 1.2 Hz, ArH) 7.05

(8H, m, ArH), 4.89 (2H, s, CH=NCH₂); ¹³C NMR: δ 158.7

(CH=N), 139.0, 135.2, 133.1, 131.6, 129.7, 128.6, 128.3, 128.0,

127.1, 127.0 (ArC); 65.3 (CH=NCH₂).

mmol) were added. An inert atmosphere was created by degassing

through evacuating and refilling with argon three times, followed

by addition of dry MeCN (5 ml) and capping. The resulting

reaction mixture was then irradiated at a set temperature of 90^oC

and a power of 120 Watts for 7 h. After completion the solvent

was evaporated to give a brown oily residue, which was purified

by column chromatography (as described in synthetic procedure

C) to afford the desired 1-substituted-5-aryl-1H-imidazoles.

5.2.25.

(E)-N-(3-Methoxybenzylidene)-1-phenylmethanamine

(15c). Yield: 98% (Method B) as a pale yellow oil;¹H NMR: δ

8.29 (1H, s, CH=N), 7.31 (1H, d, J = 8.4 Hz, ArH), 6.91-7.29 (8H,

m, ArH), 4.72 (2H, s, CH=NCH₂), 3.76 (3H, s, ArOCH₃); ¹³C

NMR: δ 161.9 (CH=N), 159.8 (ArCOCH₃), 139.1, 137.5, 129.5,

128.4, 127.9, 126.9, 121.9, 117.5, 115.6 (ArC), 64.9 (CH=NCH₂),

55.3 (ArCOCH₃).

5.3.4. 1-Butyl-5-(4-fluorophenyl)-1H-imidazole (16a). Yield: 38%

1

(Method C), 55% (Method D) as a brown oil; H NMR: δ 7.53

(1H, s, 2-CH), 7.29-7.32 (2H, m, ArH), 7.08-7.13 (2H, m, ArH),

7.00 (1H, s, 4-CH) 3.88 (2H, t, J =7.0 Hz, NCH₂), 1.53-161 (2H,

m, NCH₂CH₂), 1.16-1.25 (2H, m, NCH₂CH₂CH₂), 0.79 (3H, t, J =

7.4 Hz, NCH₂CH₂CH₂CH₃); ¹³C NMR: δ 163.0 (d, J_C,F= 248.6 Hz,

5.2.26.

(E)-N-(2,4-Dimethoxybenzylidene)-1-phenylmethan-

amine (15d). Yield: 90% (Method B) as a yellow oil; ¹H NMR: δ

8.89 (1H, s, CH=N), 8.11 (1H, d, J= 8.4 Hz, ArH), 7.36-7.47 (5H,

m, ArH), 6.5-6.66 (2H, m, ArH), 4.97 (2H, s, CH=NCH₂), 3.95

and 3.97 (6H, 2 x s, 2 x ArOCH₃); ¹³C NMR: δ 163.0 and 160.1 (2

x ArCOCH₃), 157.4 (CH=N), 139.9, 128.6, 128.5, 128.3, 127.9,

117.9, 105.3, 98.0 (ArC), 65.5 (CH=NCH₂), 55.4 and 55.3 (2 x

ArCOCH₃).

3

para-CF), 138.0 (2-CH), 131.8 (C-5), 130.6 (d, J_F,C= 8.1 Hz,

4

ortho-C), 128.1 (4-CH), 126.2 (d, J_F,C= 4.0 Hz, 1’-C), 115.7 (d,

²J_F,C= 21.1 Hz, meta-C), 44.9 (NCH₂), 32.8 (NCH₂CH₂), 19.6

(NCH₂CH₂CH₂), 13.4 (NCH₂CH₂CH₂CH₃); FTIR ν_max/cm^-1(KBr):

3388, 3104, 2960, 2873, 1682, 1599, 1516, 1456, 1448, 1221,

1110, 1023, 915, 853, 755, 695, 658, 603, 537; HRMS (ESI-TOF)

m/z: [M+H]⁺Calcd for C₁₃H₁₆FN₂219.1292, found 219.1294.

5.2.27.

(E)-N-(Benzo[d][1,3]dioxol-5-ylmethylene)-1-phenyl-

methanamine (15e). Yield: 95% (Method B) as a white solid; Mp:

5.3.5. 1-Butyl-5-(2-chlorophenyl)-1H-imidazole (16b). Yield:

20% (Method C), 26% (Method D) as a brown oil; H NMR: δ

1

64-70^oC (lit.⁵⁹); H NMR: δ 8.18 (1H, s, CH=N), 7.04-7.34 (8H,

1

m, ArH), 6.73 (1H, d, J = 8.8 Hz, ArH), 5.90 (2H, s, ArOCH₂O),

4.96 (2H, s, CH=NCH₂); ¹³C NMR: δ 161.0 (C=N), 149.9 and

148.2 (2 x ArCOCH₂O), 139.4, 131.0, 128.4, 127.9, 126.9, 124.5,

108.0, 106.7 (ArC), 101.4 (ArCOCH₂O), 64.7 (C=NCH₂).

7.58 (1H, s, 2-CH), 7.33-7.50 (4H, m, ArH), 7.02 (1H, s, 4-CH),

3.78 (2H, t, J =7.2 Hz, NCH₂), 1.50-1.57 (2H, m, 7-CH₂), 1.13-

1.22 (2H, m, NCH₂CH₂CH₂), 0.77 (3H, t, J = 7.2 Hz,

NCH₂CH₂CH₂CH₃); ¹³C NMR: δ 137.5 (2-CH), 135.0 (5-C),

132.8, 130.2, 129.8, 129.7, 129.3 (ArC), 128.9 (4-CH), 126.8

(ArC), 45.1 (NCH₂), 32.7 (NCH₂CH₂), 19.5 (NCH₂CH₂CH₂), 13.4

(NCH₂CH₂CH₂CH₃); FTIR ν_max/cm^-1(KBr): 3401, 2959, 2873,

1727, 1633, 1557, 1458, 1367, 1285, 1220, 1145, 1114, 1035, 916,

767, 659, 537; HRMS (ESI-TOF) m/z: [M+H]⁺Calcd for

C₁₃H₁₆ClN₂235.0995, found 235.1002.

5.3 Synthesis of 1-substituted-5-aryl-1H-imidazoles (16-20)

5.3.1. Synthetic method C: stepwise van Leusen general

N-arylidene alkylamine (1.93 mmol), TosMIC (2.89 mmol)

and K₂CO₃(2.51 mmol) were placed in a dried three-necked flask

equipped with a refluxing condenser under inert atmosphere

(degassed by evacuating and refilling with argon three times),

followed by addition of dry acetonitrile (5 ml). The resulting

reaction mixture was then heated at reflux for 72 h under argon.

Upon completion the solvent was evaporated in vacuo to afford a

brown oily crude material which was purified by column

chromatography [eluents: hexane- ethyl acetate c (1:2), followed

by 100% ethyl acetate] to afford the desired pure 1-substituted-5-

aryl-1H-imidazoles.

5.3.6. 1-Butyl-5-(3-methoxyphenyl)-1H-imidazole (16c). Yield:

60% (Method D) as a yellow oil; ¹H NMR: δ 7.54 (1H, s, 2-CH),

7.31 (1H, t, J = 6.8 Hz, ArH), 7.04 (1H, s, 4-CH), 6.89-6.95 (3H,

m, ArH), 3.94 (2H, t, J = 7.4 Hz, NCH₂), 3.83 (3H, s, ArOCH₃),

1.57-1.65 (2H, m, NCH₂CH₂), 1.18-1.28 (2H. m, NCH₂CH₂CH₂),

0.82 (3H, t, J = 7.4 Hz, NCH₂CH₂CH₂CH₃); ¹³C NMR: δ 159.7

(ArCOCH₃), 138.0 (2-CH), 132.8 (5-C), 131.5, 129.7 (ArC), 128.1

(4-CH), 121.1, 114.5, 113.3 (ArC), 55.3 (ArCOCH₃), 45.1

(NCH₂), 32.9 (NCH₂CH₂), 19.6 (NCH₂CH₂CH₂), 13.4

(NCH₂CH₂CH₂CH₃); FTIR ν_max/cm^-1(KBr): 3383, 2958, 2873,

1682, 1610, 1580, 1489, 1290, 1212, 1169, 1116, 1050, 1029, 919,

845, 785, 698, 656, 570; HRMS (ESI-TOF) m/z: [M+H]⁺Calcd

for C₁₄H₁₉N₂O 231.1492, found 231.1498.

5.3.2. Synthetic method D: microwave assisted van Leusen

In a 10 ml microwave reaction vessel equipped with a

magnetic stirrer bar was introduced N-arylidene alkylamine (1.77

mmol), TosMIC (2.66 mmol) and K₂CO₃(2.30 mmol). An inert

atmosphere was created by degassing through evacuating and

refilling with argon three times, followed by addition of dry

acetonitrile (5 ml) and capping. The resulting reaction mixture was

then microwave-irradiated at a set temperature of 90^oC and a

power of 120 Watts for 7 h. After completion the solvent was

evaporated in vacuo to give a brown crude oil which was then

purified by column chromatography as described in procedure C

to afford the desired 1-substituted-5-aryl-1H-imidazoles.

5.3.7. 1-Butyl-5-(2,4-dimethoxyphenyl)-1H-imidazole (16d).

Yield: 80% (Method C), 87% (Method D), 81% (Method E) as

brown oil; ¹H NMR: δ 7.53 (1H, s, 2-CH), 7.12 (1H, d, J=8.8 Hz,

ArH), 6.92 (1H, s, 4-CH), 6.52 (2H, d, J = 7.6 Hz, ArH), 3.84 and

3.75 (6H, 2 x s, 2 x ArOCH₃), 3.74 (2H, t, J =7.2 Hz, NCH₂), 1.51-

1.58 (2H, m, NCH₂CH₂), 1.15-1.21 (2H, m, NCH₂CH₂CH₂), 0.78

(3H, t, J = 7.2 Hz, NCH₂CH₂CH₂CH₃); ¹³C NMR: δ 161.5 and

158.4 (2 x ArCOCH₃), 137.1 (2-CH), 132.9 (ArC), 129.5 (5-C),

128.2 (4-CH), 111.7, 104.4, 98.7 (Ar-C), 55.4 and 55.3 (2 x

ArCOCH₃), 45.0 (NCH₂), 32.6 (NCH₂CH₂), 19.7 (NCH₂CH₂CH₂),

13.4 (NCH₂CH₂CH₂CH₃); FTIR ν_max/cm^-1(KBr): 3383,

2958,2875, 1722, 1678, 1616, 1578, 1459, 1416, 1364, 1305,

1283, 1208, 1160, 1132, 1030, 92, 1030, 922, 834, 660, 618;

HRMS (ESI-TOF) m/z: [M+H]⁺Calcd for C₁₅H₂₁N₂O₂261.1598,

found 261.1587.

5.3.3. Synthetic method E: Microwave-assisted two step-one pot

van Leusen reaction

In order to generate the N-arylidene alkylamine in situ, aryl

aldehyde (0.90 mmol) and an aliphatic amine (0.99 mmol) were

introduced into a 10 ml microwave reaction vessel equipped with

a magnetic stirrer. The resulting reaction mixture was then

microwave-irradiated at a set temperature of 60^oC for 4 min (N.B:

in cases where aniline derivatives were used, the reaction mixture

was irradiated for 1 h). The mixture was allowed to reach room

temperature and then TosMIC (1.35 mmol) and K₂CO₃(1.17

9

5.3.8. 5-(Benzo[d][1,3]dioxol-5-yl)-1-butyl-1H-imidazole (16e).

Yield: 43% (Method C), 48% (Method D), 55% (Method E) as

a brown oil; H NMR: δ 7.48 (1H, s, 2-CH), 6.94 (1H, s, 4-CH),

755, 696, 658, 536; HRMS (ESI-TOF) m/z: [M+H]⁺Calcd for

C₁₃H₁₆N₃O₂246.1237, found 246.1238.

1

5.3.13.

1-Cyclohexyl-5-(4-fluorophenyl)-1H-imidazole

6.77-6.84 (3H, m, ArH), 5.97 (2H, s, ArOCH₂O), 3.87 (2H, t, J =

7.2 Hz, NCH₂), 1.53-1.60 (2H, m, NCH₂CH₂), 1.15-1.24 (2H, m,

NCH₂CH₂CH₂), 0.79 (3H, t, J = 7.2 Hz, NCH₂CH₂CH₂CH₃); ¹³C

NMR: δ 147.7 and 147.4 (2 x ArCOCH₂O ), 137.6 (2-CH), 132.5

(5-C), 127.7 (4-CH), 123.7, 122.6, 109.2, 108.4 (ArC), 101.2

(ArCOCH₂O), 44.8 (NCH₂), 32.7 (NCH₂CH₂), 19.5

(NCH₂CH₂CH₂), 13.3 (NCH₂CH₂CH₂CH₃); FTIR ν_max/cm^-1(KBr):

3378, 2959, 2931, 2873, 1721, 1683, 1609, 1558, 1478, 1379,

1331, 1237, 1113, 1038, 933, 879, 812, 660; HRMS (ESI-TOF)

m/z: [M+H]⁺Calcd for C₁₇H₂₅N₂O₂245.1285, found 245.1276.

(17a).Yield: 46% (Method C), 51% (Method D) as a pale yellow

solid; Mp: 111-114^oC; ¹H NMR: δ 7.64 (1H, s, 2-CH), 7.27-7.7.30

(2H, m, ArH), 7.01-7.13 (2H, m, ArH), 6.69 (1H, s, 4-CH) 3.79-

3.87

(1H,

m,

NCH),

1.18-2.01

(10H,

m,

NCHCH₂CH₂CH₂CH₂CH₂); ¹³C NMR: δ 162.5 (d, ¹J_C,F= 247.6 Hz,

3

para-CF), 134.9 (2-CH), 131.4 (5-C), 130.8 (d, J_F,C= 8.1 Hz,

ortho-C), 127.6 (4-CH), 126.3 (d, ⁴J_F,C= 3.0 Hz, 1’-C), 115.6 (d,

²J_F,C=

21.1

Hz,

meta-C),

54.5

(NCH),

34.7

(NCHCH₂CH₂CH₂CH₂CH₂), 25.6 (NCHCH₂CH₂CH₂CH₂CH₂),

25.1 (NCHCH₂CH₂CH₂CH₂CH₂); FTIR ν_max/cm^-1(KBr): 3109,

3091, 3070, 2942, 2857, 1665, 1630, 1609, 1557, 1491, 1470,

1455, 1357, 1265, 814, 664, 604, 565, 504; HRMS (ESI-TOF)

m/z: [M+H]⁺Calcd for C₁₅H₁₈FN₂245.1449, found 245.1475.

5.3.9. 1-Butyl-5-(4-tert-butylphenyl)-1H-imidazole (16f). Yield:

1

44% (Method C), 52% (Method D) as a brown oil; H NMR δ:

7.55 (1H, s, 4-CH), 7.43 (2H, J = 8.4 Hz, ArH), 7.28 (2H, J = 8.4

Hz, ArH), 7.03 (1H, s, 4-CH), 3.93 (2H, t, J =7.2 Hz, NCH₂), 1.60-

1.67 (2H, m, NCH₂CH₂), 1.35 [9H, s, C(CH₃)₃], 1.22-1.30 (2H, m,

NCH₂CH₂CH₂), 0.83 (3H, t, J= 7.4 Hz, NCH₂CH₂CH₂CH₃); ¹³C

NMR: δ 151.0 [ArCC(CH₃)₃], 137.8 (2-CH), 132.9 (NCH₂), 128.5

(ArC), 127.8 (4-CH), 127.2, 125.9 (ArC), 45.0 (NCH₂), 34.6

[ArCC(CH₃)₃], 32.9 (NCH₂CH₂), 31.2, [ArCC(CH₃)₃], 19.7

(NCH₂CH₂CH₂), 13.5 (NCH₂CH₂CH₂CH₃); FTIR ν_max/cm^-1(KBr):

3373, 2960, 2931, 2865, 1717, 1682, 1614, 1556, 1463, 1363,

1269, 1220, 1114, 1033, 916, 839, 659, 571; HRMS (ESI-TOF)

m/z: [M+H]⁺Calcd for C₁₇H₂₅N₂257.2012, found 257.2008.

5.3.14. 1-Cyclohexyl-5-(2-chlorophenyl)-1H-imidazole (17b).

Yield: 23% (Method C), 29% (Method D) as a pale yellow solid;

Mp. 131-134^oC; ¹H NMR: δ 7.69 (1H, s, 2-CH), 7.28-7.49 (4H, m,

ArH), 6.97 (1H, s, 4-CH), 3.54-3.60 (1H, m, NCH), 1.17-2.01

(10H, m, NCHCH₂CH₂CH₂CH₂CH₂); ¹³C NMR: δ 135.1 (2-C),

134.6 (5-C), 132.9, 130.2, 129.7, 129.3 (ArC), 128.0 (4-CH),

126.8 (ArC), 55.1 (NCH), 34.5 (NCHCH₂CH₂CH₂CH₂CH₂)), 25.6

(NCHCH₂CH₂CH₂CH₂CH₂), 25.1 (NCHCH₂CH₂CH₂CH₂CH₂);

FTIR ν_max/cm^-1(KBr): 3095, 2934, 2855, 1704, 1661, 1566, 1477,

1453, 1272, 1231, 1113, 1074, 1034, 923, 916, 830, 754, 666;

HRMS (ESI-TOF) m/z: [M+H]⁺Calcd for C₁₅H₁₈ClN₂261.1153,

found 261.1155.

5.3.10.

1-Butyl-5-(2,3-dihydro-1,4-benzodioxin-6-yl)-1H-

imidazole (16g). Yield: 54% (Method C) as a brown oil; ¹H NMR:

δ 7.49 (1H, s, 2-CH), 6.96 (1H, s, 4-CH), 6.79-6.90 (3H, m, ArH),

4.27 (4H, s, ArOCH₂CH₂O), 3.89 (2H, t, J =7.2 Hz, NCH₂), 1.55-

1.63 (2H, m, NCH₂CH₂), 1.90-1.25 (2H, m, NCH₂CH₂CH₂), 0.81

(3H, t, J = 7.2 Hz, NCH₂CH₂CH₂CH₃); ¹³C NMR: δ 143.5 and

143.4 (ArCOCH₂CH₂O), 137.6 (2-CH), 132.4 (5-C), 127.7 (4-

CH), 123.3, 122.0, 117.7, 117.3 (ArC), 64.3 and 64.2

(ArCOCH₂CH₂O), 44.9 (NCH₂), 32.8 (NCH₂CH₂), 19.6

(NCH₂CH₂CH₂), 13.4 (NCH₂CH₂CH₂CH₃); FTIR ν_max/cm^-1(KBr):

3374, 2962, 2931, 2874, 1720, 1584, 1503, 1460, 1362, 1286,

1250, 1123, 1067, 921, 893, 815, 749, 658; HRMS (ESI-TOF)

m/z: [M+H]⁺Calcd for C₁₅H₁₉N₂O₂259.1441, found 259.1434.

5.3.15. 1-Cyclohexyl-5-(3-methoxyphenyl)-1H-imidazole (17c).

Yield: 47% (Method C), 53% (Method D) as a pale yellow oil;

¹H NMR: δ 7.64 (1H, s, 2-CH), 7.31-7.35 (1H, m, ArH), 7.00 (1H,

s, 4-CH), 6.86-6.92 (3H, m, ArH), 3.93-3.99 (1H, m, NCH), 3.78

(3H, s, ArOCH₃), 1.22-2.04 (10H, m, NCHCH₂CH₂CH₂CH₂CH₂);

¹³C NMR: δ 159.6 (ArCOCH₃), 135.0 (2-C), 132.4 (5-C), 131.6,

129.6, 127.5 (4-CH), 121.3, 114.7, 113.3 (ArC), 55.2 (ArCOCH₃),

54.5

(NCH),

34.8

(NCHCH₂CH₂CH₂CH₂CH₂),

25.6

(NCHCH₂CH₂CH₂CH₂CH₂), 25.1 (NCHCH₂CH₂CH₂CH₂CH₂);

FTIR ν_max/cm^-1(KBr): 2934, 2856, 1679, 1605, 1580, 1482, 1450,

1352, 1318, 1225, 1117, 1168, 1087, 1052, 1036, 998, 860, 844,

814, 699, 659; HRMS (ESI-TOF) m/z: [M+H]⁺Calcd for

C₁₆H₂₁N₂O 257.1648, found 257.1668.

5.3.11. 1-Butyl-5-(2-nitrophenyl)-1H-imidazole (16h). Yield:

15% (Method D) as a brown oil; ¹H NMR: δ 8.00 (1H, , t, J =

4 Hz, ArH), 7.57-7.68 (3H, m, 2-CH and ArH), 7.42 (1H, , t, J

= 7.2 Hz, ArH), 6.96 (1H, s, 4-CH), 3.71 (2H, t, J =7.2 Hz,

NCH₂), 1.51-1.57 (2H, m, NCH₂CH₂), 1.15-1.23 (2H, m,

NCH₂CH₂CH₂), 0.79 (3H, t, J = 7.2 Hz, NCH₂CH₂CH₂CH₃);

¹³C NMR: δ 149.5 (ArCNO₂), 138.0 (2-CH), 133.6 (5-C), 132.7

(ArC), 129.9 (4-CH), 128.6, 127.3, 124.6, 124.5 (ArC), 45.1

(NCH₂), 32.4 (NCH₂CH₂), 19.5 (NCH₂CH₂CH₂), 13.3

(NCH₂CH₂CH₂CH₃); FTIR ν_max/cm^-1(KBr): 3400, 3108, 2959,

2873, 1727, 1633, 1557, 1456, 1367, 1367, 1284, 1219, 1146,

1114, 1083, 1035, 916, 812, 768, 659, 537; HRMS (ESI-TOF)

m/z: [M+H]⁺Calcd for C₁₃H₁₆N₃O₂246.1237, found 246.1240.

5.3.16.

1-Cyclohexyl-5-(2,4-dimethoxyphenyl)-1H-imidazole

(17d). Yield: 64% (Method C), 74% (Method D),73% (Method

F) as a pale yellow solid; Mp: 99-102^oC; ¹H NMR: δ 7.64 (1H, s,

2-CH), 7.13 (1H, dd, J = 2.9 Hz, ArH), 6.89 (1H, s, 4-CH), 6.49-

6.51 (2H, t, J = 4.8 Hz, ArH), 3.85 and 3.75 (6H, 2 x s, 2 x

ArOCH₃), 3.55-3.62 (1H, m, NCH ), 1.52-2.00 (10H, m,

NCHCH₂CH₂CH₂CH₂CH₂); ¹³C NMR: δ 161.5 and 158.4 (2 x

ArCOCH₃), 134.5(2-C), 133.1 (5-C), 129.1 (ArC), 127.3 (4-CH),

111.7, 104.5, 98.7 (ArC), 55.4 and 55.2 (2 x ArCOCH₃), 54.9

(NCH),

34.7

(NCHCH₂CH₂CH₂CH₂CH₂),

25.8

(NCHCH₂CH₂CH₂CH₂CH₂), 25.3 (NCHCH₂CH₂CH₂CH₂CH₂);

FTIR ν_max/cm^-1(KBr): 3096, 3002, 2957, 2932, 2857, 1614, 1581,

1553, 1495, 1435, 1307, 1289, 1265, 1211, 1161, 1136, 1055,

1028, 925, 817, 796; HRMS (ESI-TOF) m/z: [M+H]⁺Calcd for

C₁₇H₂₃N₂O₂287.1754, found 287.1770.

5.3.12. 1-Butyl-5-(4-nitrophenyl)-1H-imidazole (16i). Yield: 13%

(Method D) as a brown oil; ¹H NMR: δ 8.28-8.31 (2H, m, ArH),

7.64 (1H, s, 2-CH), 7.54-7.56 (2H, m, ArH), 7.21 (1H, s, 4-CH),

4.02 (2H, t, J=7.4 Hz, NCH₂), 1.60-1.65 (2H, m, NCH₂CH₂), 1.21-

1.29 (2H, m, NCH₂CH₂CH₂), 0.83 (3H, t, J = 7.4 Hz,

NCH₂CH₂CH₂CH₃); ¹³C NMR: δ 147.03 (ArCNO₂), 139.8 (2-CH),

136.7 (5-C), 130.8, 130.2 (ArC), 128.6 (4-CH), 124.2 (ArC), 45.6

(NCH₂), 32.8 (NCH₂CH₂), 19.6 (NCH₂CH₂CH₂), 13.4

(NCH₂CH₂CH₂CH₃); FTIR ν_max/cm^-1(KBr):): 3388, 3103, 2960,

2873, 1682, 1599, 1516, 1456, 1368, 1221, 1110, 1023, 916, 854,

5.3.17. 5-(Benzo[d][1,3]dioxol-5-yl)-1-cyclohexyl-1H-imidazole

(17e). Yield: 35% (Method C), 54% (Method D), 50% (Method

E) as pale yellow solid; Mp: 85-88^oC; ¹H NMR: δ 7.58 (1H, s, 2-

CH), 6.90 (1H, s, 4-CH), 6.72-6.83 (3H, m, ArH), 5.96 (2H, s,

ArOCH₂O), 3.81-3.87 (1H, m, NCH), 1.18-1.98 (6H, m,

NCHCH₂CH₂CH₂CH₂CH₂); ¹³C NMR: δ 147.6 and 147.4

10

(ArCOCH₂O), 134.6 (2-C), 132.0 (5-C), 127.2 (4-CH), 123.8,

122.8, 109.5, 108.4 (ArC), 101.1 (ArCOCH₂O), 54.3 (NCH), 34.6

(NCHCH₂CH₂CH₂CH₂CH₂), 25.5 (NCHCH₂CH₂CH₂CH₂CH₂),

25.0 ( NCHCH₂CH₂CH₂CH₂CH₂); FTIR ν_max/cm^-1(KBr): 3126,

3100, 3070, 2944, 2894, 2848, 2783, 1670, 1556, 1501, 1475,

1445, 1265, 1232, 1118, 1099, 1042, 943, 862, 878, 808, 659, 626;

HRMS (ESI-TOF) m/z: [M+H]⁺Calcd for C₁₆H₁₉N₂O₂271.1481,

found 271.1464.

5.3.23.

1-Cyclopropyl-5-(2,4-dimethoxyphenyl)-1H-imidazole

1

(18d). Yield: 68% (Method D) as a brown oil; H NMR: δ 7.49

(1H, s, 2-CH), 7.10 (1H, d, J = 8.8 Hz, ArH), 6.87 (1H, s, 4-CH),

6.47 (2H, J = 6.8 Hz, ArH), 3.73 and 3.80 (6H, 2 x s, 2 xArOCH₃),

3.18-3.22 (1H, m, NCH), 0.66-0.70 (4H, m, NCH₂CH₂); ¹³C NMR:

δ 161.2 and 158.3 (2 x ArCOCH₃), 137.6 (2-CH), 132.0 (5-C),

131.0 (ArC), 127.9 (4-CH), 111.9, 104.1, 98.4 (ArC), 55.2 and

55.1 (2 x ArCOCH₃), 26.6 (NCH), 5.84 ( NCH₂CH₂); HRMS (ESI-

TOF) m/z: [M+H]⁺Calcd for C₁₄H₁₇N₂O₂245.1285, found

245.1290.

5.3.18. 1-Cyclohexyl-5-(4-tert-butylphenyl)-1H-imidazole (17f).

Yield: 29% (Method C), 50% (Method D) as a pale orange solid;

5.3.24. 1-Benzyl-5-(4-fluorophenyl)-1H-imidazole (19a). Yield:

26% (Method D) as a brown oil. ¹H NMR; δ 7.57 (1H, s, 2-CH),

7.21-7.34 (5H, m, ArH), 7.09 (1H, s, 4-CH), 6.97-7.05 (4H, m,

ArH), 5.10 (2H, s, NCH₂); ¹³C NMR: δ 162.8 (d, ¹J_C,F= 248.6 Hz,

para-CF), 138.6 (2-CH), 136.5 (5-C), 132.4 130.8, 130.7 (ArC),

1

Mp:113-116^oC; H NMR: δ 7.65 (1H, s, 2-CH), 7.44 (2H, d, J =

8.0 Hz, ArH), 7.25 (2H, d, J = 8.4 Hz, ArH), 6.99 (1H, s, 4-CH)

3.92-4.00

(2H,

m,

NCH),

1.20-2.07

[19H,

m,

NCHCH₂CH₂CH₂CH₂CH₂and C(CH₃)₃]; ¹³C NMR: δ 150.9

[ArCC(CH₃)_], 134.9 (2-CH), 132.5 (5-C), 128.7 (ArC), 127.3 (4-

CH), 125.6 (ArC), 54.4 (NCH), 34.9 (NCHCH₂CH₂CH₂CH₂CH₂),

4

128.8 (d, J_F,C= 3.0 Hz, 1’-C), 128.2 (4-CH) 128.0 (ArC), 126.5

(d, ³J_F,C= 8.1 Hz, ortho-C), 115.6 (d, ²J_F,C=21.1 Hz, meta-C), 48.6

(NCH₂); HRMS (ESI-TOF) m/z: [M+H]⁺Calcd for C₁₆H₁₄FN₂

253.1136, found 253.1138.

34.6

[ArCC(CH₃)₃],

31.6

[ArCC(CH₃)₃],

25.7

(NCHCH₂CH₂CH₂CH₂CH₂), 25.2 ( NCHCH₂CH₂CH₂CH₂CH₂);

FTIR ν_max/cm^-1(KBr): 3130, 3065, 3035, 2931, 2860, 1669, 1613,

1548, 1474, 1463, 1454, 1403, 1360, 1268, 1218, 1117, 993, 917,

956, 817, 808, 665, 579; HRMS (ESI-TOF) m/z: [M+H]⁺Calcd

for C₁₇H₂₇N₂283.2169, found 283.2190.

5.3.25. 1-Benzyl-5-(3-methoxyphenyl)-1H-imidazole (19c). Yield

24%; (Method D) as a brown oil; ¹H NMR: δ 7.58 (1H, s, 2-CH),

7.27-7.35 (4H, m, ArH), 6.87-7.15 (5H, m, ArH), 6.79 (1H, s, 4-

CH), 5.17 (2H, s, NCH₂) 3.69 (3H, s, ArOCH₃); ¹³C NMR: δ 159.6

(ArCOCH₃), 138.7 (2-CH), 136.8 (5-C), 133.3, 130.8 (ArC), 129.7

(4-CH), 128.9, 128.1, 127.9, 126.6, 121.2, 114.1, 113.9 (ArC),

55.1 (ArCOCH₃), 48.7 (NCH₂); HRMS (ESI-TOF) m/z: [M+H]⁺

Calcd for C₁₇H₁₇N₂O₂265.1335, Found 265.1329.

5.3.19. 1-Cyclohexyl-5-(2-nitrophenyl)-1H-imidazole (17h).

Yield: 12% (Method D) as a brown oil; ¹H NMR δ: 8.02 (1H,

s, 2-CH), 7.58-7.59 (3H, m, ArH), 7.41-7.42 (1H, m,

ArH), 6.92 (1H, s, 4-CH), 3.45-35.2 (1H, m, NCH), 1.21-1.18

- 2.02 (10H, m, NCHCH₂CH₂CH₂CH₂CH₂);¹³C NMR δ: 147.5,

135.1(2-CH), 134.6 (5-C), 133.0, 130.2, 129.3, (ArC), 128.0

5.3.26. 1-Benzyl-5-(2,4-dimethoxyphenyl)-1H-imidazole (19d).

Yield; 74% (Method D) as a brown oil; ¹H NMR: δ 7.55 (1H, s,

2-CH), 7.28-7.31 (3H, m, ArH), 7.03-7.12 (4H, m, ArH and 4-

CH), 6.50-6.53 (2H, m, ArH), 4.98 (2H, s, NCH₂), 3.86 and 3.71

(6H, 2 x s, 2 x ArOCH₃); ¹³C NMR; δ 161.5 and 158.3 (2 x

ArCOCH₃), 137.6 (2-C), 136.8 (5-C), 133.0, 130.0 (ArC), 128.5

(4-C), 128.2, 127.6 ,127.2, 111.1, 104.4, 98.6 (ArC), 55.3 and 55.2

(2 x ArCOCH₃), 48.8 (NCH₂); HRMS (ESI-TOF) m/z: [M+H]⁺

Calcd for C₁₈H₁₉N₂O₂295.1441, found 295.1408.

(4-CH),

126.8

(ArC),

55.1

(NCH),

34.5

25.6

(

(NCHCH₂CH₂CH₂CH₂CH₂),

25.1

NCHCH₂CH₂CH₂CH₂CH₂); HRMS (ESI-TOF) m/z: [M+H]⁺

Calcd for C₁₅H₁₈N₃O₂272.1394, found 272.1393.

5.3.20. 1-Cyclohexyl-5-(4-nitrophenyl)-1H-imidazole (17i).

Yield: 10% (Method D) as a brown oil; ¹H NMR: δ 8.30 (2H, d, J

=8.4 Hz, ArH), 7.77 (1H, s, 2-CH), 7.49 (2H, d, J = 8.8 Hz, ArH),

7.15 (1H, s, 4-CH), 3.92-3.98 (1H, m, NCH), 1.25-2.07 (10H, m,

NCHCH₂CH₂CH₂CH₂CH₂); ¹³C NMR: δ 147.2 (ArCNO₂), 136.7

(2-CH), 136.6 (5-C), 130.6 (ArC), 129.1 (4-CH), 124.2 (ArC),

5.3.27.

5-(Benzo[d][1,3]dioxol-5-yl)-1-benzyl-1H-imidazole

(19e). Yield 22% (Method D) as a pale yellow solid; Mp: 84-87^oC;

¹H NMR; δ 7.53 (1H, s, 2-CH), 7.27-7.33 (3H, m, ArH), 6.74-7.06

(5H, m, ArH), 6.72 (1H, s, 4-CH), 5.97 (2H, s, ArOCH₂O), 5.11

(2H, s, NCH₂); ¹³C NMR; δ 147.8 and 147.6 (ArCOCH₂O), 138.3

(2-CH), 136.7 (5-C), 133.1, 128.9 (ArC), 127.9, 126.6, 123.2,

122.9, 109.4, 108.5 (4-CH and ArC), 101.2 (ArCOCH₂O), 48.6

(NCH₂); HRMS (ESI-TOF) m/z: [M+H]⁺Calcd for C₁₇H₁₅N₂O₂

279.1128, found 279.1143.

55.5

(NCH),

34.8

(NCHCH₂CH₂CH₂CH₂CH₂),

25.6

(NCHCH₂CH₂CH₂CH₂CH₂), 25.0 ( NCHCH₂CH₂CH₂CH₂CH₂);

HRMS (ESI-TOF) m/z: [M+H]⁺Calcd for C₁₅H₁₈N₃O₂272.1394,

found 272.1382.

5.3.21. 1-Cyclopropyl-5-(4-fluorophenyl)-1H-imidazole (18a).

Yield: 23 % (Method D) as a brown oil; ¹H NMR: δ 7.58 (1H, s,

2-CH), 7.46-7.50 (2H, m, ArH), 7.09-7.13 (2H, m, ArH), 7.03 (1H,

s, 4-CH), 3.28-3.34 (H, m, NCH), 0.83-0.97 (4H, m,NCH₂CH₂);

¹³C NMR: δ 162.4 (¹J_F,C= 245.9 Hz, para-CF), 138.6 (2-CH),

5.3.28.

1-(4-Bromophenyl)-5-(4-fluorophenyl)-1H-imidazole

(20a). Yield: 68% (Method E) as pale yellow solid; Mp:130-

133^oC; H NMR: δ 7.67 (1H, s, 2-CH), 7.50-7.53 (2H, m, ArH),

1

7.23 (1H, s, 4-CH), 6.95-7.11 (6H, m, ArH);¹³C NMR: δ 162.3 (d,

J_C,F= 248.6 Hz, para-CF), 138.5 (2-CH), 135.4 (5-C), 132.7, 132.0

3

133.4 (5-C), 129.8 (d, J_F,C= 8.1 Hz, ortho-C), 127.6 (4-CH),

126.2(d, ⁴J_F,C= 3.0 Hz, 1’-C), 115.6 (d, ²J_F,C= 21.1 Hz, meta-C),

27.3 (6-C), 7.48 (NCH₂CH₂); HRMS (ESI-TOF) m/z: [M+H]⁺

Calcd for C₁₂H₁₂FN₂203.0974, found 203.0979.

3

(ArC), 130.0 (d, J_F,C= 8.1 Hz, ortho-C), 128.8, (ArC), 127.0 (4-

CH), 125.0 (d, ³J_C,F= 3.0 Hz, 1’-C), 122.1 (ArC), 115.7 (d, ²J_F,C

=

22.1 Hz, meta-C); HRMS (ESI-TOF) m/z: [M+H]⁺Calcd for

C₁₅H₁₁⁷⁹BrFN₂317.0090, found 317.0115.

5.3.22. 1-Cyclopropyl-5-(3-methoxyphenyl)-1H-imidazole (18c).

Yield: 40% (Method D) as a pale yellow oil; ¹H NMR: δ 7.58 (1H,

s, 2-CH), 7.31 (1H, t, J = 7.8 Hz, ArH), 6.88-7.13 (5H, overlapping

multiplets and singlet, ArH and 4-CH), 3.84 (3H, s, ArOCH₃),

3.33-3.39 (1H, m, NCH), 0.86-1.01 (4H, m, NCH₂CH₂); ¹³C NMR:

δ 159.6 (ArCOCH₃), 138.7 (2-CH), 134.2 (5-C), 131.3, 129.5

(ArC), 127.9 (4-CH), 120.5, 113.9, 112.9 (ArC), 55.1 (ArCOCH₃),

27.5 (NCH), 7.56 ( NCH₂CH₂); HRMS (ESI-TOF) m/z: [M+H]⁺

Calcd for C₁₃H₁₅N₂O 215.1179, found 215.1188.

5.3.29.

1-(4-Bromophenyl)-5-(2,4-dimethoxyphenyl)-1H-

imidazole (20d). Yield: 38% (Method E) as a pale yellow oil; ¹H

NMR: δ 7.70 (1H, s, 2-CH), 7.43-7.45 (2H, m, ArH), 7.13-7.18

(1H, d, J =12 Hz, ArH), 7.12 (1H, s, 4-CH), 6.98-7.00 (2H, m,

ArH), 6.31-6.50 (2H,m, ArH), 3.80 and 3.34 (3H, 2 x s, 2 x

ArOCH₃); ¹³C NMR: δ 161.5 and 157.5 (2 x ArCOCH₃), 137.2 (2-

CH), 136.8 (5-C), 134.5, 132.1, 132.0 (ArC), 129.8 (4-CH), 125.6,

120.9, 111.0, 104.6, 98.7 (ArC), 55.3 and 54.7 (2 x ArCOCH₃);

11

HRMS (ESI-TOF) m/z: [M+H]⁺Calcd for C₁₇H₁₆⁷⁹BrN₂O₂

359.0395, found 359.0394.

¹H NMR: δ 7.90-7.93 (1H, m, ArH), 7.85 (2H, d, J = 8.8 Hz, ArH),

7.52-7.69 (3H, m, ArH and 2-CH), 7.38 (2H, d, J = 8.8 Hz, ArH),

6.39 (1H, d, J = 7.2 Hz, ArH), 5.32 (1H, J = 6 Hz, 5-CH), 5.30

(1H, J = 26.0 Hz, 4-CH), 2.46 (3H, s, ArCH₃); ¹³C NMR: δ 159.0

(2-CH), 148.2, 145.9, 133.4, 132.9, 130.8, 130.4, 129.9, 129.7,

129.6, 125.2 (ArC), 91.8 (4-CH), 77.03 (5-C), 21.8 (ArCCH₃);

FTIR ν_max/cm^-1(KBr): 2922, 2868, 1702, 1672, 1599, 1524, 1347,

1317, 1224, 1133, 1036, 1011, 855, 815, 687, 569.

5.3.30.

5-(Benzo[d][1,3]dioxol-5-yl)-1-(4-bromophenyl)-1H-

imidazole (20e). Yield: 62% (Method E) as a pale yellow solid;

¹H NMR: 7.60 (2H, m, ArH), 7.18 (4-CH), 7.05-7.07 (2H, m,

ArH), 6.57-6.74 (3H, m, ArH), 5.92 (2H, s, ArOCH₂O);¹³C NMR:

δ 147.7 and 147.5 (ArCOCH₂O), 138.1 (2-CH), 135.4 (5-C), 132.7

ArC), 128.3 (4-CH), 127.0, 122.6, 122.4, 122.1, 108.7, 108.5,

(ArC), 101.3 (ArCOCH₂O); HRMS (ESI-TOF) m/z: [M+H]⁺

Calcd for C₁₆H₁₂⁷⁹BrN₂O₂343.0082, found 343.0099.

5.4.6. (4R,5R)/(4S,5S)-5-Phenyl-4-tosyl-4,5-dihydrooxazole (22j).

Yield: 76% (Method F) as a white solid; ¹H NMR: δ 7.84 (2H, d,

J= 8.8 Hz, ArH), 7.32-7.43 (7H, m, ArH), 7.21 (1H, d, J = 1.6 Hz,

, 2-CH), 6.05 (1H, d, J = 6.0 Hz, 5-CH), 5.03 (1H, dd, J = 2.6 Hz,

4-CH), 2.46 (3H, s, ArCH₃); ¹³C NMR: δ 159.4 (2-CH ), 145.7,

137.7, 133.2, 129.9, 129.5, 129.1, 129.0, 125.2 (ArC), 92.6 (4-

CH), 76.4 (5-CH), 21.7 (ArCCH₃); FTIR ν_max/cm^-1(KBr): 2894,

2875, 1622, 1316, 1304, 1148, 1102, 968, 814.

5.4 Synthesis of 5-aryl-1,3-oxazoles and 4,5-dihydro-1,3-

oxazoles (21-22)

5.4.1. Synthetic method F: Microwave assisted reaction in MeCN

To a 10 ml microwave reaction vessel equipped with a

magnetic stirrer was introduced aryl aldehyde (1.34 mmol),

TosMIC (1.47 mmol) and K₂CO₃(2.68 mmol). An inert

atmosphere was then created by degassing through evacuating and

refilling with argon three times, followed by introduction of

anhydrous MeCN (5 ml). The vessel was capped and resulting

mixture reaction was microwave irradiated at a set temperature of

90^oC and a power of 120 Watts for 12 min. Upon completion of

the reaction and conditions maintained until consumption of the

TosMIC reagent), the solvent was then evaporated and the crude

material was then stirred at 0^oC in a mixture containing MeOH (4

ml) and water (0.5 ml) until a precipitate formed. The precipitate

was collected, washed with cold water and dried under high

vacuum to give 5-aryl-4-tosyl-4,5-dihydro-1,3-oxazoles.

5.4.7. 5-(4-Fluorophenyl)-1,3-oxazole (21a). Yield: 84% (Method

H) as a brown oil. ¹H NMR: δ 7.89 (1H, s, 2-CH), 7.61-7.64 (2H,

m, ArH), 7.29 (1H, s, 4-CH), 7.10 (2H, t, J = 7.6 Hz, ArH); ¹³C

1

NMR: δ 162.3 (d, J_C,F= 249.6 Hz, para-CF), 150.7 (5-C), 150.4

(2-CH), 126.3 (d, ³J_C,F= 8.1 Hz, ortho-C), 124.0 (d, ⁴J_C,F= 5.0 Hz,

1-C), 121.1 (4-CH), 116.1 (22.1 Hz, meta-C); HRMS (ESI-TOF)

m/z: [M+H]⁺Calcd for C₉H₇FNO 164.0506, found 164.0508.

5.4.8. 5-(2-Chlorophenyl)-1,3-oxazole (21b). Yield: 74%

(Method H) as a pale yellow solid; Mp: 44-46^oC; ¹H NMR: δ 7.93

(1H, s, 2-CH), 7.42-7.78 (2H, m, ArH), 7.21-7.33 (3H, m, ArH),

NMR δ: 150.3 (2-CH), 148.0 (5-C), 130.7, 130.6, 129.1, 127.8,

127.0, 126.5 (ArC), 126.3 (4-CH ); HRMS (ESI-TOF) m/z:

[M+H]⁺Calcd for C₉H₇ClNO 180.0211, found 180.0245.

5.4.2. Synthetic method G: Aromatization in toluene

A mixture of 5-aryl-4-tosyl-4,5-dihydro-1,3-oxazole (1.06

mmol) in toluene (5 ml) was heated at reflux for 1 h. After

completion of the reaction, the mixture was concentrated in vacuo

and the crude material purified by column chromatography

[eluting with hexane and ethyl acetate (4:1)] to produce 5-aryl-1,3-

oxazoles.

5.4.9. 5-(3-Methoxyphenyl)-1,3-oxazole (21c). Yield: 60%

(Method H) as a pale yellow oil; ¹H NMR: δ 7.85 (1H, s, 2-CH),

7.55 (1H, dd, J = 4.8 Hz, 2 Hz, ArH), 7.21 (1H, s, 4-CH), 6.92 (2H,

d, J = 8.8 Hz, ArH), 3.81 (3H, s, ArOCH₃); ¹³C NMR; δ 159.8

(ArCOCH₃), 151.5 (5-C), 149.8 (2-CH), 125.8, 120.5, 119.8,

114.3 (ArC), 55.2 (ArCOCH₃); HRMS (ESI-TOF) m/z: [M+H]⁺

Calcd for C₁₀H₁₀NO₂176.0706, found 176.0736.

5.4.3. Synthetic method H Microwave assisted van Leusen in

MeOH

Aryl aldehyde (1.21 mmol), TosMIC (1.33 mmol) and K₂CO₃

(2.41 mmol) were placed in a 10 ml microwave reaction vessel

equipped with a magnetic stirrer. An inert atmosphere was then

created by degassing through evacuating and refilling with argon

three times, followed by addition of anhydrous MeOH (5 ml). The

resulting capped reaction mixture was then microwave irradiated

at a set temperature of 90^oC and a power of 120 Watts for 7 min.

Upon reaction completion (consumption of TosMIC), the solvent

was evaporated in vacuo. The crude material was purified by flash

column chromatography [elution with ethyl acetate and hexane

ratio (1:4)] to afford 5-aryl-1,3-oxazole derivatives.

5.4.10. 5-(2,4-Dimethoxyphenyl)-1,3-oxazole (21d). Yield: 44%

1

(Method H) as a pale yellow solid; Mp: 104-106^oC; H NMR: δ

7.85 (1H, s, 2-CH), 7.66 (1H, J = 8.8 Hz, ArH), 7.42 (1H, s, 4-

CH), 6.53-6.59 (2H, m, ArH), 3.83 and 3.92 (6H, 2 x s, 2 x

ArOCH₃); ¹³C NMR: δ 160.9 and 156.9 (2 x ArCOCH₃), 148.9 (2-

CH ), 148.0 (5-C), 126.9 (ArC), 123.5 (4-CH), 110.3, 104.9, 98.5

(ArC), 55.4 and 55.8 (2 x ArCOCH₃); HRMS (ESI-TOF) m/z:

[M+H]⁺Calcd for C₁₁H₁₂NO₃206.0812, found 206.0839.

5.4.11. 5-(Benzo[d][1,3]dioxol-5-yl)-1,3-oxazole (21e). Yield:

43% (Method H), 69% (Method G) as a yellow solid; Mp: 44-

46^oC (lit.^60-61); ¹H NMR: δ 7.86 (1H, s, 2-CH), 7.21 (1H, s, 4-CH),

7.10-7.18 (2H, m, ArH), 6.85 (1H, d, J = 7.6 Hz, ArH), 6.00

(ArOCH₂O); ¹³C NMR: δ 149.9 (2-CH), 148.0 (5-C), 148.2 (2 x

ArCOCH₂O), 121.9 (4-CH), 120.3, 118.6, 108.8, 105.0 (ArC),

101.4 (ArCOCH₂O); HRMS (ESI-TOF) m/z: [M+H]⁺Calcd for

C₁₀H₈NO₃190.0499, found 190.0528.

5.4.4. (4R,5R)/(4S,5S)-5-(Benzo[d][1,3]dioxol-5-yl))-4-tosyl-4,5-

dihydrooxazole (22e). Yield: 80% (Method F) as a white solid; ¹H

NMR: δ 7.83(2H, d, J = 8.8 Hz, ArH), 7.37(2H, d, J = 8.8 Hz,

ArH), 7.18 (1H, d, J = 1.2 Hz, 2-CH), 6.74-6.81 (3H, m, ArH), 597

(2H, s, ArOCH₂O), 5.94 (1H, d, J = 6.0 Hz, 5-CH), 4.99 (1H, dd,

J =4.4 Hz, 1.6 Hz, 4-CH), 2.45 (3H, s, ArCH₃); ¹³C NMR: δ 159.3

(2-CH ), 148.4 and 148.3 (2 x ArCOCH₂O), 145.7, 133.1, 131.4,

129.9, 129.5, 119.5, 108.6, 105.6 (ArC), 101.4 (ArCOCH₂O), 92.4

(4-CH), 79.4 (5-C), 21.7 (ArCCH₃); FTIR ν_max/cm^-1(KBr): 3314,

1675, 1596, 1503, 1447, 1303, 1257, 1141, 1062, 1039, 938, 816.

5.4.12. 5-(4-tert-Butylphenyl)-1,3-oxazole (21f). Yield: 59%

(Method H) as a sticky brown material; ¹H NMR: δ 7.90 (1H, s,

2-CH), 7.58-7.60 (2H, m, ArH), 7.44-7.44-7.47 (2H, m, ArH),

7.31 (1H, s, 4-CH) 1.34 [9H, s, ArC(CH₃)₃]; ¹³C NMR:δ 151.9 (2-

CH), 150.2 (5-C), 125.9, 125.0, 124.2 (ArC), 120.9 (4-CH), 34.7

[ArCC(CH₃)₃], 31.2, {ArC(CH₃)₃}; HRMS (ESI-TOF) m/z:

[M+H]⁺Calcd for C₁₃H₁₆NO 202.1242, found 202.1234.

5.4.5.

(4R,5R)/(4S,5S)-5-(2-Nitrophenyl)-4-tosyl-4,5-

dihydrooxazole (22h). Yield: 65% (Method F) as an orange solid;

12

5.4.13. 5-(2-Nitrophenyl)-1,3-oxazole (21i). Yield: 68% (Method

H), 63% (Method G) as a pale yellow solid; Mp: 70-72^oC (lit. ⁶¹);

¹H NMR:δ 7.97 (1H, s, 2-CH), 7.84-7.86 (1H, m, ArH), 7.64-7.72

(2H, m, ArH), 7.51-7.56 (1H, m, ArH), 7.39 (1H, s, 4-CH); ¹³C

NMR: δ 151.6 (2-CH), 147.5 (5-C), 146.5 (ArCNO₂), 132.5,

129.8, 129.6, 125.8, 124.4 (ArC), 121.6 (4-CH); HRMS (ESI-

TOF) m/z: [M+H]⁺Calcd for C₉H₇N₂O₃191.0451, found

191.0475.

a Corning® Costar® 96-Well Cell Culture Plates (Sigma Aldrich,

USA) for testing.^47-48The plate was placed into the incubator to

equilibrate to 37°C and 5 % CO₂. During this time, test compounds

were made up in in RPMI media containing 10 % heat inactivated

FCS (10% RPMI solution) in a serial dilution from 100 µM to 1.56

µM. A total of 100 µl compound was added to wells containing

cells and mixed to ensure the solution was homogeneous with the

cells. The plate was placed into the 37°C incubator for 5 days. On

the 5^thday 10 µl of MTS was added and mixed. The plates were

then incubated for a further 4 h, and read at 490 nm (xMARK^TM

,

5.4.14. 5-(4-Nitrophenyl)-1,-3-oxazole (21h). Yield: 73%

(Method H) as a yellow solid; Mp: 137-139 °C (lit. ^62-64), ¹H NMR:

δ 8.29-8.31 (2H, m, ArH), 8.02 (1H, s, 2-CH), 7.80-7.83 (2H, m,

ArH), 7.56 (1H, s, 4-CH); ¹³C NMR: δ 151.8 (2-CH), 149.5 (5-C),

147.4 (ArCNO₂), 133.4, 124.8, 124.7 (ArC), 124.5 (4-CH); HRMS

(ESI-TOF) m/z: [M+H]⁺Calcd for C₉H₇N₂O₃191.0451, found

191.0458.

Bio-Rad, USA.) The data analysis was completed on Origin 8.1

with the log value of the concentration plotted against the

absorbance level to determine a dose curve. From the curve, a half

maximal cytotoxicity CC₅₀for compounds was determined.

Controls used included auranofin, raltegravir and CX05168.

5.5.1.4. Antiviral assay ⁴⁷

MT-4 cells (obtained through the NIH AIDS Reagent Program,

Division of AIDS, NIAID, NIH: MT-4 catalogue number 120)

were seeded the day before antiviral testing at 3 x 105 cells per ml.

The following day the viability was checked and 2 x 105 cells per

ml were placed into a 50 ml conical tube and HIV-1NL4-3 stock

added. The cells were spinoculated at 3000 x g for 90 min. Cells

were subsequently washed four times with 0.01 M PBS to remove

any unbound virus. A control set of cells were spinoculated

without virus and washed four times with 0.01 M PBS to replicate

the test cells. A total of 10 ml 10 % RPMI media was then added

to the cells and 100 µl of cells were added to each well of a

Corning® Costar® 96-Well Cell Culture Plates (Sigma Aldrich,

USA). The plate was placed into the 37°C, 5 % CO₂incubator to

equilibrate for one hour. During the incubation compounds were

made up in RPMI media containing 10 % heat inactivated FCS.

The compounds were evaluated at 10 µM. A total of 100 µl of

compound solution was added to the wells containing cells and

mixed to ensure they were homogeneous. The plate was placed

into a 37°C, 5 % CO₂incubator for five days.

A Biomerieux Vironostika HIV-1 Ab/Ag micro ELISA system

was used to test for p24. All buffers were prepared according to

manufacturer’s specifications and the manufacturer’s protocol was

followed. Briefly, 145 μl of disruption buffer was added to each

well including control wells and incubated at 37°C for one hour.

A total volume of 5 μl of each test specimen was added to the

disruption buffer and control samples were added. The plate was

then incubated at 37°C for 60 minutes and wells were then washed

with 1x wash buffer for 30 seconds. Washing was repeated six

times. Once washed 100 μl of 3,3',5,5'-tetramethylbenzidine

(TMB) substrate mix (1:1 ratio of TMB substrate A and B) was

added and incubated for 5-30 minutes in the dark until sufficient

colour had developed. The reaction was stopped by the addition of

100 μl 1 M sulphuric acid. The plates were then read on a

multiplate reader at 450nm (xMARKTM, Bio-Rad, USA).

5.4.15. 5-Phenyl-1,3-oxazole (21j). Yield; 68% (Method G) as a

pale yellow solid; Mp: 38-41^oC (lit.⁶²); ¹H NMR: δ 7.91 (1H, s, 2-

CH), 7.64-7.67 (2H, m, ArH), 7.40-7.44 (2H, m, ArH), 7.32-7.35

(2H, m, 4-CH and ArH); ¹³C NMR: δ 151.5 (2-CH), 150.4 (5-C),

128.9, 128.7, 128.6, 127.7, 124.3 (ArC), 121.4 (4-CH); HRMS

(ESI-TOF) m/z: [M+H]⁺Calcd for C₉H₇NO 145.0528, found

145.0521.

5.5. Biological Evaluation Experiments

5.5.1. HIV-1 IN-LEDGF/p75 biological evaluations

5.5.1.1. HIV-1 IN-LEDGF/p75 AlphaScreen^TMAssay⁴⁵

For the HIV-1 IN-LEDGF/p75 AlphaScreen^TMassay, HIV-1

IN was incubated with 10 μM of each compound for 30 min at

26°C with slight shaking and subsequently 0.3 μM LEDGF/p75

was added and incubated for an hour. Nickel donor beads (Perkin

Elmer, USA) and Nickel acceptor beads (Perkin Elmer, USA)

were added to a final concentration of 10 μg/ml and incubated at

30°C in the dark with gentle shaking. Once incubation was

complete the plate was read between 520-620nm on the EnSpire^TM

plate reader (Perkin Elmer, USA). Controls included: HIV-1 IN

and LEDGF/p75 protein and CX05168 (6); which is a known HIV-

1 IN-LEDGF/p75 inhibitor that was used as the control compound

to validate the assay. Compounds displaying inhibition of above

50% benchmark were analyzed and were subsequently tested in

ten serially diluted doses ranging from 0.39μM to 200 μM to

determine an IC₅₀for each compound. This was carried out in

duplicate on the plate and in three separate experiments.

5.5.1.2 AlphaScreen TruHits counter assay protocols⁴⁶

The AlphaScreen Triuhits counter assay was performed according

to the manufacturer’s protocol (Perkin Elmer, Benelux). Reactions

were performed using the TruHits kit contains streptavidin donor

beads and biotinylated acceptor beads, which bind to each other

without any further reagents added. In total, 30 µL TruHits kit

bead premix was dispensed into each well of an optical microplate-

96 and incubated for 30 min at room temperature. Subsequent to

this, test compounds (final concentration of 10 µM) were

transferred and the mixture was incubated for 1 h at room

temperature. The assay microplates were read using the Enspire

multimode plate Reader (PerkinElmer). All data were processed

using Excel (Microsoft Corp) and visualized using origin 8.1

software.

5.5.2 Minimum inhibitory concentration (MIC) assay

5.5.2.1 Preparation of stock solution for antimicrobial testing

The stock solutions of the synthesized compounds used for the

minimum inhibitory concentration (MIC) assays were prepared by

dissolving the sample in 10% DMSO, followed by dilution with

sterile water in order to make a concentration of 1 mg/ml. The

concentration of 0.01 mg/ml of ciprofloxacin and 0.1 mg/ml

nystatin were also prepared and were used as positive controls for

bacteria and yeast respectively.

A

solution of p-

5.5.1.3. Cellular toxicity assay

iodonitrotetrazolium chloride (INT) (0.08 g) in 200 ml of sterile

water was prepared as the indicator

The day of cytotoxicity testing, MT-4 cells (obtained through the

NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH:

MT-4 catalogue number 120) were counted and seeded at 1 x 10⁶

cells per ml. A total of 100 µl of cells were added to each well of

13

5.5.2.2. Screening of synthesized compounds against pathogens

The synthesized compounds were assessed for their

antimicrobial effects against two Gram-positive bacterial species

(S. aureus ATCC 25923 and B. cereus ATCC 11779), two Gram-

negative bacterial species (E. coli ATCC 8739 and P. aeruginosa

ATCC 27858) and one yeast (C. tropicalis ATCC 750). Tryptone

Soya broth (100 μl) was added into each of the 96 well microtiter

plates, followed by the addition 100 μl of each tested compound

solution including positive and negative controls. Serial dilutions

were performed to provide the following nine concentrations: 250,

125, 62.5, 31.25, 15.6, 7.8, 3.91, 1.95, and 0.98 μg/ml or lower

when necessary. Prior to the addition of culture to the microtiter

plates, 1 ml of each culture was sub-cultured in 100 μl Tryptone

soya broth to obtain a 0.5 McFarland standard. A sub-cultured

volume of 100 μl was then added to all wells of microtiter plates,

which contained the compounds and broth. Each plate was sealed

with a sterile adhesive sealing film to avoid evaporation of test

samples, followed by incubation at 37°C for 24 h for bacteria

species and for 48 h for the yeast. After incubation, 40 μl INT was

added to each well of the microtiter plates.^49-51Each plate was read

to determine the MIC. The wells with clear or no visible microbial

growth at the lowest concentration was noted as the MIC. The

assessments were carried out in duplicate and triplicate if any

variability was noted.

After validation of the pharmacophore model using small

molecules which bind at the LEDGF/p75-binding site promoting

aberrant multimerization of the IN, a nine point hypothesis model

was then used as a query to screen the best six identified hits 16c,

16f, 17c, 17f, 20a and 20d.⁶⁶Prior to screening, hits were prepared

by the ligPrep protocol. A phase ligand screening protocol was

then utilized to position the best six hits onto a nine-point

pharmacophore model. The compounds were then scored and

ranked relative to active reference ligand 5 with respect to their

match ligand site, aligned score, vector score, volume score,

fitness score and phase screen score (see Table 5 in the

supplementary information).

Acknowledgments

We acknowledge with thanks financial support from the

National Research Foundation Professional Development

Program (NRF-PDP) of South Africa, Mintek and the University

of the Witwatersrand.

Supplementary Material

1

Supplementary information, containing the H and ¹³C NMR

spectra of all synthesized compounds and docking scores for active

compounds, is available.

5.6. Generation of Ligand-Based Pharmacophore Model

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Design, synthesis and biological evaluation of imidazole and oxazole fragments as

HIV-1 integrase-LEDGF/p75 disruptors and inhibitors of microbial pathogens

Thompho J. Rashamuse, Angela T. Harrison, Salerwe Mosebi, Sandy van Vuuren, E. Mabel Coyanis,* and Moira

L. Bode*

17

Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships that

could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered as potential

competing interests:

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Design, synthesis and biological evaluation of imidazole and oxazole fragments as HIV-1 integrase-LEDGF/p75 disruptors and inhibitors of microbial pathogens

DOI: 10.1016/j.bmc.2019.115210

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