Antimicrobial and cytotoxic activity of agelasine and agelasimine analogs

Bioorganic & Medicinal Chemistry 15 (2007) 4016–4037

Antimicrobial and cytotoxic activity of agelasine

and agelasimine analogs

a

b

c

a

d

Anders Vik, Erik Hedner, Colin Charnock, Linda W. Tangen, Ørjan Samuelsen,

e

b

a,

*

Rolf Larsson, Lars Bohlin and Lise-Lotte Gundersen

a

Department of Chemistry, University of Oslo, PO Box 1033, Blindern, N-0315 Oslo, Norway

Division of Pharmacognosy, Department of Medicinal Chemistry, Biomedical Centre, Uppsala University,

PO Box 574, S-751 23, Sweden

b

c

Faculty of Health Sciences, Oslo University College, PO Box 4, St. Olavs plass, N-0130 Oslo, Norway

University Hospital of North Norway, Department of Microbiology and Infection Control, Tromsø, Norway

d

e

Department of Medicinal Sciences, Uppsala Academic Hospital, S-751 85 Uppsala, Sweden

Received 22 January 2007; revised 23 March 2007; accepted 30 March 2007

Available online 2 April 2007

Abstract—Agelasine and agelasimine derivatives with substantially less complicated terpenoid side chains compared to the naturally

occurring compounds have been synthesized and their ability to inhibit growth of microorganisms and cancer cells has been studied.

Compounds with excellent activity against cancer cell lines (MIC ca. 1 lM for the most potent compounds), including a drug resis-

tant renal cell line, have been identiﬁed. Most compounds studied also exhibited broad spectrum antimicrobial activity including

activity against Mycobacterium tuberculosis.

ꢀ

1

. Introduction

asine D and close analogs display a broad spectrum of

antibacterial activities including eﬀect on M. tuberculosis,

Gram-positive and Gram-negative bacteria (both

aerobes and anaerobes), and that the same compounds

also display profound cytotoxic activity against several

cancer cells including a multi-drug resistant renal cell

1

2

Agelasines and agelasimines are isolated from marine

sponges (Agelas sp.) and these natural products are

associated with various bioactivities including antimi-

crobial and cytotoxic eﬀects. More than ten agelasines

3

4

5

6

7

6b

are currently known, and agelasine A, B, C, D, E,

line. In order to obtain structure–activity relationship

8

and (±) agelasine F have been synthesized. The struc-

tures of some agelasines are shown in Figure 1. Agelasi-

mines are also purine derivatives carrying a diterpenoid

side chain. However, these compounds are neutral 3,7-

dialkylated purines. Only agelasimine A and B (Fig. 1)

have been found in nature and both have been prepared

(SAR) knowledge on agelasines, and analogs as antimi-

crobials and cytotoxic compounds, with the ultimate

goal to develop analogs with better selectivity against

bacteria or cancer cells, we have synthesized several ana-

logs of agelasines and agelasimines and evaluated their

ability to inhibit bacterial growth and their cytotoxicity

against certain cancer cell lines.

9

synthetically.

Activity against Mycobacterium tuberculosis has been re-

1

0

ported for agelasine F. We have found only modest

antimycobacterial activity for agelasine E, but several

agelasine analogs exhibited profound inhibitory activity

2

. Synthesis

We have utilized the same strategy for the preparation

of the agelasine analogs 3 and 5, as for our previously

7

against M. tuberculosis. We have also shown that agel-

6

,7

reported agelasines, and the synthetic route is outlined

in Scheme 1. 9-Alkyl-6-chloropurines 1 were converted

Keywords: Antimicrobial agent; Anti-cancer agent; Adenine derivative;

Natural product.

6

to the N -alkoxypurines 2 and subsequently reacted with

*

Corresponding author. Tel.: +47 22857019; fax: +47 22855507;

e-mail: l.l.gundersen@kjemi.uio.no

allylic halides. Ammonia was present in the eluent, when

compounds 3 were puriﬁed chromatographically. Thus,

0

doi:10.1016/j.bmc.2007.03.086

A. Vik et al. / Bioorg. Med. Chem. 15 (2007) 4016–4037

4017

Figure 1. Structures of some agelasines and agelasimines.

0

00

Scheme 1. Reagents and conditions: (a) R ONH

2

ÆHCl, Et

3

N, n-BuOH, D; (b) R CH

2

Br, DMA, 50 ꢁC; (c) Zn, AcOH, MeOH, H O, 60 ꢁC.

2

compounds 3 were generally isolated as betaines. The

desired compounds 3 were formed together with various

amounts of N -alkylated compounds 4. The regiochem-

6

istry was improved when N was sterically shielded (i.e.,

compounds 2b, 2c, 2g or 2j). Reductive removal of the

6

N -alkoxy groups gave the agelasine analogs 5. In order

to gain increased knowledge on SAR of antimicrobial

and cytotoxic agelasine analogs, compounds with vari-

ous substituents at N , N-7, and N-9 were prepared.

6

2 3

Scheme 2. Reagents and condition: (a) K CO , MeOH; (b) Zn, AcOH,

MeOH, H

2

O, 75 ꢁC.

We wanted to compare biological activities of the agel-

asine analogs 3 and 5 with activities found for related

structures without a permanently charged imidazole

ring. Compounds 6 and 7 were prepared as shown in

Scheme 2. Compounds 3n and 3o were treated with

K CO in methanol in order to remove the N-9 substitu-

compound 6b gave 7-geranylgeranyladenine 7. An alter-

native route to compound 7 is shown in Scheme 3. 6-

Chloro-7-geranylgeranylpurine 9b was formed, together

with the isomer 9a, when 6-chloropurine 8 was reacted

with geranylgeranyl bromide in the presence of base.

2

3

ent, and reductive cleavage of the benzyloxy group in

4018

A. Vik et al. / Bioorg. Med. Chem. 15 (2007) 4016–4037

0

Scheme 3. Reagents and condition: (a) R CH

R CH

2

Br, K

, DMA; (c) NH

2

CO

3

, DMF; (b)

, t-BuOH,

00

2

Br, methylaquacobaloxime, K

2

CO

3

100 ꢁC.

Selective N-7 alkylation was achieved when 6-chloropu-

rine 8 was reacted with geranylgeranyl bromide in the

0

1

Scheme 6. Reagents and condition: (a) R CH Br, DMA, 50 ꢁC; (b)

presence of methylaquacobaloxime, and compound

b was isolated in 40% yield when DMA was used as sol-

2

3 4 2

CH I, DMA; (c) NaBH , MeOH, H O.

9

vent. Chloropurine 9b was easily converted to the ade-

nine derivative 7 when reacted with ammonia. t-BuOH

was used as solvent to avoid nucleophilic substitution

from solvent attack, in the purine 6-position.

nine 14 was alkylated at N-7 to give compounds 15.

The agelasimine A analog 16 was prepared by N -meth-

ylation of 15b. Sodium borohydride mediated reduction

of 15b followed by N-1 methylation of the dihydropu-

rine 17 gave the agelasimine B analog 18.

6

In order to evaluate the importance of an intact purine

ring for antibiotic activity of agelasine analogs, we also

prepared the simple imidazolium and benzimidazolium

bromides 11 from N-methylimidazole 10a or N-ethyl-

benzimidazole 10b (Scheme 4).

3. Antimicrobial and cytotoxic activity

Antibacterial activities as well as cytotoxicity toward

cancer cell lines were examined for the agelasine analogs

3, 5–7, 9b, 11, and 13, and the agelasimine analogs 15–

18. The results are summarized in Tables 1–6. Com-

pounds 4 were only isolated in minor amounts and we

have previously demonstrated that related structures

were only weak inhibitors of M. tuberculosis. Hence bio-

activity of compounds 4 was not determined in the pres-

ent study.

6

When the N alkoxy substituent in compounds 2 is pres-

ent, the second N-alkyl substituent is directed to N-7

(

see Scheme 1 above). N-Alkylation of 9-methyladenine

2 with benzyl bromide or geranylgeranyl bromide gave

the 1,9-dialylated purines 13, which can be regarded as

1

regioisomers of agelasines (Scheme 5).

Since analogs of agelasines carrying a simple terpenoid

side chain (i.e., geranylgeranyl) are shown to exhibit

7

antimycobacterial activity, we also wanted to synthe-

size geranylgeranyl analogs of agelasimines and evaluate

their potential as antibiotics (Scheme 6). 3-Methylade-

Biological activities found for agelasines 3 and 5 are

shown in Table 1. A comparison of compounds 3 carry-

ing N-7 substituents of various lengths reveals that the

00

Scheme 4. Reagents and condition: (a) R CH Br, DMA, 50 ꢁC.

2

00

Scheme 5. Reagents and condition: (a) R CH Br, DMA, 50 ꢁC.

2

A. Vik et al. / Bioorg. Med. Chem. 15 (2007) 4016–4037

4021

Table 2. Antibacterial activity of compound 3i against Streptococcus pyogenes, Enterococcus faecalis, and Pseudomonas aeruginosa

a

b

c

Compound

MIC (lg/mL) S. pyogenes

MIC (lg/mL) E. faecalis

MIC (lg/mL) P. aeruiginosa

3i

4.0

8.0

>32

a

b

c

MIC gentamycin 1–2 lg/mL.

MIC gentamycin >4 lg/mL.

MIC gentamycin 0.13 lg/mL.

Table 3. Antibacterial activity of compounds 6–9 against Mycobacterium tuberculosis, Staphylococcus aureus and Escherichia coli, and cytotoxic

activity against the cell lines U-937 GTB (lymphoma), RPMI 8226/s (myeloma), CEM/s (leukemia), and ACHN (renal)

0

00

Compound R

R

% Inhib.

MIC

M. tuberculosis M. tuberculosis S. aureus E. coli U-937

IC50 (lM) IC50 (lM) IC50

RPMI (lM)

IC50

a

b

c

(lM)

CEM/s ACHN

d

e

f

g

6

.25 lg/mL

(lg/mL)

(lg/mL) (lg/mL) GTB

8226/s

h

6

7

9

a

–CH

—

3

74

40

99

33

n.d.

>32

3.31

3.00

5.69

18.1

27.4

2

b

2

Ph

n.d.

1.56

n.d.

11.2

10.9

2.78

5.20

20.4

3.60

5.07

2.88

6.79

7.24

b

—

6.75

a

b

c

MIC rifampicin 0.25 lg/mL.

MIC gentamycin 0.1 lg/mL.

MIC gentamycin 0.5 lg/mL.

IC50 doxorubicin 0.11 lM, IC50 cisplatin 2.56 lM, IC50 palitaxel 0.0059 lM.

IC50 doxorubicin 0.13 lM, IC50 cisplatin 14.8 lM, IC50 palitaxel 0.007 lM.

IC50 doxorubicin 0.18 lM, IC50 cisplatin 2.48 lM, IC50 palitaxel 0.007 lM.

IC50 doxorubicin 14.2 lM, IC50 cisplatin 17.8 lM, IC50 palitaxel 31.5 lM.

Not determined.

d

e

f

g

h

chain length of the 7-substituent is (extremely) impor-

tant for both antibacterial and cytotoxic activities. 7-Al-

lyl (3a), 7-benzyl (3b–d), and 7-geranyl derivatives (3e)

were more or less inactive against the bacteria and can-

cer cell lines examined. (2E,6E)-Farnesyl (3f) and the

isomeric (2E,6Z)-farnesyl derivatives (3g–h), on the

other hand, exhibited profound inhibitory activity

against M. tuberculosis and Staphylococcus aureus.

These compounds were also active against Escherichia

coli and even against Bacteroides fragilis and Bacteroides

thetaiotaomicron (anaerobe). The geometry of the terpe-

noid side chain appears to have no signiﬁcant inﬂuence

on antibacterial activity. Compounds 3f–h also dis-

played inhibitory activity against the cancer cell lines

examined. When the N-7 terpenoid side chain was ex-

tended even further to geranylgeranyl, cytotoxicity to-

ward the cancer cells increased whereas, the

antibacterial activities remained essentially unchanged

pound 3i were also determined (Table 2). Geranylgera-

nylpurine 3i was also active against Streptococcus

pyogenes and Enterococcus facealis, but not against

Pseudomonas aeruiginosa, in the concentration range

examined. Reduced inhibitory activity, especially

against bacteria, was found for the phytyl derivatives

3t–u compared to the geranylgeranylpurines 3i–j, indi-

cating that unsaturations in the side chain are important

for the bioactivities studied.

6

The identity of the N -substituent in compounds 3

also inﬂuences bioactivity. Comparison of the far-

nesyl derivatives 3g–h, the geranylgeranyl compounds

3i–k, and the phytylpurines 3t–u revealed that cyto-

toxicity toward cancer cells generally decreases when

a larger alkoxy substituent, benzyloxy or tert-butyl-

6

oxy, is introduced at N , whereas the antimycobacte-

rial activity is virtually unchanged and there are

only minor variations in activities against other

bacteria.

(

geranylgeranyl derivative 3i compared with (E,E)-far-

nesyl derivative 3f and (E,Z)-farnesyl derivative 3g).

Compound 3i exhibited profound activity against the

cancer cell lines and this agelasine analog was also a po-

tent inhibitor of ACHN (renal adenocarcinoma cells)

growth. Renal cell carcinomas (RCC) are quite resistant

6

When the N -alkoxy groups were reductively removed

(Scheme 1), compounds 5 were formed. These adenini-

um salts are structurally more closely related to natu-

rally occurring agelasines than compounds 3.

However, with the exception of the phytyl derivative

5e, substantially reduced activity toward both bacteria

and cancer cells was observed for compounds 5 com-

pared to analogous compounds 3.

1

2

to chemotherapy; hence it was especially intriguing to

ﬁnd that compound 3i was more eﬀective against the pri-

1

3

mary multidrug-resistant (MDR) cell line ACHN than

any of the anticancer drugs used as positive controls.

Antibacterial activities against other bacteria for com-

A. Vik et al. / Bioorg. Med. Chem. 15 (2007) 4016–4037

4023

We also examined if variations in the purine 9-substitu-

ent would aﬀect biological activities. The geranylgeranyl

derivative 3i, carrying a methyl group in the purine 9-

position, just as naturally occurring agelasines, was

compared with compounds 3l–3n, 3p, and 3q. With re-

spect to antimycobacterial activity, a variety of N-9

substituents were well tolerated and the 9-isopropyl

and 9-benzyl derivatives, 3l and 3p, are among the most

active antimycobacterials examined (MICs 1.56 lg/mL).

A negative inﬂuence on other antibacterial activity,

especially against E. coli, may be observed when the

N-9 substituent is increased. Cytotoxicity against cancer

cells is also generally high for the compounds in this ser-

ies. The isopropyl (3l), allyl (3m), benzyl (3p), and diphe-

nylmethylpurine 3q are all excellent inhibitors of ACHN

cell growth (IC₅₀< 5 lM). As mentioned above, this re-

nal cell line is regarded as drug resistant. The 9-cyano-

ethyl derivatives 3n and 3o were not among the most

active antimycobacterials. However, the cyanoethyl

group is easily removed, at least under basic conditions

(see Scheme 2 above). If this 9-substituent also is

cleaved to some extent during the antimycobacterial

testing, the less active compounds 6a and 6b (Table 3)

are formed.

Naturally occurring agelasines are 7,9-dialkylated puri-

nium salts just as are the analogs 3 and 5. We were curi-

ous to see if the mono-alkylated neutral purines 6, 7, and

9b also exhibited similar biological activities as the cat-

ionic species. The antibacterial and cytotoxic activities

found for these compounds are shown in Table 3. Some

antimycobacterial activity was observed at 6.25 lg/mL

for compounds 6 and 9b, and 7-geranylgeranyladenine

7

is actually among the most active inhibitors of M.

tuberculosis growth described in this study (MIC

.56 lg/mL). It is interesting to note that for the purini-

1

um salts 3 and 5, the free NH -group found in com-

2

pounds 5 generally resulted in low antimycobacterial

activity, whereas the primary amine 7 is substantially

more active than the N alkoxy derivatives 6. It is possi-

6

ble that a cationic charge is required for antimycobacte-

rial activity and that compound 7, but not the less basic

compounds 6, is protonated in the cell culture. However,

none of the compounds 6, 7 or 9b were active against S.

aureus or E. coli at 32 lg/mL. Compounds 6, 7, and 9b

exhibited signiﬁcant inhibitory activity against the can-

cer cell lines examined, but none of these mono N-alkyl-

ated purines could compete with the most active

geranylgeranyl purinium salts 3 (see Table 1).

Compounds 11 were designed to reveal if simple imi-

dazolium or benzimidazolium salts might exhibit the

same biological proﬁle as the purinium salts 3 and 5. Ta-

ble 4 displays antibacterial and anticancer activities

found for compounds 11. The N-benzyl-N-methyl

(

benz)imidazolium bromides 11a and 11b were only very

weak antimycobacterials, just as the benzyl derivatives

b–d and 5a, and 11a and 11b were not screened against

3

other bacteria or against cancer cells. The geranylgera-

nyl derivatives 11c and 11d displayed moderate antibac-

terial activity and fairly good activity against most of the

cancer cells. The benzimidazole 11d was generally the

most potent of these compounds.

4024

A. Vik et al. / Bioorg. Med. Chem. 15 (2007) 4016–4037

Compounds 13a and 13b (Table 5) are isomers of the

purinium salts 5a and 5d (Table 1). It was not unex-

pected that also the benzyl derivative 13a, just like all

other N-benzyl derivatives studied herein, was inactive

against the bacteria examined. The geranylgeranyl deriv-

ative 13b was found to be slightly more active against M.

tuberculosis and S. aureus than the isomer 5d, and far

less active against all cancer cells examined.

C. krusei, often in the same range as the reference drug

amphotericine B. Again the length of the terpenoid side

chain appears to be important for activity; the farnesyl

derivative 3f displays a somewhat higher MIC value

than the geranylgeranyl derivatives 3i–k. The detailed

structure of the heterocycle seems to be less important

since both agelasine derivatives 3i–k, imidazole and

benzimidazole analogs 11c–d, and the agelasimine deriv-

ative 15b are found to be strong inhibitors of fungal

growth.

Since we have demonstrated that analogs of agelasines

carrying much less complicated terpenoid side chains

may still be highly potent inhibitors of bacterial growth

and growth of cancer cells, we found it interesting to

examine if the same simpliﬁcation of agelasimine struc-

ture also would lead to compounds with potent biolog-

ical activity. Hence we prepared analogs of agelasimines

The mode of action for the agelasines and agelasimines

as antibacterials or cytotoxic compounds has not been

shown. However, our hypothesis is that these amphi-

philic compounds, carrying a hydrophilic ‘head’ (purine)

and a hydrophobic ‘tail’ (terpenoid chain), may very

well interact with cell membranes. We could detect no

changes in the morphology of C. krusei cells after treat-

ment with the compounds studied (antifungal results,

see Table 7). The morphology of yeast cells taken from

wells containing 50% of the MIC of each test substance

was investigated. Examination by phase contrast

microscopy revealed no diﬀerences between these cells

and those grown in the absence of agelasine analogs.

This may indicate that agelasines exert their eﬀect(s) in

a way, which does not aﬀect the gross cell morphology,

but a mechanism of action involving interactions with

cell membranes cannot be excluded. Several classes of

amphiphilic molecules, especially naturally occurring

(

compounds 15–18) and examined their ability to act as

antibacterials and inhibitors of cancer cells (Table 6). All

geranylgeranyl derivatives were profound inhibitors of

M. tuberculosis and S. aureus, and except for compound

1

8, the geranylgeranyl derivatives were also active

against E. coli in the concentration range examined.

Compounds 15b and 18 were also strong inhibitors of

cancer cells, whereas compounds 16 and 17 appear to

be much less cytotoxic.

It has been reported that agelasine B displays activity

against certain yeasts (Candida neoformans and to a les-

1

g

ser extent Candida albicans). To investigate if the agel-

asine and agelasimine analogs described herein possess

antimycotic activity, the control strain ATCC 6258 of

the clinically important yeast Candida krusei was in-

cluded in the study (Table 7). Although C. albicans is

considered to be the most pathogenic species, C. krusei

is increasingly associated with infections of both immu-

1

6

peptides and analogs, but also for instance cationic

1

7

stereoids, interact with cell membranes. It is claimed

that antibiotics that interrupt cell membranes may es-

cape most drug resistance mechanisms compared with

classical antibiotics that must penetrate the target

1

6b

cell.

Compounds that discriminate suﬃciently be-

1

4

nocompromised and non-immunocompromised hosts.

tween prokaryote and eukaryotes are highly interesting

as antibacterials. It is known that the lipid composition

in bacteria and eukaryote cells diﬀers signiﬁcantly. For

instance, bacterial membranes are negatively charged,

whereas the outer leaﬂets of mammalian cells are neutral

at physiological pH, and bacterial membranes do not

contain sterols. These facts may account for the selectiv-

ity observed for some of the agelasine and agelasimine

analogs described herein. Increased knowledge of inter-

action between the compounds studied and cell mem-

branes may eventually lead to development of

agelasines or agelasimine analogs with increased selec-

tivity. Of the molecules described herein, 7-geranylger-

anyladenine 7 is among the most interesting, with a

MIC = 1.56 lg/mL against M. tuberculosis. Further

modiﬁcations of the structure of 7 may indeed lead to

an improved antimycobacterial compound with less

cytotoxicity. It is also interesting to note that compound

C. krusei can show intrinsic and acquired resistance to

azole antifungals (ﬂuconazole and itraconazole); The

widespread use of ﬂuconazole has probably led to an in-

crease in C. krusei infections and patients receiving

ﬂuconazole prophylaxis are at risk of developing infec-

tions caused by ﬂuconazole-resistant C. krusei. Except

for compounds 5d and 7, both carrying free primary

amino group in the purine 7-positions, the compounds

examined displayed profound inhibitory activity against

1

5

a

Table 7. Antifungal activity of selected compounds against Candida

krusei

b

Compound

MIC C. krusei (lg/mL)

3

f

i

j

8.0

2.0

1.0

2.0

3k

7

is inactive against S. aureus and E. coli in the concen-

5

7

d

>16

4.0

tration range examined (MIC > 32 lg/mL for both bac-

teria). For treatment of mycobacterial infections,

11c

1

8

1

1d

5b

8

2.0

organism-speciﬁc agents are recommended.

16

a

4. Conclusions

Structures of compounds 3 and 5, see Scheme 1; compound 7, see

Scheme 2; compounds 11, see Scheme 4 and compounds 15 and 18,

see Scheme 6.

We have demonstrated that agelasine and agelasimine

derivatives with substantially less complicated terpenoid

b

MIC amphotericin B 1.0 lg/mL.

A. Vik et al. / Bioorg. Med. Chem. 15 (2007) 4016–4037

4025

6

b,29

side chains compared to the naturally occurring com-

pounds may still exhibit very interesting biological activ-

ities. These analogs are much easier to synthesize

compared to the natural products and they are therefore

more interesting as potential drugs. Several agelasine

analogs with excellent activity against cancer cell lines,

including a drug resistant renal cell line, have been iden-

tiﬁed. Furthermore, determination of the mechanism by

which these compounds act as antibiotics and further

functionalization of the agelasine analogs may also re-

sult in compounds with more speciﬁc antimicrobial

activity. The seemingly unavoidable development of

resistance against any antibacterial compound used clin-

ically calls for the development of new agents with novel

P. aeruginosa (ATCC 27853),

b

B. fragilis (DSM

6b

6

2151), B. thetaiotaomicron (DSM 2255), and C. kru-

3

0

sei (ATCC 6258), and activity against cancer cell lines

6

b

(U-937 GTB, RPMI 8226/s, CEM/s, and ACHN) were

determined as reported before.

5.1. (2E,6E)-1-Bromo-3,7,11-trimethyl-2,6,10-

dodecatriene

trans,trans-Farnesol (667 mg, 3.00 mmol) was dissolved

in dry diethyl ether (15 mL) under N before phospho-

2

rus tribromide (0.35 mL, 3.6 mmol) was added. The

mixture was stirred for 3 h at 0 ꢁC, diluted with diethyl

ether (25 mL), washed with satd aq NaHCO (2·

3

1

8

mechanisms and chemical structures. It is especially

intriguing to ﬁnd compounds with activity against M.

tuberculosis, since there have been launched no new

drugs to treat tuberculosis for approximately 40 years,

even though the disease claims ca. 2 million lives every

year, and infections with multidrug resistant strains

10 mL) and satd aq NaCl (10 mL), dried (MgSO₄),

and evaporated in vacuo; yield 630 mg (74%), yellow

oil. The crude allylic bromide was used directly in the

1

next step. H NMR (CDCl , 200 MHz) d 1.64 (s, 6H,

3

2· CH ), 1.72 (s, 3H, CH ), 1.78 (s, 3H, CH ), 1.90–

3

2.20 (m, 8H, 4· CH ), 4.06 (d, J = 8.4 Hz, 2H, CH Br),

2

1

9

are an increasing problem.

5.13 (m, 2H, 2· CH@), 5.53 (t, J = 8.4 and 1.3 Hz, 1H,

CH@).

5. Experimental

5.2. (2E,6Z)-1-Bromo-3,7,11-trimethyl-2-dodecatriene

1

Melting points are uncorrected. The H NMR spectra

were recorded at 500 MHz with a Bruker Avance

DRX 500 instrument, 300 at 300 MHz with a Bruker

Avance DPX 300, or at 200 MHz with a Bruker Avance

cis-Nerolidol (7.0 mmol, 1.56 g) was dissolved in dry

diethyl ether (25 mL) and pyridine (7.0 mmol,

0.57 mL) at ꢀ35ꢁC under N , before phosphorus tribro-

2

mide (9.8 mmol, 0.93 mL) was added dropwise, and the

solution was stirred for 1 h. The mixture was diluted

with diethyl ether (80 mL) and aq HCl (10%, 30 mL).

The phases were separated and the organic phase was

washed with aq HCl (10%, 30 mL) and satd aq NaHCO₃

1

3

DPX 200 instrument. The decoupled C NMR spectra

were recorded at 125, 75 or 50 MHz using the instru-

ments mentioned above. All spectra were obtained at

ambient temperature. Chemical shifts (d) are given in

ppm downﬁeld from tetramethylsilane. Mass spectra un-

der electron impact conditions were recorded with a VG

Prospec instrument at 70 eV ionizing voltage and are

presented as m/z (% rel. int.). Electrospray MS spectra

were recorded with a Bruker Apex 47e FT-ICR mass

spectrometer. Elemental analyses were performed by

Ilse Beetz Mikroanalytisches Laboratorium, Kronach,

Germany. As is typical of the class, some of the agela-

sine and agelasimine analogs prepared in this work ana-

(30 mL), dried with MgSO , ﬁltered, and evaporated in

4

vacuo; yield 1.762 g (88%), pale yellow oil (2E/2Z ratio

1

3:1). H NMR (CDCl , 200 MHz) d 1.65 (s, 3H, CH ),

3

1.65 (s, 6H, 2· CH ), 1.81 (s, 3H, CH ), 1.95–2.15 (m,

3

8H, 4· CH ), 4.06 (d, J = 8.5 Hz, 2H, CH Br), 5.14

2

(m, 2H, 2· CH@), 5.69 (m, 1H, CH@).

5.3. (E)-1-Bromo-3,7,11,15-tetramethyl-2-hexadecene

6

,7

lyze poorly by this microanalytical method; however,

NMR analysis demonstrated that these compounds

were greater than 97% pure. DMA, DMF, and n-buta-

nol were distilled from BaO and stored over molecular

sieves (3 A). Dry diethyl ether was obtained by distilla-

tion from Na/benzophenone. Triethylamine, acetoni-

trile, and pyridine were distilled from CaH and stored

2

˚

over molecular sieves (3 A). Methanol was saturated

with NH by bobbling NH (g) in the methanol at ambi-

3

An isomeric mixture of phytol (about 65:35 mixture of E

and Z isomers) was puriﬁed by ﬂash chromatography on

silica gel eluting with hexane/EtOAc/EtOH (50:3:1).

Fractions containing more than 90% pure (E)-isomers

were combined and puriﬁed once more using the same

ﬂash chromatography system. Pure fractions were col-

lected to give (E)-3,7,11,15-tetramethyl-2-hexadecen-1-

˚

ol, (E/Z ratio 99:1; GC). From 1.6 g isomeric mixture,

0.57 g pure E isomers were obtained. H NMR (CDCl ,

1

3

ent temperature for ca. 5 min. Silica gel for ﬂash chro-

matography was purchased from Merck, Darmstadt,

Germany (Merck No. 09385). O-tert-Butylhydroxyl-

amine hydrochloride was synthesized according to the

literature from N-(tert-butoxy)phthalimide. Com-

pounds available by literature methods: (2E,6E,10E)-1-

300 MHz) d 0.81–0.85 (m, 12H, CH ), 1.04–1.50 (m,

3

13H), 1.64 (s, 3H, CH ), 1.95 (t, J = 7.7 Hz, 1H, CH ),

4.12 (d, J = 6.7 Hz, 2 H, CH OH), 5.40 (t, J = 6.7 Hz,

3

2

1H, CH@). The isomeric mixture of (E)-phytol was dis-

solved in diethyl ether (4 mL) and stirred at 0 ꢁC under

2

0

21

N . Phosphorus tribromide (0.063 mL, 0.68 mmol) in

2

7

bromo-3,7,11,15-tetramethyl-2,6,10,14-hexadecatetraene,

1

hexane (1 mL) was added, and the resulting mixture

was stirred for 20 min. The colorless precipitate was re-

moved by ﬁltration and washed with hexane (25 mL).

The organic phase was washed with aq HCl (2 M,

1

7

22

23

24

25

methylaquacobaloxime,

1a, 1b,

1c, 1d,

1e,

6b

f, 2a, 2b, , 2c, 2g, 2j, 3a, 3b, 3c, 3d,

2

6

7

6b

24

7

6b

1

7

27

3

M. tuberculosis H Rv (ATCC 27294),

e, 3i, 3s, 5d, 12. Antimicrobial activities against

7

,28

S. pyogenes

6b,29

E. faecalis (ATCC 700802),

5 mL), satd aq NaHCO (5 mL) and satd aq NaCl (2·

37

b,29

3

6

(

ATCC 19615),

10 mL), dried over MgSO , ﬁltered, and evaporated in

4

4026

A. Vik et al. / Bioorg. Med. Chem. 15 (2007) 4016–4037

vacuo to give the crude allylic bromide; yield 463 mg

(

61.78 (CH O), 119.0 (CN and C-5), 139.08 (C-8)

3

1

95%), which was used directly in the next step. H

141.77 (C-4/C-6), 141.80 (C-4/C-6), 145.11 (C-2); MS

EI m/z (rel.%) 218 (27, M ), 188 (56), 173 (31), 135

+

NMR (CDCl , 200 MHz) d 0.87–0.92 (m, 12H, CH ),

3

1

J = 7.6 Hz, 2H, CH ), 4.07 (d, J = 8.4 Hz, 2H, CH Br),

.0–1.7 (m, 19H), 1.75 (s, 3H, CH₃), 2.09 (t,

(100), 108 (39); Anal. Calcd for C H N O: C, 49.54;

9

H, 4.62; N, 38.51. Found: C, 49.54; H, 4.67; N, 38.44%.

10

6

2

5

.57 (m, J = 8.4 and 1.3 Hz, 1H, CH@).

5

.7. 9-Benzyl-N-methoxy-9H-purin-6-amine (2h)

5

.4. N-Methoxy-9-isopropyl-9H-purin-6-amine (2d)

The product was prepared from 9-benzyl-6-chloro-9H-

purine 1e (600 mg, 2.50 mmol) as described for com-

pound 2d above; yield 370 mg (64%), mp 216–219 ꢁC, col-

orless crystals. The compound exists as a mixture of imino

and amino tautomers in DMSO, NMR resonances for the

A mixture of 6-chloro-9-isopropyl-9H-purine 1b (200 mg,

.00 mmol), triethylamine (1.5 mL, 10.8 mmol), and

O-methylhydroxylamine hydrochloride (400 mg,

.00 mmol) in dry n-butanol (10 mL) was stirred at reﬂux

1

5

1

for 17 h and evaporated in vacuo. The product was puri-

ﬁed by ﬂash chromatography on silica gel eluting with

EtOH/EtOAc/CH Cl (1:5:4); yield 119 mg (57%), mp

imino form are given below. H NMR (DMSO-d₆,

500 MHz) d 3.74 (s, 3H, CH ), 5.26 (s, 2H, CH ), 7.24–

7.34 (m, 5H, Ph), 7.57 (s, 1H, H-2), 7.95 (s, 1H, H-8),

3

2

1

3

1

78–180 ꢁC. The compound exists as a mixture of imino

and amino tautomers in DMSO, NMR resonances for

11.20 (s, 1H, NH); C NMR (DMSO-d , 125 MHz) d

6

46.00 (CH ), 60.89 (CH ), 118.12 (C-5), 127.36 (CH in

2

3

1

the imino form are given below. H NMR (DMSO-d ,

Ph), 127.72 (CH in Ph), 128.67 (CH in Ph), 137.12 (C in

Ph), 138.41 (C-8), 141.03 (C-4/C-6), 141.23 (C-4/C-6),

144.22 (C-2); MS EI m/z (rel.%) 255 (18, M ), 224 (42),

6

3

OCH ), 4.61 (m, 1H, i-Pr), 7.71 (s, 1H, H-2), 8.01

00 MHz) d 1.46 (d, J = 6.8 Hz, 6H, i-Pr), 3.74 (s, 3H,

+

3

1

3

(

s, 1H, H-8), 11.0 (br s, NH); C NMR (DMSO-d₆,

210 (7), 91 (100); HRMS C H N O requires 255.1120,

13

5

1

25 MHz) d 22.32 (CH in i-Pr), 46.51 (CH in i-Pr),

found 255.1116.

3

6

and C-6), 146.59 (C-2); MS EI m/z (rel.%) 207 (62, M ),

1.41 (OCH ), 118.97 (C-5), 138.61 (C-5), 143.61 (C-4

3

+

5.8. 9-(Diphenylmethyl)-N-methoxy-9H-purin-6-amine

(2i)

1

77 (31), 165 (14), 162 (25), 150 (12), 135 (100), 120

31), 108 (59); HRMS C H N O requires 207.1120,

(

found 207.1119.

9

13

5

The product was prepared from 6-chloro-9-(diphenylm-

ethyl)-9H-purine 1f (642 mg, 2.00 mmol) as described

for compound 2d above and puriﬁed by ﬂash chroma-

tography on silica gel eluting with EtOAc; yield 83 mg

(43%), mp 232–234 ꢁC, colorless crystals. The com-

pound exists as a mixture of imino and amino tautomers

5

.5. 9-Allyl-N-methoxy-9H-purin-6-amine (2e)

The product was prepared from 9-allyl-6-chloro-9H-

purine 1c (427 mg, 2.17 mmol) as described for com-

pound 2d above; yield 245 mg (55%), mp 183–185 ꢁC.

The compound exists as a mixture of imino and amino

in DMSO, NMR resonances for the imino form is given

1

below. H NMR (DMSO-d , 300 MHz) d 3.75 (s, 3H,

6

tautomers in DMSO, NMR resonances for the imino

form are given below. H NMR (DMSO-d , 500 MHz)

CH O), 6.94 (s, 1H, CHPh ), 7.19 (m, 4H, Ph), 7.36

(m, 6H, Ph), 7.55 (s, 1H, H-2), 7.62 (s, 1H, H-8), 11.23

3

2

1

6

1

3

d 3.74 (s, 3H, CH ), 4.67 (br d, J = 5.4 Hz, 2H,

(br s, 1H, NH); C NMR (DMSO-d , 75 MHz) d

3

6

NCH ), 4.98 (dd, J = 17.1 and 1.3 Hz, 1H in @CH ),

60.86 (CHPh ), 60.91 (CH O), 118.33 (C-5), 127.97 (4·

2

3

5

.16 (dd, J = 10.3 and 1.3 Hz, 1H in @CH ), 5.96–6.07

CH, Ph), 128.06 (2· CH, Ph), 128.77 (4· CH, Ph)

137.28 (C-8), 138.84 (2· C, Ph), 140.94 (C-6), 141.35

(C-4), 144.29 (C-2); MS EI m/z (rel.%) 331 (21, M ),

2

(

m, 1H, @CH), 7.55 (br s, 1H, H-2), 7.80 (s, 1H, H-8),

13

+

1

1.18 (br s, 1H, NH);

C

NMR (DMSO-d ,

6

25 MHz) d 45.00 (CH ), 60.88 (CH ), 117.36 (@CH ),

301 (15), 167 (100), 165 (28), 152 (15); HRMS

C H N O requires 331.1433, found 331.1430.

2

3

2

17.96 (C-5), 133.58 (@CH), 138.23 (C-8), 141.06 (C-4/

1

9

17

5

C-6), 141.16 (C-4/C-6), 144.05 (C-2); MS EI m/z

+

0

(

rel.%) 205 (79, M ), 190 (6), 175 (54), 174 (100), 160

45), 159 (15), 148 (21), 147 (24); HRMS C H N O

requires 205.0964, found 205.0957.

5.9. (2 E,6 E)-6-Methoxyamino-9-methyl-7-(3,7,11-tri-

methyl-2,6,10-dodecatrienyl)-7H-purinium (3f) and

9

11

5

0

(2 E,6 E)-N-methoxy-9-methyl-N-(3,7,11-trimethyl-

,6,10-dodecatrienyl)-9H-purin-6-amine (4f)

2

5

.6. 6-Methoxyamino-9H-purine-9-propanenitrile (2f)

(2E,6E)-1-Bromo-3,7,11-trimethyl-2-dodecene (630 mg,

2.10 mmol) was added to a solution of purine 2a

(179 mg, 1.00 mmol) in dry DMA (2 mL) and the result-

The product was prepared from 6-chloro-9H-purine-9-

propanenitrile 1d (620 mg, 3.00 mmol) as described for

compound 2d above and puriﬁed by ﬂash chromatogra-

phy on silica gel eluting with EtOH/EtOAc (1:4) fol-

lowed by recrystallization from EtOAc; yield 340 mg

ing mixture was stirred at 50 ꢁC under N overnight.

2

The solvent was removed in vacuo and the residue was

puriﬁed by ﬂash chromatography on silica gel eluting

with EtOAc followed by EtOH/EtOAc (1:15) and

CH Cl /MeOH (satd NH ) (10:1).

(51%), mp 184–185 ꢁC, colorless crystals. The com-

pound exists as a mixture of imino and amino tautomers

2

3

in DMSO, NMR resonances for the imino form is given

1

below. H NMR (DMSO-d , 300 MHz) d 3.11 (m, 2H,

Compound 3f: Yield 205 mg (53%), yellow wax.

H

6

CH ), 3.79 (s, 3H, CH O), 4.34 (m, 2H, CH ), 7.62 (br

NMR (CDCl , 300 MHz) d 1.60 (s, 6H, CH ), 1.70 (s,

3H, CH ), 1.82 (s, 3H, CH ), 1.97–2.20 (m, 8H), 3.78

3 3

(s, 3H, CH ), 3.83 (s, 3H, CH ), 5.06 (m, 4H, CH N

3 3 2

2

3

2

1

3

s, 1H) 7.91 (br s, 1H), 11.2 (br s, 1H, NH); C NMR

DMSO-d , 75 MHz) d 19.38 (CH ), 38.69 (CH ),

(

6

2

A. Vik et al. / Bioorg. Med. Chem. 15 (2007) 4016–4037

4027

1

and 2· CH@), 5.45 (t, J = 6.8 Hz, 1H, CH@), 7.81 (s,

Compound 4g: Yield 111 mg (29%), colorless oil. H

NMR (CDCl , 300 MHz) d 1.56 (s, 3H, CH ), 1.62 (s,

1

3

1

7

2

3

H, H-2), 8.17 (s, 1H, H-8);

C NMR (CDCl ,

3

5 MHz) d 16.48 (CH ), 17.23 (CH ), 18.08 (CH ),

3H, CH ), 1.65 (s, 3H, CH ), 1.77 (s, 3H, CH ), 1.9–

2.2 (m, 8H, 4· CH ), 3.82 (s, 3H, NCH ), 3.93 (s, 3H,

3

6.08 (CH ), 26.44 (CH ), 27.07 (CH ), 31.51 (NCH ),

9.86 (CH ), 40.12 (CH ), 48.11 (NCH ), 61.92

3

2

3

2

3

OCH ), 4.73 (d, J = 6.8 Hz, 2H, CH N), 5.05 (m, 2H,

3 2

2

(

1

OCH ), 109.98 (C-5), 116.40 (CH@), 123.59 (CH@),

2· CH@), 5.43 (t, J = 6.8 Hz, 1H, CH@), 7.80 (s, 1H,

3

1

3

24.44 (CH@), 129.91 (C-8), 131.92 (C@), 136.33

H-2), 8.47 (s, 1H, H-8); C NMR (CDCl , 75 MHz) d

3

(

C@), 145.36 (C-4), 146.05 (C@), 147.56 (C-6), 157.05

16.86 (CH ), 18.01 (CH ), 23.69 (CH ), 26.07 (CH ),

26.06 (CH ), 26.49 (CH ), 26.94 (CH ), 30.13 (NCH ),

2 2 2 3

3

(C-2); HRMS (ESI) C H N O requires 384.2757,

found 384.2773.

2

33

5

32.32 (CH ), 40.31 (CH ), 48.67 (CH N), 62.92

2 2 2

(

OCH ), 118.92 (CH@), 119.63 (C-5), 124.68 (CH@),

3

1

Compound 4f: Yield 112 mg (29%), colorless oil. H

NMR (CDCl , 300 MHz) d 1.57 (s, 3H, CH ), 1.59 (s,

125.07 (CH@), 131.86 (C@), 135.67 (C@), 140.75

(C@), 141.31 (C-2), 152.09 (C-4), 152.69 (C-8), 156.40

(C-6); MS EI m/z (rel.%) 383 (3, M ), 353 (10), 352

3

+

3

H, CH ), 1.68 (s, 3H, CH ), 1.79 (s, 3H, CH ), 1.90–

3

2

OCH ), 4.76 (d, J = 6.8 Hz, 2H, NCH ), 5.07 (m, 2H,

.11 (m, 8H, CH ), 3.84 (s, 3H, NCH ), 3.95 (s, 3H,

(19), 246 (98), 216 (100), 179 (38), 162 (21), 150 (26),

149 (33); HRMS (ESI) C H N O requires 383.2685,

found 383.2671.

2

3

2

22 33

5

CH@), 5.46 (t, J = 6.8 Hz, 1H, CH@), 7.82 (s, 1H, H-8),

1

3

8

.49 (s, 1H, H-2); C NMR (CDCl , 75 MHz) d 16.36

3

0

(

CH ), 16.92 (CH ), 18.04 (CH ), 26.06 (CH ), 26.27

CH ), 26.69 (CH ), 27.08 (CH ), 30.16 (NCH ), 40.03

2 2 2 3

5.11. (2 E,6 Z)-6-Benzyloxyamino-9-methyl-7-(3,7,11-

trimethyl-2,6,10-dodecatrienyl)-7H-purinium (3h) and

3

0

(

CH ), 40.05 (CH ), 48.65 (NCH ), 62.92 (OCH ),

2

(2 E,6 Z)-N-benzyloxy-9-methyl-N-(3,7,11-trimethyl-

2,6,10-dodecatrienyl)-9H-purin-6-amine (4h)

2

3

1

18.84 (CH@), 119.55 (C-5), 124.26 (CH@), 124.72

(

CH@), 131.62 (C@), 135.57 (C@), 140.87 (C@), 141.35

C-8), 152.06 (C-4), 152.64 (C-2), 156.28 (C-6); MS EI

The products were prepared from purine 2b (393 mg,

1.63 mmol) and (2E,6Z)-1-bromo-3,7,11-trimethyl-

2,6,10-dodecatriene (695 mg, 2.44 mmol; 2E/2Z-ratio

75:25) as described for compounds 3f and 4f above.

The products were separated by ﬂash chromatography

on silica gel eluting with EtOAc/hexane (4:1) followed

by CH Cl /MeOH (satd NH ) (12:1). Compound 3f

+

m/z (rel.%) 386 (6, M ), 352 (16), 247 (17), 246 (100),

216 (20), 215 (20), 179 (30), 162 (5), 150 (10), 149 (12);

HRMS (EI): C H N O requires 383.2685, found

3

8

2

33

5

83.2686. Anal. Calcd for C H N O: C, 68.90; H,

22 33 5

.67. Found: C, 68.14; H, 8.40%.

2

3

0

5.10. (2 E,6 Z)-6-Methoxyamino-9-methyl-7-(3,7,11-tri-

methyl-2,6,10-dodecatrienyl)-7H-purinium (3g) and

was further puriﬁed by ﬂash chromatography eluting

with CH Cl /MeOH (satd NH ) (12:1), and compound

4f by ﬂash chromatography eluting with EtOAc/hexane

2

3

0

(

2

2 E,6 Z)-N-methoxy-9-methyl-N-(3,7,11-trimethyl-

,6,10-dodecatrienyl)-9H-purin-6-amine (4g)

(4:1).

(

2

(

2E,6Z)-1-Bromo-3,7,11-trimethyl-2-dodecene (570 mg,

.00 mmol) was added to a solution of purine 2a

179 mg, 1.00 mmol) in dry DMA (2 mL) and the result-

Compound 3h: Yield 427 mg (59%), yellow wax, 2E/

1

2Z-ratio 75:25. H NMR (CDCl , 300 MHz) d 1.60 (s,

3

3H, CH ), 1.67 (s, 3H, CH ), 1.69 (s, 3H, CH ), 1.79

(s, 3H, CH ), 1.95–2.22 (m, 8H, CH ), 3.80 (s, 3H,

3 2

3

ing mixture was stirred at 50 ꢁC under N overnight.

The solvent was removed in vacuo and the products

were separated by ﬂash chromatography on silica gel

eluting with EtOAc followed by EtOAc/EtOH/MeOH

(

NH ) (160:10:5). Compound 3g was puriﬁed further by

ﬂash chromatography eluting with CH Cl /MeOH/

2

CH ), 4.98 (s, 2H, CH O), 4.99 (m, 2H, NCH ), 5.08

3 2 2

(m, 2H, CH@), 5.44 (t, J = 7.0 Hz, 1H, CH@), 7.16

(m, 2H, Ph), 7.28 (m, 3H, Ph), 7.81 (s, 1H, H-2), 9.31

1

3

satd NH ) (200:10:5) and CH Cl /MeOH/MeOH (satd

(s, 1H, H-8); C NMR (CDCl , 75 MHz) d 17.27

3

2

3

(CH ), 18.07 (CH ), 23.84 (CH ), 26.14 (CH ), 26.30

(CH ), 26.95 (CH ), 31.66 (NCH ), 32.40 (CH ), 40.16

2 2 3 2

3

2

MeOH (satd NH ) (150:10:5) and compound 4g was

3

(CH ), 48.00 (CH N), 76.22 (OCH ), 110.23 (C-5),

2 2 2

puriﬁed by ﬂash chromatography eluting with EtOH/

EtOAc (1:25).

116.76 (CH@), 124.53 (2· CH@), 127.59 (CH in Ph),

128.25 (2· CH in Ph), 128.80 (2· CH in Ph), 132.05

(

C@), 134.28 (C-8), 136.24 (C@), 138.59 (C in Ph),

1

Compound 3g: Yield 218 mg (57%), yellow wax. H

143.03 (C-6), 143.74 (C-4), 145.61 (C@), 153.60 (C-2);

MS EI m/z (rel.%) 459 (10, M ), 322 (40), 284 (21),

+

NMR (CDCl , 300 MHz) d 1.59 (s, 3H, CH ), 1.66 (s,

3

2

H, CH ), 1.69 (s, 3H, CH ), 1.79 (s, 3H, CH ), 1.9–

.2 (m, 8H, 4· CH ), 3.76 (s, 3H, NCH ), 3.83 (s, 3H,

255 (48), 238 (28), 216 (85), 174 (42), 149 (27), 134

(14), 107 (24), 91 (100); HRMS (EI) C H N O requires

459.2998, found 459.3018.

3

2

3

28 37

5

OCH ), 5.05 (m, 4H, CH N and 2· CH@), 5.43 (t,

3

2

J = 7.5 Hz, 1H, CH@), 7.80 (s, 1H, H-2), 7.95 (s, 1H,

1

3

H-8); C NMR (CDCl , 75 MHz) d 17.15 (CH ),

Compound 4h: Yield 113 mg (15%), colorless oil, 2E/

3

1

2

4

8.04 (CH ), 23.83 (CH ), 26.10 (CH ), 26.23 (CH ),

6.91 (CH ), 31.43 (NCH ), 32.39 (CH ), 40.11 (CH ),

2Z-ratio 75:25. H NMR (CDCl , 300 MHz) d 1.58 (s,

3

2

3

3H, CH ), 1.63 (s, 3H, CH ), 1.67 (s, 3H, CH ), 1.72

(s, 3H, CH ), 1.95–2.15 (m, 8H, CH ), 3.84 (s, 3H,

3 2

2

3

2

3

8.09 (CH N), 61.87 (OCH ), 109.88 (C-5), 116.50

2

3

(

1

CH@), 124.42 (CH@) 124.45 (CH@), 129.37 (C-8),

NCH ), 4.67 (d, J = 7.0 Hz, 2H, NCH ), 5.10 (m, 2H,

3 2

32.13 (C@), 136.47 (C@), 145.58 (C-4), 145.70 (C@),

CH@), 5.17 (s, 2H, CH O), 5.45 (t, J = 7 Hz, 1H,

2

148.21 (C-6), 157.48 (C-2); HRMS (ESI) C H N O+H

requires 384.2757, found 384.2771.

CH@), 7.32–7.43 (m, 3H, Ph), 7.55–7.60 (m, 2H, Ph),

7.83 (s, 1H, H-8), 8.52 (s, 1H, H-2); C NMR (CDCl₃,

2

33

5

1

3

4028

A. Vik et al. / Bioorg. Med. Chem. 15 (2007) 4016–4037

7

5 MHz) d 16.92 (CH ), 18.01 (CH ), 23.72 (CH ), 26.10

for C H N O: C, 75.10; H, 8.59; N, 13.27. Found:

33 45 5

3

(

CH ), 26.54 (CH ), 26.97 (CH ), 30.16 (NCH ), 32.34

C, 75.01; H, 8.60; N, 13.22%.

3

2

3

(

1

(

1

CH ), 40.37 (CH ), 49.87 (NCH ), 77.65 (CH O),

2 2 2 2

0

18.98 (CH@), 119.97 (C-5), 124.72 (CH@), 125.11

5.13. (2 E,6E,10 E)-6-tert-Butoxyamino-9-methyl-7-

(3,7,11,15-tetramethyl-2,6,10,14-hexadecatetraenyl)-

7H-purinium (3k)

CH@), 128.11 (2· CH, Ph), 130.11 (2· CH, Ph),

30.19 (CH, Ph), 131.89 (C@), 135.70 (C@), 136.45

(

C@), 140.58 (C@), 141.29 (C-8), 152.17 (C-4), 152.70

C-2), 157.00 (C-6); MS EI m/z (rel.%) 459 (16, M ),

+

The product was prepared from purine 2c (73 mg,

0.33 mmol) and (2E,6E,10E)-1-bromo-3,7,11,15-tetra-

methyl-2,6,10,14-hexadecatetraene (140 mg, 0.40 mmol)

as described for compound 3f above, and puriﬁed by

ﬂash chromatography on silica gel eluting with

CH Cl /MeOH (satd NH ) (10:1); yield 113 mg (69%),

3

(

52 (14), 323 (27), 322 (100), 255 (39), 238 (15), 223

19), 216 (31), 164 (21), 91 (45); HRMS (EI):

C H N O requires 459.2998, found 459.3019.

2

8

37

5

0

5

(

.12. (2 E,6E,10 E)-6-Benzyloxyamino-9-methyl-7-

3,7,11,15-tetramethyl-2,6,10,14- hexadecatetraenyl)-7H-

2

3

1

colorless wax. H NMR (CDCl , 300 MHz) d 1.26 (s,

3

0

purinium (3j) and (2 E,6 ,10 E)-N-benzyloxy-9-methyl-N-

3,7,11,15-tetramethyl-2,6,10,14-hexadecatetraenyl)-9H-

purin-6-amine (4j)

9H, t-Bu), 1.56 (s, 9H, CH ), 1.64 (s, 3H, CH ), 1.78

(s, 3H, CH ), 1.95–2.08 (m, 12H, CH ), 3.84 (s, 3H,

3 2

3

(

NCH ), 5.09 (m, 5H, NCH and 3· CH@), 5.52 (t,

3

2

J = 6.8 Hz, 1H, CH@), 7.80 (s, 1H, H-2), 8.56 (s, 1H,

1

3

The products were prepared from purine 2b (153 mg,

0

methyl-2,6,10,14-hexadecatetraene (338 mg, 1.09 mmol)

as described for compounds 3f and 4f above, and

puriﬁed by ﬂash chromatography on silica gel eluting

with EtOAc/hexane (1:1) followed by EtOAc/MeOH

H-8); C NMR (CDCl , 50 MHz) d 15.96, 16.03,

3

.60 mmol) and (2E,6E,10E)-1-bromo-3,7,11,15-tetra-

16.92, 17.63, 25.64, 26.04, 26.59, 26.69, 27.66 (3· CH ,

3

t-Bu), 31.18 (NCH ), 39.42, 39.66, 39.68, 47.61

3

(NCH ), 77.10 (C, t-Bu), 110.67 (C-5), 116.23 (CH@),

2

123.20 (CH@), 123.91 (CH@), 124.26 (CH@),

130.33 (C-8), 131.24 (C@), 135.1 (C@), 135.88 (C@),

144.20 (C-6), 144.40 (C-4), 145.48 (C@), 155.87 (C-2);

HRMS (ESI) C H N O requires 494.3853, found

(

satd NH ) (20:1) and CH Cl /MeOH (satd NH )

2

3

2

3

15:1).

3

0

47

5

4

94.3849.

1

Compound 3j: Yield 222 mg (70%), yellow wax.

H

0

NMR (CDCl , 300 MHz) d 1.60 (s, 9H, CH ), 1.68 (s,

5.14. (2 E,6E,10 E)-6-Methoxyamino-9-isopropyl-7-

(3,7,11,15-tetramethyl-2,6,10,14-hexadecatetraenyl)-

7H-purinium (3l)

3

CH ), 4.94 (d, J = 7.3 Hz, 2H, NCH ), 5.04 (s, 2H,

H, CH ), 1.95–2.20 (m, 12H, CH ), 3.61 (s, 3H,

3

2

3

2

CH O), 5.09 (m, 3H, CH@), 5.37 (t, J = 7.2 Hz, 1H,

2

CH@), 7.28 (m, 3H, Ph), 7.43 (d, J = 7.2 Hz, 2H, Ph),

The product was prepared from purine 2d (119 mg,

0.60 mmol) and (2E,6E,10E)-1-bromo-3,7,11,15-tetra-

methyl-2,6,10,14-hexadecatetraene (274 mg, 0.80 mmol)

as described for compound 3f above, and puriﬁed by

ﬂash chromatography on silica gel eluting with

CH Cl /MeOH (satd NH ) (17:1). Compound 4l was

1

3

7

.81 (s, 1H), 7.83 (s, 1H); C NMR (CDCl , 75 MHz)

3

d 16.42 (CH ), 16.52 (CH ), 17.13 (CH ), 18.08 (CH ),

3

2

3

4

6.09 (CH ), 26.51 (CH ), 27.04 (CH ), 27.14 (CH ),

3 2 2 2

1.22 (NCH ), 39.84 (CH ), 40.11 (CH ), 40.16 (CH ),

3

2

7.92 (NCH ), 75.95 (CH O), 109.94 (C-5), 116.51

2

3

(

1

CH@), 123.64 (CH@), 124.32 (CH@), 124.70 (CH@),

27.52 (CH, Ph), 128.31 (2· CH, Ph), 128.91 (2· CH,

also formed but not isolated in pure form.

1

Compound 3l: Yield 138 mg (48%), yellow oil. H NMR

Ph), 129.10 (C-8), 131.71 (C@), 135.60 (C@), 136.35

(

C@), 140.13 (C@), 145.60 (C-4), 145.78 (C@), 148.46

(CDCl , 300 MHz) d 1.48–1.58 (m, 15H), 1.69–1.73 (m,

3

(

C-6), 157.70 (C-2); HRMS (ESI) C H N O requires

28.3696, found 528.3715.

7H, i-Pr), 1.95–2.04 (m, 12H), 3.73 (s, 3H, OCH ), 5.01–

5.07 (m, 4H), 5.39 (m, 1H), 7.75 (s, 1H, H-8), 8.07 (s,

3

45

5

3

5

1

3

1

H, H-2); C NMR (CDCl , 75 MHz) d 16.37 (2· C),

3

1

Compound 4j: Yield 45 mg (14%), colorless oil.

NMR (CDCl , 300 MHz) d 1.57 (s, 3H, CH ), 1.59 (s,

3

(

NCH ), 4.67 (d, J = 7.0 Hz, 2H, NCH ), 5.10 (m, 3H,

H

17.28 (2· C), 18.03, 22.50 (CH , i-Pr), 26.05, 26.78,

3

27.01, 39.88, 40.08 (2· C), 48.01 (NCH ), 61.79

3

2

H, CH ), 1.61 (s, 3H, CH ), 1.68 (s, 3H, CH ), 1.75

(OCH ), 110.41 (C-5), 123.30 (2· CH@), 125.15 (2·

3

s, 3H, CH ), 1.90–2.12 (m, 12H, CH ), 3.85 (s, 3H,

CH@), 131.61, 135.40 (2· C), 136.30, 145.30 (C-4),

3

2

149.03, 157.56 (C-2); HRMS (ESI) C H N O+H re-

quires 480.3696, found 480.3690.

3

2

29 45

5

3

· CH@), 5.45 (t, J = 7.0 Hz, 1H, CH@), 7.35–7.42

m, 3H, Ph), 7.58 (m, 2H, Ph), 7.83 (s, 1H, H-8), 8.52

(

1

3

0

s, 1H, H-2); C NMR (CDCl , 75 MHz) d 16.38 (2·

5.15. (2 E,6E,10 E)-9-Allyl-6-methoxyamino-7-

(3,7,11,15-tetramethyl-2,6,10,14-hexadecatetraenyl)-

7H-purinium (3m)

3

CH ), 16.98 (CH ), 18.07 (CH ), 26.08 (CH ), 26.77

(

3

CH ), 27.02 (CH ), 27.07 (CH ), 27.16 (CH ), 30.14

2 2 2 2

(

NCH ), 40.06 (CH ), 40.11 (CH ), 49.84 (NCH ),

3 2 2 2

7

7.65 (CH O), 118.91 (CH@), 119.96 (C-5), 124.33

The product was prepared from purine 2e (200 mg,

0.98 mmol) and (2E,6E,10E)-1-bromo-3,7,11,15-tetra-

methyl-2,6,10,14-hexadecatetraene (465 mg, 1.40 mmol)

as described for compound 3f above, and puriﬁed

by ﬂash chromatography on silica gel eluting with

CH Cl /MeOH (satd NH ) (17:1). Compound 4m

2

(

CH@), 124.62 (CH@), 124.80 (CH@), 128.74 (3· CH,

Ph), 130.11 (2· CH, Ph), 131.61 (C@), 135.27 (C@),

1

8

35.81 (C@), 136.46 (C, Ph), 140.66 (C@), 141.29 (C-

), 152.17 (C-4), 152.69 (C-2), 156.99 (C-6); MS CI m/

+

z (rel.%) 527 (2, M ), 458 (3), 322 (36), 284 (11), 255

11), 216 (100), 162 (14), 150 (17), 91 (11); Anal. Calcd

2

3

(

was also formed but not isolated in pure form.

A. Vik et al. / Bioorg. Med. Chem. 15 (2007) 4016–4037

4029

1

Compound 3m: Yield 224 mg (48%), yellow oil.

H

459 (7), 285 (93), 255 (100), 218 (35), 189 (18), 135

(14); HRMS (EI) C H N O requires 490.3420, found

490.3409.

NMR (CDCl , 300 MHz) d 1.52–1.63 (m, 15H), 1.78

3

29 42

6

(

s, 3H, CH ), 1.98–2.12 (m, 12H), 3.77 (s, 3H, OCH ),

3

4

5

.67 (m, 2H, @CH ), 5.01–5.04 (m, 4H, 4· CH@),

2

0

.30 (m, 1H, CH@), 5.93 (m, 1H, CH@), 7.73 (s, 1H,

5.17. (2 E,6E,10 E)-6-Benzyloxyamino-9-cyanoethyl-6-

methoxyamino-7-(3,7,11,15-tetramethyl-2,6,10,14-hexa-

1

3

H-8), 7.75 (s, 1H, H-2); C NMR (CDCl , 75 MHz) d

3

0

1

3

5

1

5.87, 16.71, 17.52, 25.55, 26.21, 26.55, 26.99, 39.36,

9.60 (2· C), 46.75 (NCH ), 61.31 (OCH ), 109.45 (C-

decatetraenyl)-7H-purinium (3o) and (2 E,6 ,10 E)-N-

benzyloxy-9-cyanoethyl-N-(3,7,11,15-tetramethyl-

2,6,10,14-hexadecatetraenyl)-9H-purin-6-amine (4o)

2

3

), 123.82 (2· CH@), 127.79 (@CH ), 130.02 (C-8),

2

31.13 (C@), 134.99 (2· C@), 136.35 (C@), 145.32

(

C-6), 148.21 (C-4), 157.31 (C-2); MS ESI m/z (rel.%)

78 (16, M+1), 434 (17), 206 (100).

The products were prepared from purine 2g (630 mg,

1.80 mmol) and (2E,6E,10E)-1-bromo-3,7,11,15-tetra-

methyl-2,6,10,14-hexadecatetraene (430 mg, 1.20 mmol)

as described for compounds 3f and 4f above, and puri-

ﬁed by ﬂash chromatography on silica gel eluting with

CH Cl /MeOH (satd NH ) (16:1). Compound 4o was

4

0 0

.16. (2 E,6E,10 E)-9-Cyanoethyl-6-methoxyamino-7-

3,7,11,15-tetramethyl-2,6,10,14-hexadecatetraenyl)-7H-

5

(

0

purinium (3n) and (2 E,6 ,10 E)-9-cyanoethyl-N-meth-

oxy-N-(3,7,11,15-tetramethyl-2,6,10,14-hexadecatetrae-

nyl)-9H-purin-6-amine (4n)

2

3

puriﬁed further by ﬂash chromatography eluting with

EtOAc/hexane (1:1).

1

Compound 3o: Yield 410 mg (61%), yellow wax. H

The products were prepared from purine 2f (153 mg,

0

.70 mmol) and (2E,6E,10E)-1-bromo-3,7,11,15-tetra-

NMR (CDCl , 200 MHz) d 1.63 (s, 9H, CH ), 1.70

3

methyl-2,6,10,14-hexadecatetraene (430 mg, 1.20 mmol)

as described for compounds 3f and 4f above, and puriﬁed

by ﬂash chromatography on silica gel eluting with

CH Cl /MeOH (satd NH ) (12:1). Compound 4n was

(s, 3H, CH ), 1.78 (s, 3H, CH ), 1.97–2.13 (m, 12H,

CH ), 2.88 (t, J = 6.2 Hz, 2H, CH CN), 4.24 (t,

J = 6.2 Hz, 2H, NCH ), 4.99 (d, J = 7.4 Hz, 2H,

2

3 3

2

NCH ), 5.04 (s, 2H, CH O), 5.13 (m, 3H, CH@),

2

3

2

puriﬁed further by ﬂash chromatography eluting with

EtOAc.

5.46 (t, J = 7.4 Hz, 1H, CH@), 7.20–7.34 (m, 3H,

Ph), 7.40–7.44 (m, 2H, Ph), 7.77 (s, 1H, H-2), 8.41

1

3

(

s, 1H, H-8); C NMR (CDCl , 50 MHz) d 15.98

3

1

Compound 3n: Yield 140 mg (41%), colorless oil. H

(CH ), 16.07 (CH ), 16.76 (CH ), 17.64 (CH ), 18.21

3 3 3 3

NMR (CDCl , 300 MHz) d 1.60 (s, 6H, CH ), 1.62

(CH ), 25.65 (CH ), 26.12 (CH ), 26.63 (CH ), 26.70

2 3 2 2

(CH ), 39.45 (CH ), 39.67 (CH ), 39.70 (CH ), 40.54

2 2 2 2

3

(

s, 3H, CH ), 1.68 (s, 3H, CH ), 1.83 (s, 3H, CH ),

3

1

3

.90–2.12 (m, 8H, CH ), 2.12–2.16 (m, 4H, CH ),

2

.08 (t, J = 6.4 Hz, 2H, CH CN), 3.83 (s, 3H,

2

(NCH ), 47.78 (NCH ), 75.68 (CH O), 109.58 (C-5),

2 2 2

2

115.44 (CH@), 116.32 (CN), 123.14 (CH@), 123.94

(CH@), 124.27 (CH@), 127.36 (CH, Ph), 128.01 (2·

CH, Ph), 128.60 (2· CH, Ph), 129.27 (C-8), 131.26

(C@), 135.12 (C@), 135.95 (C@), 139.31 (C, Ph),

144.63 (C-4), 146.20 (C@), 147.84 (C-6), 157.13

(C-2); HRMS (ESI) C H N O requires 567.3805,

OCH ), 4.47 (t, J = 6.4 Hz, 2H, NCH ), 5.10 (m,

3

2

5

H), 5.49 (t, J = 6.9 Hz, 1H), 7.78 (s, 1H, H-2), 8.42

1

3

(

s, 1H, H-8); C NMR (CDCl , 75 MHz) d 16.41

3

(

CH ), 16.50 (CH ), 17.31 (CH ), 18.08 (CH ), 18.87

CH CN), 26.09 (CH ), 26.53 (CH ), 27.06 (CH ),

2 3 2 2

3

35 46

6

2

4

7.15 (CH ), 39.92 (CH ), 40.11 (CH ), 40.14 (CH ),

2

found 567.3824.

2

1.31 (CH ), 48.55 (CH ), 62.00 (CH O), 110.30

2

3

1

(

1

(

1

C-5), 115.68 (CH@), 116.73 (CN), 123.53 (CH@),

24.37 (CH@), 124.71 (CH@), 129.62 (C-8), 131.70

C@), 135.57 (C@), 136.46 (C@), 145.13 (C-4),

Compound 4o: Yield 12 mg (18%), colorless oil.

H

NMR (CDCl , 300 MHz) d 1.53 (s, 3H, CH ), 1.55

3

(s, 3H, CH ), 1.57 (s, 3H, CH ), 1.65 (s, 3H,

3 3

CH ), 1.70 (s, 3H, CH ), 1.90–2.05 (m, 12H, CH ),

3 3 2

3.00 (t, J = 6.5 Hz, 2H), 4.46 (t, J = 6.5 Hz, 2H),

4.63 (d, J = 6.9 Hz, 2H, NCH₂), 5.05 (m, 3H,

47.12 (C@), 147.59 (C-6), 157.25 (C-2); HRMS

+

(

ESI) C H N O+H

91.3513.

requires 491.3492, found

2

9

42

6

4

CH@), 5.06 (s, 2H, CH O), 5.41 (t, J = 6.5 Hz, 1H,

2

1

Compound 4n: Yield 86 mg (25%), pale yellow oil. H

NMR (CDCl , 300 MHz) d 1.58 (s, 6H, CH ), 1.60 (s,

CH@), 7.31–7.38 (m, 3H, Ph), 7.56 (m, 2H, Ph),

1

3

7.91 (s, 1H), 8.43 (s, 1H);

C NMR (CDCl ,

3

2

3

4

5

1

H, CH ), 1.71 (s, 3H, CH ), 1.80 (s, 3H, CH ), 1.90–

.20 (m, 12H, CH ), 3.03 (t, J = 6.5 Hz, 2H, CH CN),

75 MHz) d 16.39 (2· CH ), 17.00 (CH ), 18.08

3

(CH ), 19.27 (CH CN), 26.09 (CH ), 26.77 (CH ),

3 2 3 2

2

.95 (s, 3H, OCH ), 4.50 (t, J = 6.5 Hz, 2H, NCH ),

3

27.02 (CH ), 27.16 (CH ), 40.06 (CH ), 40.11 (2·

2

.75 (d, J = 6.8 Hz, 2H, NCH ), 5.09 (m, 3H, CH@),

CH ), 40.19 (CH ), 49.53 (NCH ), 77.72 (CH O),

2 2 2 2

2

.45 (t, J = 6.8 Hz, 1H, CH@), 7.93 (s, 1H), 8.45 (s,

117.10 (CN), 118.66 (CH@), 120.11 (C-5), 124.29

(CH@), 124.61 (CH@), 124.79 (CH@), 128.30 (2·

CH, Ph), 128.88 (CH, Ph), 130.15 (2· CH, Ph),

131.64 (C@), 135.30 (C@), 135.65 (C@), 136.20 (C,

Ph), 140.14 (C-8), 140.95 (C@), 151.42 (C-4), 152.88

(C-2), 156.94 (C-6); MS EI m/z (rel.%) 566 (4,

1

3

H); C NMR (CDCl , 75 MHz) d 16.39 (2· CH ),

3

6.96 (CH ), 18.07 (CH ), 19.24 (CH CN), 26.08

3

2

(

CH ), 26.74 (CH ), 27.01 (CH ), 27.15 (CH ), 40.06

3 2 2 2

(

2· CH₂), 40.10 (CH₂), 40.21 (NCH₂), 48.44

(

1

NCH ), 63.02 (OCH ), 117.02 (CN), 118.57 (CH@),

2

3

+

19.72 (C-5), 124.25 (CH@), 124.59 (CH@), 124.78

M ), 391 (9), 361 (28), 323 (12), 294 (15), 255

(

CH@), 131.63 (C@), 135.30 (C@), 135.66 (C@),

(100), 201 (19), 91 (23); Anal. Calcd for

C H N O: C, 74.17; H, 8.18; N, 14.83. Found: C,

74.10; H, 8.10; N, 14.93%.

140.17 (C-8), 141.18 (C@), 151.33 (C-4), 152.84 (C-

2), 156.19 (C-6); MS EI m/z (rel.%) 490 (2, M ),

3

5

46

6

+

4030

A. Vik et al. / Bioorg. Med. Chem. 15 (2007) 4016–4037

0

5

(

7

.18. (2 E,6E,10 E)-9-Benzyl-6-methoxyamino-7-

3,7,11,15-tetramethyl-2,6,10,14- hexadecatetraenyl)-

H-purinium (3p)

NCH ), 5.07 (m, 3H, CH@), 5.44 (dt, J = 6.5 and

2

1.0 Hz, 1H, CH@), 7.11–7.17 (m, 4H, Ph), 7.15 (s, 1H,

CHPh ), 7.28–7.37 (m, 6H, Ph), 7.65 (s, 1H, H-8), 8.44

2

1

3

(

s, 1H, H-2); C NMR (CDCl , 75 MHz) d 15.99

3

The product was prepared from purine 2h (128 mg,

.50 mmol) and (2E,6E,10E)-1-bromo-3,7,11,15-tetra-

(CH ), 16.00 (CH ), 16.55 (CH ), 17.67 (CH ), 25.68

3 3 3 3

(CH ), 26.38 (CH ), 26.62 (CH ), 26.75 (CH ), 39.67

2 2 2 2

0

methyl-2,6,10,14-hexadecatetraene (237 mg, 0.70 mmol)

as described for compound 3f above, and puriﬁed by

ﬂash chromatography on silica gel eluting with

CH Cl /MeOH (satd NH ) (17:1). Compound 4p was

(CH ), 39.69 (CH ), 39.70 (CH ), 48.22 (NCH ), 61.26

2 2 2 2

(CHPh ), 62.68 (CH O), 118.42 (CH@), 119.39 (C-5),

2

3

123.92 (CH@), 124.20 (CH@), 124.39 (CH@), 128.11

(4· CH, Ph), 128.39 (2· CH, Ph), 128.94 (4· CH, Ph),

131.22 (C@), 134.90 (C@), 135.23 (C@), 138.44 (2· C,

Ph), 139.95 (C-8), 140.56 (C@), 151.41 (C-4), 152.42

2

3

also formed but not isolated in pure form.

1

+

Compound 3p: Yield 115 mg (44%), yellow oil.

H

(C-2), 156.23 (C-6); MS EI m/z (rel.%) 603 (2, M ),

NMR (CDCl , 300 MHz) d 1.47–1.67 (m, 15H), 1.83–

572 (5), 398 (68), 368 (34), 167 (100), 165 (19); Anal.

Calcd for C H N O: C, 77.57; H, 8.18; N, 11.60.

Found: C, 77.46; H, 8.23; N, 11.36%.

3

1

5

7

.96 (m, 12H), 3.71 (s, 3H, CH ), 4.95–5.00 (m, 4H),

.22 [s, 2H, N(9)CH ], 5.28–5.32 (m, 1H, CH@), 7.31–

3

39 49

5

2

1

3

.35 (m, 5H, Ph), 7.73 (1H, H-8), 8.05 (1H, H-2);

C

0

NMR (CDCl , 75 MHz) d 15.81, 15.83, 16.67 (2· C),

5.20. (2 E,6E,10 E)-6-Benzyloxyamino-9-(diphenylmeth-

yl)-7-(3,7,11,15-tetramethyl-2,6,10,14-hexadecatetrae-

3

2

5.48, 26.00, 26.41, 26.53, 39.27, 39.50, 47.61, 61.22

0

(

CH ), 109.33 (C-5), 123.01 (2· C), 124.13 (2· C),

nyl)-7H-purinium (3r) and (2 E,6 ,10 E)-N-benzyloxy-9-

(diphenylmethyl)-N-(3,7,11,15-tetramethyl-2,6,10,14-

hexadecatetraenyl)-9H-purin-6-amine (4r)

3

1

4

5

28.23 (CH, Ph), 128.72 (CH, Ph), 129.07 (CH, Ph),

31.03, 133.76, 134.90, 135.65, 144.92 (C-8), 147.73 (C-

), 156.90 (C-2); HRMS (ESI) C H N O+H requires

+

3

45

5

28.3696, found 528.3666.

The products were prepared from purine 2j (338 mg,

0

.83 mmol) and (2E,6E,10E)-1-bromo-3,7,11,15-tetra-

0

5

7

.19. (2 E,6E,10 E)-9-(Diphenylmethyl)-6-methoxyamino-

-(3,7,11,15-tetramethyl-2,6,10,14-hexadecatetraenyl)-

methyl-2,6,10,14-hexadecatetraene (353 mg, 1.00 mmol)

as described for compounds 3f and 4f above, and puri-

ﬁed by ﬂash chromatography on silica gel eluting with

EtOAc/hexane (1:4) followed by EtOAc/EtOH (10:1).

The isolated bromide of 3r was dissolved methanol sat-

0

H-purinium (3q) and (2 E,6 ,10 E)-9-(diphenylmethyl)-

N-methoxy-N-(3,7,11,15-tetramethyl-2,6,10,14-hexa-

decatetraenyl)-9H-purin-6-amine (4q)

urated with NH (5 mL) and puriﬁed by ﬂash chroma-

3

The products were prepared from purine 2i (196 mg,

0

tography eluting with CH Cl /MeOH (satd NH ) (10:1).

3

2

.90 mmol) and (2E,6E,10E)-1-bromo-3,7,11,15-tetra-

1

methyl-2,6,10,14-hexadecatetraene (441 mg, 1.25 mmol)

as described for compounds 3f and 4f above, and the

products were separated by ﬂash chromatography on

silica gel eluting with CH Cl /MeOH (satd NH )

Compound 3r: Yield 340 mg (60%), yellow oil. H NMR

(CDCl , 300 MHz) d 1.58 (s, 3H, CH ), 1.60 (s, 9H, 3·

3

CH ), 1.71 (s, 3H, CH ), 1.90–2.16 (m, 12H, 6· CH ),

3

2

4.98 (d, J = 7.2 Hz, 2H, CH N), 5.07–5.12 (m, 5H,

CH O and 3· CH@), 5.29 (t, J = 7.2 Hz, 1H, CH@),

2

3

2

(12:1). Compound 4q was puriﬁed further by ﬂash chro-

matography eluting with EtOAc.

2

7.05 (s, 1H, CHPh ), 7.08–7.11 (m, 4H, Ph), 7.16–7.31

2

(

m, 3H, Ph), 7.36.7.46 (m, 9H, Ph and H-8), 7.84 (s,

1

13

Compound 3q: Yield 221 mg (50%), yellow wax. H

NMR (CDCl , 300 MHz) d 1.53 (s, 3H, CH ), 1.55 (s,

1H, H-2); C (CDCl , 75 MHz) d 15.96 (2· CH ),

16.77 (CH ), 17.71 (CH ), 25.72 (CH ), 26.44 (CH ),

26.63 (CH ), 26.77 (CH ), 39.42 (CH ), 39.70 (CH ),

2 2 2 2

3

2

6

H, CH ), 1.63 (s, 3H, CH ), 1.66 (s, 3H, CH ), 1.89–

3

2

.05 (m, 12H, CH ), 3.80 (s, 3H, CH O), 5.02 (m, 3H,

2

39.74 (CH ), 47.80 (CH N), 62.01 (CHPh ), 75.49

2 2 2

3

CH@), 5.07 (d, J = 7.1 Hz, 2H, NCH ), 5.33 (t,

(CH O), 110.23 (C-5), 115.87 (CH@), 123.00 (CH@),

2

J = 7.1 Hz, 1H, CH@), 7.03 (s, 1H, CHPh ), 7.05–7.10

123.93 (CH@), 124.26 (CH@), 126.85 (CH in Ph),

127.26 (C-8), 127.83 (2· CH in Ph), 127.96 (4· CH in

Ph), 128.21 (2· CH in Ph), 129.01 (2· CH in Ph),

129.22 (4· CH in Ph), 131.25 (C@), 135.01 (C@),

135.91 (C@), 136.60 (2· C in Ph), 140.00 (C in Ph),

145.67 (C-4), 145.73 (C@), 149.00 (C-6), 157.68 (C-2);

2

(

7

m, 4H, Ph), 7.30–7.36 (m, 6H, Ph), 7.50 (s, 1H, H-8),

1

3

.78 (s, 1H, H-2); C NMR (CDCl , 75 MHz) d 15.94

3

(

CH ), 15.95 (CH ), 16.83 (CH ), 17.60 (CH ), 25.61

3 3 3 3

(

CH ), 26.33 (CH ), 26.51 (CH ), 26.66 (CH ), 39.39

3 3 2 2

(

CH ), 39.58 (CH ), 39.63 (CH ), 47.83 (NCH ), 61.51

2 2 2 2

+

(

1

CH O), 62.16 (CHPh ), 110.14 (C-5), 115.80 (CH@),

HRMS (ESI) C H N O+H requires 680.4322, found

3

2

45 53

5

22.94 (CH@), 123.90 (CH@), 124.24 (CH@), 127.78

680.4305. Anal. Calcd for C H N O: C, 79.49; H,

45 53 5

7.86; N, 10.30. Found: C, 78.90; H, 7.93; N, 10.79%.

(

1

C-8), 127.96 (4· CH, Ph), 129.01 (2· CH, Ph), 129.21

4· CH, Ph), 131.21 (C@), 135.01 (C@), 135.93 (C@),

1

36.54 (2· C, Ph), 145.62 (C-4), 145.87 (C@), 147.73

Compound 4r: Yield 66 mg (12%), colorless oil.

H

+

(C-6), 157.32 (C-2); HRMS (ESI) C H N O+H

39 49 5

requires 604.4009, found 604.4019.

NMR (CDCl , 200 MHz) d 1.63 (s, 9H, 3· CH ), 1.72

3

(s, 3H, CH ), 1.78 (s, 3H, CH ), 1.93–2.20 (m, 12H, 6·

3

CH ), 4.70 (d, J = 6.9 Hz, 2H, CH N), 5.16 (m, 3H, 3·

2

1

Compound 4q: Yield 148 mg (34%), colorless oil. H

NMR (CDCl , 300 MHz) d 1.55 (s, 9H, CH ), 1.67 (s,

CH@), 5.23 (s, 2H, CH in Bn), 5.51 (t, J = 6.9 Hz,

2

1H, CH@), 7.19–7.24 (m, 5H), 7.35–50 (m, 9H), 7.59–

7.68 (m, 2H) 7.75 (s, 1H), 8.52 (s, 1H); C NMR

3

1

3

CH ), 3.93 (s, 3H, OCH ), 4.72 (d, J = 6.8 Hz, 2H,

H, CH ), 1.76 (s, 3H, CH ), 1.90–2.15 (m, 12H,

3

(CDCl , 75 MHz) d 15.96 (2· CH ), 16.56 (CH ),

2

3

A. Vik et al. / Bioorg. Med. Chem. 15 (2007) 4016–4037

4031

1

2

7.64 (CH ), 25.65 (CH ), 26.36 (CH ), 26.58 (CH ),

3

(OCH ), 118.62 (CH@), 119.66 (C-5), 141.25 (C@),

3

2

3

6.72 (CH ), 39.67 (3· CH ), 49.04 (CH N), 61.19

141.31 (C-8), 152.11 (C-4), 152.73 (C-2), 156.44 (C-6);

MS EI m/z (rel.%) 457 (4, M ), 428 (7), 427 (40), 426

2

+

(

1

CHPh ), 77.33 (CH O), 118.41 (CH@), 119.79 (C-5),

2

23.91 (CH@), 124.18 (CH@), 124.36 (CH@), 128.11

(100), 246 (8), 216 (9), 179 (38), 174 (8), 149 (13); Anal.

Calcd for C H N O: C, 70.85; H, 10.35; N, 15.30.

Found: C, 70.80; H, 10.36; N, 15.20%.

(

4· CH in Ph), 128.30 (2· CH in Ph), 128.33 (3· CH

in Ph), 128.89 (4· CH in Ph), 129.72 (2· CH in Ph),

31.16 (C in Ph), 134.83 (C@), 135.17 (C@), 135.89

C@), 138.47 (2· C in Ph), 139.94 (C-8), 140.32 (C@),

51.49 (C-4), 152.30 (C-2), 156.74 (C-6); HRMS (ESI)

C H N O+H requires 680.4322, found 680.4336.

2

7

47

5

1

(

1

5.22. (E)-6-Benzyloxyamino-9-methyl-7-(3,7,11,15-tetra-

methyl-2-hexadecenyl)-7H-purinium (3u) and (E)-N-ben-

zyloxy-9-methyl-N-(3,7,11,15-tetramethyl-2-hexadece-

nyl)-9H-purin-6-amine (4u)

4

5

53

5

.21. (E)-6-Methoxyamino-9-methyl-7-(3,7,11,15-tetra-

methyl-2-hexadecenyl)-7H-purinium (3t) and (E)-N-

methoxy-9-methyl-N-(3,7,11,15-tetramethyl-2-hexadece-

nyl)-9H-purin-6-amine (4t)

The products were prepared from purine 2b (117 mg,

0.46 mmol) and (E)-1-bromo-3,7,11,15-tetramethyl-2-

hexadecene (215 mg, 0.60 mmol) as described for com-

pounds 3t and 4t above, and puriﬁed by ﬂash chroma-

tography on silica gel eluting with CH Cl /MeOH

(satd NH ) (15:1). Compound 4u was puriﬁed further

3

by ﬂash chromatography eluting with EtOAc/hexane

(1:2).

Purine 2a (197 mg, 1.10 mmol) was dissolved in dry DMA

and stirred at 50 ꢁC under N . A solution of (E)-1-bromo-

2

3,7,11,15-tetramethyl-2-hexadecene (610 mg, 1.70 mmol)

in diethyl ether (1 mL) was added and the ﬂask was

ﬂushed with N to remove the diethyl ether. The resulting

2

1

Compound 3u: Yield 122 mg (55%), yellow wax. H

mixture was stirred at 50 ꢁC overnight, the solvent was

removed in vacuo, and the products were puriﬁed by ﬂash

chromatography on silica gel eluting with CH Cl /

NMR (CDCl , 500 MHz) d 0.84–0.88 (m, 12H,

3

CH ), 1.00–1.60 (m, 18H, CH and CH), 1.72 (s,

3H, CH₃), 2.00 (t, J = 7.8 Hz, 2H, CH₂), 3.63

(s, 3H, NCH ), 4.95 (d, J = 7.5 Hz, 2H, NCH ), 5.04

2

3

2

MeOH (satd NH ) (15:1). Compound 4t was puriﬁed

3

further by ﬂash chromatography eluting with EtOAc/

hexane (1:1).

3

2

(s, 2H, CH O), 5.37 (t, J = 7.5 Hz, 1H, CH@), 7.17–

2

7

.28 (m, 3H, Ph), 7.36–7.42 (m, 2H, Ph), 7.81 (s,

1

Compound 3t: Yield 271 mg (54%), colorless oil. H

13

1H, H-2), 7.99 (s, 1H, H-8); C NMR (CDCl₃,

125 MHz, double set of signals due to diastereomers

are marked ‘d’) d 16.63, 19.58 (d), 19.62 (d), 22.57,

22.66, 24.44, 24.74, 25.10, 27.91, 30.80 (NCH₃),

32.64, 32.73, 36.80 (d), 37.24 (d), 37.34 (d), 37.38

NMR (CDCl , 500 MHz) d 0.84–0.86 (m, 6H, 2·

3

CH ), 0.87 (d, J = 6.6 Hz, 6H, 2· CH ), 1.08–1.50 (m,

3

1

8H), 1.53 [sept, J = 6.6 Hz, 1H, CH(CH ) ], 1.80 (s,

3 2

3

NCH ), 3.85 (s, 3H, OCH ), 5.08 (d, J = 7.4 Hz, 2H,

H, CH ), 2.10 (t, J= 7.7 Hz, 2H, CH ), 3.78 (s, 3H,

3

2

(d), 39.31, 39.83, 47.48 (NCH₂), 75.52 (CH O),

2

3

NCH ), 5.44 (t, J = 7.4 Hz, 1H, CH@), 7.82 (s, 1H, H-

109.43 (C-5), 115.92 (CH@), 127.09 (CH, Ph), 127.86

(2· CH, Ph), 128.48 (2· CH, Ph), 129.04 (C-8),

139.60 (C@), 145.06 (C-4), 145.58 (C, Ph), 147.92

(C-6), 157.10 (C-2); Anal. Calcd for C H N O: C,

2

1

3

2

), 7.84 (s, 1H, H-8); C NMR (CDCl , 125 MHz, dou-

3

ble set of signals due to diastereomers are marked ‘d’) d

7.17 (CH ), 20.01 (d, CH ), 20.06 (d, CH ), 23.01

1

3

33 51

5

(

CH ), 23.10 (CH ), 24.87 (CH ), 25.18 (CH ), 25.53

3

CH ), 28.36 (CH), 31.45 (CH), 31.51 (NCH ), 33.07

2 3

74.25; H, 9.36; N, 13.12. Found: C, 74.00; H, 9.56;

N, 12.99%.

3

2

(

3

CH), 33.17 (CH), 37.16 (d, CH ), 37.25 (d, CH ),

2 2

1

7.68 (d, CH ), 37.71 (d, CH ), 39.75 (CH ), 40.29

2

Compound 4u: Yield 31 mg (13%), colorless oil.

H

2

(

CH ), 48.12 (NCH ), 61.91 (OCH ), 109.92 (C-5),

2

NMR (CDCl , 500 MHz) d 0.76–0.84 (m, 12H,

2

3

116.09 (CH@), 128.93 (C-4), 145.68 (C@), 146.55 (C-

8), 148.40 (C-6), 157.66 (C-2); HRMS (ESI)

CH ), 0.95–1.40 (m, 17H, CH and CH), 1.49 [sept,

J = 6.6 Hz, 1H, CH(CH ) ], 1.67 (s, 3H, CH ), 1.91

3 2 3

3

2

C H N O requires 458.3853, found 458.3839; Anal.

2

(t, J = 7.7 Hz, CH ), 3.81 (s, 3H, NCH ), 4.63 (d,

2 3

7

47

5

Calcd for C H N O: C, 70.85; H, 10.35; N, 15.30.

5

J = 6.9 Hz, 2H, NCH ), 5.13 (s, 2H, CH O), 5.39

2 2

2

7

47

Found: C, 70.00; H, 10.36; N, 15.20%.

(m, 1H, CH@), 7.30-7.38 (m, 3H, Ph), 7.53-7.57 (m,

13

2H, Ph), 7.79 (s, 1H, H-8), 8.48 (s, 1H, H-2);

NMR (CDCl , 125 MHz, double set of signals due

C

1

Compound 4t: Yield 193 mg (38%), colorless oil. H

3

NMR (CDCl , 500 MHz) d 0.76–0.79 (m, 6H, 2·

to diastereomers are marked ‘d’) d 16.40, 19.58 (d),

3

CH ), 0.82 (d, J = 6.6 Hz, 6H, 2· CH ), 0.95–1.41 (m,

19.70 (d), 22.59, 22.68, 24.42 (CH ), 24.75 (CH ),

25.09 (CH ), 27.93, 29.71 (NCH ), 32.65, 32.72,

2 3

3

2

1

8H, 9· CH ), 1.48 [sept, J = 6.6 Hz, 1H, CH(CH ) ],

2

3 2

1

NCH ), 3.90 (s, 3H, OCH ), 4.71 (d, J = 6.8 Hz, 2H,

.73 (s, 3H, CH ), 2.00 (m, 2H, CH ), 3.80 (s, 3H,

36.75 (d, CH ), 37.25 (d, CH ), 37.30 (d, CH ),

2 2 2

37.35 (d, CH ), 39.33 (CH ), 40.01 (CH ), 49.44

2 2 2

3

2

3

NCH ), 5.39 (dt, J = 6.8 and 1.1 Hz, 1H, CH@), 7.78

(NCH ), 77.20 (CH O), 118.27 (CH@), 119.54 (C-5),

2

1

3

s, 1H, H-8), 8.44 (s, 1H, H-2); C NMR (CDCl ,

(

128.31 (3· CH, Ph), 129.67 (2· CH, Ph), 136.04 (C,

3

1

25 MHz, double set of signals due to diastereomers

are marked ‘d’) d 16.83, 20.10 (d), 20.14 (d), 23.03,

3.85, 25.20 (CH ), 25.50 (CH), 25.52 (CH ), 28.35

Ph), 140.59 (C@), 140.85 (C-8), 151.73 (C-4), 152.27

(C-2), 156.58 (C-6); MS EI m/z (rel.%) 533 (26, M ),

+

2

516 (38), 426 (85), 255 (78), 216 (100), 202 (42), 174

(63), 150 (40), 149 (47), 91 (23); Anal. Calcd for

C H N O: C, 74.25; H, 9.63; N, 13.12. Found: C,

2

[

CH ), 37.18 (d, CH ), 37.68 (d, CH ), 37.74 (d, CH ),

CH(CH ) ], 30.14 (NCH ), 33.06, 33.18, 37.08 (d,

3 2 3

2

33 51

5

3

9.75 (CH ), 40.39 (CH ), 48.69 (NCH ), 62.90

2

74.29; H, 9.64; N, 13.12%.

2

4032

A. Vik et al. / Bioorg. Med. Chem. 15 (2007) 4016–4037

5

(

.23. 6-Amino-7-benzyl-9-methyl-7H-purinium chloride

5a)

CH), 5.25 (d, J = 6.9 Hz, 2H, CH N), 5.45 (t,

2

J = 6.9 Hz, 1H, CH@), 8.00 (br s, 2H, NH ), 8.45 (s,

2

1

3

1

H, H-2), 9.79 (br s, 1H, H-8); C NMR (DMSO-d ,

6

Betaine 3c (107 mg, 0.31 mmol) and zinc (182 mg,

.79 mmol) were dissolved in methanol (5 mL) and

75 MHz) d 17.50 (CH ), 18.33 (CH ), 23.97 (CH ),

26.28 (CH ), 26.37 (CH ), 26.87 (CH ), 32.26 (CH ),

2 3 2 2

3 3 3

2

water (1 mL). Concd acetic acid (0.2 mL) was added

and the resulting mixture was stirred at 60 ꢁC overnight.

The mixture was ﬁltered, the solid washed with metha-

nol (25 mL), and satd aq NaCl (3 mL) was added to

the combined ﬁltrates. The resulting mixture was stirred

at ambient temperature for 1.5 h and evaporated in va-

cuo. Satd aq NaCl (5 mL) and water (15 mL) were

added to the residue and the mixture was extracted with

32.34 (NCH ), 40.15 (CH ), 47.83 (CH N), 110.02 (C-

3 2 2

5), 116.35 (CH@), 124.90 (CH@), 125.22 (CH@),

131.81 (C@), 135.80 (C@), 141.81 (C-8), 145.99 (C@),

149.83 (C-4), 153.22 (C-6), 156.27 (C-2); HRMS (ESI)

þ

C

21

H

32N requires 354.2652, found 354.2651.

5

5.26. (E)-6-Methoxyamino-9-methyl-7-(3,7,11,15-tetra-

methyl-2-hexadecenyl)-7H-purinium (5e)

EtOH/CHCl (1:2; 4· 30 mL). The combined organic

3

extracts were dried (MgSO ) and evaporated in vacuo,

The product was prepared from betaine 3t (225 mg,

0.56 mmol) essentially as described for the synthesis of

compound 5a above, except that the reaction tempera-

ture was 70 ꢁC, and the product was puriﬁed by ﬂash

chromatography on silica gel eluting with MeOH/

CH Cl (1:7), yield 175 mg (69%), mp 172–174 ꢁC, col-

4

and the product was puriﬁed by ﬂash chromatography

on a short silica gel column eluting with CH Cl /

MeOH/MeOH (satd NH ) (6:1:1); yield 74 mg (87%),

2

3

1

mp 249–250 ꢁC, colorless crystals. H NMR (DMSO-

d , 200 MHz) d 3.90 (s, 3H, CH ), 6.03 (s, 2H, CH ),

6

3

2

1

7

1

7

1

.33–7.52 (m, 5H, Ph), 7.97 (br s, 2H, NH ), 8.43 (s,

orless crystals. H NMR (DMSO-d , 300 MHz) d

2

6

1

H, H-2), 9.97 (s, 1H, H-8); C NMR: (DMSO-d₆,

3

0.79–1.03 (m, 12 H, CH ), 1.06–1.23 (m, 21H), 1.77 (s,

3H, CH ), 2.04 (t, J = 7.2 Hz, CH ), 3.88 (s, 3H, CH ),

3

5 MHz) d 32.48 (CH ), 52.20 (CH ), 109.70 (C-5),

3

2

3

2

3

28.71 (2· CH, Ph), 129.56 (CH, Ph), 129.80 (2· CH,

5.24 (d, J = 7.0 Hz, NCH ), 5.43 (t, J = 7.0 Hz, CH@),

2

13

Ph), 134.89 (C, Ph), 143.26 (C-8), 150.00 (C-4), 152.93

(

7.99 (br s, 2H, NH ), 8.43 (s, 1H), 9.76 (s, 1H); C

NMR (DMSO-d , 75 MHz, double set of signals due

2

+

C-6), 156.41 (C-2); MS CI m/z (rel.%) 240 (20, M ),

6

1

78 (8), 164 (8), 151 (10), 150 (100), 149 (78), 122 (10),

05 (7); HRMS (ESI) C13

to diastereomers are marked ‘d’) d 16.55, 19.43 (d),

19.49 (d), 22.42, 22.51, 23.76 (CH ), 24.10 (CH ),

2 2

þ

H

14

N

requires 240.1243,

5

found 240.1232.

24.36 (CH ), 27.33, 31.34 (NCH ), 31.98, 32.01, 36.11

2 3

(

d), 36.58 (d), 36.68 (d), 36.69 (d), 38.73 (CH ), 39.21

2

0

5

2

.24. (2 E,6 E)-6-Amino-9-methyl-7-(3,7,11-trimethyl-

,6,10-dodecatrienyl)-7H-purinium chloride (5b)

(CH ), 46.97 (NCH ), 109.15 (C-5), 115.18 (CH@),

140.94 (C-8), 145.47 (C@), 148.93 (C-4), 152.33 (C-6),

155.36 (C-2); HRMS (ESI)

428.3747, found 428.3758.

2

þ

C

26

H

46

N

requires

5

The product was prepared from betaine 3f (115 mg,

.30 mmol) essentially as described for the synthesis of

compound 5a above, and the product was puriﬁed by

ﬂash chromatography on silica gel eluting with

MeOH/CH Cl (1:5), yield 94 mg (80%), colorless wax.

0

5.27. (2 E,6E,10 E)-6-Methoxyamino-7-(3,7,11,15-tetra-

methyl-2,6,10,14-hexadecatetraenyl)-7H-purine (6a)

2

1

H NMR (DMSO-d , 300 MHz) d 1.54 (s, 3H, CH ),

A mixture of betaine 3n (160 mg, 0.33 mmol) and K CO

2

6

3

1

CH ), 1.88–2.22 (m, 8H, CH ), 3.89 (s, 3H, NCH ),

.56 (s, 3H, CH ), 1.62 (s, 3H, CH ), 1.80 (s, 3H,

(320 mg, 2.30 mmol) in MeOH (15 mL) was stirred

3

vigorously for 2 h at ambient temperature. The mixture

was ﬁltered, the solid was washed with MeOH (10 mL),

and the combined ﬁltrates were evaporated in vacuo.

The product was puriﬁed by ﬂash chromatography on

silica gel eluting with MeOH/CH Cl (1:11); yield

3

2

3

5

8

7

2

3

1

.05 (m, 2H, CH@), 5.23 (d, J = 7.0 Hz, 2H, NCH₂),

.44 (t, J = 7.0 Hz, 1H, CH), 8.00 (br s, 2H, NH ),

2

1

.43 (s, 1H), 9.74 (s, 1H); C NMR (DMSO-d₆,

3

5 MHz) d 16.70 (CH ), 17.57 (CH ), 18.42 (CH ),

3

2

1

6.35 (CH ), 26.45 (CH ), 27.03 (CH ), 32.24 (NCH ),

9.84 (CH ), 40.02 (CH ), 47.84 (NCH ), 110.07 (C-5),

135 mg (95%), mp 111–112 ꢁC, colorless crystals.

H

3

2

3

NMR (CDCl , 300 MHz) d 1.60 (s, 9H, CH ), 1.68 (s,

2

3

16.15 (CH@), 124.28 (CH@), 124.90 (CH@), 131.55

3H, CH ), 1.80 (s, 3H, CH ), 1.95–2.20 (m, 12H, CH ),

3 3 2

3.86 (s, 3H, OCH ), 4.89 (d, J = 7.2 Hz, 2H, NCH ),

3 2

(

C

C@), 135.78 (C@), 141.74 (C-8), 146.17 (C@), 149.82

C-4), 153.22 (C-6), 156.28 (C-2); HRMS (ESI)

5.10 (m, 3H), 5.43 (dt, J = 7.2 and 1.0 Hz, 1H, CH@),

7.59 (s, 1H, H-8), 7.68 (s, 1H, H-2), 9.29 (br s, 1H, NH);

þ

21

H

32N requires 354.2652, found 354.2635.

5

1

3

C NMR (CDCl , 75 MHz) d 16.39 (CH ), 16.44

3

0

5

2

.25. (2 E,6 Z)-6-Amino-9-methyl-7-(3,7,11-trimethyl-

,6,10-dodecatrienyl)-7H-purinium chloride (5c)

(CH ), 17.02 (CH ), 18.07 (CH ), 26.08 (CH ), 26.62

(CH ), 26.98 (CH ), 27.15 (CH ), 39.89 (CH ), 40.05

3 3 3 3

2

(CH ), 40.11 (CH ), 45.65 (NCH ), 62.13 (OCH ),

2 2 2 3

The product was prepared from betaine 3h (194 mg,

.42 mmol) essentially as described for the synthesis of

110.70 (C-5), 118.57 (CH@), 123.80 (CH@), 124.52

(CH@), 124.77 (CH@), 131.65 (C@), 135.38 (C@),

136.15 (C@), 139.80 (C-8), 140.72 (C-6), 142.17 (C-2),

142.71 (C@), 150.43 (C-4); MS EI m/z (rel.%) 437 (28,

0

compound 5a above, and the product was puriﬁed by

ﬂash chromatography on silica gel eluting with

MeOH/CH Cl (1:4), yield 122 mg (74%), colorless

+

M ), 407 (18), 368 (15), 338 (10), 203 (33), 166 (100),

2

1

wax. H NMR (DMSO-d , 300 MHz) d 1.57 (3H,

165 (66), 136 (62), 135 (68); HRMS (EI) C H N O re-

26 39

6

5

CH ), 1.62 (6H, 2· CH ), 1.79 (3H, CH ), 1.91–2.23

quires 437.3154, found 437.3140; Anal. Calcd for

C H N O: C, 71.36.; H, 8.98. Found: C, 71.31; H, 8.98.

3

(

m, 8H, 4· CH ), 3.90 (s, 3H, NCH ), 5.08 (m, 2H, 2·

2

3

26 39

5

A. Vik et al. / Bioorg. Med. Chem. 15 (2007) 4016–4037

4033

0

5.28. (2 E,6E,10 E)-6-Benzyloxyamino-7-(3,7,11,15-tet-

ramethyl-2,6,10,14- hexadecatetraenyl)-7H-purine (6b)

was evaporated in vacuo and the residue puriﬁed by

ﬂash chromatography on silica gel eluting with

CH Cl /EtOH (7:1); yield 106 mg (62%), colorless crys-

2

The product was prepared from betaine 3o (350 mg,

0

tals. Data see above.

.62 mmol) essentially as described for the synthesis

0

of compound 5a above, except that the reaction mix-

ture was stirred overnight, and the product was puri-

ﬁed by ﬂash chromatography on silica gel eluting with

EtOH/CH Cl (1:19), yield 302 mg (95%), colorless

5.30. (2 E,6E,10 E)-6-Chloro-9-(3,7,11,15-tetramethyl-

2,6,10,14-hexadecatetraenyl)-9H-purine (9a) and

0

(2 E,6E,10 E)-6-chloro-7-(3,7,11,15-tetramethyl-

2,6,10,14-hexadecatetraenyl)-7H-purine (9b)

2

1

wax. H NMR (CDCl , 300 MHz) d 1.54 (s, 9H,

3

CH ), 1.62 (s, 3H, CH ), 1.69 (s, 3H, CH ), 1.91–

2

Method A: A mixture of 6-chloropurine 8 (53 mg,

0.34 mmol), (2E,6E,10E)-1-bromo-3,7,11,15-tetrameth-

yl-2,6,10,14-hexadecatetraene (242 mg, 0.68 mmol, crude,

purity 92%), and potassium carbonate (140 mg,

1.02 mmol) in dry DMF (2 mL) was stirred under N₂

for 20 h, before the mixture was ﬁltered and evaporated

in vacuo. The products were separated by ﬂash chroma-

tography on silica gel eluting with EtOAc/hexane (2:3).

3

.10 (m, 12H, CH₂), 4.76 (d, J = 7.0 Hz, 2H,

NCH ), 4.99 (s, 2H, CH O), 5.05 (m, 3H, CH@),

2

5

and H-8), 7.80 (s, 1H, H-2), 10.11 (s, 1H, NH);

.28 (t, J = 7.0 Hz, 1H, CH@), 7.21–7.36 (m, 6H, Ph

13

C

NMR (CDCl , 75 MHz) d 15.93 (CH ), 15.98 (CH ),

3

1

2

6.53 (CH ), 17.60 (CH ), 25.61 (CH ), 26.20 (CH ),

3 3 3 2

6.53 (CH ), 26.68 (CH ), 39.39 (CH ), 39.59, 39.63

2

(

1

CH ), 45.12 (NCH ), 76.03 (CH O), 110.08 (C-5),

2 2 2

18.40 (CH@), 123.40 (CH@), 124.06 (CH@), 124.31

1

Compound 9a: Yield 82 mg (57%), colorless oil.

H

(

1

CH@), 127.90 (CH, Ph), 128.28 (2· CH, Ph),

NMR (CDCl , 300 MHz) d 1.54 (s, 6H, CH ), 1.62 (s,

3H, CH ), 1.80 (s, 3H, CH ), 1.90–2.11 (m, 12H,

3 3

CH ), 2.07 (s, 3H, CH ), 4.82 (d, J = 7.0 Hz, 2H,

3

28.58 (2· CH, Ph), 131.15 (C@), 134.90 (C@),

35.59 (C@), 137.69 (C, Ph), 139.27 (C-8), 140.54

1

2

(

C-6), 141.76 (C@), 142.59 (C-2), 150.09 (C-4); MS

NCH ), 5.05 (m, 3H, CH@), 5.40 (t, J = 6.9 Hz, 1H,

2

+

13

EI m/z (rel.%) 513 (38, M ), 445 (14), 407 (30), 242

90), 203 (47), 136 (88), 135 (53), 91 (65), 69 (100);

Anal. Calcd for C H N O: C, 74.82; H, 8.44; N,

CH@), 8.06 (s, 1H, H-8), 8.69 (s, 1H, H-2); C NMR

(

(75 MHz, CDCl ) d 16.40, 16.45, 17.03, 18.08, 26.09,

3

26.45, 26.93, 27.13, 39.83, 40.04, 40.10, 42.17, 116.96

(CH@), 123.57 (CH@), 124.40 (CH@), 124.73 (CH@),

31.65 (C@), 132.04 (C-5), 135.43 (C@), 136.34 (C@),

3

2

43

5

1

3.63. Found: C, 74.88; H, 8.45; N, 13.54%.

1

0

5

2

.29. (2 E,6E,10 E)-6-Amino-7-(3,7,11,15-tetramethyl-

,6,10,14-hexadecatetraenyl)-7H-purine (7)

144.35 (C@), 145.11 (C-8), 151.25 (C-4), 152.09 (C-6),

152.24 (C-2); MS EI m/z (rel.%) 428/426 (12/33, M ),

+

392 (24), 391 (84), 222 (25), 157 (32), 155 (100), 136

(6), 135 (19), 93 (20); HRMS (EI) C H ClN requires

426.2550, found 426.2536.

Method A: Compound 6b (250 mg, 0.47 mmol) and

zinc (330 mg, 2.79 mmol) were dissolved in methanol

2

5

35

4

(

20 mL) and water (2 mL). Concd acetic acid

0.5 mL) was added and the resulting mixture was

Compound 9b: Yield 37 mg (27%), mp 50–53 ꢁC, color-

1

stirred at 75 ꢁC overnight. The mixture was ﬁltered,

the ﬁltrate was evaporated in vacuo, and the product

was puriﬁed by ﬂash chromatography on silica gel

eluting with CH Cl /EtOH (7:1); yield 95 mg (50%),

mp 117.0–117.5 ꢁC, colorless crystals.

(

less solid. H NMR (CDCl , 300 MHz) d 1.55 (s, 9H,

3

CH ), 1.63 (s, 3H, CH ), 1.79 (s, 3H, CH ), 1.90–2.10

(m, 12H, CH ), 5.04 (m, 5H, CH and CH@), 5.42 (t,

3

2

J = 7.3 Hz, 1H, CH@), 8.20 (s, 1H, H-8), 8.83 (s, 1H,

2

1

13

H

NMR

H-2); C NMR (75 MHz, CDCl ) d 16.40, 16.47,

3

CDCl , 300 MHz) d 1.56 (s, 9H, CH ), 1.64 (s,

17.19, 18.07, 26.09, 26.45, 26.92, 27.13, 39.81, 40.02,

40.10, 45.64, 117.41 (CH@), 122.93 (C-5), 123.49

(CH@), 124.39 (CH@), 124.74 (CH@), 131.66 (C@),

135.44 (C@), 136.48 (C@), 143.50 (C-4), 144.42 (C@),

148.71 (C-8), 152.71 (C-2), 162.52 (C-6); MS EI m/z

3

H, CH ), 1.81 (s, 3H, CH ), 1.87–2.08 (m, 8H,

3 3

CH₂), 2.13 (m, 4H), 4.88 (d, 2H, J = 6.0 Hz,

NCH ), 5.05 (m, 3H, CH@), 5.30 (br s, 2H, NH ),

5

1

7

1

2

.41 (dt, J = 6.0 and 0.75 Hz, 1H, CH@), 7.92 (s,

13

+

H, H-8), 8.45 (s, 1H, H-2);

C NMR (CDCl ,

(rel.%) 428/426 (5/14, M ), 222 (13), 157 (31), 155 (98),

135 (43), 121 (24), 107 (27), 93 (57); HRMS (EI)

C H ClN requires 426.2550, found 426.2569; Anal.

3

5 MHz) d 15.98 (CH ), 16.05 (CH ), 16.85 (CH ),

3

7.65 (CH₃), 25.66 (CH₃), 26.01 (CH₂), 26.51

25

35

4

(

3

CH₂), 26.72 (CH₂), 39.32 (CH₂), 39.64 (CH₂),

9.68 (CH ), 45.69 (NCH ), 112.10 (C-5), 119.20

Calcd for C H ClN : C, 70.32; H, 8.26. Found: C,

25 35 4

70.61; H, 8.01%.

2

(

CH@), 122.79 (CH@), 123.79 (CH@), 124.29

CH@), 131.28 (C@), 135.13 (C@), 136.37 (C@),

Method B: A mixture of 6-chloropurine 8 (182 mg,

1.18 mmol) and methylaquacobaloxime (419 mg,

1.30 mmol) in dry DMA (20 mL) was stirred at ambient

temperature under N for 5 min before K CO (195 mg,

1

4

3

1

7

43.19 (C@), 145.04 (C-8), 150.81 (C-6), 153.17 (C-

+

), 161.19 (C-2); MS EI m/z (rel.%) 407 (51, M ),

38 (20), 284 (10), 271 (16), 203 (77), 136 (100),

35 (86), 93 (31); Anal. Calcd for C H N : C,

3.67; H, 9.15; N, 17.18. Found: C, 73.62; H, 9.14;

2

3

1.30 mmol) was added. The resulting mixture was

stirred for 20 min in the dark before (2E,6E,10E)-1-

bromo-3,7,11,15-tetramethyl-2,6,10,14-hexadecatetraene

2

5

37

5

N, 17.03%.

(500 mg, 1.42 mmol) in DMA (2 mL) was added. The

Method B: Compound 9b (180 mg, 0.42 mmol) and

t-BuOH (40 mL) saturated with NH (g) were stirred

at 100 ꢁC in a sealed container for 48 h. The mixture

reaction mixture was stirred in the dark for 43 h and

evaporated in vacuo. The residue was dissolved in

CHCl₃(200 mL) and washed with 2 M NaOH

3

4034

A. Vik et al. / Bioorg. Med. Chem. 15 (2007) 4016–4037

(

100 mL), brine (100 mL), and 1 M HCl (100 mL), dried

MgSO ), and evaporated in vacuo. The product was

122.98 (CH@), 123.47 (C-4), 123.89 (CH@), 124.22

(CH@), 131.17 (C@), 134.98 (C@), 135.92 (C@),

137.22 (C-2), 146.47 (C@); MS (ESI) m/z 355

4

isolated by ﬂash chromatography on silica gel eluting

with EtOAc/hexane (2:1); yield 203 mg (40%) of com-

pound 9b, data see above. Compound 9a was not

formed.

+

(M ꢀBr).

0

5.34. (2 E,6E,10 E)-3-Methyl-1-(3,7,11,15-tetramethyl-

,6,10,14-hexadecatetraenyl)benzimidazolium bromide (11d)

2

5

.31. 1-Benzyl-3-methylimidazolium bromide (11a)

The product was prepared from 1-methylbenzimidazole

10b (86 mg, 0.65 mmol) and (2E,6E,10E)-1-bromo-

3,7,11,15-tetramethyl-2,6,10,14-hexadecatetraene (410 mg,

1.16 mmol) as described for the synthesis of compound

11a above. The product was puriﬁed by ﬂash chroma-

tography on silica gel eluting with MeOH/CH Cl

A

2

mixture of 1-methylimidazole 10a (164 mg,

.00 mmol) and benzyl bromide (0.48 mL, 4.00 mmol)

in dry DMA (5 mL) was stirred at 50 ꢁC under N over-

night and evaporated in vacuo. The product was puri-

ﬁed by ﬂash chromatography on silica gel eluting with

MeOH/CH Cl (1:6); yield 465 mg (92%), colorless

2

1

(1:10); yield 283 mg (90%), colorless wax. H NMR

(CDCl , 300 MHz) d 1.55 (s, 6H, CH ), 1.58 (s, 3H,

2

1

wax. H NMR (DMSO-d , 300 MHz) d 4.08 (s, 3H,

6

3

CH ), 5.45 (s, 2H, CH ), 7.40–7.46 (m, 5H, Ph), 7.74

(

CH ), 1.66 (s, 3H, CH ), 1.90–2.12 (m, 12H, CH ),

3 3 2

1.94 (s, 3H, CH ), 4.31 (s, 3H, NCH ), 5.07 (m, 3H,

3 3

3

2

1

s, 1H), 7.93 (s, 1H), 9.32 (s, 1H); C NMR (DMSO-

3

d , 75 MHz) d 36.77 (NCH ), 52.64 (CH ), 123.19

CH@), 5.21 (d, J = 7.0 Hz, 2H, NCH ), 5.41 (t,

6

3

2

(

CH, Ar), 124.84 (CH, Ar), 129.19 (2· CH, Ph),

J = 7.0 Hz, 1H, CH@), 7.62–7.68 (m, 3H, Ar), 7.74 (m,

1

1H, Ar), 11.38 (s, 1H, H-2); C NMR (CDCl ,

3

1

29.58 (CH, Ph), 129.83 (2· CH, Ph), 135.79 (C, Ph),

3

þ

37.52 (C-2); HRMS (ESI) C H N

73.1073, found 173.1081.

requires

75 MHz) d 15.90 (CH ), 15.99 (CH ), 17.05 (CH ),

11

13

2

3

17.59 (CH ), 25.60 (CH ), 25.90 (CH ), 26.49 (CH ),

6.64 (CH ), 33.70 (NCH ), 39.33 (CH ), 39.55 (CH ),

2 3 2 2

3 3 2 2

2

5

.32. 1-Benzyl-3-methylbenzimidazolium bromide (11b)

39.61 (CH ), 45.87 (NCH ), 112.80 (CH, Ar), 113.24

2 2

(CH, Ar), 115.20 (CH@), 122.99 (CH@), 123.95

The product was prepared from 1-methylbenzimidaz-

ole 10b (132 mg, 1.00 mmol) and benzyl bromide

(CH@), 124.23 (CH@), 127.01 (CH, Ar), 127.12 (CH,

Ar), 130.92 (C@), 131.18 (C@), 132.21 (C-3a), 134.92

(C@), 135.81 (C@), 142.91 (C-2), 145.19 (C-7a); HRMS

(ESI) C H N requires 405.3264, found 405.3268.

(

0.24 mL, 2.00 mmol) as described for the synthesis

of compound 11a above. The product was puriﬁed

by ﬂash chromatography on silica gel eluting with

MeOH/CH Cl (1:8); yield 291 mg (96%), colorless

2

8

41

2

5.35. 6-Amino-1-benzyl-9-methyl-1H-purinium bromide

(13a)

2

1

wax. H NMR (DMSO-d , 300 MHz) d 4.09 (s, 3H,

6

CH ), 5.80 (s, 2H, CH ), 7.27–7.41 (3H, Ar), 7.51–

3

2

7

2

7

1

.54 (2H, Ar), 7.64–7.70 (m, 2H, Ar), 7.94–8.05 (m,

A mixture of 9-methyladenine 12 (149 mg, 1.00 mmol)

and benzyl bromide (0.18 mL, 1.5 mmol) in DMA

(5 mL) was stirred overnight at 50 ꢁC. The mixture

was evaporated in vacuo and the residue puriﬁed by

ﬂash chromatography on silica gel using CH Cl /MeOH

1

H, Ar), 9.92 (s, 1H, H-2); C NMR (DMSO-d₆,

3

5 MHz) d 34.28 (NCH ), 50.59 (CH ), 114.55 (Ar),

3

2

14.63 (CH, Ar), 127.42 (CH, Ar), 127.48 (CH,

Ar), 129.14 (2· CH, Ph), 129.56 (CH, Ph), 129.91

2

(

2· CH, Ph), 131.55 (C, Ar), 132.91 (C, Ar), 134.94

C, Ph), 143.81 (C-2); MS (ESI) m/z 223 (100,

(6:1); yield 79 mg (25%), mp 250–254 ꢁC, colorless solid.

1

H NMR (DMSO-d , 300 MHz) d 3.84 (s, 3H, CH ),

6

3

+

ꢀ

M ꢀBr ).

5.63 (s, 2H, CH ), 7.27–7.42 (m, 5H, Ph), 8.50 (s, 1H),

8

2

1

.91 (s, 1H), 9.51 (br s, 2H, NH₂). C NMR (DMSO-

3

0

5.33. (2 E,6E,10 E)-3-Methyl-1-(3,7,11,15-tetramethyl-

2,6,10,14-hexadecatetraenyl)imidazolium bromide (11c)

d , 75 MHz) d 30.19 (CH ), 51.73 (CH ), 118.77 (C-5),

6

3

2

127.00 (2· CH in Ph), 128.25 (CH in Ph), 128.82 (2·

CH in Ph), 133.90 (C in Ph), 145.17 (C-8), 147.15 (C-

þ

The product was prepared from 1-methylimidazole

0a (53 mg, 0.65 mmol) and (2E,6E,10E)-1-bromo-

,7,11,15-tetramethyl-2,6,10,14-hexadecatetraene (410 mg,

.16 mmol) as described for the synthesis of com-

4), 147.59 (C-2), 150.06 (C-6); HRMS (ESI): C13

requires 240.1243, found 240.1240.

H

14

N

5

1

3

1

0

5.36. (2 E,6E,10 E)-6-Amino-9-methyl-1-(3,7,11,15-tet-

ramethyl-2,6,10,14- hexadecatetraenyl)-1H-purinium

bromide (13b)

pound 11a above. The product was puriﬁed by ﬂash

chromatography on silica gel eluting with MeOH/

CH Cl (1:9); yield 219 mg (78%), pale yellow wax.

2

1

H NMR (CDCl , 300 MHz) d 1.57 (s, 9H, CH ),

A mixture of 9-methyladenine 12 (134 mg, 0.90 mmol)

and (2E,6E,10E)-1-bromo-3,7,11,15-tetramethyl-2,6,10,14-

hexadecatetraene (576 mg, 1.63 mmol) in DMA (8 mL)

3

1.65 (s, 3H, CH ), 1.82 (s, 3H, CH ), 1.90 (m,

12H, CH ), 4.11 (s, 3H, CH ), 4.92 (d, J = 7.5 Hz,

2

3 3

2

3

H, NCH₂), 5.05 (m, 3H, CH@), 5.38 (t,

was stirred under N at 50 ꢁC overnight. The mixture

2

J = 7.5 Hz, 1H, CH@), 7.25 (m, 1H), 7.54 (m, 1H),

was evaporated in vacuo and the residue puriﬁed by

ﬂash chromatography on silica gel eluting with

MeOH/CH Cl (1:7); yield 147 mg (33%), colorless

1

3

1

0.34 (br s, 1H, H-2); C NMR (CDCl , 75 MHz)

3

d 15.91 (CH ), 16.00 (CH ), 16.82 (CH ), 17.58

3

2

1

(

2

CH₃), 25.59 (CH₃), 25.97 (CH₂), 26.50 (CH₂),

6.63 (CH ), 36.71 (NCH ), 39.35 (CH ), 39.59 (2·

wax. H NMR (CDCl , 300 MHz) d 1.58 (s, 6H,

3

CH ), 1.60 (s, 3H, CH ), 1.67 (s, 3H, CH ), 1.89

(s, 3H, CH ), 1.95–2.22 (m, 12H, CH ), 3.90 (s, 3H,

3 2

2

3

2

3

CH ), 47.32 (NCH ), 115.06 (CH@), 121.03 (C-5),

2

A. Vik et al. / Bioorg. Med. Chem. 15 (2007) 4016–4037

4035

NCH ), 5.09 (m, 3H, CH@), 5.28 (d, J = 7.4 Hz, 2H,

(s, 3H, CH ), 1.92–2.21 (m, 12H, CH ), 3.72 (s, 3H,

3 2

3

NCH ), 5.57 (t, J = 5.7 Hz, 1H, CH@), 8.30 (s, 1H, H-

NCH ), 5.09 (m, 3H, CH@), 5.19 (d, J = 7.2 Hz,

2

3

8

1

2

3

), 9.45 (s, 1H, H-2), 10.64 (br s, 1H, NH), 10.76 (br s,

NCH ), 5.49 (t, J = 7.2 Hz, 1H, CH@), 7.62 (s, 1H, H-

2

1

3

13

H, NH); C NMR (CDCl , 75 MHz) d 15.96 (CH ),

8), 7.74 (s, 1H, H-2); C NMR (CDCl , 75 MHz) d

3

6.09 (CH ), 17.29 (CH ), 17.63 (CH ), 25.63 (CH ),

6.04 (CH ), 26.53 (CH ), 26.70 (CH ), 30.66 (NCH ),

16.41 (CH ), 16.46 (CH ), 17.08 (CH ), 18.08 (CH ),

26.10 (CH ), 26.56 (CH ), 26.97 (CH ), 27.14 (CH ),

3 2 2 2

3

2

3

9.58 (2· CH ), 39.66 (CH ), 50.11 (NCH ), 114.04

34.92 (NCH ), 39.88 (CH ), 40.07 (CH ), 40.11 (CH ),

3 2 2 2

2

(

1

CH@), 118.17 (C@), 123.06 (CH@), 124.00 (CH@),

24.26 (CH@), 131.23 (C@), 134.99 (C@), 136.15

45.48 (NCH ), 113.22 (C-5), 117.93 (CH@), 123.82

2

(CH@), 124.50 (CH@), 124.75 (CH@), 131.68 (C@),

135.40 (C@), 136.19 (C@), 139.87 (C-8), 143.80 (C@),

145.43 (C-4), 145.66 (C-2), 156.28 (C-6); MS EI m/z

(

C@), 144.96 (C-2), 147.11 (C-4 and C-8), 148.05

C@), 150.03 (C-6); HRMS (ESI) C H N requires

(

2

6

40

5

+

4

22.3278, found 422.3266.

(rel.%) 421 (7, M ), 352 (13), 285 (5), 284 (27), 217

(22), 216 (100), 150 (19); Anal. Calcd for C H N : C,

26 39 5

5

.37. 3-Methyl-3H-adenine (14)

74.07; H, 9.32; N, 16.61. Found: C, 73.85; H, 9.20; N,

6.20%.

1

A mixture of adenine (4.00 g, 29.6 mmol), methyl-p-tol-

uenesulfonate (13 mL), and DMA (25 mL) was stirred

at 100 ꢁC for 2 h. The mixture was evaporated in vacuo

and the resulting syrup was subjected to ﬂash chroma-

tography on silica gel eluting with CH Cl followed by

0

6

5.40. (2 E,6E,10 E)-3,N -Dimethyl-7-(3,7,11,15-tetra-

methyl-2,6,10,14-hexadecatetraenyl)-3H-purine-6(7H)-

imine (16)

2

CH Cl /MeOH (6:1). The fractions containing a com-

2

A mixture of compound 15b (93 mg, 0.20 mmol) and

iodomethane (0.125 mL, 2.00 mmol) in DMA (2 mL)

was stirred in a sealed ﬂask at ambient temperature

for 5 h. The mixture was evaporated in vacuo and the

residue was dissolved in 10% aq NaOH (5 mL) and ex-

tracted with CH Cl (5· 5 mL). The combined organic

2

pound with R < 0.20 (TLC: SiO , eluent CH Cl /

2

f

2

MeOH 6:1) were combined and evaporated in vacuo.

The residue was recrystallized twice from EtOH to give

approximately 3.7 g of the tosylate salt of 3-methylade-

nine. The salt was recrystallized from aq NH to give the

3

2

desired product; yield 1.08 g (24%), mp 282–290 ꢁC (dec)

extracts were evaporated and the product was isolated

by ﬂash chromatography on silica gel eluting with

MeOH/CH Cl (1:12); yield 33 mg (38%), pale yellow

3

1

(

3

2

lit. 310–313 ꢁC) colorless solid. H NMR (DMSO-d ,

6

00 MHz) d 3.91 (s, 3H, CH ), 7.79 (s, 1H), 7.85 (br s,

H, NH ), 8.31 (s, 1H); MS CI m/z (rel.%) 149 (100,

3

2

1

wax. H NMR (CDCl , 300 MHz) d 1.59 (s, 9H,

2

3

+

M ), 121 (29), 94 (19).

CH ), 1.67 (s, 3H, CH ), 1.79 (s, 3H, CH ), 1.95–2.12

(m, 12H, CH ), 3.25 (s, 3H, NCH ), 3.77 (s, 3H,

3

2 3

5

.38. 7-Benzyl-3-methyl-3H-purine-6(7H)-imine (15a)

NCH ), 5.08 (m, 3H, CH@), 5.29 (d, J = 7.0 Hz, 2H,

3

NCH ), 5.42 (t, J = 7.0 Hz, 1H, CH@) 7.62 (s, 1H, H-

2

1

3

A mixture of 3-methyladenine 14 (75 mg, 0.50 mmol)

and benzyl bromide (0.71 mL, 0.60 mmol) in DMA

2), 7.88 (br s, 1H, H-8); C NMR (CDCl , 75 MHz) d

3

15.94 (CH ), 16.00 (CH ), 16.65 (CH ), 17.61 (CH ),

25.62 (CH ), 26.09 (CH ), 26.52 (CH ), 26.69 (CH ),

3 2 2 2

3

(

3 mL) was stirred at 50 ꢁC under N for 4 h, and evap-

2

orated in vacuo. The product was puriﬁed by ﬂash chro-

matography on silica gel eluting with MeOH (satd

NH )/CH Cl (1:7); yield 72 mg (60%), mp 159–161

32.82 (NCH ), 34.64 (NCH ), 39.44 (CH ), 39.61

3 3 2

(CH ), 39.65 (CH ), 45.58 (CH N), 113.21 (C-5),

2

117.30 (CH@), 123.39 (CH@), 124.04 (CH@), 124.29

(CH@), 131.19 (C@), 134.94 (C@), 135.72 (C@),

138.58 (br s, C-8), 143.74 (br s, C-4), 144.63 (C@),

145.61 (C-2), 150.89 (C-6); HRMS (ESI) C H N +H

3

2

1

ꢁ

C, colorless crystals. H NMR (CD OD, 300 MHz) d

3

.70 (s, 3H, CH ), 5.69 (s, 2H, CH ), 7.26–7.56 (m,

3 2

H, Ph), 7.81 (s, 1H, H-2), 8.00 (s, 1H, H-8);

1

3

5

C

2

7

41

5

NMR (CD OD, 75 MHz) d 33.89 (CH ), 49.85 (CH ),

requires 436.3434, found 436.3427.

3

2

1

12.70 (C-5), 127.83 (2· CH, Ph), 128.23 (CH, Ph),

0

28.91 (2· CH, Ph), 137.14 (C, Ph), 141.67 (C-8),

45.27 (C-5), 146.77 (C-2), 156.76 (C-6); MS EI m/z

5.41. (2 E,6E,10 E)-2,3-Dihydro-3-methyl-7-(3,7,11,15-

tetramethyl-2,6,10,14-hexadecatetraenyl)-3H-purine-

6(7H)-imine (17)

+

(

rel.%) 239 (93, M ), 238 (100), 224 (10), 162 (47), 91

31); Anal. Calcd for C H N : C, 65.25; H, 5.48; N,

(

1

3

13

5

2

9.27. Found C, 65.12; H, 5.83; N, 29.03%.

Compound 15b (256 mg, 0.61 mmol) was dissolved in

70% aq MeOH (10 mL), NaBH₄(92 mg, 2.4 mmol)

0

5

2

.39. (2 E,6E,10 E)-3-Methyl-7-(3,7,11,15-tetramethyl-

,6,10,14-hexadecatetraenyl)-3H-purine-6(7H)-imine (15b)

was added in small portions, and the resulting mixture

was stirred at ambient temperature for 2.5 h. The mix-

ture was evaporated in vacuo and the residue transferred

to a separatory funnel using CH Cl (50 mL) and satd

The compound was prepared from 3-methyladenine 14

100 mg, 0.67 mmol) and (2E,6E,10E)-1-bromo-

2

(

aq K CO (30 mL). The layers were separated and the

2 3

3

0

1

,7,11,15-tetramethyl-2,6,10,14-hexadecatetraene (330 mg,

.93 mmol) as described for the synthesis of compound

5a above. The product was puriﬁed by ﬂash chroma-

aq phase was extracted with CH Cl (5· 25 mL). The or-

2

ganic phases were combined, dried (K CO ), and evap-

2

3

orated in vacuo. The product was puriﬁed by ﬂash

chromatography on silica gel eluting with MeOH/

CH Cl (1:6); yield 143 mg (56%), pale yellow oil. H

tography on silica gel eluting with MeOH/EtOAc

1:4) followed by MeOH (satd NH )/CH Cl (1:7);

1

(

3

2

1

yield 153 mg (54%), yellow wax. H NMR (CDCl₃,

00 MHz) d 1.59 (s, 9H, CH ), 1.67 (s, 3H, CH ), 1.79

NMR (CDCl , 300 MHz) d 1.60 (s, 9H, CH ), 1.71 (s,

3

3H, CH ), 1.77 (s, 3H, CH ), 1.92–2.15 (m, 12H), 2.88

3 3

3

4036

A. Vik et al. / Bioorg. Med. Chem. 15 (2007) 4016–4037

(

4

s, 3H, NCH ), 4.30 (s, 2H, CH ), 4.60 (br s, 1H, NH),

3

use. Antibiotic solutions were prepared fresh for each

experimental run. After the samples were thawed, serial

twofold dilutions were prepared in Mueller–Hinton

broth (Oxoid). Working concentrations ranging from

1.25 to 320 lg/mL were made to obtain ﬁnal antibiotic

concentrations of 0.125–32 lg/mL after dilution in

broth. A direct suspension of colonies was prepared in

10 mL physiological saline. The turbidity of the suspen-

sion was adjusted spectrophotometrically to match a 0.5

McFarland standard. This suspension was further di-

luted 1:10 with Mueller–Hinton broth to obtain the

2

.73 (d, J = 6.5 Hz, 2H, NCH ), 5.09 (m, 3H, CH@),

2

5

NH could not be observed;

.37 (t, J = 6.5 Hz, 1H, CH@), 7.28 (s, 1H, H-8), one

1

3

C NMR (CDCl₃,

7

1

2

3

1

5 MHz) d 15.97 (CH ), 16.01 (CH ), 16.63 (CH ),

3 3 3

7.64 (CH ), 25.65 (CH ), 26.13 (CH ), 26.55 (CH ),

6.72 (CH ), 34.22 (NCH ), 39.34 (CH ), 39.64 (CH ),

3

2

3

2

9.68 (CH ), 44.94 (NCH ), 65.95 (C-2), 105.10,

2

18.84 (CH@), 123.16 (CH@), 124.93 (CH@), 124.33

(

CH@), 131.23 (C@), 134.99 (C@), 135.93 (C@),

38.06 (C-8), 142.30 (C@), 152.90 (C-6), 157.05 (C-4);

MS EI m/z (rel.%) 423 (2, M ), 354 (8), 352 (7), 284

1

+

7

inoculum having a concentration of about 10 CFU/

mL. Susceptibility testing was performed using the broth

(

17), 218 (27), 216 (100), 150 (30), 149 (12); HRMS

EI) C H N requires 423.3361, found 423.3344.

3

2

microdilution technique.

Dilutions (100 lL) were

2

6

41

5

added in triplicate to the wells of a microtiter plate.

To each well was added 5 lL of the inoculum. Broth

alone, inoculated broth, and broth amended with anti-

microbial without inoculate were used as controls. After

inoculation, plates were lidded and incubated aerobi-

cally at 35 ꢁC for 20 h. The MIC value was read as the

lowest concentration of antimicrobial agent that com-

pletely inhibits growth of the organism. Tests were re-

peated at least once with fresh samples and a fresh

inoculum. The MIC values for gentamycin sulfate and

0

5

.42. (2 E,6E,10 E)-2,3-Dihydro-1,3,-dimethyl-7-

3,7,11,15-tetramethyl-2,6,10,14-hexadecatetraenyl)-3H-

(

purine-6(7H)-imine (18)

A mixture of compound 17 (156 mg, 0.37 mmol) and

iodomethane (0.090 mL, 1.47 mmol) in DMA (1.5 mL)

was stirred in a sealed ﬂask at ambient temperature

for 2.5 h. The mixture was evaporated in vacuo and

the residue puriﬁed by ﬂash chromatography on silica

gel eluting with MeOH/CH Cl (1:12); yield 83 mg

streptomycin sulfate were for both strains within the

32,33

2

1

(

51%), pale yellow oil. H NMR (CDCl , 300 MHz) d

range previously reported,

providing a quality con-

3

1

.55 (s, 9H, CH ), 1.64 (s, 3H, CH ), 1.73 (s, 3H,

trol for the analysis. Compounds 3a–b, 3e–g, 3i–j, 3t,

5a–c, and 9b were examined against E. coli (ATCC

25922) and S. aureus (ATCC 25923) as described

3

CH₃), 1.90–2.12 (m, 12H, CH₂), 2.83 [s, 3H,

N(3)CH ], 2.92 [s, 3H, N(1)CH ], 4.08 (s, 2H, CH ),

3

2

9

4

5

.85 (d, J = 6.8 Hz, 2H, NCH ), 5.05 (m, 3H, CH@),

before.

5.44. Determination of cytotoxic activities

Fluorometric Microculture Cytotoxity Assay

2

.26 (t, J = 5.8 Hz, 1H, CH@), 7.18 (s, 1H, H-8), NH

1

3

could not be observed; C NMR (CDCl , 75 MHz) d

3

15.96 (CH ), 15.99 (CH ), 16.55 (CH ), 17.64 (CH ),

25.66 (CH ), 26.19 (CH ), 26.53 (CH ), 26.68 (CH ),

3

3 3 3 3

A

3

2

3.40 [N(1)CH ], 35.24 [N(3)CH ], 39.40 (CH ), 39.62

3

(FMCA) was used to determine cytotoxicity of the agel-

asine analogs 3, 5–7, 9b, 11, and 13, and the agelasimine

3

2

(

1

CH ), 39.66 (CH ), 44.82 (NCH ), 71.21 (C-2), 107.27

2

C-5), 118.36 (CH@), 123.42 (CH@), 124.08 (CH@),

24.31 (CH@), 131.21 (C@), 134.90 (C@), 135.60

2

3

4

analogs 15–18. The procedure was conducted as out-

6

lined in our previous publication.

b

(

C@), 137.36 (C-8), 141.80 (C@), 154.35 (C-4), 155.57

+

C-6); MS EI m/z (rel.%) 437 (5, M ), 394 (15), 368

(

7), 300 (10), 232 (100), 230 (13), 164 (9), 122 (34), 81

21); HRMS (EI) C H N requires 437.3518, found

Acknowledgments

2

7

43

5

4

37.3499.

The Norwegian Research Council is greatly acknowl-

edged for a scholarship to A.V. (KOSK program, Grant

149339/431) as well as for partial ﬁnancing of the Bruker

Avance instrument used in this study. The work has also

been supported by the Swedish Research for Environ-

ment, Agricultural Science and Spatial Planning. Anti-

mycobacterial data were provided by the Tuberculosis

Antimicrobial Acquisition and Coordinating Facility

(TAACF) through a research and development contract

with the U.S. National Institute of Allergy and Infec-

tious Diseases; we are grateful for all help provided by

Dr. C. Kwong and his co-workers. M. C. Olsen at Uni-

versity Hospital of North Norway is acknowledged for

technical assistance with some of the antibacterial sus-

ceptibility assays.

5.43. Determination of antibacterial activity against

E. coli (ATCC 25922) and S. aureus (ATCC 25923)

The reference strains tested were E. coli (ATCC 25922)

and S. aureus (ATCC 25923). The strains were main-

tained as thick suspensions in sterile deionized water

at ꢀ80 ꢁC. Prior to experiment strains were grown for

2

4 h at 35 ꢁC on Tryptone-soya agar (Oxoid, Basing-

stoke, Hampshire, UK). Reagent grade gentamycin sul-

fate and streptomycin sulfate (Sigma–Aldrich, St. Louis,

MO, USA) and agelasine or agelasimine analogs were

prepared as stock solutions of 5120 lg/mL according

to the recommendations of the National Committee

3

2

for Clinical Laboratory Standards: sterile deionized

water (the reference antibiotics) or dimethylsulfoxide

(

agelasine or agelasimine analogs) was used as the sol-

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(

Article Doi

Antimicrobial and cytotoxic activity of agelasine and agelasimine analogs

DOI: 10.1016/j.bmc.2007.03.086

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Full text of DOI:10.1016/j.bmc.2007.03.086

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