Total synthesis and cytotoxicity of Leucetta alkaloids

Accepted Manuscript

Total Synthesis and Cytotoxicity of Leucetta Alkaloids

Panduka B. Koswatta, Sabha Kasiri, Jayanta Das, Arunoday Bhan, Heather M.

Lima, Beatriz Garcia-Barboza, Nicole N. Khatibi, Muhammed Yousufuddin,

Subhrangsu S. Mandal, Carl J. Lovely

PII:

S0968-0896(17)30097-4

DOI:

http://dx.doi.org/10.1016/j.bmc.2017.01.024

BMC 13503

Reference:

Bioorganic & Medicinal Chemistry

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Revised Date:

Accepted Date:

7 September 2016

12 January 2017

16 January 2017

Please cite this article as: Koswatta, P.B., Kasiri, S., Das, J., Bhan, A., Lima, H.M., Garcia-Barboza, B., Khatibi,

N.N., Yousufuddin, M., Mandal, S.S., Lovely, C.J., Total Synthesis and Cytotoxicity of Leucetta Alkaloids,

Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.01.024

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Total synthesis and cytotoxicity of Leucetta

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alkaloids

Panduka B. Koswatta, Sabha Kasiri, Jayanta Das, Arunoday Bhan, Heather M. Lima, Beatriz Garcia-Barboza, Nicole N. Khatibi, Muhammed

Yousufuddin, Subhrangsu S. Mandal and Carl J. Lovely

Department of Chemistry and Biochemistry, University of Texas at Arlington, Arlington, TX76019-0065, USA

Bioorganic & Medicinal Chemistry

Total Synthesis and Cytotoxicity of Leucetta Alkaloids

Panduka B. Koswatta, Sabha Kasiri, Jayanta Das, Arunoday Bhan, Heather M. Lima, Beatriz Garcia-Barboza, Nicole N. Khatibi, Muhammed Yousufuddin,

Subhrangsu S. Mandal and Carl J. Lovely

Department of Chemistry and Biochemistry, University of Texas at Arlington, Arlington, TX76019-0065, USA

ARTICLE INFO

ABSTRACT

Article history:

Received

Received in revised form

Accepted

The total synthesis of a number of representative natural products isolated from Leucetta and

Clathrina sponges containing a polysubstituted 2-aminoimidazole are described. These

syntheses take advantage of the site specific metallation reactions of 4,5-diiodoimidazoles

resulting in the syntheses of three different classes of Leucetta derived natural products. The

cytotoxicities of these natural products, along with several precursors in MCF7 cells were

determined through an MTT growth assay. For comparative purposes a series of

naphthimidazole-containing family members are included.

Available online

Keywords:

2-Aminoimidazole

Heterocycles

Metallation

Reduction

Naphthimidazole

1. Introduction

needed reliable synthetic strategies that ideally were short and

readily amenable to variation thus providing both the opportunity

The Leucetta and Clathrina families of marine sponges have

been investigated as sources of novel bioactive secondary

metabolites for several years.¹Broadly speaking, these sponges

produce either polyunsaturated fatty acid derivatives or a

growing family of 2-aminoimidazole containing alkaloids (e.g.,

1-7, Figure 1).²These alkaloids vary in complexity from the

simple monobenzyl substituted derivatives such as clathridine A

(1) to the highly condensed kealiiquinone (6)³or the highly

oxidized spiroleucettadine (7) (Figure 1).⁴In between these

extremes are the two isomeric dibenzyl substituted systems

exemplified by isonaamidine E (2)⁵or naamidine H (5).⁶The

vast majority of these natural products are described as

possessing “biological activity”, but rarely are either the assays

or the cell types the same from one isolation group to the next.⁷

Furthermore, it is often times difficult to obtain extremely pure

materials from natural isolates which influences both the

magnitude and in some cases the reliability of these data. Lastly,

the ability to conduct SAR studies is limited to derivatization of

the natural material, which often can only be obtained in limited

supply. To date, only the biological activity of naamidine A (3)

has been extensively investigated by the Ireland lab which has

demonstrated that it promotes caspase-dependent apoptosis in

cultured tumor cells.⁸An Australian team has reported a modest

exploration of the SAR of the naamine/naamidine A framework.⁹

More recently, the Looper lab¹⁰and our group¹¹have begun to

evaluate the biological activity of synthetic versions of various

other family members, specifically the naphthimidazole group.

Very recently Jiang and coworkers have applied our synthetic

methods to access the naamine framework to prepare derivatives

at the 2-amino group with activity against P-glycoprotein

mediated drug resistance in various cancer cell lines.¹²In order

to pursue a medicinal chemistry campaign of these molecules, we

to access natural products but also permitting the construction of

analogues around the imidazole core.

Several years ago, we began a synthetic program directed

towards the total synthesis of several of the more complex

members of the Leucetta alkaloids, specifically calcaridine A

(10)¹³and spirocalcaridines A (8) and (9),¹⁴using a broadly

15

bioinspired-synthetic strategy.^1a,

As a result we needed to

access the basic framework found in the naamine group of

alkaloids to perform a variety of biomimetic rearrangements. As

we considered our options, it became clear that any strategy

along these lines would provide an opportunity for the

preparation of a selection of different family members. In this

manuscript, we detail this strategy with the total synthesis of an

example from each major class of Leucetta alkaloids. In

addition, we describe the cytotoxicity of the synthesized natural

products and selected precursors using an MTT based cell

viability assay in MCF7 breast cancer cells, thereby obtaining

direct comparative biological data for several members of the

Leucetta alkaloids and their precursors for the first time.

Figure 2: Strategic approaches to the Leucetta alkaloids

Our initial target centered on the construction of the simple

trisubstituted system clathridine

A (1) and the related

preclathridine A (19) (Scheme 1).¹⁹Our synthesis began with the

preparation of 4,5-diiodo-1-methylimidazole (14);²⁰while this

material is commercially available through various sources, it can

be prepared easily and in large quantities through the iodination

of imidazole in basic solution and then N-methylation. No

chromatography is required for either step; simply washing with

solvent provides material of sufficient purity for further use. In

order to access clathridine A (1), the 4-iodoimidazole 15 was

required, this can be obtained by treatment of 14 with

ethylmagnesium bromide and quenching with water.²¹At this

stage we were in position to install the benzylic substituent.

Previous experience in our lab had demonstrated that this would

be difficult to achieve directly by using a benzyl halide with

electron rich imidazoles due to competitive N-alkylation^13aand

therefore we employed an aldehyde and reduced the

corresponding alcohol. In practice this involved metallation of

C4 of 15 by treatment with ethylmagnesium bromide followed by

treatment with piperanal, which delivered the doubly benzylic

alcohol 16 in excellent yield. Ionic reduction occurred smoothly

upon treatment of 16 with Et₃SiH in a mixture of TFA and

dichloromethane. At this stage the introduction of the 2-amino

group was necessary and this was accomplished through a two-

step sequence involving C2-metallation of 17 with n-BuLi and

trapping with TsN₃.²²The resulting 2-azido derivative 18 was

subjected to catalytic hydrogenation (Pd-C/H₂) affording

preclathridine A (19). Clathridine A (1’) was obtained by

introduction of the methyl parabanic acid fragment using a

literature procedure involving exposure of 19 to the silylated

parabanic acid derivative 20.²³The resulting synthetic material

had spectroscopic properties that nicely matched those reported

for the natural material. In addition, the synthetic natural product

was crystalline and so an X-ray crystal structure was obtained

which nicely illustrated the location of all of the substituents and

confirmed the structure of the natural product (Figure 3).

Interestingly, the crystalline material obtained was tautomeric to

the reported structure of clathridine A (1) (Figure 1) but this may

simply reflect crystal packing forces.

Figure 1: Characteristic members of the Leucetta alkaloids

2. Results and Discussion

Strategy: As noted above, we wished to develop an approach

to this group of molecules which was flexible, would readily

deliver analogs and above all would be scalable. Several general

strategies can be envisioned for the preparation of the

polysubstituted imidazoles, either through the intermediacy of –

substituted ketones (12→11), hydroamination of alkynes^{10b, 16}or

through the functionalization of pre-formed imidazoles

(13→11).¹⁷In each case, the strategies are executed to avoid

introducing the polar 2-amino substituent until the end of the

synthetic sequence. In this context we pursued the latter

approach, relying on halogen-Grignard exchange reactions of

4,5-diiodoimidazole derivatives and subsequent electrophilic

quench.¹⁸Based on literature reports, it was known that C5

would undergo exchange first, followed by C4, the remaining

unsubstituted position at C2 could then be deprotonated with n-

BuLi allowing the sequential and selective polyfunctionalization

of the heterocycle. The majority of examples described in the

literature prior to our work involved imidazoles with stabilizing

N-substituents, whereas our substrates by necessity would have

either methyl or benzyl groups which would not stabilize any

adjacent negative charge character to any appreciable extent. As

a consequence, when we initiated this effort it was uncertain

whether this technique could be extrapolated to such systems.

Figure 3: X-ray crystal structure of clathridine

With the synthesis of the two clathridine derivatives complete,

we turned our attention to the isonaamidine series.²⁴In this

subfamily, the imidazole nitrogen is substituted with a benzyl

moiety and thus the synthesis commenced with the 4,5-

diiodoimidazole by alkylation with p-methoxybenzyl chloride

resulting in the formation of 22 (Scheme 2). Treatment of 22

with ethylmagnesium bromide followed by quenching with water

provided the 4-iodoimidazole 23.²⁵

Exposure of 23 to

ethylmagnesium bromide and treatment of the metallated

imidazole with benzaldehyde 24 gave a mixture of the desired

alcohol 25 and the corresponding ketone. Treatment of this

unpurified mixture with NaBH₄reduced the ketone to the

alcohol, which upon purification delivered 73% of the required

product 25. Reduction of the doubly benzylic alcohol with

Et₃SiH and TFA provided 26. At this stage we were in a position

to introduce the C2-amine via metallation with n-BuLi and

trapping with TrisN₃, however there was some concern vis à vis

chemoselectivity as it is known that N-benzylimidazoles are

prone to deprotonation at the benzylic position.²⁶Fortunately,

when 26 was exposed to n-BuLi followed by reaction with

TrisN₃the 2-azido derivative 27 was obtained in good yield.

Reduction with Pd-C/H₂delivered isonaamine C (28) which upon

treatment with the silylated parabanic acid derivative 20 afforded

isonaamidine E (2) in good yield (Scheme 2).

Scheme 2: Total synthesis of isonaamine (28) and isonaamidine

E (2)

With approaches to examples of 4-substituted imidazoles

secured, we turned our attention to the 4,5-disubstituted

derivatives. Strategically, we anticipated that both benzyl

moieties could be installed sequentially through metallation and

trapping with the appropriate aldehydes. As initial targets, our

attention was directed to naamidine G (4),²⁷one of several

molecules isolated by Pietra and coworkers.²⁸The crude extract

containing these molecules was described as being cytotoxic, but

no details were described for the individual purified molecules

and thus access to synthetic versions would help address the

question of which of these several derivatives were responsible

for the cytotoxicity. The synthesis commenced from the

diiodoimidazole 14 which was subjected to transmetallation with

EtMgBr. Exposure of the resulting organometallic to p-

anisaldehyde delivered the corresponding alcohol 29; ionic

reduction of the doubly benzylic alcohol using Et₃SiH and

trifluoroacetic acid provides 30 (Scheme 3). In the context of our

calcaridine A (10) synthesis we had attempted to introduce the

second benzylic substituent by reaction of the Grignard derived

from 30 and a protected benzaldehyde derivative, but this was not

very efficient.¹³We were able to circumvent this problem by

formylating first and then using an aryl Grignard reagent.

Accordingly, formation of the Grignard reagent was

accomplished by treatment of 30 with EtMgBr and then reaction

with N-methyl formanilide (31) which delivered the aldehyde 32

Scheme 1: Total synthesis of preclathridine A (19) and

clathridine A (1‘)

in excellent yield.

Reaction of the aldehyde with p-

from the activation of the parabanic acid leads to competitive

ionization of the methyl ether and non-productive side reactions.

anisylmagnesium bromide provided the corresponding alcohol 33

in good yield. Ionic reduction of the benzylic alcohol 33 was

readily accomplished on exposure to Et₃SiH and TFA. The

synthesis naamidine G (5) was completed in short order by

metallation of imidazole 34 at C2 with n-BuLi and reaction of the

thus formed organolithium with TsN₃. The resulting azido

derivative 35 was hydrogenated with Pd-C to provide the 2-

amino derivative 36 which upon treatment with the activated

parabanic acid derivative 20 afforded naamidine G (4) (Scheme

3). Compound 36 has not been reported as a natural product, but

the desmethyl derivative (naamidine D) and the dimethyl

derivative (N,N-dimethylnaamidine D)²have been described.

Scheme 4: Total synthesis of 14‘-Methoxynaamidine

The last targets that we pursued were naamine G and

naamidine H as these derivatives were reported to possess

cytotoxicity against several cancer cell lines in the low

micromolar range.²⁹Our synthesis began from 14 and proceeded

through halogen-metal exchange and subsequent reaction with

the Bn-protected aldehyde 41 resulting in the formation of

alcohol 42. Attempts to reduce this alcohol under ionic

conditions were not especially efficient, delivering less than 30%

yield of the benzylic derivative 43. In addition to the required

product, the reductive debenzylation product 43 was obtained in

a similar yield. However, rather than searching for conditions to

optimize this reduction, we chose to introduce the second

benzylic substituent and then remove both hydroxyl groups

simultaneously.

We were able to introduce this second

substituent through similar chemistry to that employed above

wherein the imidazolyl aldehyde 45 was prepared first and then

reaction with p-anisylmagnesium bromide to deliver diol 46 as a

mixture of diastereomers. With the crude diol in hand it was

subjected to the standard conditions that had been successful

previous for the ionic reduction of related derivatives, however,

rather than reduction, the diol underwent an intramolecular

Friedel-Crafts reaction followed by elimination to provide the

naphthimidazole 47. Presumably, a carbocation is formed and

then this is trapped by the more electron rich aromatic system,

Scheme 3: Total synthesis of naamidine G (4)

We noted that alcohol 33 might serve as a precursor to the

C14-oxygenated

derivatives

exemplified

by

14-

methoxynaamidine G (40) (Scheme 4). Accordingly, alcohol 33

was converted to the methyl ether by treatment with TFA and

methanol followed by C2-azidation and reduction to give the

corresponding amine. Unfortunately, we were unable to obtain

the naamidine derivative in useful amounts as the introduction of

the parabanic acid fragment was not efficient using standard

literature methods for its introduction. Presumably, residual BSA

dehydration then affords 47.

The initial formation and

rearrangement of an ipso addition adduct cannot be ruled out.

Indeed, in recently published work we have evidence of such

rearrangements resulting in the formation of naphthimidazoles.¹⁴

While clearly in the context of our targets this outcome was not

useful, the framework produced in this reaction maps very nicely

onto other members of the Leucetta alkaloids, most notably the

kealiinines and kealiiquinones. Indeed, we have used related

chemistry to access both families of natural products.^{11, 15}

4,5-diiodoimidazole was used as a starting material. Sequential

treatment of 49 with EtMgBr then CuCN.2LiCl provided the

corresponding cuprate which was then reacted with p-

methoxybenzyl bromide to provide 50 in 65% yield. Essentially

the same sequence of reactions was used to obtain the dibenzyl

substituted imidazole this time using benzyl bromide 48 as the

electrophile. At this stage, we chose to perform the protecting

group exchange sequence by first converting 51 to the methyl

imidazolium salt 52 by treatment with methyl triflate.

Subsequent reaction with benzylamine in acetonitrile at reflux

provided the N-methyl substituted derivative 53 in 90% yield for

the two step sequence. At this point all that remained to be done

was to introduce the 2-amino group and the parabanic acid

moiety. Interestingly, it was found that metalation at C2

followed by treatment with TsN₃resulted in sulfonylation, but

substitution with TrisN₃delivered the 2-azidoimidazole 55.

Catalytic hydrogenation resulted in reduction of the azido moiety

to the corresponding amine and hydrogenolysis of the benzyl

protecting group resulting in the synthesis of naamine G (56) in

excellent yield. Treatment with activated N-methyl parabanic

acid then delivered naamidine H (5) in good yield. The

spectroscopic data for both synthetic naamine G and synthetic

naamidine H were in good agreement with that obtained for the

isolated materials.

Scheme 5: First generation approach to naamidine H (5)

Given the propensity of this electron-rich system to undergo

cyclization rather than reduction chemistry, we needed to develop

an approach to circumvent this problem. An obvious way to

accomplish this task was to avoid the generation of these

benzylic carbocations. One way that this can be achieved is to

use benzyl halides as electrophiles rather than aldehydes. We

attempted to execute such a strategy with N-methylimidazole

derivatives, e.g., 14+4843. Unfortunately this chemistry was

not successful and led to the formation of monoiodinated

imidazolium salts On the other hand, Lindell has demonstrated

that C-alkylation can be feasible using dimethylsulfamoyl

(DMAS)-protecting group via the corresponding organocuprate.

While this was encouraging, this meant that an exchange of

imidazole N-substituents was necessary and that this had to be

accomplished in a position selective manner. Fortunately,

Robiette,³⁰Sames³¹and more recently Knochel^17b

have

demonstrated that the DMAS-protecting group can be removed

with a methyl group via the intermediacy of an imidazolium salt

formed by reaction with methyl triflate or Meerwein’s salt. Given

this encouraging precedent, the corresponding DMAS-protected

Scheme 6: Second generation total syntheses of naamine G (56)

and naamidine G (5)

With this group of natural products in hand they were

subjected to cytotoxicity testing using an MTT assay with MCF7

cells; the outcomes of these experiments are reported in Table 1

(entries 1-10). In addition to the naamine/naamidine group of

compounds, we also include the naphthimidazole based family

that we have reported previously for comparative purposes

(entries 12-23, Table 1).¹¹Generally speaking for the synthetic

versions of the natural products, the systems substituted with N-

methyl parabanic acid are substantially more active than the

corresponding 2-amino derivatives or desamino derivatives.

Interestingly, there appears to be broad tolerance with respect to

the identity and location of the other substituents around the

imidazole moiety. In cases where cytotoxicity has been

determined on material isolated from natural sources, our

synthetic materials display similar values – typically IC₅₀< 10

µM. One notable exception was with isonaamine C (28) (entry

4), for which the naturally occurring material was reported to

have an IC₅₀value of 2-5 µM, although this was obtained in

different cell lines, whereas our synthetic material had an IC₅₀

>100 µM. Our data are consistent with respect to the naamine vs

naamidine activities, with the latter being more active. In the

cases of the naphthimidazole derivatives, the cytotoxicities were

not determined on natural materials, only on the synthetic

32

materials.^11,

In general terms the presence of a C2-amino

substituent increases their cytotoxicity as evidenced by the

kealiinines, but the presence of quinone in the C-ring as found in

kealiiquinone (6) and 2-deoxy-2-aminokealiiquinone (68)

appears to reduce activity. Interestingly, kealiinine C (63) is

almost devoid of activity, whereas the isomeric congener 65

(entry 19) is about equipotent with the other two family members

59 and 61 (entries 13 and 15). At one point in our synthetic

studies we were concerned that the structure of kealiinine C had

been misassigned, however, HPLC studies suggested that the

original assignment does in fact appear to be correct.^{11, 32a}

Table 1. Cytotoxicity data for several Leucetta-derived alkaloids and analogs in MCF7 cells using an MTT growth assay.

Entry

Compound

IC50/μMol Entry

Compound

Kealiinine A (59)

60

IC50/μMol

1

>100

13

14

15

16

23.41.9

17

2

3

4

>100

28.01.3

28.81.2

99.80.9

Preclathridine A (19)

5.780.04

Clathridine A (1)

Kealiinine B (61)

>100

62

Isonaamine C (28)

5

17

>100

4.40.2

Kealiinine C (63)

Isonaamidine E (2)

6

18

19.51.5

68.21.6

36

64

7

19

5.080.05

22.71.0

Naamidine G (4)

Isokealiinine C (65)

8

20

16.30.4

29.90.3

39

2-Deamino-2-oxokealiinine C (66)

9

21

76.90.97

41.10.1

2-Deoxykealiiquinone (67)

Naamine G (56)

10

4.940.12

22

91.90.5

Kealiiquinone (6)

Naamidine H (5)

11

23

92.02.2

43.80.4

57

2-Deoxy-2-aminokealiiquinone (68)

12

>100

58

Conclusion

this was followed by ionic reduction to deliver the net C-

alkylated product. This strategy worked well for the production a

number of representative members of the Leucetta alkaloids,

however, in at least one case the ionic reduction chemistry was

Our initial approach to these molecules relied on the formation

of Grignards by in situ metal-halogen exchange reactions

followed by trapping with carbonyl derivatives. In most cases

compromised by the presence of an electron rich aromatic ring

that led to an unanticipated Friedel-Craft’s pathway and the

formation of a naphthimidazole. To circumvent this pathway, an

electron poor imidazole derivative was alkylated at carbon

thereby avoiding the non-productive cyclization pathway and

ultimately delivering naamidine H. This latter strategy, while

requiring the use of an N-protecting group, results in an overall

shorter synthetic sequence and would appear to represent the best

approach to this family of natural products. In general terms,

synthetic variants of the natural products containing the methyl

parabanic acid moiety exhibited higher cytotoxicities.

NaHCO₃solution until effervescence ceased. The resulting

mixture was extracted with CH₂Cl₂several times and the

combined extracts were dried (Na₂SO₄) and concentrated. The

residue was purified by chromatography (acetone/EtOAc, 3/7) to

isolate a pale yellow semi-solid: 17 (3.55 g, 72%); ¹H NMR: δ =

7.27 (s, 1H), 6.71 (s, 1H), 6.67 (s, 2H), 6.46 (s, 1H), 5.82 (s, 2H),

3.76 (s, 2H), 3.52 (s, 3H); ¹³C NMR: δ = 147.6, 145.8, 142.8,

137.4, 134.4, 121.6, 117.0, 109.4, 108.2, 101.0, 34.7, 33.2; IR

(neat, cm^-1): = 3367, 2900, 2778, 1492, 1441, 1246, 1185, 1038,

928, 815, 773; HR-ESIMS (m/z): Calcd. for C₁₂H₁₃N₂O₂[M+H]⁺

217.0972, found 217.0991.

2-Azido-4-(benzo[1,3]dioxol-5-yl)methyl-1-methyl-1H-

imidazole (18): n-Butyl lithium (1.4 M solution in hexanes,

11.34 mL, 15.9 mmol) was added dropwise to a solution of 17

(3.12 g, 14.4 mmol) in THF at -78 °C and TsN₃(3.41 g, 17.3

mmol) were used to afford the crude product, which was purified

by chromatography (15% → 20% EtOAc in hexanes) to isolate

Experimental

Chemicals were used as received unless indicated otherwise.

NMR spectra were obtained at either 500 MHz (for H NMR

1

spectra) and 125 MHz (for ¹³C NMR spectra) in CDCl₃unless

indicated otherwise. Infrared (IR) spectra were obtained on neat

samples (ATR spectroscopy) or using either KBr pellets for

solids or neat films on NaCl plates for liquids (transmission).

Analytical thin-layer chromatography (TLC) was performed on

silica gel plates, 200 mesh with F254 indicator.

Visualization was accomplished by UV light (254 nm), and/or a

10% ethanol solution of KMnO₄. Flash column chromatography

was performed with 230−400 silica gel. High resolution mass

spectra (HRMS) were recorded by electrospray ionization (ESI-

TOF) or atmospheric-pressure chemical ionization (APCI-TOF)

unless otherwise indicated.

1

18 (2.93 g, 78%) as a reddish brown oil: H NMR: δ = 6.76 (s,

1H), 6.72 (s, 2H), 6.24 (s, 1H), 5.90 (s, 2H), 3.74 (s, 2H), 3.31 (s,

3H); ¹³C NMR: δ = 147.7, 146.0, 140.2, 140.0, 133.6, 121.7,

116.1, 109.5, 108.2, 100.9, 34.8, 31.5; IR (neat, cm^-1): = 2896,

2154, 2125, 1494, 1441, 1245, 1039, 929, 812; HR-ESIMS (m/z):

Calcd. for C₁₂H₁₂N₅O₂[M+H]⁺258.0986, found 258.0987.

2-Amino-4-(benzo[1,3]dioxol-5-yl)methyl-1-methyl-1H-

imidazole (Preclathridine A) (19): Azide 18 (2.80 g, 10.9

mmol) was dissolved in EtOH (30 mL) and stirred under a

hydrogen atmosphere (1 atm) in the presence of 10% Pd-C on

charcoal (0.30 g) at rt overnight to afford 19 (2.59 g, quant) as a

yellow solid: m.p. = 116-118 °C; ¹H NMR (CDCl₃): δ = 6.73 (s,

1H), 6.69 (s, 2H), 6.08 (s, 1H), 5.86 (s, 2H), 4.14 (br, 2H), 3.63

(s, 2H), 3.26 (s, 3H); ¹³C NMR δ = 148.0, 147.5, 145.8, 137.1,

134.5, 121.7, 112.8, 109.5, 108.1, 100.8, 34.7, 31.3; IR (KBr, cm^-

¹): = 3432, 3299, 3109, 1643, 1547, 1505, 1442, 1244, 1034, 921,

814; HR-ESIMS (m/z): Calcd. for C₁₂H₁₄N₃O₂[M+H]⁺232.1081,

found 232.1094.

4-Iodo-1-methyl-1H-imidazole (15):

EtMgBr (3.0 M

solution in ether, 11.00 mL, 32.9 mmol) was added to a solution

of 14 (10.00 g, 29.9 mmol) in dry CH₂Cl₂(150 mL) at rt. over

~15 min. The resulting mixture was stirred for 20 min and water

(20 mL) was added to the reaction mixture after running a TLC

experiment to confirm all the starting material is consumed.

Then, the layers were separated. The aqueous layer was extracted

with CH₂Cl₂(3x50 mL) and the combined organic layers were

dried (Na₂SO₄) and concentrated to isolate 15 as pale yellow oil

(5.86 g, 94%).

2-(3-Methylimidazolidine-2,4-dione)imino-4-(4-

benzo[1,3]dioxol-5-ylmethyl-1-methyl-1H-imidazole

(Clathridine A) (1’): N,O-Bis(trimethysilyl)acetamide (5.18 mL,

21.2 mmol) was added to a solution of 1-methylparabanic acid

(2.26 g, 17.7 mmol) in dry CH₃CN (30 mL) under an N₂

atmosphere and the resulting mixture was heated at reflux

temperature for 1.5 h. The solvent was removed by vacuum

distillation, and to the resulting yellow residue 20 was dissolved

in dry toluene (5 mL) and then added amine 19 (817 mg, 3.5

mmol) under N₂atmosphere. After, this mixture was heated at 85

ºC overnight (product starts to form after 10 min as a yellow

solid at the bottom of the flask), the product was filtered and

solid was washed with methanol (10 mL) to deliver 1’ as a

yellow solid (730 mg, 61%): m.p. = 253-254 ºC (lit.⁷m.p. = 260-

262 ºC); ¹H NMR: δ = 6.75 (d, J = 8.3 Hz, 1H), 6.71 (s, 1H), 6.70

(d, J = 8.3 Hz, 1H), 6.52 (s, 1H), 5.93 (s, 2H), 3.80 (s, 2H), 3.71

(s, 3H), 3.18 (s, 3H); ¹³C NMR: δ = 162.0, 155.0, 147.8, 147.1,

146.3, 144.3, 139.7, 132.7, 121.7, 117.7, 109.3, 108.4, 101.0,

34.5, 32.2, 24.8; IR (KBr, cm^-1): = 3204, 3122, 2914, 1789, 1738,

1665, 1444, 1391, 1323, 1249, 1209, 1114, 1034, 925, 732; HR-

ESIMS (m/z): Calcd. for C₁₆H₁₆N₅O₄[M+H]⁺342.1197, found

342.1201.

4-(Benzo[1,3]dioxol-5-yl)hydroxymethyl-1-methyl-1H-

imidazole (16): EtMgBr (3.0 M solution in ether, 9.76 mL, 29.3

mmol) was added to a solution of 15 (5.80 g, 27.9 mmol) in dry

THF (100 mL) at 0 ºC over ~10 min. The resulting mixture was

stirred at rt. for 30 min and piperonal (4.65 mL, 30.7 mmol) was

added to the reaction slowly at rt. After stirring overnight at rt.,

satd. aq. NH₄Cl (20 mL) was added to the reaction and the layers

were separated. The aqueous layer was extracted with EtOAc (3

x 50 mL) and the combined organic extracts were dried (Na₂SO₄)

and concentrated to provide the crude product, which was

purified by flash column chromatography on silica gel (100%

EtOAc → 100% Acetone) to isolate 16 (5.82 g, 90%) as an

1

orange semi-solid: H NMR: δ = 7.35 (s, 1H), 6.90 (s, 1H), 6.87

(d, J = 7.8 Hz, 1H), 6.72 (d, J = 7.8 Hz, 1H), 6.45 (s, 1H), 5.88

(s, 2H), 5.66 (s, 1H), 5.27 (br, 1H), 3.54 (s, 3H); ¹³C NMR: δ =

147.5, 146.8, 146.0, 137.8, 137.4, 120.1, 117.2, 107.9, 107.4,

101.0, 70.0, 33.5; IR (neat, cm^-1): = 3114, 2891, 1493, 1442,

1243, 1037, 927, 794, 756; HR-ESIMS (m/z): Calcd. for

C₁₂H₁₃N₂O₃[M+H]⁺233.0921, found 233.0925.

4,5-Diiodo-1-(4-methoxybenzyl)-1H-imidazole (22): 4,5-

Diiodo-1H-imidazole 21 (0.50 g, 1.6 mmol) in THF ( 15 mL)

was cooled to 0 °C and NaH (60% suspension in mineral oil)

(0.10 g, 2.5 mmol) was added slowly. The reaction was allowed

to come to rt and stir under N₂for 30 min. 4-Methoxybenzyl

chloride (0.28 mL, 2.0 mmol) was added and stirred at 50 °C for

18 h. The reaction was allowed to cool to rt, quenched with water

4-(Benzo[1,3]dioxol-5-yl)methyl-1-methyl-1H-imidazole

(17): TFA (7.00 mL, 91.4 mmol) and Et₃SiH (17.00 mL, 106.4

mmol were added to a solution of 16 (5.31 g, 22.9 mmol) in dry

CH₂Cl₂(150 mL) at rt. The resulting mixture was stirred for 48 h;

the reaction mixture was then quenched by the addition of sat.

(10 mL) and extracted with EtOAc (2 x 10 mL). The combined

organic solutions were washed with water (10 mL), brine (10

mL), dried (anhyd. Na₂SO₄) and concentrated. The resulting

crude residue was purified by column chromatography (4:6

EtOAc/Hexane) to give 22 as a yellow solid (0.63 g, 91%). m.p.

(d, J = 1.7 Hz, 1H), 6.78 (d, J = 1.7 Hz, 1H), 6.77 (s, 1H), 6.49

(d, J = 1.2 Hz, 1H), 4.93 (s, 2H), 3.83 (s, 5H), 3.81 (s, 3H), 3.78

(s, 3H); ¹³C NMR: δ = 159.6, 148.8, 147.4, 143.2, 136.8, 133.1,

128.9, 128.3, 120.8, 116.0, 114.3, 112.2, 111.2, 56.0, 55.8, 55.4,

50.4, 34.8; IR (cm^-1): 2998, 2933, 2834, 1611, 1510, 1463, 1246,

1175, 1137, 1025, 818, 764, 686; HR-ESIMS (m/z): Calcd. for

[M+H]⁺C₂₀H₂₃N₂O₃is 339.1703 found 339.1718.

1

= 158-160 °C; H NMR: δ = 7.55 (s, 1H), 7.10 (d, J = 8.6 Hz,

2H), 6.88 (d, J = 8.6 Hz, 2H), 5.07 (s, 2H), 3.80 (s, 3H); ¹³C

NMR: δ = 159.9, 141.2, 129.2, 126.7, 114.5, 96.1, 82.4, 55.4,

53.1; IR (cm-1): 3099, 2929, 2832, 1611, 1510, 1433, 1298,

1241, 1174, 1024, 816, 760, 629; HR-ESIMS (m/z): Calcd. for

[M+H]⁺C₁₁H₁₁N₂OI₂is 440.8955 found 440.8956.

2-Azido-4-(3,4-dimethoxybenzyl)-1-(4-methoxybenzyl)-1H-

imidazole (27): Imidazole 26 (0.10 g, 0.30 mmol) in THF (15

mL) was cooled to -78 °C under N₂. n-BuLi (1.6 M in hexane)

(0.22 mL, 0.36 mmol) was added and stirred at -78 °C for 7 min.

TrisN₃(0.11 g, 0.36 mmol) was added in one portion and then the

mixture was stirred at -78 °C for 30 min. The reaction was

quenched by the addition of water (15 mL) and extracted with

EtOAc (2 x 10 mL). The combined organic extracts were washed

with brine, dried (anhyd. Na₂SO₄), and concentrated. The

resulting residue was purified by column chromatography (35%

4-Iodo-1-(4-methoxybenzyl)-1H-imidazole

(23):^25b

Diiodoimidazole 22 (16.2 g, 36.8 mmol) in CH₂Cl₂(250 mL) was

cooled to 0 °C under N₂and EtMgBr (3.0 M in Et₂O) (12.3 mL,

36.8 mmol) was added slowly. The reaction mixture was allowed

to come to rt and stirred for 1.5 h. Water (5 mL) was slowly

added and then the mixture was stirred at rt for an additional 1 h.

The reaction was partitioned with water and the organic layer

separated. The aqueous layer was extracted with CH₂Cl₂(2 x 100

mL). The combined organic extracts were washed with brine,

dried (anhyd. Na₂SO₄), and concentrated. The resulting residue

was purified by column chromatography (1:1 EtOAc/Hexane) to

1

EtOAc in hexane) to give 27 as a yellow oil (0.072 g, 65%). H

NMR: δ = 7.08 (d, J = 8.6 Hz, 2H), 6.84 (d, J = 8.6 Hz, 2H), 6.80

(s, 1H), 6.78 (m, 2H), 6.24 (s, 1H), 4.72 (s, 2H), 3.85 (s, 3H),

3.83 (s, 3H), 3.78 (s, 3H), 3.77 (s, 2H); ¹³C NMR: δ = 159.5,

148.8, 147.5, 140.7, 139.6, 132.2, 128.9, 128.2, 120.8, 114.9,

114.3, 112.3, 111.2, 56.0, 55.9, 55.4, 48.2, 34.8; IR (cm^-1): 2934,

2907, 2128, 1734, 1611, 1512, 1244, 1175, 1137, 1027; HR-

ESIMS (m/z): Calcd. for [M+H]⁺C₂₀H₂₂N₅O₃is 380.1717 found

380.1727.

1

give 23 as a pale yellow solid (9.6 g, 83%). m.p. = 49-51 °C; H

NMR (CDCl₃+DMSO-d₆): δ = 7.37 (d, J = 1.7 Hz, 1H), 7.08, (d,

J = 8.6 Hz, 2H), 6.91 (d, J = 1.7 Hz, 1H), 6.85 (d, J = 8.6 Hz,

2H), 4.97 (s, 2H), 3.76 (s, 3H); ¹³C NMR (CDCl₃+DMSO-d₆): δ

= 159.9, 138.7, 129.2, 127.2, 124.7, 114.5, 81.9, 55.4, 50.8; IR

(cm^-1): 3127, 3005, 2959, 2836, 1612, 1512, 1441, 1244, 1223,

1179, 1095, 1033, 934, 837, 813, 760, 619.

2-Amino-4-(3,4-dimethoxybenzyl)-1-(4-methoxybenzyl)-

1H-imidazole (28): Isonaamine C 10% Pd/C (0.020 g, 0.018

mmol) was added to azide 26 (0.069 g, 0.18 mmol) in MeOH (5

mL) and stirred under H₂(1 atm) at rt for 20 h. The catalyst was

filtered through Celite and washed with CH₂Cl₂. The filtrate was

reduced and the resulting residue purified by column

chromatography using Biotage KP-NH Silica (100% EtOAc to

1% MeOH in EtOAc) to give 28 as a yellow solid (0.055 g,

86%). m.p. = 103-106 °C; ¹H NMR: δ = 7.06 (d, J = 8.6 Hz, 2H),

6.85 (d, J = 8.6 Hz, 2H), 6.80 (d, J = 2.0 Hz, 1H), 6.79 (d, J = 2.0

Hz, 1H), 6.78 (s, 1H), 6.16 (s, 1H), 4.74 (s, 2H), 3.83 (s, 3H),

3.83 (s, 3H), 3.78 (s, 3H), 3.70 (s, 2H); ¹³C NMR: δ = 159.4,

148.8, 147.5, 147.4, 137.4, 132.9, 128.3, 128.1, 120.8, 114.5,

112.5, 112.3, 111.2, 56.0, 55.9, 55.4, 48.2, 34.6; IR (cm^-1): 3367,

3075, 2997, 2939, 2838, 1612, 1587, 1512, 1442, 1232, 1151,

1024, 803; HR-ESIMS (m/z): Calcd. for [M+H]⁺C₂₀H₂₄N₃O₃is

354.1812, found 354.1824.

4-[Hydroxy-(3,4-dimethoxyphenyl)]methyl-1-(4-

methoxybenzyl)-1H-imidazole (25): Iodoimidazole 23 (1.0 g,

3.2 mmol) in THF (15 mL) was added EtMgBr (3.0 M in Et₂O)

(1.28 mL, 3.83 mmol) under N₂and stirred at rt for 30 min. The

resulting Grignard solution was then added to aldehyde 24 (0.53

g, 3.2 mmol) in THF (30 mL) and the mixture was stirred at rt for

18 h. The reaction was quenched with water (30 mL) and the

aqueous phase was extracted with EtOAc (2 x 20 mL). The

combined organic extracts were washed with brine, dried (anhyd.

Na₂SO₄), and concentrated. The crude residue was dissolved in

MeOH (10 mL) and NaBH₄(0.060 g, 1.6 mmol) was added and

stirred at rt for 3 h. The reaction was quenched with water (10

mL) and extracted with CH₂Cl₂(2 x 15 mL). The combined

organics were dried (anhyd. Na₂SO₄) and concentrated. The

resulting residue was purified by column chromatography (5%

MeOH in EtOAc) to give 25 as an off-white solid (0.82 g, 73%).

4-(3,4-dimethoxybenzyl)-1-(4-methoxybenzyl)-2-(3-

methylimidazolidine-2,4-dione)imino-1H-imidazole

1

m.p. = 115-117 °C; H NMR: δ = 7.45 (d, J = 1.2 Hz, 1H), 7.07

(Isonaamidine E) (2): 1-Methylparabanic acid (0.11 g, 0.85

mmol) in dry CH₃CN (8 mL) was added N,O-

bis(trimethylsilyl)acetamide (0.25 mL, 1.0 mmol) and heated to

reflux for 1.5 h. The solvent was removed by vacuum distillation

and the resulting residue was dissolved in dry toluene (5 mL).

Amine 28 (0.060 g, 0.17 mmol) was added and stirred at 85 °C

under N₂for 18 h. The reaction was quenched with water (10

mL) and extracted with EtOAC (2 x 10 mL). The combined

organic extracts were dried (anhyd. Na₂SO₄) and concentrated.

The resulting residue was purified by column chromatography

(1:1 EtOAc/Hexane) to give 2 as an orange solid (0.078 g, 51%).

(d, J = 8.6 Hz, 2H), 7.02 (d, J = 2.3 Hz, 1H), 6.96 (dd, J = 2.3,

8.0 Hz, 1H), 6.85 (d, J = 8.6 Hz, 2H), 6.82 (d, J = 8.0 Hz, 1H),

6.51 (s, 1H), 5.72 (s, 1H), 4.93 (s, 2H), 3.86 (s, 3H), 3.84 (s, 3H),

3.79 (s, 3H); ¹³C NMR: δ = 159.7, 148.9, 148.5, 146.3, 137.0,

135.6, 129.0, 127.8, 119.0, 116.0, 114.4, 110.9, 109.9, 70.6, 56.0,

55.9, 55.4, 50.6; IR (cm^-1): 3125 (br), 2838, 1612, 1505, 1417,

1302, 1248, 1134, 792, 746, 630; HR-ESIMS (m/z): Calcd. for

[M+H]⁺C₂₀H₂₃N₂O₄is 355.1652 found 355.1642.

4-(3,4-Dimethoxybenzyl)-1-(4-methoxybenzyl)-1H-

imidazole (26): Alcohol 25 (0.82 g, 2.3 mmol) in CHCl₃(25

mL) was added TFA (1.10 mL, 13.9 mmol) followed by Et₃SiH

(1.85 mL, 11.6 mmol) and heated to 55 °C under N₂for 30 h. The

reaction was quenched with 2M Na₂CO₃until neutral and then

extracted with CH₂Cl₂(2 x 20 mL), dried (anhyd. Na₂SO₄), and

concentrated. The resulting residue was purified by column

chromatography (100% EtOAc to 5% MeOH in EtOAc) to give

26 as a colorless oil (0.67 g, 86%). ¹H NMR: δ = 7.42 (d, J = 1.2

Hz, 1H), 7.07 (d, J = 8.6 Hz, 2H), 6.84 (d, J = 8.6 Hz, 2H), 6.79

1

m.p. = 121-124 °C; H NMR: δ = 7.16 (d, J = 8.6 Hz, 2H), 6.83

(d, J = 8.6 Hz, 2H), 6.80-6.74 (m, 3H), 6.47 (s, 1H), 5.19 (s, 2H),

3.85 (s, 3H), 3.83 (s, 3H), 3.80 (s, 2H), 3.77 (s, 3H), 3.18 (s, 3H);

¹³C NMR: δ = 162.0, 159.5, 155.1, 149.0, 147.5, 147.7, 146.8,

144.6, 140.0, 131.3, 129.5, 128.4, 120.8, 116.1, 114.3, 112.1,

111.3, 56.0, 55.9, 55.4, 48.4, 34.4, 24.8; IR (cm^-1): 3001, 2936,

2839, 1789, 1728, 1661, 1609, 1512, 1440, 1389, 1343, 1245,

1226, 1137, 1105, 1025, 822, 603; HR-ESIMS (m/z): Calcd. for

3.30 (s, 3H); ¹³C NMR: δ = 158.3, 157.9, 139.0, 136.7, 133.2,

130.4, 129.6, 129.0, 125.7, 114.1, 113.9, 55.3, 33.1, 31.7, 28.3;

IR (neat, cm^-1): = 3000, 2908, 2835, 1610, 1509, 1461,

1245,1178, 1034,819; HR-ESIMS (m/z): Calcd. for C₂₀H₂₃N₂O₂

[M+H]⁺323.1754, found 323.1774.

[M+H]⁺C₂₄H₂₆N₅O₅is 464.1928 found 464.1915.

4-Iodo-5-(4-methoxybenzyl)-1-methyl-1H-imidazole (30):

Et₃SiH (13.9 mL, 87.2 mmol) and TFA (5.60 mL, 72.6 mmol)

were added to a solution of benzyl alcohol 29 (5.00 g, 14.5

mmol) in anhydrous CH₂Cl₂(100 mL) at rt, then the resulting

mixture was heated at reflux for 60 h under N₂atmosphere. After

cooling to rt, the reaction was quenched by the addition of sat.

aq. solution of NaHCO₃. The resulting mixture was extracted

with CH₂Cl₂several times and the combined extracts were dried

(Na₂SO₄) and concentrated. The residue was purified by

chromatography (hexane/EtOAc, 1/3) to isolate 30 (4.65 g, 97%)

asa pale yellow solid: m.p. = 97-99 ºC; ¹H NMR (Acetone–D6): δ

= 7.50 (s, 1H), 7.09 (d, J = 8.7 Hz, 2H), 6.85 (d, J = 8.7 Hz, 2 H),

3.92 (s, 2H), 3.74 (s, 3H), 3.51 (s, 3H); ¹³C NMR: δ = 158.6,

139.4, 133.4, 129.3, 129.1, 114.3, 84.9, 55.4, 32.6, 30.0; IR (KBr,

cm^-1): = 3005, 3001, 2957, 2834, 1608, 1508, 1444, 1280, 1247,

1176, 1030, 981, 821, 770, 694; HR-ESIMS (m/z): Calcd. for

C₁₂H₁₄IN₂O [M+H]⁺329.0145, found 329.0142.

2-Azido-4,5-bis(4-methoxybenzyl)-1-methyl-1H-imidazole

(35): n-Butyl lithium (1.4 M solution in hexanes, 1.50 mL, 2.1

mmol) was added dropwise to a stirred solution of 34 (613 mg,

1.9 mmol) in dry THF (15 mL) at -78 °C. The reaction was

stirred for 1 h at the same temperature. The cooling bath was

removed for 10 min, then the reaction mixture was re-cooled to -

78 °C, and TsN₃(0.80 g, 4.1 mmol) in THF (3 mL) was added

dropwise. After stirring for 40 min at -78 °C, the reaction mixture

was allowed to come to rt. and was quenched by the addition of

satd. aq. NH₄Cl (3 mL). The aqueous layer was extracted with

EtOAc (3x20 mL), and the combined organic extracts were dried

(Na₂SO₄) and concentrated to provide a pale brown oil, which

was purified through a short column of silica gel (hexane/EtOAc,

1

4:1) to isolate 35 (615 mg, 89%) as a pale yellow oil: H NMR: δ

= 7.17 (d, J = 8.3 Hz, 2H), 6.90 (d, J = 8.3 Hz, 2H), 6.79 (d, J =

8.3 Hz, 2H), 6.77 (d, J = 8.3 Hz, 2H), 3.84 (s, 2H), 3.79 (s, 2H),

3.76 (s, 6H), 3.06 (s, 3H); ¹³C NMR: δ = 158.3, 158.0, 139.0,

136.3, 132.8, 130.2, 129.5, 129.0, 125.0, 114.1, 113.9, 55.3, 32.8,

29.5, 28.7; IR (neat, cm^-1): = 3000, 2944, 2835, 2132, 1609,

1508, 1247, 1176, 1034, 821; HR-ESIMS (m/z): Calcd. for

C₂₀H₂₂N₅O₂[M+H]⁺364.1773, found 364.1768.

5-(4-Methoxybenzyl-1-methyl-1H-imidazole-4-

carbaldehyde (32): EtMgBr (3.0 M in ether, 5.2 mL, 15.7

mmol) was added into a solution of 30 (4.90 g, 14.9 mmol) in dry

THF (50 mL) at 0 ºC, and the resulting mixture was stirred at rt.

for 2 h. Then, N-methylformanilide (31) (2.23 mL, 17.9 mmol)

was added at 0 ºC and the resulting mixture was stirred at rt

overnight. Saturated aq. NH₄Cl (20 mL) was added to quench the

reaction and the organic layer was extracted with EtOAc, dried

(Na₂SO₄) and concentrated to provide the crude product, which

was purified through a short plug of silica gel (EtOAc) to isolate

32 (3.10 g, 90%) as a white solid: m.p. = 120-121 °C; ¹H NMR: δ

= 9.99 (s, 1H), 7.40 (s, 1H), 7.04 (d, J = 8.3 Hz, 2H), 6.79 (d, J =

8.3 Hz, 2H), 4.32 (s, 2H), 3.3.75 (s, 3H), 3.45 (s, 3H); ¹³C NMR:

δ = 187.7, 158.6, 138.8, 138.4, 137.7, 129.3, 128.5, 114.4, 55.3,

31.6, 28.5; IR (KBr, cm^-1): = 3105, 3022, 2957, 2818, 2770,

1669, 1552, 1511, 1344, 1244,1022, 824, 789; HR-ESIMS (m/z):

Calcd. for C₁₃H₁₅N₂O₂[M+H]⁺231.1128, found 231.1139.

2-Amino-4,5-bis(4-methoxybenzyl)-1-methyl-1H-imidazole

(36): Azide 35 (475 mg, 1.3 mmol) was dissolved in EtOH (10

mL) and stirred overnight under a hydrogen atmosphere (1 Atm)

in the presence of 10% Pd-C on charcoal (47 mg) at rt. The

catalyst was removed by filtration through a pad of Celite and the

filtrate was concentrated to isolate amine, 36 (440 mg, quant) as a

pale yellow solid: m.p. = 175-176 °C; ¹H NMR: δ = 7.09 (d, J =

8.3 Hz, 2H), 6.93 (d, J = 8.3 Hz, 2H), 6.77 (d, J = 8.3 Hz, 2H),

6.74 (d, J = 8.3 Hz, 2H), 5.79 (br, 2H), 3.75 (s, 2H), 3.74 (s, 3H),

3.70 (s, 3H), 3.68 (s, 2H), 3.03 (s, 3H); ¹³C NMR: δ = 158.4,

158.0, 147.4, 132.4, 130.4, 130.4, 129.6, 128.9, 121.0, 114.1,

113.9, 55.3, 55.3, 31.8, 29.5, 28.5; IR (KBr, cm^-1): = 3374, 3296,

3123, 2954, 2838, 1764, 1610, 1550, 1510, 1462, 1249, 1178,

1033, 819, 758; HR-ESIMS (m/z): Calcd. for C₂₀H₂₄N₃O₂

[M+H]⁺338.1863, found 338.1871.

4-[Hydroxy-(4-methoxyphenyl)]methyl-5-(4-methoxy-

benzyl)-1-methyl-1H-imidazole (33): p-Anisylmagnesium

bromide was prepared from p-bromoanisole (6.67 mL, 52.1

mmol) and magnesium turnings (1.25 g, 52.1 mmol) in THF. A

solution of 32 (3.00 g, 13.0 mmol) in THF (20 mL) was added

dropwise. to afford a crude product, which was purified through

a short plug of silica gel (EtOAc) to isolate 33 (4.34 g, 98%) as

4,5-Bis(4-methoxybenzyl)-1-methyl-2-(3-methyl-

imidazolidine-2,4-dione)imino-1H-imidazole (Naamidine G)

(4): N,O-Bis(trimethysilyl)acetamide (1.00 mL, 4.1 mmol) was

added to a solution of 1-methylparabanic acid (526 mg, 4.1

mmol) in dry CH₃CN under an N₂atmosphere and the resulting

mixture was heated at reflux for 1 h. Then, the solvent was

removed by distillation and to the resulting yellow residue in

toluene (3 mL) was added amine 36 (277 mg, 0.8 mmol) under

N₂. After, this mixture was heated at 85 ºC overnight, water (5

mL) was added and the organic layer was extracted in to EtOAc.

The dried organic layer (Na₂SO₄) was concentrated to afford a

yellow residue, which was purified over silica gel

(EtOAc/hexanes, 3/7) to provide 4 (287 mg, 78%) as a yellow

1

an off-white solid: m.p. = 134-135 °C; H NMR: δ = 7.33 (d, J =

8.7 Hz, 2H), 7.33 (s, 1H), 6.88 (d, J = 8.7 Hz, 2H), 6.81 (d, J =

8.7 Hz, 2H), 6.75 (d, J = 8.7 Hz, 2H), 5.74 (s, 1H), 3.81 (s, 2H),

3.75 (s, 6H), 3.30 (s, 3H); ¹³C NMR: δ = 158.5, 158.1, 141.4,

136.8, 136.7, 130.2, 129.2, 127.8, 126.2, 113.9, 113.4, 69.4, 55.1,

55.1, 31.4, 28.0; IR (KBr, cm^-1): = 3112 (br), 2835, 2710, 1608,

1582, 1511, 1461, 1298, 1250, 1173, 1042, 846, 820, 787; HR-

ESIMS (m/z): Calcd. for C₂₀H₂₃N₂O₃[M+H]⁺339.1703, found

339.1701.

4,5-Bis(4-methoxybenzyl)-1-methyl-1H-imidazole

(34):

Et₃SiH (1.88 mL, 11.8 mmol) and TFA (0.73 mL, 9.5 mmol)

were added to a solution of 33 (800 mg, 2.4 mmol) in anhydrous

CH₂Cl₂(20 mL) at rt, then the resulting mixture was stirred

overnight. The reaction was quenched by the addition of satd. aq.

solution of NaHCO₃and the resulting mixture was extracted with

CH₂Cl₂several times. The combined extracts were dried

(Na₂SO₄), concentrated and the residue was purified by

chromatography (acetone) to provide 34 (615 mg, 81%) as a pale

yellow oil: ¹H NMR: δ = 7.32 (s, 1H), 7.16 (d, J = 8.7 Hz, 2H),

6.90 (d, J = 8.7 Hz, 2H), 6.78 (d, J = 8.7 Hz, 2H), 6.77 (d, J = 8.7

Hz, 2H), 3.86 (s, 2H), 3.85 (s, 2H), 3.75 (s, 3H), 3.74 (s, 3H),

1

powder: m.p. = 195-196 ºC (lit.²⁸m.p. = 94 ºC); H NMR: δ =

10.04 (br, 1H), 7.10 (d, J = 8.7 Hz, 2H), 6.90 (d, J = 8.7 Hz, 2H),

6.80 (d, J = 8.7 Hz, 2H), 6.77 (d, J = 8.7 Hz, 2H), 3.89 (s, 2H),

3.88 (s, 2H), 3.76 (s, 6H), 3.47 (s, 3H), 3.15 (s, 3H); ¹³C NMR: δ

= 162.3, 158.6, 158.3, 155.7, 146.5, 145.0, 135.7, 131.5, 129.4,

129.1, 129.0, 127.0, 114.3, 114.1, 55.4, 32.3, 30.0, 28.7, 24.7; IR

(KBr, cm-1): = 3241, 2931, 2835, 1788, 1738, 1656, 1511, 1451,

1302, 1248, 1177, 1036, 744, 697; HR-ESIMS (m/z): Calcd. for

C₂₄H₂₆N₅O₄[M+H]⁺448.1979, found 448.1984.

1

2-Azido-5-[4-methoxybenzyl]-4-[methoxy-(4-methoxy-

phenyl)]methyl-1-methyl-1H-imidazole (38): TFA (0.60 mL,

7.8 mmol) was added to a solution of 33 (1.31 g, 3.9 mmol) in

anhyd MeOH (20 mL) at rt. and the mixture was then heated at

55 °C overnight. Satd. aq. solution of NaHCO₃was used to

neutralize the above reaction mixture, and the aqueous layer was

extracted with EtOAc (3x 30 mL). Combined organic layers were

washed with water and brine, then dried (Na₂SO₄) and

concentrated to provide 37 (1.37 g, >95%) as a pale yellow oil

which was used directly in the next step: ¹H NMR: δ = 7.34 (d, J

= 8.7 Hz, 2H), 7.23 (s, 1H), 6.86 (d, J = 8.7 Hz, 2H), 6.77 (d, J =

8.7 Hz, 2H), 6.71 (d, J = 8.7 Hz, 2H), 5.26 (s, 1 H), 3.93 (s, 2H),

3.75 (s, 6H), 3.34 (s, 3H), 3.29 (s, 3H). Following the general

procedure for azidation, n-Butyl lithium (1.4 M solution in

hexanes, 2.66 mL, 3.7 mmol), 37 (1.45 g, 3.4 mmol) in dry THF

(20 mL) and TsN₃(0.80 g, 4.1 mmol) were used to obtain the

crude product, which was purified through a short column of

silica gel (hexane/EtOAc, 4:1) to isolate 38 (891 mg, 67%) as a

95%) as a white solid: m.p. = 173-175 ºC; H NMR: δ = 7.44 (d,

J = 7.8 Hz, 2H), 7.31-7.24 (m, 4H), 6.57 (s, 2H), 5.98 (s, 1H),

5.18 (s, 1H), 4.98 (s, 2H), 3.75 (s, 6H), 3.43 (s, 3H); ¹³C NMR: δ

= 153.6, 141.1, 137.8, 136.6, 135.9, 135.2, 128.6, 128.2, 127.9,

102.7, 84.6, 75.0, 67.1,56.3, 33.5; IR (neat, cm^-1): = 3253 (br),

3104, 1585, 1501, 1411, 1338, 1226, 1140, 1033, 908 ; HR-

DARTMS (m/z): Calc. for C₂₀H₂₁IN₂O₄[M]⁺480.0546; found:

480.0546; Calc. for C₂₀H₂₂IN₂O₄[M+H]⁺481.0624, found

481.0624.

5-(4-Benzyloxy-3,5-dimethoxybenzyl)-4-iodo-1-methyl-1H-

imidazole (43): Et₃SiH (1.00 mL, 6.2 mmol) and TFA (0.40

mL, 5.2 mmol) were added to a solution of 42 (0.50 g, 1.0 mmol)

in anhydrous CHCl₃(20 mL) at rt. and the resulting mixture was

heated at reflux temperature for 24 h under nitrogen atmosphere.

After cooling to rt., reaction was quenched by the addition of

satd. aq. NaHCO₃. The aqueous layer was extracted several times

with CHCl₃and the combined organic extracts were dried

(Na₂SO₄) and concentrated. The residue was purified by

chromatography (EtOAc) to isolate 43 (0.14 g, 28%) as a pale

1

reddish brown oil: H NMR: δ = 7.41 (d, J = 8.7 Hz, 2H), 6.90

(d, J = 8.7 Hz, 2H), 6.84 (d, J = 8.7 Hz, 2H), 6.76 (d, J = 8.7 Hz,

2H), 5.23 (s, 1 H), 3.85 (s, 2H), 3.78 (s, 3H), 3.76 (s, 3H), 3.35

(s, 3H), 3.03 (s, 3H); ¹³C NMR: δ = 159.0, 158.4, 139.8, 136.9,

133.4, 129.9, 129.1, 128.4, 126.1, 114.1, 113.7, 79.1, 56.8, 55.3,

55.3, 29.4, 28.6; IR (neat, cm^-1) = 2937, 2834, 2138, 1508, 1246,

1174, 1087, 1033, 829; HR-ESIMS (m/z): Calcd. for

C₂₁H₂₃N₅NaO₃[M+Na]⁺416.1693, found 416.1677.

1

brown semi solid: H NMR: δ = 7.44 (d, J = 6.9 Hz, 2H), 7.36 (s,

1H), 7.30 (t, J = 6.9 Hz, 2H), 7.25 (d, J = 6.9 Hz, 1H), 6.31 (s,

2H), 4.95 (s, 2H), 3.87 (s, 2H), 3.73 (s, 6H), 3.40 (s, 3H); ¹³C

NMR: δ = 153.8, 139.6, 137.9, 135.9, 133.1, 128.5, 128.2, 127.9,

105.2, 85.0, 75.1, 56.3, 32.7, 31.0; IR (neat, cm-1): = 3107, 2939,

1588, 1494, 1460, 1420, 1237, 1215, 1187, 1100, 978,758 ; HR-

ESIMS (m/z): Calc. for C₂₀H₂₂IN₂O₃[M+H]⁺465.0670, found

465.0669.

2-Amino-5-(4-methoxybenzyl)-4-[methoxy-(4-methoxy-

phenyl)]methyl-1-methyl-1H-imidazole (39): Following the

general procedure for catalytic reduction, azide 38 (800 mg, 2.0

mmol) in EtOH (10 mL) and 10% Pd-C on charcoal (80 mg) at 1

atm hydrogen were used to afford amine 39 (745 mg, quant) as a

5-[(3,5-Dimethoxy-4-hydroxy)benzyl]-4-iodo-1-methyl-1H-

imidazole (44): A second product was obtained from above

reaction in which the O-benzyl protecting group has been

removed 44 (0.11 g, 27%) as a pale yellow solid: m.p. = 210-212

ºC; ¹H NMR (DMSO-D₆): δ = 7.40 (s, 1H), 6.34 (s, 2H), 5.47 (br,

1H), 3.90 (s, 2H), 3.82 (s, 6H), 3.45 (s, 3H); ¹³C NMR: δ = 148.6,

140.6, 134.7, 133.7, 128.4, 106.1, 85.1, 56.5, 32.6, 30.0; IR (neat,

cm^-1): = 3107, 2936 (br), 1595, 1514, 1499, 1413, 1242, 1214,

1113, 1037, 838, 811, 765, 737; HR-ESIMS (m/z): Calc. for

C₁₃H₁₅IN₂O₃[M+H]⁺

1

pale yellow solid: m.p. = 139-140 °C; H NMR (CD₃OD): δ =

7.29 (d, J = 8.7 Hz, 2H), 6.95 (d, J = 8.7 Hz, 2H), 6.82 (d, J = 8.7

Hz, 2H), 6.78 (d, J = 8.7 Hz, 2H), 5.21 (s, 1H), 3.86 (s, 2H), 3.74

(s, 3H), 3.73 (s, 3H), 3.30 (s, 3H), 3.08 (s, 3H); ¹³C NMR: δ =

159.0, 158.4, 148.9, 133.8, 131.8, 130.7, 128.7, 127.9, 123.0,

113.6, 113.1, 78.5, 55.4, 54.4, 54.4, 28.0, 27.7; IR (KBr, cm^-1) =

3129, 1671, 1510, 1459, 1248, 1176, 1084, 1031, 830, 746; HR-

ESIMS (m/z): Calcd. for C₂₁H₂₆N₃O₃[M+H]⁺368.1969, found

368.1970.

5-(4-Benzyloxy-3,5-dimethoxyphenyl)hydroxymethyl-1-

methyl-1H-imidazole-4-carbaldehyde (45): EtMgBr (3.0 M in

ether, 13.8 mL, 41.4 mmol) was added into a solution of 42 (9.03

g, 18.8 mmol) in dry THF (200 mL) at rt., and the resulting

mixture was stirred for 20 min. Then, N-methylformanilide (30)

(2.78 mL, 22.6 mmol) was added to the reaction mixture

followed by stirring for 33 h. Half saturated NH₄Cl (30 mL) was

added to quench the reaction and the organic layer was extracted

with EtOAc, dried (Na₂SO₄) and concentrated to provide the

crude product, which was purified through a short plug of silica

gel (EtOAcAcetone) to isolate 45 as a pale yellow solid (5.52

g, 77%): m.p. = 132-134 ºC; ¹H NMR: δ = 9.82 (s, 1H), 7.42 (d, J

= 7.3 Hz, 2H), 7.38 (s, 1H), 7.29 (t, J = 7.3 Hz, 2H), 7.25 (m,

1H), 6.50 (s, 2H), 6.23 (s, 1H), 4.94 (s, 2H), 3.71 (s, 6H), 3.47 (s,

3H); ¹³C NMR: δ = 188.4, 153.8, 141.7, 139.9, 138.0, 137.7,

136.6, 136.2, 128.5, 128.2, 127.9, 103.4, 75.1, 66.7, 56.3, 33.1;

IR (neat, cm^-1): = 3253 (br), 3106, 2938, 1680, 1587, 1502, 1450,

1415, 1230, 1100, 1056, 824 ; HR-DARTMS (m/z): Calc. for

C₂₁H₂₃N₂O₅[M+H]⁺383.1601, found 383.1597.

5-(4-Methoxybenzyl)-4-[methoxy-(4-methoxy-

phenyl)]methyl-1-methyl-2-(3-methylimidazolidine-2,4-

dione)imino-1H-imidazole (4-Methoxynaamidine G) (40):

Following

the

general

procedure,

N,O-

Bis(trimethysilyl)acetamide (0.33 mL, 1.4 mmol) and 1-

methylparabanic acid (174 mg, 1.4 mmol) in dry CH₃CN (15

mL) were used to produce a yellow residue, which was treated

with amine 39 (100 mg, 0.3 mmol) to obtain the product 40 (13

mg, 11%) as a yellow solid after the purification over silica gel

1

(EtOAc/hexanes, 2/3): H NMR: δ = 7.34 (d, J = 8.8 Hz, 2H),

6.92 (d, J = 8.8 Hz, 2H) , 6.87 (d, J = 8.8 Hz, 2H) , 6.78 (d, J =

8.8 Hz, 2H) , 5.29 (s, 1H), 3.98 (s, 2H), 3.79 (s, 3H), 3.77 (s, 3H),

3.45 (s, 3H), 3.35 (s, 3H), 3.17 (s, 3H);

5-(4-Benzyloxy-3,5-dimethoxyphenyl)hydroxymethyl-4-

iodo-1-methyl-1H-imidazole (42): EtMgBr (3.0 M solution in

ether, 7.54 mL, 22.6 mmol) was added to a solution of 14 (7.19

g, 21.5 mmol) in dry CH₂Cl₂(100 mL) at rt. over ~10 min. After

stirring at rt. for 20 min, aldehyde 41 (6.46 g, 23.7 mmol) was

added to the reaction followed by 48 h stirring. Then, satd. aq.

NH4Cl (10 mL) was added to the reaction and the resulting pale

yellow solid was filtered and the filtrate was partitioned with

CH₂Cl₂. The organic layer was dried (Na₂SO₄) and concentrated

to provide a pale yellow solid. The resulting solid was triturated

with hexanes, recrystallized with CH₂Cl₂to isolate 42 (9.68 g,

6-Benzyloxy-5,7-dimethoxy-4-(4-methoxyphenyl)-1-

methyl-1H-naphtho[2,3-d]imidazole (47): A Grignard reagent

was prepared from p-bromoanisole, (7.23 mL, 56.5 mmol),

magnesium turnings (1.35 g, 56.5 mmol) and a small crystal of

iodine in THF (100 mL) and a solution of 45 (5.40 g, 14.1 mmol)

in THF (50 mL) were used to obtain a crude product, which was

purified through a short plug of silica gel (EtOAc) to isolate 46

(4.38 g, 63%) as a pale yellow oil, which was used in the next

step directly without further characterization. Et₃SiH (11.41 mL,

71.4 mmol) and TFA (4.81 mL, 62.5 mmol) were added to a

solution of 45 (4.38 g, 8.9 mmol) in anhydrous CH₂Cl₂(100 mL)

at rt. and the resulting mixture was stirred for 24 h under nitrogen

atmosphere. Then, the reaction was quenched by the addition of

satd. aq. solution of NaHCO₃. The aqueous layer was extracted

with CH₂Cl₂several times and the combined extracts were dried

(Na₂SO₄), and concentrated. The residue was purified by

chromatography (EtOAc  acetone) to provide 47 (3.30 g, 81%)

crude oil, which was purified with a gradient column

(EtOAc:hexanes, 3:1EtOAc:acetone; 1:1) to isolate 53 (2.96 g,

90%) as a light brown oil: ¹H NMR: δ = 7.45 (d, J = 7.3 Hz, 2H),

7.40 (s, 1H), 7.31 (t, J = 7.3 Hz, 2H), 7.27 (t, J = 7.3 Hz, 1H),

7.19 (d, J = 8.7 Hz, 2H), 6.77 (d, J = 8.7 Hz, 2H), 6.15 (s, 2H),

4.96 (s, 2H), 3.88 (s, 2H), 3.87 (s, 2H), 3.75 (s, 3H), 3.64 (s, 6H),

3.34 (s, 3H); ¹³C NMR: δ = 157.9, 153.8, 138.8, 137.9, 136.8,

135.5, 134.1, 133.1, 129.5, 128.5, 128.2, 127.9, 125.5, 113.9,

105.1, 75.1, 56.1, 55.3, 32.8, 31.9, 29.5; IR (neat, cm^-1): = 2929,

2857, 1691, 1507, 1391, 1251, 1176, 1150, 910; HR-ESIMS

(m/z): Calc. for C₂₈H₃₁N₂O₄[M+H]⁺459.2278, found 459.2278.

1

as a pale brown solid; m.p. = 194-197 ºC; H NMR: δ = 7.87 (s,

1H), 7.61 (s, 1H), 7.53 (d, J = 8.3 Hz, 2H), 7.45 (d, J = 8,7 Hz,

2H), 7.36 (t, J = 7.3 Hz, 2H), 7.30 (t, J = 7.3 Hz, 1H), 7.08 (s,

1H), 7.05 (d, J = 8.7 Hz, 2H), 5.10 (s, 2H), 3.96 (s, 3H), 3.89 (s,

3H), 3.74 (s, 3H), 3.37 (s, 3H); ¹³C NMR: δ = 158.2, 152.1,

150.6, 146.6, 142.9, 139.8, 138.0, 134.4, 132.4, 131.6, 130.9,

129.7, 128.4, 128.3, 127.9, 119.6, 112.7, 104.0, 102.3, 75.2, 60.9,

55.8, 55.4, 31.0; IR (neat, cm^-1): = 2929, 2831, 1607, 1514, 1451,

1330, 1274, 1236, 1145, 1075, 1027, 826, 740; HR-DARTMS

(m/z): Calc. for C₂₈H₂₇N₂O₄[M+H]⁺455.1971, found 455.1983.

2-Azido-5-(4-benzyloxy-3,5-dimethoxybenzyl)-4-(4-

methoxybenzyl)-1-methyl-1H-imidazole (55): n-Butyl lithium

(1.6 M solution in hexanes, 1.31 mL, 2.1 mmol) was added

dropwise to a stirred solution of 52 (870 mg, 1.9 mmol) in dry

THF (20 mL) at -78 °C, and the reaction was stirred for 1 h. The

cooling bath was removed for 10 min, then the reaction mixture

was re-cooled to -78 °C, and then TrisN₃(706 mg, 2.3 mmol)

was added. After stirring for an additional 45 min at -78 °C, the

reaction mixture was quenched by the addition of satd. aq. NH₄Cl

(5 mL). The aqueous layer was extracted with EtOAc (3x15 mL),

and the combined organic extracts were dried (Na₂SO₄) and

concentrated to provide a pale brown oil, which was purified

through a short column of silica gel (hexane/EtOAc, 7:3) to

isolate azide 55 (600 mg, 63%) as a pale brown oil: ¹H NMR: δ =

7.46 (d, J = 7.4 Hz, 2H), 7.38-7.26 (m, 3H), 7.21 (d, J = 8.5 Hz,

2H), 6.79 (d, J = 8.5 Hz, 2H), 6.16 (s, 2H), 4.95 (s, 2H), 3.85 (s,

2H), 3.80 (s, 2H), 3.76 (s, 3H), 3.64 (s, 6H), 3.08 (s, 3H); ¹³C

NMR: δ = 158.0, 153.7, 139.2, 137.9, 136.5, 135.5, 134.0, 132.9,

129.5, 128.6, 128.2, 127.9, 124.6, 113.9, 105.0, 75.0, 56.1, 55.3,

32.7, 30.0, 29.6; IR (neat, cm-1): = 2929, 2857, 2129, 1691,

1507, 1391, 1252, 1150, 1124, 909, 836, 779 ; HR-ESIMS (m/z):

Calc. for C₂₈H₃₀N₅O₄[M+H]⁺500.2292, found 500.2290.

1-(N,N-dimethylaminosulfonyl)-4-iodo-5-(4-methoxy-

benzyl)-1H-imidazole (50): EtMgBr (3.0 M solution in ether,

8.60 mL, 25.8 mmol) was added to a solution of 49 (7.19 g, 21.5

mmol) in dry CH₂Cl₂(150 mL) at rt. The resulting mixture was

stirred at rt. for 20 min and 1.0 M solution of CuCN.2LiCl in dry

THF (26.0 mL, 26.0 mmol) was added followed by p-

methoxybenzyl bromide (3.80 mL, 25.8 mmol). The orange

reaction solution was stirred at rt. for 48 h and poured into half

sat. NH₄Cl containing 2% concentrated NH₃(50 mL). After

stirring for 20 min, the resulting solid was filtered off and the

filtrate was partitioned with CH₂Cl₂(3x50 mL). The organic

layer was dried (Na₂SO₄), concentrated and purified by

chromatography (EtOAc/hexane, 3:7) to afford 50 (6.41 g, 65%)

1

as a pale yellow solid: m.p. = 76-78 ºC; H NMR: δ = 7.87 (s.

1H), 6.99 (d, J = 8.7 Hz, 2H), 6.77 (d, J = 8.7 Hz, 2H), 6.37 (s,

2H), 4.09 (s, 2H), 3.71 (s, 3H), 2.49 (s, 6H); ¹³C NMR: δ = 158.5,

139.7, 137.9, 132.5, 129.1, 114.0, 90.6, 55.4, 37.6, 29.9; IR (neat,

cm^-1): = 3111, 2919, 1514, 1459, 1415, 1240, 1173, 1174, 1095,

960, 802 ; HR-DARTMS (m/z): Calc. for C₁₃H₁₇IN₃O₃S [M+H]⁺

422.0030, found 422.0047.

2-Amino-5-(3,5-dimethoxy-4-hydroxybenzyl)-4-(4-

methoxybenzyl)-1-methyl-1H-imidazole (Naamine G) (56):

Azide 55 (600 mg, 1.2 mmol) was dissolved in EtOH (15 mL)

and stirred overnight under a hydrogen atmosphere (55 psi) in the

presence of 20% Pd(OH)₂on charcoal (100 mg) at rt. The

catalyst was filtered through a pad of Celite and the filtrate was

concentrated to isolate naamine G, (56) (430 mg, 95%) as a

4-(4-Benzyloxy-3,5-dimethoxybenzyl)-1-(N,N-dimethyl-

sulfonyl)-5-(4-methoxybenzyl)-1H-imidazole (51): EtMgBr

(3.0 M solution in ether, 4.98 mL, 14.9 mmol), 50 (5.72 g, 13.6

mmol) in dry CH₂Cl₂(150 mL), 1.0 M solution of CuCN.2LiCl

in dry THF (16.3 mL, 16.3 mmol) and 206 (6.87 g, 20.4 mmol)

were used to synthesize 51 (5.18 g, 70%) as a pale yellow oil

after the purification by chromatography (EtOAc:hexane, 1:1):

¹H NMR: δ = 7.94 (s. 1H), 7.48 (d, J = 7.3 Hz, 2H), 7.33 (t, J =

7.3 Hz, 2H), 7.27 (t, J = 7.33 Hz, 1H), 6.94 (d, J = 8.7 Hz, 2H),

6.77 (d, J = 8.7 Hz, 2H), 6.37 (s, 2H), 4.94 (s, 2H), 4.14 (s, 2H),

3.78 (s, 2H), 3.75 (s, 3H), 3.71 (s, 6H), 2.57 (s, 6H); ¹³C NMR: δ

= 158.4, 153.4, 141.3, 138.1, 138.0, 135.6, 134.8, 130.3, 129.9,

128.9, 128.5, 128.2, 127.8, 114.0, 106.0, 75.1, 56.1, 55.4, 37.5,

34.0, 28.1; IR (neat, cm^-1): = 2929, 2857, 1691, 1507, 1393,

1252, 1124, 909, 836, 779 ; HR-DARTMS (m/z): Calc. for

C₂₉H₃₄N₃O₆S [M+H]⁺552.2163, found 552.2180.

1

greenish-yellow solid; m.p. = 218-220 °C; H NMR (CD₃OD): δ

= 7.17 (d, J = 8.7 Hz, 2H), 6.84 (d, J = 8.7 Hz, 2H), 6.34 (s, 2H),

3.92 (s, 2H), 3.84 (s, 2H), 3.74 (s, 3H), 3.69 (s, 6H), 3.23 (s, 3H);

¹³C NMR: δ = 158.8, 148.3, 146.5, 134.3, 129.8, 129.2, 127.3,

122.8, 122.4, 114.0, 105.2, 55.5, 54.5, 28.8, 28.3, 27.9; IR (neat,

cm^-1): = 3244 (br), 3004, 2836, 1667, 1654, 1609, 1500, 1461,

1429, 1245, 1216, 1110, 1022; HR-DARTMS (m/z): Calc. for

C₂₁H₂₆N₃O₄[M+H]⁺384.1918, found 384.11907.

5-(3,5-Dimethoxy-4-hydroxybenzyl)-4-(4-methoxybenzyl)-

1-methyl-2-(3-methylimidazolidine-2,4-dione)imino-1H-

imidazole (Naamidine H) (5): Following the general procedure

for this reaction, N,O-Bis(trimethysilyl)acetamide (0.63 mL, 2.6

mmol) and 1-methylparabanic acid (331 mg, 4.1 mmol) in dry

CH₃CN (10 mL) were used to produce 3-trimethylsilyl-1-

methylparabanic acid. After removing the solvent, naamine G, 1

h (198 mg, 0.5 mmol) was added and the mixture was heated at

80 ºC overnight in dry toluene (5 mL). Usual workup and

purification over silica gel (EtOAc/hexanes, 4/6) provided

naamidine H (5) as a yellow amorphous solid (205 mg, 80%):

5-(4-Benzyloxy-3,5-dimethoxybenzyl)-4-(4-methoxy-

benzyl)-1-methyl-1H-imidazole

(53):

Methyl

trifluoromethanesulfonate (0.95 mL, 8.7 mmol) was added

dropwise to a solution of 51 (3.96 g, 7.2 mmol) in CH₂Cl₂(50

mL), at 0 ºC under N₂and stirred for 4 h at the same temperature.

Then, the solvent was evaporated under reduced pressure and the

crude pale yellow oil was dissolved in dry acetonitrile (30 mL),

and benzylamine (0.95 mL, 8.7 mmol) was added to it. After

heating at 80 ºC for 10 h, the solvent was evaporated to provide a

1

m.p. = 204-205 ºC; H NMR: δ = 7.14 (d, J = 8.7 Hz, 2H), 6.98

(br, 1H), 6.78 (d, J = 8.7 Hz, 2H), 6.14 (s, 2H), 3.89 (s, 2H), 3.88

(s, 2H), 3.75 (s, 3H), 3.69 (s, 6H), 3.49 (s, 3H), 3.47 (s, 3H), 3.16

(s, 3H); ¹³C NMR: δ = 162.3, 158.3, 155.5, 147.4, 146.6, 144.7,

136.1, 133.7, 131.7, 129.4, 128.1, 126.7, 114.1, 104.7, 56.3, 55.4,

32.3, 30.0, 29.7, 24.8; IR (neat, cm^-1): = 3501, 3212, 2929, 2837,

1784, 1718, 1652, 1511, 1392, 1113, 1039, 1020, 918 ; HR-

DARTMS (m/z): Calc. for C₂₅H₂₈N₅O₆[M+H]+ 494.2034, found

494.2049.

convert MTT to formazan. Then the media was discarded and

formazan crystals were dissolved by adding 100 µL of DMSO

and incubating 2 h with continuous shaking. The absorbance of

the lysates was measured directly at 560 nm using a micro plate

reader (Fluostar-omega, BMG Labtech). The percent viable cells

(calculated based on absorbance of the untreated control sample)

were plotted as a function of concentration to obtain the IC50

values. Each experiment was repeated at least twice (with 5

replicates each time).

Cytotoxicity assay (IC₅₀measurement): Human breast

adenocarcinoma cell line (MCF7, ATCC) was maintained in

growth medium containing Dulbeco’s modified Eagles media

(DMEM, Sigma) with 10% heat inactivated fetal bovine serum

(FBS, Sigma), 2 mM L-glutamine, 100 units of penicillin, and

0.1 mg/mL streptomycin. Cells were incubated in a water-

jacketed humidified CO2 incubator with 5% CO2 and at 37 °C.

To measure the cytotoxicity of the test compounds,

approximately 10,000 MCF7 cells were cultivated in each well of

a 96-well microtiter plate and maintained overnight in 150 180

μL of the growth medium and the conditions described above.

Each compound was initially dissolved in DMSO and then was

serially dilutedion also made (in DMSO) for (10 μL for each

concentration). 10 μL andof each diluted stock was further then

diluted within 990 μL of above growth medium to have desired

concentration. To treat the cells, 50 20 μL of growth medium

containing the appropriate amount of each compound was added

to each well to obtain required final concentration (200 μL final

volume and 0.1 μL of DMSO in each well). An equivalent

volume of the vehicle (0.1 % DMSO) was added to each control

well. After 96 h of incubation, the cell viability was analyzed by

using the MTT assay. In brief, 20 μL of MTT solution (stock 5

mg/ml in PBS) was added into each well and incubated for 2 h

under normal growth conditions to allow the viable cell to

Acknowledgments

We thank Paromita Deb for performing the evaluation of

compound 17 (entry 1, Table 1). This research has been

supported by The Robert A. Welch Foundation (Y-1362; CJL)

and the NIH (1R15 ES019129-01; SSM and in part GM65503;

CJL). NMR spectrometers were purchased with funds provided

by the NSF (CHE-0234811 and CHE-0840509).

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Article Doi

Total synthesis and cytotoxicity of Leucetta alkaloids

DOI: 10.1016/j.bmc.2017.01.024

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Full text of DOI:10.1016/j.bmc.2017.01.024

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