Novel polycyclic 'cage'-1,2-diamines as potential anti-tuberculosis agents

Article abstract of DOI:10.1016/j.ejmech.2012.03.041

A series of polycyclic 'cage' derivatives of N-geranyl-1,2 diamines were synthesized and screened for their anti-mycobacterial activity against H ₃₇Rv, multidrug resistant (MDR) and extensively drug-resistant (XDR) strains of tuberculosis. By substituting the adamantyl skeleton of SQ109 with trishomocubanyl (9), oxa-pentacycloundecyl (14, 16), pentacycloundecyl, PCU, (10, 15) and azapentacycloundecyl (22, 23), the effect of other polycyclic "cage" skeletons could be investigated. Compound 9 (trishomocubanyl moiety) proved to be the most active (MICs: 0.5-2 μg/mL) while PCU hydroxyl derivatives (15 and 23), oxa-pentacycloundecyl and azapentacycloundecyl derivatives displayed similar activity to SQ109 (MICs: 0.5-4 μg/mL) against all three strains of TB used in this study.

Full text of DOI:10.1016/j.ejmech.2012.03.041

European Journal of Medicinal Chemistry 54 (2012) 1e9
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Novel polycyclic ‘cage’-1,2-diamines as potential anti-tuberculosis agents
a
b
a
,
a
b
*
Oluseye K. Onajole , Yacoob Coovadia , Hendrik G. Kruger , Glenn E.M. Maguire , Melendhran Pillay ,
c
,
**
Thavendran Govender
School of Chemistry, University of KwaZulu-Natal, Durban, South Africa
Microbiology, National Health Laboratory Services (NHLS), Inkosi Albert Luthuli Central Hospital, Durban, South Africa
School of Pharmacy and Pharmacology, University of KwaZulu-Natal, Durban, South Africa
a
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 10 February 2012
Received in revised form
A series of polycyclic ‘cage’ derivatives of N-geranyl-1,2 diamines were synthesized and screened for their
anti-mycobacterial activity against H37Rv, multidrug resistant (MDR) and extensively drug-resistant
(
(
XDR) strains of tuberculosis. By substituting the adamantyl skeleton of SQ109 with trishomocubanyl
9), oxa-pentacycloundecyl (14, 16), pentacycloundecyl, PCU, (10, 15) and azapentacycloundecyl (22, 23),
22 March 2012
Accepted 22 March 2012
Available online 4 April 2012
the effect of other polycyclic “cage” skeletons could be investigated. Compound 9 (trishomocubanyl
moiety) proved to be the most active (MICs: 0.5e2 g/mL) while PCU hydroxyl derivatives (15 and 23),
oxa-pentacycloundecyl and azapentacycloundecyl derivatives displayed similar activity to SQ109 (MICs:
.5e4 g/mL) against all three strains of TB used in this study.
Ó 2012 Elsevier Masson SAS. All rights reserved.
m
Keywords:
SQ109
Polycyclic “cage”
Tuberculosis
Anti-tuberculosis
0
m
1
. Introduction
anti-TB drug candidates such as diarylquinolone (TMC207), nitro-
imidazole (OPC67683 and PA824), pyrrole (LL3858), diamine
(SQ109) are in different stages of clinical trials [2e5].
Tuberculosis (TB) is a highly contagious and insidious disease
with a high infection rate that has been present in humans since
antiquity. TB is predominantly caused by Mycobacterium tubercu-
losis which is a slow growing bacterium. The causal organism can
remain dormant in the host (human) for a very long time and may
only become active when the person falls sick or has a back drop in
his/her immune system. This event in particular has become most
prominent in immuno-compromised patients such as those living
with HIV (Human Immunodeficiency Virus). The 2011 global
tuberculosis control report of the World Health Organisation
SQ109 (2) first reported by Lee et al. [6] shares the same 1,2
ethylenediamine pharmacophore with ethambutol (1). SQ109
possesses remarkable activity against MDR-TB which includes the
EMB resistant strain suggesting that SQ109 is an anti-TB agent with
a new mechanism [7]. SQ109 induces a synergistic effect when used
in combination with other first line drugs such as isoniazid and
rifampicin, however an additive effect is observed when used with
streptomycin [8].
The incorporation of polycyclic “cage” compounds (such as
adamantane and pentacycloundecane, PCU) into drugs with phar-
maceutical applications has enjoyed much attention from
researchers for several decades starting with the discovery of
amantadine, an anti-viral drug [9e11]. Polycyclic “cage”
compounds have the ability to improve drug lipophilicity, thus
serving as a transport aid in carrying such drugs across cell
membranes. It has been reported that ‘cage’ moieties such as ada-
mantane and PCU are able to cross the Blood Brain Barrier (BBB)
and the Central Nervous System (CNS) [9,12,13]. It also helps to
reduce the bio-degradation of drugs in biological systems thus
prolonging their pharmaceutical effect in the body [9,14,15]. SQ109
(
WHO), estimated that about 8.8 million incident cases of TB
occurred globally. An estimated 1.1 million (13%) patients are living
with HIV, and 82% of TB cases among people living with HIV
occurred in the African region [1].
An urgent need for highly potent, more effective drugs
with fewer or no side effects and shorter treatment periods to
combat the increasing TB pandemic is therefore apparent. Potential
*
Corresponding author. Tel.: þ27 31 2602181; fax: þ27 31 2603091.
*
* Corresponding author. Tel.: þ27 31 2608212; fax: þ27 31 2603091.
(2) and SQ117 (3) both have a lipophilic moiety, namely the 2-
E-mail addresses: kruger@ukzn.ac.za (H.G. Kruger), govenderthav@ukzn.ac.za
adamantyl moiety in SQ109 (log P; 5.26) and a diphenyl moiety
in SQ117 (log P; 5.50). However, different MIC values were reported
(
T. Govender).
URL: http://ggkm.ukzn.ac.za
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.03.041

2
O.K. Onajole et al. / European Journal of Medicinal Chemistry 54 (2012) 1e9
for each [7]. SQ109 was five-fold more active than SQ117 suggesting
that the nature of the lipophilic group does play a role in their
activities even though SQ117 possesses a higher log P value [7]
Fig. 1.
literature [13,26]. Compound 11 was debenzylated using 10% Pd/C
under hydrogen gas at atmospheric pressure to obtain 12 in 60.8%
yield [27]. Compound 12 was reacted with chloroacetyl chloride to
obtain compound 13 (62% yield), which was reacted with ger-
anylamine [17] to obtain compound 14 (58.5% yield). Compound 14
was reduced with a strong agent (LAH) at a 1:4 ratio in dry THF
under reflux conditions to obtain a compound with m/z of 357.2897
In the course of our research for novel anti-tubercular
compounds [16,17], we recently demonstrated the importance
of the length, saturation and di-substitution of the alkene chain
of some SQ109 derivatives [17,18]. This present study aims at
investigating the possibility of further enhancing/improving the
anti-TB activity of the diamine via substitution of the adamantyl
moiety with other polycyclic compounds such as trishomocubane
and pentacycloundecane. Based on this; seven novel diamine
based compounds bearing the lipophilic pentacycloundecane,
oxa-pentacycloundecane aza-pentacycloundecane and trisho-
mocubane cages were synthesized and screened for activity
against drug sensitive (H37Rv) and drug-resistant strains of
tuberculosis.
þ
(M þ H , 33.7% yield). Compound 15 was proposed and this
structure was confirmed using 2D NMR experiments. A through
space interaction of H-11 (3.86 ppm) with H-1 (2.68 ppm), H-3
(2.30 ppm) and H-10 (2.36 ppm) was observed thus proving that
the hydroxyl group was at an endo position. H-8 also displayed
a NOESY interaction with H-5 (2.34 ppm), H-7 (2.80 ppm), H-9
0
0
(2.51 ppm) and H-1 /2 (ethylene protons at 3.18 ppm) while the
0
0
methylene protons, H-1 /2 , displayed NOESY interactions with
methyl protons (1.61 ppm), H-7, H-8 and H-9 respectively. These
assignments confirm the endo positioning of the isoprenyl diamine
moiety on the PCU cage as a result of the opening of the ether
bridge.
2
. Results and discussion
Attempts to synthesize the H-8 endo-orientated compound 10
prove unsuccessful; however an exo positioning of the R-group
would be obtained if an oxa-pentacycloundecyl moiety is used, this
led to the design and successful synthesis of compound 16. This was
achieved by using a milder reducing reagent (65% Red-Al in
toluene) on compound 14 to obtain 16 (45.2% yield). NMR spec-
troscopic analysis of compound 16 showed the successful synthesis,
as no extra methine carbon was observed at position 57.4 ppm and
a quaternary carbon (C-8) at position 110.1 ppm was observed. C-8
2
.1. Chemistry
Cookson’s dione 4 [19,20] was the starting material for the
synthesis of trishomocubanone 5 and the PCU-monoketone 6.
Trishomocubanone was synthesized via a six step reaction pathway
[21,22] and PCU monoketone was synthesized via a five step reac-
tion pathway as reported in literature [21e23] (Scheme 1). Geranyl
bromide 7 was reacted with excess ethane-1,2-diamine in dry
dichloromethane at ꢀ78 C to afford (E)-N -(3,7-dimethylocta-2,6-
dienyl)ethane-1,2-diamine 8 (86% yield). Isoprenyl ethane-1,2-
diamine 8 was reacted with trishomocubanone, PCU monoketone
and 2-adamantanone via reductive amination; the resulting imines
0
ꢁ
0
displays a HMBC correlation with methylene protons (H-1 ) at
3.02 ppm while H-11 (4.58 ppm) also displayed NOESY interactions
with H-1, H-3 and H-10 respectively, thus confirming the successful
synthesis of compound 16 Scheme 3.
Replacement of the oxo-bridge of 16 with an aza-bridge to give
the isomeric hemiaminal 23 was also achieved (Scheme 4) and this
compound was screened for anti-TB activity. Mono-protection of
Cookson’s diketone 4 was carried out to obtain the ethylene ketal
were reduced with NaBH
diamines were converted to their HCl salts to obtain compounds 9
4
to obtain polycyclic-diamines; the
0
[
N-geranyl-N -(11-trishomocubanyl)ethane-1,2-diamine
hydro-
0
chloride] and SQ109 [N-geranyl-N -(2-adamantyl)ethane-1,2-
diamine hydrochloride] (55e57% yield). The reaction with PCU
monoketone to yield 10 [N-geranyl-N -(8-pentacycloundecyl)
ethane-1,2-diamine hydrochloride] was successful. The expected
orientation of the nitrogen atom on the PCU skeleton upon
reductive amination with NaBH
17 in 74% yield and condensation with benzylamine gave the imine
0
18. Reduction with NaBH , followed by hydrolysis resulted in the
4
formation of the racemic hexacyclic cage amine 19. Benzyl depro-
tection of 19 led to compound 20 (44% yield). Geranylamine was
reacted with chloroacetyl chloride to afford (E)-2-chloro-N-(3,7-
dimethylocta-2,6-dienyl)acetamide (21, 81% yield) which was
4
is the endo-form (10) [24,25]. This
orientation was confirmed by a NOESY experiment as H-8
2
,6 3,10 5,9 8,11
reacted with 4-azahexacyclo [5.4.1.0 .0 .0 .0 ] dodecan-3-ol
20) to obtain 22 (81% yield). Reduction of 22 to yield the desired
(
(
2.67 ppm) displayed through space interaction with H-5 and H-9
2.16e2.18 ppm) Scheme 2.
(
compound 23 (72% yield) was achieved using 65% Red-Al in
toluene.
It was anticipated that the presence of a hydroxyl group (endo/
exo positioning) on the cage moiety might contribute significantly
to its activity; this led to the design and successful synthesis of
compound 15 and 23. 8-Benzylamino-8, 11-oxapentacyclo-
The successful syntheses of all novel compounds were
1
13
confirmed using H, C, 2D NMR experiments (COSY, HSQC, HMBC,
5.4.0.0 .03,10.05,9]undecane 11 was synthesized as reported in
2
,6
ROESY and NOESY), IR and HR-MS.
[
2.2. Anti-tubercular activity
All compounds (except SQ109 which is achiral) were tested
as a racemate. The in vitro anti-mycobacterial activity of all
the compounds against M. tuberculosis was carried out using
the Mycobacteria Growth Indicator Tube system (MGIT). The
minimum inhibitory concentration (MIC, mg/mL) was detected by
BACTEC 960TB (Becton Dickinson). All compounds were screened
against H37Rv (ATCC No: 25177), MDR and XDR strains of tuber-
culosis and the results are summarized in Table 1. Compounds 14
and 22 did not show any promising anti-TB activity implying that
the carbonyl group contributes negatively to their inhibitory effect.
Compounds 15 (endo-positioned hydroxyl group) and 23 (exo-
positioned hydroxyl group) displayed similar activity as SQ109
Fig. 1. Structure of ethambutol (1), SQ109 (2) and SQ117 (3).
against both MDR and XDR strains of TB at a MIC of 2 and 4 mg/mL

O.K. Onajole et al. / European Journal of Medicinal Chemistry 54 (2012) 1e9
3
Scheme 1. Synthetic route for the synthesis of trishomocubanone 5 and the PCU monoketone 6.
respectively. On the other hand, compound 10 (similar to 15)
3. Conclusion
without the hydroxyl group displayed reduced activity against
all tested TB stains; it appears that the hydroxyl group not with-
standing its positioning is essential for anti-tubercular activity. The
oxa-pentacycloundecyl derivative 16 proved to be more active
We have synthesized a series of novel polycyclic ‘cage’ diamine
derivatives with potent anti-TB activity. As observed, the posi-
tioning of the isoprenyl diamine on the PCU moiety either endo
or exo does influence its anti-TB activity while the hydroxyl group
(
two-fold) than 10 suggesting that the exo positioning of the iso-
prenyl diamine moiety on the pentacycloundecanyl moiety is
essential for the anti-TB activity. Compound 9 proves to be the
most active in this series; with a twofold increase in activity
against MDR and XDR-TB when compared to 10, 15, 16, 23 and
SQ109. The D -trishomocubyl derivative 9 displayed significantly
3
higher activity than its pentacycloundecyl and adamantyl
counterparts.
This result suggests that the nature or type of polycyclic “cage”
compounds is important for activity. As observed by some
researchers in the field [9,11] the substitution of the polycyclic
is also essential for efficacy. Compound 9 (trishomocubyl
moiety) was identified as the most potent against MDR and XDR
strains of TB used with a two-fold increase in activity than 10, 15
(pentacycloundecyl), 16 (oxa-pentacycloundecyl), 23 (azapentacy-
cloundecyl), and SQ109 (2-adamantyl). This suggests that the type
of polycyclic ‘cage’ moiety used has an influence on the activity of
this compound. Further studies are ongoing in our laboratory to
derivatise the lead compound with the possibility of enhancing its
anti-TB activity.
“cage” moiety (adamantyl) with similar moieties such as trisho-
4. Experimental
mocubane, pentacycloundecane, oxa-pentacycloundecane etc. in
most cases maintains the activity of such compounds. Of all
reported polycyclic ‘cage’ compounds only trishomocubane have
The NMR spectroscopic data were recorded on Bruker AVANCE
III 400 MHz and 600 MHz instruments using CDCl as a solvent. All
3
a unique D
3
stereochemistry which could have contributed to its
chemical shifts (
d) were quoted in parts per million downfield from
increased activity in this series.
TMS and the coupling constants (J) recorded in Hertz. Splitting
Scheme 2. Reagents and conditions: (a) ethylene diamine (100:1), ꢀ78 ꢁC, dry DCM; (b) MeOH, polycyclic ‘cage’ monoketone (1.2:1), N
2
4
atmosphere, 2 h, NaBH , overnight; then
HCl, MeOH.

4
O.K. Onajole et al. / European Journal of Medicinal Chemistry 54 (2012) 1e9
ꢁ
ꢁ
Scheme 3. Reagents and conditions: (a) benzylamine, THF, 0 Ce5 C, 20 min; azeotropic distillation, benzene, 1 h, NaBH
pressure; (c) chloroacetyl chloride, dry DCM, K CO , reflux for 12 h; (d) geranylamine (1:2) mole ratio, K CO , dry THF, reflux; (e) LAH (1:5) mole ratio, dry THF, reflux, N
atmosphere, 12 h; then HCl, MeOH. (f) Red-Al (1:5) mole ratio, dry THF, reflux, N atmosphere; then HCl, MeOH.
4 2
, 24 h; (b) 10% Pd/C (1:1) mass ratio, H gas, atmospheric
2
3
2
3
2
2
pattern abbreviations are as follows: s ¼ singlet, d ¼ doublet,
t ¼ triplet, m ¼ multiplet, br ¼ broad. Reactions were monitored
using thin layer chromatography (TLC, Merck Kieselgel 60, F254).
Most purifications were achieved with column chromatography
95% solution A and 5% solution B which changes linearly over
25 min to 20% solution A and 80% solution B at 15 mL/min, detected
on a UVeVIS detector at 215 and 254 nm. Mass Spectra were
obtained using a Bruker MicroTOF QII Time of Flight mass spec-
trometer while melting point analysis was performed on a Stuart
Scientific digital melting point apparatus SMP3. Melting points
results were uncorrected. Tetrahydrofuran was freshly distilled
3 3 4
using Fluka Kieselgel 60 (70e230 mesh) and CHCl :CH OH:NH OH
(
88:10:2) as the eluent (solvent mixture). Purification of compound
15 were done via semi-preparative HPLC on a Shimadzu, LC-6AD
instrument using water (solvent A) and acetonitrile (solvent B)
before use from a flask containing sodium benzophenone under N
2
while methanol (as Solvent B) was used for compound 23. An ACE
atmosphere while dichloromethane was dried using phosphorus
pentoxide prior to use.
5
C18 150 ꢂ 21.2 mm column was used. A gradient elution system of
ꢁ
Scheme 4. Reagent and conditions: (a) ethylene glycol, p-toluenesulfonic acid (cat.), toluene, reflux, DeaneStark Conditions; (b) benzylamine, EtOH, 100 C, 18 h; (c) NaBH
4
, EtOH,
rt, 8 h; (d) acetone, 4 M HCl, 12 h, basified with 1M NaOH; (e) MeOH, 10% Pd/C, H
2
atm; (f) 21, K
2
CO
3
, THF, reflux, H
2
atm; (g) Red-Al (1:5) mole ratio, dry THF, reflux, N
2
atmosphere;
then HCl, MeOH.

O.K. Onajole et al. / European Journal of Medicinal Chemistry 54 (2012) 1e9
5
Table 1
atmosphere was dropwise added geranyl bromide 7 (2 g, 9.2 mmol)
in dichloromethane (1200 mL) over 4 h. The reaction mixture was
left to attain room temperature with stirring over night. The solu-
tion mixture was reduced in vacuo to about 500 mL and washed
with water to remove excess ethane-1,2 diamine, the organic
The MICs of the target compounds against M. tuberculosis (H37Rv, MDR and XDR)
strains.
Compound
Structure
MIC (
37Rv
mg/mL)
H
MDR
XDR
2
extract was dried over Mg
crude residue was purified using column chromatography [eluent;
CHCl :CH OH:NH OH (88:10:2)] to give a light yellow oil (1.56 g,
86%, R , 400 MHz]: 1.36 (NH), 1.56 (3H, s,
¼ 0.45). H NMR [CDCl
CH ), 1.61 (3H, s, CH ), 1.64 (3H, s, CH ), 1.97 (2H, m, CH ), 2.05 (2H,
m, CH ), 2.78 (2H, t, J ¼ 5.7, 6.1 Hz, CH ),
), 2.63 (2H, t, J ¼ 5.6 Hz, CH
.21 (2H, d, J ¼ 6.8 Hz, CH
2 4
SO and concentrated in vacuo. The
H
N
R
3
3
4
N
9
0.5
1
1
H
d
H
H
f
3
.
2HCl
3
3
3
2
2
2
2
H
N
3
2
), 5.06 (1H, J ¼ 6.9 Hz, CH), 5.23 (1H,
R
13
N
J ¼ 6.8 Hz, CH). C NMR [CDCl
5.7 (CH ), 26.5 (CH ), 39.6 (CH
22.8 (CH), 124.1 (CH), 131.5 (C), 137.7 (C).
, 100 MHz]:
d
C
16.3 (CH
), 17.6 (CH
),
),
3
3
3
1
1
0
4
H
1
4
4
8
8
H
2
1
3
2
2
), 41.8 (CH
2
), 47.1 (CH ), 52.1 (CH
2
2
.
2HCl
0
4
.2. Synthesis of N-geranyl-N -(trishomocubanyl)ethane-1,2-
O
HN
R
0
diamine (9), N-geranyl-N -(pentacycloundecyl)ethane-1,2-diamine
10), N-geranyl-N -(2-adamantyl)ethane-1,2-diamine (SQ109)
1
0
(
NH
O
A mixture of isoprenyl diamine 8 (1.2 mol) and polycyclic ‘cage’
H
monoketone (1.0 mol) in methanol (15 mL) was stirred for 2 h at
room temperature under a nitrogen atmosphere. The resulting
H
4
imine was reduced with solid NaBH (1.2 mol) which was added
slowly over 15 min and the mixture stirred overnight. Additional
methanol (15 mL) was added to the reaction vessel after which
H
1
1
5
6
N
0.5
2
2
4
4
R
N
H
OH
H
water (20 mL) was added to quench excess NaBH
extracted with ethyl acetate (2 ꢂ 50 mL) and the solution dried over
Na SO and concentration in vacuo. The crude product was purified
via column chromatography on silica gel using CHCl :CH OH:N-
OH (88:10:2) as eluent to give a yellow oil and converted to its
HCl salt.
4
. The solution was
.
2HCl
HN
R
2
4
3
3
1
H
4
NH . 2HCl
O
H
0
4
.2.1. Data for N-geranyl-N -(11-trishomocubyl)ethane-1,2-
diamine dihydrochloride (9)
A white solid (Mp. 174e178 C, 0.65 g, yield 57%, R
ꢁ
f
¼ 0.64). IR
1
ꢀ
v
max: 3388, 2949, 2871, 2736, 1585, 1443, 1034, 773 and 555 cm
MS (TOF) calculated for C23
.
22
OH
1
4
8
þ
H
37
N
2
(M þ H of free base) 341.2951,
N
1
found 341.2940. H NMR [CDCl
3
, 400 MHz]:
d
H
1.21e1.28 (3H, m, H-
),
), 1.85e1.88 (2H, m, H-1, 2),
), 1.97e2.03 (5H, m, H-5, 6, 9, CH ),
.37 (1H, s, H-3), 2.63e2.69 (4H, m, 2 ꢂ CH ), 2.91 (1H, s, H-11), 3.17
), 5.03 (1H, m, CH), 5.19 (1H, m, CH). C NMR
H
NH
R
4
a, H-7a, H-7s), 1.37 (1H, AB, d, J ¼ 10.2 Hz, H-4s), 1.53 (3H, s, CH
3
O
1.57 (3H, s, CH
1
2
(
[
3
), 1.61 (3H, s, CH
3
3
.91e1.96 (4H, m, H-8, 10, CH
2
2
2
13
2H, d, J ¼ 6.8 Hz, CH
CDCl , 100 MHz]:
2.8 (C-4), 33.4 (C-7), 39.5 (CH
2
3
d
C
16.2 (CH
3
), 17.6 (CH
3
), 25.6 (CH
3
), 26.4 (CH
2
),
2
3
OH
1
2
2
4
4
2
), 40.6 (C-8), 41.4 (C-2), 44.2 (C-3),
N
H
2 2
44.7 (C-9), 46.9 (CH ), 47.0 (C-5), 47.4 (C-6), 48.0 (CH ), 49.2 (CH
2
),
NH
R
50.4 (C-10), 51.6 (C-1), 64.2 (C-11), 122.8 (CH), 124.0 (CH), 131.3 (C),
.
2HCl
137.5 (C).
H
N
R
4
.2.2. Data for N-geranyl-N -(8-pentacycloundecyl)ethane-1,2-
diamine dihydrochloride (10)
A white solid (Mp. 158e160 C, 0.59 g, yield 55%, R
0
N
H
SQ109
0.5
.
2HCl
ꢁ
f
¼ 0.62). IR
1
ꢀ
v
max: 3140, 3048, 2948, 2694, 1444, 1405, 1034, 793 and 557 cm
MS (TOF) calculated for C23
found 341.2928. H NMR [CDCl
.
þ
H
37
N
2
(M þ H of free base) 341.2951,
1
3
, 400 MHz]:
d
H
0.94 (1H, AB,
),
), 1.64 (1H, s, H-4s), 1.83 (2H, br s,
J ¼ 11.9 Hz, H-11a), 1.13 (1H, AB, J ¼ 10.3 Hz, H-4a), 1.55 (3H, s, CH
3
SQ109 was used as the reference drug; R ¼ geranyl moiety.
1.59 (3H, s, CH
3
), 1.62 (3H, s, CH
3
Values reported were done in triplicate.
NH), 1.91e1.98 (2H, m, CH
2
2
), 2.02e2.07 (2H, m, CH ), 2.16e2.18 (3H,
m, H-3, 5, 9), 2.26 (1H, s, H-10), 2.31 (1H, AB, J ¼ 11.7 Hz, H-11s),
0
4
.1. Synthesis of (E)-N -(3,7-dimethylocta-2,6-dienyl)ethane-1,2-
2.43 (1H, m, H-6), 2.49e2.54 (2H, m, H-2, 7), 2.60e2.62 (1H, m, H-
diamine (8) [17]
1), 2.63e2.70 (5H, m, H-8, 2 ꢂ CH
.05 (1H, t, J ¼ 5.6 Hz, CH), 5.21 (1H, t, J ¼ 5.9 Hz, CH). C NMR
[CDCl , 100 MHz]: 16.2 (CH ), 17.6 (CH ), 25.6 (CH ), 26.4 (CH ),
28.7 (C-11), 34.6 (C-4), 36.2 (C-1), 37.7 (C-7), 39.6 (CH ), 40.8 (C-6),
2
), 3.19 (2H, d, J ¼ 6.8 Hz, CH
2
),
13
5
To a vigorously stirred solution of ethane-1,2-diamine (55.4 g,
3
d
C
3
3
3
2
0
.92 mol) in dichloromethane (400 mL) at ꢀ78 ꢁC under N
2
2

6
O.K. Onajole et al. / European Journal of Medicinal Chemistry 54 (2012) 1e9
4
9
1.8 (C-10), 41.9 (C-2), 44.2 (C-3/5), 44.6 (C-3/5), 46.9 (CH
2
), 47.2 (C-
2.30e2.37 (m, 3H), 2.51e2.56 (m, 2H), 2.69e2.80 (m, 3H), 4.56 (t,
13
), 48.6 (CH
2 2
), 49.3 (CH ), 61.9 (C-8), 122.6 (CH), 124.1 (CH), 131.4
1H). C NMR [CDCl
43.4 (CH ), 44.8 (CH), 47.0 (CH), 55.1 (CH), 57.4 (CH), 83.0 (CH),
06.3 (C).
3 C
, 100 MHz]: d 41.4 (CH), 42.4 (CH), 43.3 (CH),
(
C), 137.7 (C).
2
1
0
4.2.3. Data for SQ109 (N-geranyl-N -(2-adamantyl)ethane-1,2-
diamine) dihydrochloride
A white solid (Mp. 180e184 C, 0.35 g, 60% yield, R
4.4. Synthesis of compound 8-chloroacetylamine-8,11-
oxapentacyclo[5.4.0.0. .0 .0 ]-undecane (13)
ꢁ
2,6 3,10 5,9
f
¼ 0.62). IR
v
5
max: 3142, 3050, 2910, 2850, 1588, 1459, 1408, 1102, 778 and
ꢀ
1
þ
53 cm . HR-MS calculated for C22
H
39
N
2
(M þ H) 331.3108,
A mixture of compound 12 (1.62 g, 9.2 mmol), chloroacetyl
1
found 331.3135; H NMR (CDCl
J ¼ 12.5 Hz, H-4a/9a), 1.57 (3H, s, CH
CH ), 1.66e1.68 (4H, m, H-6, 8a/10a), 1.74 (1H, s, H-5), 1.80e1.82
5H, m, H-1, 3, 7, 8b/10b), 1.95 (2H, d, J ¼ 12.6 Hz, H-4b/9b),
.98e2.00 (2H, m, CH ), 2.03e2.09 (2H, m, CH ), 2.67 (1H, s, H-2),
), 3.22 (2H, d, J ¼ 6.8 Hz, CH ), 5.06 (1H, m, CH),
, 100 MHz); 16.3
), 27.6 (C-5), 27.8 (C-7), 31.3
C-4/9), 32.1 (C-1/3), 37.6 (C-8/10), 37.9 (C-6), 39.6 (CH ), 46.5
CH ), 47.1 (CH ), 49.5 (CH ), 61.9 (C-2), 122.9 (CH), 124.1 (CH),
3
, 400 MHz):
d
H
1.47 (2H, d,
chloride (0.807 mL,10.1 mmol) and K
DCM (20 mL) was stirred and heated gently at 40 C for 1 h and
2
CO
3
(2.54 g,18.4 mmol) in dry
ꢁ
3
), 1.61 (3H, s, CH
3
), 1.65 (3H, s,
3
allowed to stir overnight without heat. Purification was carried on
silica, CH Cl :CH OH (95:5) to obtain pale yellow oil which solidi-
2 2 3
(
1
2
2
fied on standing at room temperature to afford a yellowish solid
ꢁ
1
2
5
.70 (4H, s, 2 ꢂ CH
.24 (1H, t, J ¼ 6.7 Hz, CH). C NMR (CDCl
), 17.6 (CH ), 25.7 (CH ), 26.5 (CH
2
2
(Mp. 152e154 C, 1.44 g, 62%, R
f
¼ 0.67). H NMR [CDCl
3
, 400 MHz]:
13
3
d
C
d
H
1.53 (AB, J ¼ 10.6 Hz, 1H), 1.91 (AB, 10.6 Hz, 1H), 2.42 (s, 1H),
(
(
(
CH
3
3
3
2
2.59e2.62 (m, 2H), 2.74e2.79 (m, 1H), 2.89e1.98 (m, 4H), 4.02 (s,
13
2
2H), 4.73 (t, 1H). C NMR [CDCl
3 C
, 100 MHz]: d 41.4 (CH), 41.9 (CH),
2
2
2
42.6 (CH ), 43.2 (CH ), 43.6 (CH), 44.6 (CH), 44.8 (CH), 46.1 (CH),
2
2
131.5 (C), 137.6 (C).
55.0 (CH), 56.7 (CH), 83.7 (CH), 102.9 (C), 166.2 (C).
4
.2.4. 8-Benzylamino-8,11-oxapentacyclo[5.4.0.02,6.03,10.05,9
]
4.5. Synthesis of 8-[(E)-N-3,7-dimethylocta-2,6-dienylamino]
acetamide-8,11-oxapentacyclo[5.4.0.0. .0 .0 ]-undecane (14)
2
,6 3,10 5,9
undecane (11) [13]
PCU-8,11-dione (Cookson’s dione) was synthesized via photo-
cyclization of DielseAlder adducts obtained from reacting freshly
cracked cyclopentadiene with p-benzoquinone [19,20]. PCU-dione
To a solution of geranylamine (0.33 g, 3.57 mmol) in dry THF
(15 mL) was added K CO (0.37 g) and compound 13 (0.45 g,
2 3
(
(
5.0 g, 28.7 mmol) was dissolved in anhydrous tetrahydrofuran
THF, 50 mL) and cooled with stirring to 5 C with an external ice
1.8 mmol), the mixture was stirred with reflux, and the reaction
was monitored by TLC until no starting material was observed. The
reaction mixture was cooled, filtered and concentrated in vacuo, the
ꢁ
bath. Benzylamine (3.39 g, 31.6 mmol) was added slowly with
continuous stirring while maintaining the temperature. The reac-
tion mixture was stirred over 30 min and the resulting hydroxyl-
amine (a white precipitate) was filtered and washed with cold THF.
Dehydration of the hydroxylamine in dry benzene was achieved
under DeaneStark condition for 1 h or until no more water
collected in the trap. The resulting solution was concentrated in
vacuo to obtain the Schiff base (a yellow oil) which was reduced
crude product was purified on silica gel; CHCl
3
:CH
¼ 0.34). IR vmax
3308, 2966, 1672, 1514, 1002 and 747 cm . MS (TOF) calculated for
3
OH (95:5) to
obtain 14. A yellow oil (380 mg, 57.5% yield, R
f
:
ꢀ1
þ
1
C
23
H
33
N
2
O
2
d
(M þ H ) 369.2537, found 369.2537. H NMR [CDCl
3
,
400 MHz]:
(3H, s, CH
H
1.54 (1H, AB, J ¼ 10.5 Hz, H-4a), 1.57 (3H, s, CH
3
), 1.58
3
), 1.59 (3H, s, CH
3
), 1.90 (1H, AB, J ¼ 10.5 Hz, H-4s),
1.95e2.06 (4H, m, 2 ꢂ CH
m, H-5), 2.57e2.60 (1H, m, H-2), 2.69e2.73 (1H, m, H-6), 2.85e3.00
(4H, m, H-1, 7, 9, 10), 3.18 (2H, d, J ¼ 6.88 Hz, CH ), 3.19 (2H, s, CH ),
4.69 (1H, t, J ¼ 5.16 Hz, H-11), 5.02e5.06 (1H, m, CH), 5.13e5.17 (1H,
2
), 2.41 (1H, t, J ¼ 4.76 Hz, H-3), 2.53 (1H,
4
with NaBH (1.63 g, 43.05 mmol) in dry methanol (30 mL) and dry
THF (150 mL) with stirring for 24 h at room temperature. The
2
2
solution was concentration in vacuo and water (2 ꢂ 100 mL) was
1
3
added and the resulting mixture extracted with CH
2
Cl
2
(4 ꢂ 50 mL)
m, CH), 7.94 (CONH). C NMR [CDCl
(CH ), 25.6 (CH ), 26.4 (CH ), 39.5 (CH
(C-4), 43.5 (C-5), 44.6 (C-1), 44.8 (C-3), 45.9 (C-7), 47.1 (CH
(CH ), 54.8 (C-10), 56.4 (C-9), 83.5 (C-11), 102.2 (C-8), 121.7 (CH),
3
, 100 MHz]:
), 41.4 (C-2), 41.8 (C-6), 43.4
), 52.2
C 3
d 16.2 (CH ), 17.7
and the combined organic solution dried over MgSO
concentrated in vacuo to yield a yellow oil. Purification was carried
using column chromatography with solvent system; Hex-
4
and
3
3
2
2
2
2
ꢁ
ane:CH
.73 g, 36% yield, R
AB, J ¼ 10.44 Hz, 1H), 1.91 (AB, J ¼ 10.48 Hz, 1H), 2.14 (br s, 1H, NH),
.42 (t, J ¼ 4.9 Hz, 1H), 2.51e2.62 (m, 4H), 2.71e2.84 (m, 3H), 3.98
AB, J ¼ 13.36 Hz, 1H), 4.03 (AB, J ¼ 13.36 Hz, 1H), 4.66 (t, J ¼ 5.3 Hz,
H), 7.21e7.37 (m, 5H). 13C NMR [CDCl
,100 MHz]: 41.5 (CH), 42.0
CH), 43.1 (CH), 43.2 (CH ), 54.7
2
Cl
2
(1:1) to obtain 11 as a colourless solid (Mp. 79e80 C,
123.9 (CH), 131.6 (C), 139.0 (C), 172.1 (C]O).
1
2
(
2
(
f
¼ 0.20). H NMR [CDCl
3 H
, 400 MHz]: d 1.55
4.6. Synthesis of 11-hydroxylpentacyclo[5.4.0.0.2,6.03,10.05,9]-
undecane-8-aminoethyl[(E)-N-3,7-dimethylocta-2,6-dien-amine]
dihydrochloride (15)
1
3
d
C
(
(
(
2
), 44.5 (CH), 44.8 (2 ꢂ CH), 47.8 (CH
2
Compound 14 (460 mg, 1.25 mmol) dissolved in dry THF (15 mL)
was added LAH (0.24 g, 6.25 mmol) gently; the mixture was
refluxed overnight under nitrogen atmosphere. The reaction vessel
CH), 55.2 (CH), 82.5 (CH), 109.5 (C), 126.8 (CH), 127.8 (CH), 128.3
CH), 140.8 (C).
2 4
was cooled and the mixture quenched with aqueous Na SO , the
4
.3. Synthesis of 8-amino-8,11-oxapentacyclo[5.4.0.02,6.03,10.05,9
undecane (12)
]
obtained precipitate was filtered off and the filtrate concentrated to
obtain the crude product. The crude product was purified via
preparative HPLC (as specified above, retention time: 9.1 min)
sample was lyophilized to obtain 15 (150 mg, 33.7% yield) and
converted to its HCl salt to obtain a yellow slurry. IR vmax: 3149,
To a solution of compound 11 (2.73 g, 10.3 mmol) dissolved in
dry methanol (100 mL) was added 10% Pd/C (1.37 g) and stirred
under hydrogen gas at atmospheric pressure for 16 h or until no
starting material was observed on TLC. The spent Pd/C was filtered
using celite and a sintered funnel, the solution was concentrated in
ꢀ1
3048, 2960, 2809, 1452, 1071 and 568 cm MS (TOF) calculated for
þ
1
C
23
H
37
N
2
O (M þ H of free base) 357.2900, found 357.2897. H NMR
, 400 MHz]:
1.08 (1H, AB, J ¼ 10.8 Hz, H-4a), 1.53 (3H, s,
),
[CDCl
CH
3
d
H
vacuo and the crude product purified on silica using CH
2
Cl
2
:CH
3
OH
3
), 1.61e1.63 (7H, m, H-4s, 2 ꢂ CH
3
), 1.98e2.01 (4H, m, 2 ꢂ CH
2
ꢁ
(
95:5) to obtain 12 in pure form. A white solid (Mp. 143e145 C,
2.30e2.38 (3H, m, H-3, 5, 10), 2.50e2.55 (2H, m, H-2, 9), 2.64e2.69
1
1.66 g, yield 60.8% yield, R
f
¼ 0.52). H NMR [CDCl
3
, 400 MHz]:
d
H
(2H, m, H-1, 6), 2.80 (1H, s, H-7), 2.94 (s, 1H, H-8), 3.18 (4H, s,
1.50 (AB, J ¼ 10.44 Hz, 1H), 1.86 (AB, J ¼ 10.48 Hz, 1H), 2.09 (NH
2
),
2 ꢂ CH
2
), 3.49 (2H, d, J ¼ 7.2 Hz, CH ), 3.86 (1H, t, J ¼ 3.2 Hz, H-11),
2

O.K. Onajole et al. / European Journal of Medicinal Chemistry 54 (2012) 1e9
7
4
.98 (1H, t, J ¼ 5.2 Hz, CH), 5.22 (1H, t, J ¼ 6.7 Hz, CH). 13C NMR
concentrated in vacuo, water (20 mL) was added and the interme-
diate was extracted with CH Cl
(3 ꢂ 15 mL). The combined organic
solution was washed with brine (15 mL) dried (MgSO ) and
concentrated in vacuo. To the crude product was added acetone
(30 mL) and aqueous 4 M HCl (20 mL) with stirring at room
temperature for 12 h. Water was added (250 mL) and the solution
was basified to pH 14 with aqueous 1 M NaOH and extracted with
[CDCl
3
, 100 MHz]:
d
C
16.4 (CH
3
), 17.6 (CH
3
), 25.6 (CH
3
), 26.1 (CH
2
),
2
2
34.0 (C-4), 34.9 (C-7), 37.8 (C-1), 39.5 (CH
6), 42.1 (C-9), 43.0 (C-3), 43.1 (CH ), 43.2 (CH
44.8 (C-5/10), 45.3 (CH ), 57.4 (C-8), 70.4 (C-11), 114.2 (CH), 123.3
2
), 39.7 (C-2), 40.2 (C-
4
2
2
), 44.7 (C-5/10),
2
(
CH), 131.9 (C), 145.2 (C).
4
8
.7. Synthesis of 8,11-oxapentacyclo[5.4.0.0.2,6.03,10.05,9]undecane-
-aminoethyl[(E)-N-3,7-dimethylocta-2,6-dien-amine]
CH
MgSO
2
Cl
2
(3 ꢂ 25 mL). The combined organic extract was dried over
4
and concentrated to obtain crude product, which was
dihydrochloride (16)
recrystallised from isopropanol to yield the desired product 19
ꢁ
1
(
0.35 g, 29% yield, Mp. 158e159 C), H NMR [DMSO-d , 400 MHz]:
6
To a solution of compound 14 (0.38 g, 1.03 mmol) in dry THF
10 mL) at 0 C was added slowly 65% Red Al in toluene (1.02 mL,
d
H
1.43 (AB, J ¼ 10.2 Hz, 1H), 1.76 (AB, J ¼ 10.2 Hz, 1H), 2.27 (t,
ꢁ
(
5
J ¼ 4.7 Hz,1H), 2.46e2.51 (m, 4H), 2.60e2.69 (m, 2H), 2.77e2.80 (m,
.16 mmol) and kept at this temperature for 10 min. The reaction
1H), 3.17 (t, J ¼ 5.0 Hz, 1H), 3.23 (br, 1H), 3.61 (d, J ¼ 13.3 Hz, 1H),
ꢁ
13
was kept at 35 C for an hour and then refluxed. The reaction was
5.76 (s, OH), 7.17e7.31 (m, 5H). C NMR [DMSO-d
6
, 100 MHz]
d
C
monitored until no starting material was observed on TLC. THF
41.2 (CH), 41.5 (CH), 41.6 (CH), 41.8 (CH
2
), 42.5 (CH), 44.7 (CH), 45.1
(
20 mL) was added to the reaction mixture and quenched with 5 N
NaOH (10 mL), the organic layer was separated, dried over Na SO
and concentrated in vacuo. The crude product was purified on silica
gel; the column was first flushed with 100 mL of CHCl :CH OH
95:5) after which a CHCl :CH OH:NH OH (88:10:2) as eluent
mixture was introduced to obtain 16 (165 mg, 45.2%, R
¼ 0.57) in
pure form and converted to its HCl salt to obtain a yellow slurry. IR
(CH), 50.4 (CH), 50.8 (CH ), 55.3 (CH), 65.2 (CH), 105.9 (C), 126.2
(CH), 127.9 (CH), 128.4 (CH), 140.6 (C).
2
2
4
4.10. Synthesis of 4-azahexacyclo[5.4.1.0.2,6.03,10.05,9.08,11]dodecan-
3-ol (20)
3
3
(
3
3
4
f
To a solution of compound 18 (2.2 g, 8.3 mmol) in of dry CH
(30 mL) was added gently 10% Pd/C (1.1 g) and stirred under H
3
OH
at
ꢀ
1
v
max: 3359, 2960, 1449, 1370, 1009 and 556 cm . MS (TOF) calcu-
2
þ
lated for C23
3
4
H
35
N
2
O (M þ H of free base) 355.2744, found
atmospheric pressure for 16 h or until no starting material was
observed on TLC. The spent Pd/C was filtered using celite and
a sintered funnel, the solution was concentrated in vacuo and the
1
55.2744. H NMR [CDCl
a), 1.57 (3H, s, CH
3
, 400 MHz]:
), 1.62 (3H, s, CH ), 1.65 (3H, s, CH
), 2.39 (1H, t, H-3), 2.46
d
H
1.52 (1H, AB, J ¼ 10.5 Hz, H-
3
3
3
), 1.88 (1H, AB,
J ¼ 10.5 Hz, H-4s), 1.97e2.07 (4H, m, 2 ꢂ CH
2
crude product purified on silica using CHCl
3
:CH
3
OH:NH
4
OH
ꢁ
(
1H, t, J ¼ 4.7 Hz, H-5), 2.50e2.59 (3H, m, H-2, 7, 9), 2.66e2.69 (1H,
(88:10:2) to obtain 19 in pure form as a white solid (Mp. 81e83 C,
1
m, H-6), 2.73e2.81 (4H, m, H-1, 10, CH
2
), 3.00 (2H, t, J ¼ 5.4, 5.9 Hz,
0.64 g, yield 44.1%, R
f
¼ 0.30). H NMR [CDCl
3 H
, 400 MHz]: d 1.48
CH
.03e5.07 (1H, m, CH), 5.22e5.26 (1H, m, CH). C NMR [CDCl
00 MHz]: 16.4 (CH ), 17.7 (CH ), 25.7 (CH ), 26.5 (CH ), 39.6
CH ), 41.5 (C-2), 41.9 (C-6), 42.3 (CH ), 43.1 (C-5), 43.2 (C-4), 44.5
C-1, 7), 44.8 (C-3), 46.3 (CH ), 49.3 (CH ), 54.7 (C-10), 55.1 (C-9),
2.5 (C-11), 110.1 (C-8), 120.2 (CH), 124.0 (CH), 131.8 (C), 140.4 (C).
2
), 3.29 (2H, d, J ¼ 6.96 Hz, CH
2
), 4.58 (1H, t, J ¼ 5.2 Hz, H-11),
(AB, J ¼ 10.5 Hz, 1H), 1.83 (AB, J ¼ 10.4 Hz, 1H), 2.37e2.46 (m, 3H),
13
13
5
1
(
(
8
3
,
2.57e2.62 (m, 2H), 2.67e2.82 (m, 3H), 3.53 (t, J ¼ 5.0 Hz, 1H).
C
d
C
3
3
3
2
NMR [CDCl , 100 MHz]: 41.6 (CH), 41.8 (CH ), 42.6 (CH), 43.4
3
d
C
2
2
2
(CH), 44.9 (CH), 45.8 (CH), 46.1 (CH), 54.5 (CH), 55.2 (CH), 60.5 (CH)
105.9 (C).
2
2
4
.11. Synthesis of (E)-2-chloro-N-(3,7-dimethylocta-2,6-dienyl)
4
.8. Synthesis of pentacyclo[5.4.0.0.2,6.03,10.05,9] undecane-8,11-
acetamide (21)
dione ethylene acetal (17) [28e30]
A mixture of chloroacetyl chloride (0.88 g, 7.8 mmol), geranyl-
A mixture of PCU-dione 4 (10.0 g, 57 mmol), ethylene glycol
4.5 mL, 80 mmol) and p-toluenesulfonic acid (0.33 g, 1.9 mmol)
was dissolved in toluene (45 mL) and refluxed using a DeaneStark
apparatus for three days. The reaction mixture was allowed to cool
down followed by addition of cold aqueous solution of 10% (m/v)
amine (1.0 g, 6.5 mmol) and K
(30 mL) was refluxed for 16 h. The solution was filtered and
concentrated in vacuo and purified on silica using CHCl :EtOAc
(70:30) to afford (E)-2-chloro-N-(3,7-dimethylocta-2,6-dienyl)
2 3
CO (1.08 g, 7.8 mmol) in dry THF
(
3
1
acetamide [20, 1.20 g, 81% yield, R
400 MHz]: 1.56 (s, 3H) 1.64 (s, 6H), 1.97e2.08 (m, 4H), 3.86 (t,
J ¼ 6.24 Hz, 2H), 4.00 (s, 2H), 5.03 (t, J ¼ 6.72 Hz, 1H), 5.16 (t,
f
¼ 0.80]. H NMR [CDCl
3
,
Na
2
CO
3
(60 mL) and extracted with dichloromethane (3 ꢂ 25 mL).
d
H
The organic layer was dried using anhydrous Na
2
SO . The mixture
4
13
was filtered and the solvent evaporated in vacuo. The crude product
was purified via silica gel column chromatography (40:60; EtOAc:
J ¼ 6.96 Hz, 1H). C NMR [CDCl
(CH ), 25.6 (CH ), 26.2 (CH ), 37.7 (CH
118.9 (CH), 123.6 (CH), 131.8 (C), 140.7 (C), 165.5 (C]O).
3
, 100 MHz]:
d
C
16.2 (CH
3
), 17.6
),
3
3
2
2
), 39.3 (CH
2
), 42.5 (CH
2
ꢁ
Hexane) and obtained as a white solid (Mp. 71 C; 10.3 g, 80%,
1
R
1
2
3
(
f
¼ 0.55). H NMR [CDCl
3
, 400 MHz]:
d
H
1.55 (AB, J ¼ 11.0 Hz, 1H),
.84 (AB, J ¼ 11.0 Hz, 1H), 2.37e2.38 (m, 1H), 2.44e2.47 (m, 1H),
4.12. Synthesis of 4-[(E)-N-3,7-dimethylocta-2,6-dienyl]acetamide-
2
,6 3,10 5,9 8,11
.51e2.63 (m, 3H), 2.74e2.79 (m, 2H), 2.89e2.94 (m, 1H),
4-azahexacyclo[5.4.1.0. .0 .0 .0 ]dodecan-3-ol (22)
13
.80e3.91 (m, 4H). C NMR [CDCl
), 41.3 (CH), 41.4 (CH), 42.2 (CH), 42.8 (CH), 45.8 (CH), 50.7 (CH),
2.9 (CH), 64.4 (CH ), 65.6 (CH ), 113.8 (C), 215.2 (C).
3 C
, 100 MHz] d 36.3 (CH), 38.7
CH
2
To a stirred solution of compound 20 (0.3 g,1.7 mmol) in dry THF
(15 mL) was added (E)-2-chloro-N-(3,7-dimethylocta-2,6-dienyl)
5
2
2
acetamide (21, 0.39 g,1.7 mmol) and K
mixture was then refluxed overnight under a N
2
CO
3
(0.35 g, 2.55 mmol). The
atmosphere. The
4
.9. Synthesis of 4-benzyl-4-azahexacyclo[5.4.1.0.2,6.03,10.05,9.08,11
]
2
dodecan-3-ol (19)
reaction was allowed to cool to room temperature, filtered and
concentrated in vacuo. The crude product was purified on silica
A mixture of compound 17 (1.0 g, 4.59 mmol) and benzylamine
0.59 g, 5.5 mmol) in EtOH (10 mL) was heated at 100 C using
using CHCl
oil (445 mg, 72% yield, R
3
:CH
3
OH (90:10) to afford the product 22 as light yellow
¼ 0.53). IR vmax: 3230, 2966, 2868, 1661,
ꢁ
(
f
ꢀ1
a sealed high pressure glass tube for 16 h. The solution was cooled
and NaBH (0.35 g, 9.17 mmol) was added gradually and the
mixture was stirred at room temperature for 8 h. The solution was
33 2 2
1562, 1348 and 728 cm . MS (TOF) calculated for C23H N O
þ
1
4
(M þ H ) 369.2537, found 369.2539. H NMR [CDCl
3
, 400 MHz]:
d
H
1.49 (3H, s, CH ), 1.52 (1H, m, H-12a), 1.56 (3H, s, CH
3
3
), 1.57 (3H, s,

8
O.K. Onajole et al. / European Journal of Medicinal Chemistry 54 (2012) 1e9
CH
3
), 1.84 (1H, m, H-12s), 1.87e1.98 (4H, m, 2 ꢂ CH
2
), 2.56 (1H, s, H-
phosphate buffer, 0.05% tween 80 with glass beads (5 mm diam-
eter) by vortexing. Once the clumps were allowed to settle for
45 min, the supernatant was aspirated and adjusted to a McFarland
7
(
3
), 2.66e2.70 (2H, m, H-8, 1), 2.78e2.84 (3H, m, H-11, 10, 2), 2.95
1H, s, H-6), 3.06 (1H, s, H-9), 3.39 (br s,1H), 3.71 (d, J ¼ 15.4 Hz,1H),
.75 (2H, m, CH
), 4.05 (1H, t, J ¼ 4.8 Hz, H-5), 4.97 (1H, t, J ¼ 5.6 Hz,
CH), 5.08 (1H, t, J ¼ 6.3 Hz, CH), 7.93 (s, CONH). C NMR [CDCl
00 MHz]: 16.1 (CH ), 17.5 (CH ), 25.4 (CH ), 26.2 (CH ), 37.5
CH ), 39.3 (CH ) 40.4 (C-9), 41.1 (C-12), 41.4 (C-8, 11), 43.2 (C-10),
3.8 (C-1), 46.2 (C-7), 47.5 (CH ), 49.6 (C-6), 52.8 (C-2), 65.9 (C-5),
11.8 (C-3), 119.1 (CH), 123.6 (CH), 131.4 (C), 139.5 (C), 164.5 (C]O).
7
2
No. 1 standard, equivalent to a 10 colony forming units CFU/mL.
1
3
3
,
0.5 mL of the standardised inoculum was diluted tenfold to obtain
5
1
(
4
1
d
C
3
3
3
2
a final concentration of 10 CFU/mL, after which 0.5 mL of the
2
2
standardised inoculum was added to each of the MGIT containing
the compounds. A 1:100 inoculum dilution was used to inoculate
the drug free control tubes. This represents 1% of the bacterial
population. The MGITs were loaded in the BACTEC 960 drawers and
the MIC was determined to be the lowest dilutions that were
negative by the automated system in the compound containing
tubes when the control tube showed positive. Antimycobacterial
analysis of all compounds was done in triplicate.
2
4.13. Synthesis of 4-[(E)-N-ethyl-3,7-dimethylocta-2,6-dienyl]
2
,6 3,10 5,9 8,11
amine-4-azahexacyclo[5.4.1.0 . .0 .0 .0 ]dodecan-3-ol
dihydrochloride (23)
To a solution of compound 22 (0.36 g, 0.97 mmol) in dry THF
10 mL) at 0 C was added slowly 65% Red Al in toluene (0.58 mL,
All compounds containing tubes belonging to the positive drug
free control were unloaded from the BACTEC 960 system and
a Ziehl Neelsen stain was performed to confirm the presence of
M. tuberculosis [32]. Colony counts of the test inoculum were also
ꢁ
(
2
.9 mmol) and the mixture was kept at this temperature for 10 min.
ꢁ
The reaction was kept at 35 C for an hour and refluxed; the reac-
tion was monitored until no starting material was observed on LC-
MS. THF (20 mL) was added to the reaction mixture and quenched
with 5 N NaOH (10 mL), the organic layer was separated, dried over
prepared by plating out 20
m
L onto Middlebrook 7H11 agar plates.
ꢁ
Plates were incubated aerobically at 37 C for 21 days. SQ109 was
used as the reference drug.
2 4
Na SO and concentrated in vacuo. The crude product was dissolved
in methanol (5 mL) and purified using preparative HPLC (as spec-
ified above, retention time: 13.6 min). The sample was lyophilized
to obtain 23 (200 mg, 55% yield) and converted to its HCl salt to
obtain a light yellow slurry. IR vmax: 2967, 2866, 1668, 1198, 1178,
Acknowledgement
This study was supported by Grants from the National Research
Foundation, GUN 2073251, Aspen Pharmacare, K-Rith, MRC (South
Africa) and the University of KwaZulu-Natal. We thank Dr Patrick
Govender for his kind assistence with the project.
ꢀ1
1
129, 831 and 719 cm . MS (TOF) calculated for C23H N O
35 2
þ
1
(
M þ H of free base) 355.2744, found 355.2748. H NMR [CDCl
3
,
4
2
2
2
00 MHz]:
ꢂ CH ), 1.93 (1H, d, J ¼ 10.92 Hz, H-12s), 2.00e2.07 (4H, m,
ꢂ CH ), 2.60 (1H, m, H-7), 2.73 (1H, m, H-8), 2.78 (1H, m, H-1),
.83e2.87 (3H, m, H-11, 10, 2), 2.96e2.98 (1H, m, H-6), 3.01e3.24
), 3.41 (2H, d, J ¼ 7.16 Hz, CH ), 3.90 (1H, t,
H 3
d 1.57 (3H, s, CH ), 1.60 (1H, H-12a), 1.65 (6H, s,
3
Appendix A. Supplementary material
2
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.ejmech.2012.03.041.
(
5H, m, H-9, 2 ꢂ CH
2
2
J ¼ 5.1 Hz, H-5), 5.03 (1H, m, CH), 5.21 (1H, t, J ¼ 6.5 Hz, CH), 5.61
(
(
(
OH, NH). 13C NMR [CDCl
CH ), 26.3 (CH ), 39.6 (CH
C-11), 43.5 (C-10), 44.1 (C-1), 44.3 (CH
6.3 (C-7), 50.1 (C-6), 53.5 (C-2), 67.3 (C-5), 113.5 (C-3), 117.0 (CH),
23.7 (CH), 132.1 (C), 143.4 (C).
3
, 100 MHz]:
), 41.1 (C-9), 41.2 (C-12), 41.6 (C-8), 41.7
), 44.5 (CH ), 45.9 (CH ),
C 3 3
d 16.4 (CH ), 17.7 (CH ), 25.7
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5
1
(