A new class of small molecule RNA polymerase inhibitors with activity against Rifampicin-resistant Staphylococcus aureus1

Article abstract of DOI:10.1016/j.bmc.2006.05.035

The RNA polymerase holoenzyme is a proven target for antibacterial agents. A high-throughput screening program based on this enzyme from Staphylococcus aureus had previously identified a 2-ureidothiophene-3-carboxylate as a low micromolar inhibitor. An investigation of the relationships between the structures of this class of compounds and their inhibitory- and antibacterial activities is described here, leading to a set of potent RNA polymerase inhibitors with antibacterial activity. Characterization of this bioactivity, including studies of the mechanism of action, is provided, highlighting the power of the reverse chemical genetics approach in providing tools to inhibit the bacterial RNA polymerase.

Full text of DOI:10.1016/j.bmc.2006.05.035

Bioorganic & Medicinal Chemistry 14 (2006) 5812–5832
A new class of small molecule RNA polymerase inhibitors with
activity against Rifampicin-resistant Staphylococcus aureus1
a
a
a
a
a
´
´
Francis Arhin, Odette Belanger, Stephane Ciblat, Mohammed Dehbi, Daniel Delorme,
Evelyne Dietrich,a Dilip Dixit,a Yanick Lafontaine,a Dario Lehoux,a Jing Liu,a
Geoffrey A. McKay,a Greg Moeck,a Ranga Reddy,a Yannick Rose,a
Ramakrishnan Srikumar,a Kelly S. E. Tanaka,a Daniel M. Williams,a Philippe Gros,b
Jerry Pelletier,b Thomas R. Parr, Jr.a and Adel Rafai Fara,*
aTarganta Therapeutics Inc, 7170 Frederick Banting, 2nd Floor, St. Laurent, Que., Canada H4S 2A1
bDepartment of Biochemistry, 3655 Promenade Sir William Osler, McIntyre Medical Sciences Building,
McGill University, Montreal, Que., Canada H3G 1Y6
Received 22 March 2006; revised 15 May 2006; accepted 16 May 2006
Available online 8 June 2006
Abstract—The RNA polymerase holoenzyme is a proven target for antibacterial agents. A high-throughput screening program
based on this enzyme from Staphylococcus aureus had previously identified a 2-ureidothiophene-3-carboxylate as a low micromolar
inhibitor. An investigation of the relationships between the structures of this class of compounds and their inhibitory- and antibac-
terial activities is described here, leading to a set of potent RNA polymerase inhibitors with antibacterial activity. Characterization
of this bioactivity, including studies of the mechanism of action, is provided, highlighting the power of the reverse chemical genetics
approach in providing tools to inhibit the bacterial RNA polymerase.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
complex natural products.6 Small molecule RNAP
inhibitors have been recently reported for Escherichia
coli, but their inability to eradicate the host bacterium
was also noted.7a Lately a set of inhibitors of the core-
sigma factor interaction displaying antibacterial activity
have been disclosed.7b Herein is described the develop-
ment of a new class of compounds, with antibacterial
activity, acting via the S. aureus RNAP holoenzyme.
Transcription is essential for growth and survival and is
catalyzed by the DNA-dependent RNA polymerase
(RNAP) core enzyme.2 In bacteria, this protein complex
associates with one of a variety of sigma factors to form
the RNA polymerase holoenzyme with specificity for a
given set of promoters. The essential role of the RNAP
sigma factor has been established through the intracellu-
lar production, under regulated expression of bacterio-
phage inhibitory peptides targeting the sigma factor of
Staphylococcus aureus,3 an important pathogen involved
in a number of bacteremias.4 In addition, the RNAP has
been validated as a drug target through its inhibition by
the Rifamycin class of antibacterial agents, some of
which are used clinically.5 There have been few reports
of other classes of RNA polymerase inhibitors as
antibacterial agents, but in nearly all cases, these were
The screening of a library of 250,000 commercially
available compounds against the S. aureus RNAP holo-
enzyme was performed with a functional assay measur-
ing the incorporation of a-[32P]-UTP in acid-precipitable
counts. This has allowed the identification of the substi-
tuted 2-ureidothiophene-3-carboxylate 1 as a low micro-
molar inhibitor (Fig. 1).3 It displayed good antibacterial
CO2Et
NH
S
N
Keywords: 2-Ureidothiophene-3-carboxylates; RNA polymerase; Anti-
bacterial agents; Reverse chemical genetics.
O
H
1
*
Corresponding author. Tel.: +1 514 496 6667; fax: +1 514 332
6033; e-mail: afar@targanta.com
Figure 1.
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.05.035

F. Arhin et al. / Bioorg. Med. Chem. 14 (2006) 5812–5832
5813
Table 1. Inhibitory activity against the Staphylococcus aureus RNAP enzyme in vitro and antibacterial activities of compounds 1 and 6b–r
R
NH
S
NHPh
O
1-6(b-r)
Compound
Aminothiophene
precursor
Substituent R
IC50 (lM),
S. aureus RNAP
MIC (lg/mL), S. aureus
ATCC 13709
MIC (lg/mL),
S. aureus RN4220
1
5a
5b
5c
5d
5e
5f
–CO2Et
–CO2Me
1
1
>16
0.5
>128
>16
1
6b
6c
6d
6e
6f
6g
2
i
–CO2 Pr
0.04
0.17
0.14
8
t
–CO2 Bu
i
–CO2 Bu
1
>128
>32
>16
>128
>32
>16
>128
–CO2Bn
–CO2All
5g
0.20
O
O
6h
6i
5h
5i
0.36
0.14
0.21
1.25
1.6
>16
0.5
>16
>16
1
O
O
O
6j
5j
O
O
0.5
O
6k
6l
5k
5l
>8
>8
O
OMe
O
>128
32
>128
32
O
NMe
NMe
O
6m
5m
1.24
O
6n
6o
6p
6q
6r
5n
5o
5p
5q
–COiPr
–COiBu
–CN
5
1.5
>8
>16
>128
>16
64
>8
>16
>128
>16
128
0.008
1
>100
>100
31
–CONHiPr
–CO2H
—
—
Rifampicin
Vancomycin
Linezolid
nd
nd
0.008
1
—
nd
2
1
nd, not determined.
activity against S. aureus ATCC 13709 and S. epidermi-
dis (MICs 1 and 0.25 lg/mL, respectively, Table 1) but it
lacked activity against four laboratory strains of S. aure-
us (MIC > 16 lg/mL). Compound 1 lacked Gram-nega-
tive activity and the presence of 50% human or mouse
serum or 4% human serum albumin abolished all activ-
ity. It did not display cytotoxicity in vitro against the
HeLa cell line in an MTS assay8 or against primary hu-
man hepatocytes in an ATP assay.9
catalyzed (Fischer) esterification or via a DCC coupling
(Scheme 2).11 a-Cyanoketones 3 were produced through
the condensation of the dilithium salt of cyanoacetic
acid with an acid chloride, followed by decarboxylation
(Scheme 2).12
The cyanoacetamide 4 can be prepared from ethyl cya-
noacetate by simple treatment with isopropylamine
(Scheme 3).13
These results made 1 an interesting starting point for the
development of antistaphylococcal agents.
R3
R1
R2
O
base
R3
R1
R2
+
+ "S"
NH2
CN
S
2. Chemistry
Scheme 1. The Gewald reaction.
A number of analogs of 1 can be readily prepared via
the Gewald reaction,10 a three-component condensation
route to thiophenes, involving a carbonyl compound, an
activated nitrile, and elemental sulfur (Scheme 1).
O
CO2H
CN
CO2R
CN
2(a-m)
b
a
R
CN
3(a-b)
Several such activated nitriles have been prepared for
use in this reaction. The cyanoacetic acid esters 2 were
prepared from cyanoacetic acid either through an acid
Scheme 2. Reagents and conditions: (a) PhH, H2SO4, D (60%—
quant.), or DCC, MeCN (52–79%); (b) 4 equiv nBuLi, ꢀ78 ꢁC, then
RCOCl (72%).

5814
F. Arhin et al. / Bioorg. Med. Chem. 14 (2006) 5812–5832
O
CO2iPr
CO2iPr
NH
CO2Et
CN
a
NH R1
NHiPr
CN
S
S
N
S NHPh
O
R2
O O
10
4
8(a-y)
b
d; R = iPr
i
Scheme 3. Reagent: (a) PrNH2, neat (quant.).
CO2R
NH2
The treatment of cyclooctanone with these easily acces-
sible activated nitriles, as well as with commercially
available ones, provides 2-aminothiophenes 5 (Scheme
4). Subsequent reaction of these compounds with pheny-
lisocyanate provides their parent ureas 6. The condensa-
tion to furnish compound 5q required two steps rather
than the single-step Gewald reaction. Ureidothiophene
6m was prepared from aminothiophene 5m by treatment
with phenylisocyanate, followed by Boc deprotection
and reductive methylation. Acid 6r was obtained by
the hydrolysis of the tert-butyl ester 6d.
S
5
c; R = iPr
a; R = Et
CO2Et
CO2iPr
NH
NH
S
S
S
NHPh
OBn
O
7
9
Scheme 5. Reagents and conditions: (a) phenyl thioisocyanate, pyri-
dine, 50 ꢁC (76%); (b) isocyanate, pyridine, 50 ꢁC (15–77%); or
triphosgene, Et3N, CH2Cl2 then amine (17–44%); (c) benzyl chloro-
formate, Et3N, CH2Cl2 (47%); (d) phenyl sulfamyl chloride, Et3N,
CH2Cl2 (36%).
The aminothiophene 5a with an ethyl ester substitution
at position 3 was treated with phenylthioisocyanate to
provide the thiourea 7 (Scheme 5). The aminothiophenes
5a, 5c, and 5j with varying ester substituents at position
3 were also reacted with a number of isocyanates or with
triphosgene in the presence of triethylamine,14 followed
by an amine to afford ureas 8 (Scheme 5). Treatment of
5c with benzyl chloroformate and triethylamine provid-
ed the carbamate 9, whereas reaction with phenylsul-
famidyl chloride15 in the presence of base resulted in
the sulfuric diamide 10.
CO2iPr
CO2iPr
R1
R2
R1
R2
R1
R2
a
O
b
NH
NH2
S
NHPh
S
O
11(a-k)
12(a-k)
Scheme 6. Reagents and conditions: (a) isopropyl cyanoacetate, S8,
amine base (17–89%); (b) phenylisocyanate, pyridine, 50 ꢁC (15–77%).
presence of the compounds by scintillation counting of
TCA-precipitable a[32P]-UTP using the same assay
format that was used to screen the diversity library.3,16
The Gewald reaction was also used with isopropyl
cyanoacetate and a variety of ketones to provide
aminothiophenes 11 and eventually ureidothiophenes
12 (Scheme 6).
Given the strain dependence of the antibacterial activity
of 1, analogs were screened against both the ATCC
13709 and the RN4220 strains of S. aureus.
3. In vitro evaluation of inhibitor structure–activity
relationships
The activity of the analogs of 1 differentiated by their
substituents at position 3 (compounds 1 and 6b–r) is pre-
sented in Table 1. For comparison, the antibacterial
activities on these strains for Rifampicin, Vancomycin,
and Linezolid are also indicated in this table.
To assay the inhibitory activity of the compounds
against the RNAP, the synthesis of RNA by the
S. aureus RNAP holoenzyme was measured in the
R
R
a
O
b
NH
NH2
S
NHPh
S
O
5
1-6(b-p)
c
O
O
NHiPr
NHiPr
O
NHiPr
d
b
NH
O
NH2
CN
S
NHPh
S
5q
6q
NMe
NBoc
CO2tBu
NH
CO2H
NH
O
b,e,f
O
O
e
O
S
S
NHPh
NHPh
NH
NH2
O
O
S
NHPh
S
O
5m
6m
6d
6r
Scheme 4. Reagents and conditions: (a) activated nitrile, S8, amine base (23–88%); (b) phenylisocyanate, pyridine, 50 ꢁC (25–89%); (c) 4, NH4OAc,
AcOH (16%); (d) S8, morpholine, EtOH, 70 ꢁC (91%); (e) TFA/CH2Cl2 (quant.); (f) HCO2H, CH2O, D (10%).

F. Arhin et al. / Bioorg. Med. Chem. 14 (2006) 5812–5832
5815
It is clear from this study that bulky esters at position 3
greatly favor the inhibitory activity of these compounds.
An optimum is however obtained with the isopropyl es-
ter 6c. Ketones 6n–o, the nitrile 6p, the amide 6q, and the
carboxylate 6r are inadequate replacements. The anti-
bacterial activity of this class of compounds closely
matches the inhibitory activities with all of the submi-
cromolar compounds displaying at least activity against
S. aureus ATCC 13709. Two compounds, 6c,j, stand out
as being active against both strains. Although this does
not come as a surprise for 6c, the most potent inhibitor,
the activity of 6j is slightly more difficult to explain.
Thus, the closely related 6i, with its improved potency
against RNAP, fails to display activity against the
RN4220 strain. A key difference between these two com-
pounds is their polarity with clogD values17 calculated
at pH 7.4 to be 5.84 and 5.00 for 6i and 6j, respectively.
resulted in good inhibitors with antibacterial activities
in both strains, as expected. Surprisingly, the use of a
thiazolyl substituent led to a compound that, while
maintaining the potent RNAP-inhibitory activity associ-
ated with the thiophenes, completely lacked antibacteri-
al activity.
The substituted thiophenyl ureas behaved much like the
parent aryls, in the sense that 1,4-substitution (com-
pound 8q) maintained the inhibitory potency but abol-
ished the antibacterial activity, while 1,2-substitution
(‘ortho’) (compounds 8r,s) resulted in complete loss of
inhibitory activities. The same outcome is expectedly
observed with the isoxazole substituent in 8t.
N,N0,N0-Trisubstituted ureas such as 8u and 8v display a
complete lack of inhibition of RNAP, and so does the
carbamate 9, which once more draws attention to the
role of the urea in interacting with the binding site.
From that perspective, compound 8w is a little puzzling,
as it does display some activity, perhaps pointing to the
possibility of an alternative binding mode. The crucial
impact of the urea functionality is further demonstrated
with the sulfuric diamide 10 whose complete lack of
activity clearly stems from either its weaker H-bonding
ability or the differences in geometry between the two
functionalities.
It is noteworthy that the antibacterial activity does not
respond incrementally to the structural modifications.
Compounds either display antimicrobial activities of
0.5–1 lg/mL or are without activity.
Derivatives with different substituents at position 2 were
evaluated in the same manner (Table 2). Replacement of
the urea functionality in 1 by a thiourea (compound 7) is
clearly detrimental, as is its removal (compound 5c).
Differently substituted 2-(phenylureido)thiophenes (8a–
g) display fairly similar inhibitory activities toward
RNAP, but the introduction of substituents abolishes
the antibacterial activity. The exceptions are ortho sub-
stituents, such as 8f and 8g which are clearly negatively
impacting the inhibitory activity of the compounds.
Replacement of the phenyl group in 6c with a benzyl
group (compound 8j) or by similar aliphatic groups
(compounds 8h,i) is also unfavorable.
Given the improved antibacterial activities associated
with the more polar substituents at positions 2 and 3
of the thiophenes, it was reasoned that the combination
of a thiophenyl urea as in 8n and 8o and a tetrahydrof-
uranyl ester as in 6j would result in active compounds.
Indeed, compounds 8x,y do display both good inhibito-
ry and antibacterial activities, although the weaker of
the two (8y) eradicates only S. aureus ATCC 13709.
The trend observed with the ester substituents, whereby
higher polarity resulted in improved antibacterial activ-
ity, is noticeable in this case as well. Thus, whereas com-
pounds 8a (clogD 6.27) and 8c (clogD 5.56) display
similar inhibitory activities, 8a has no antibacterial
activity, while 8c is effective against the ATCC13709
strain. Compounds 6c (clogD 5.72) and 8d (clogP
6.50) display the same tendency.
To further examine the role of the functional groups in
relation to the inhibitory and antibacterial activities of
this class of compounds, analogs 12a–k, with replace-
ments for the 8-membered carbocyclic moiety, were also
assessed (Table 3).
This table shows that the inhibitory activity of this class
of compounds relates to the ring size. The activity is
optimal with compounds 6c (8-membered ring) and
12b (9-membered ring), and decreases with compounds
12a (7-membered ring) and 12c (10-membered ring).
The antibacterial activity follows suit with 6c and 12b,
but not 12a and 12c, displaying an ability to eradicate
the two strains. The pattern observed previously, with
either good (1 lg/mL) antibacterial activity or none at
all, is particularly obvious in this case.
This trend has directed our attention to heterocyclic
substituents as replacements for the phenyl group of
6c. A 4-pyridyl group (compound 8k) results in a slight
decrease in inhibitory activity, but, interestingly, the 2-
pyridyl or 2-pirazinyl substituents (compounds 8l,m)
completely abolish all inhibition. This effect may be ex-
plained by the ability of these two substituents to form
an intramolecular hydrogen bond (Fig. 2) resulting in
a presumably unfavorable conformation. The 4-pyridyl
substituent, unable to form such a hydrogen bond, is
more flexible and can therefore adopt the conformation
required by the binding site. This clearly relates to the
earlier observation that ortho-substituted phenyl groups
are also inactive.
The contrast with the positive results, in terms of anti-
bacterial activity, obtained by the introduction of in-
creased polarity in positions 2 and 3, is fairly marked
in this case. Smaller rings with polar groups (compounds
12d,e), a heteroatom bridge (12f), non-fused rings (com-
pounds 12g,h) or acyclic groups (compounds 12i–k), all
gave a similar decrease in the inhibitory activity back to
the micromolar range. Of particular interest is the small
difference between 12j and 12k in which the removal of
The replacement of the phenyl substituent on the urea
by the bioisosteric thiophenes18 (compounds 8n,o)

5816
F. Arhin et al. / Bioorg. Med. Chem. 14 (2006) 5812–5832
Table 2. Inhibitory activities against the RNAP enzyme and antibacterial activities of compounds 7, 5c, 8(a–y), 9, and 10b
CO2R1
NH
S
R2
7, 5c, 8(a-y), 9, 10
Compound
R1
Et
R2
IC50 (lM),
S. aureus RNAP
MIC (lg/mL), S. aureus
ATCC 13709
MIC (lg/mL),
S. aureus RN4220
S
7
32
>100
0.12
0.11
0.12
0.06
0.57
3
>32
>128
>128
>128
1
>32
NHPh
5c
8a
8b
8c
8d
8e
8f
iPr
iPr
iPr
iPr
iPr
iPr
iPr
iPr
iPr
iPr
iPr
iPr
iPr
iPr
iPr
iPr
iPr
H
32
O
>128
N
H
Cl
O
>128
N
H
OMe
O
O
>128
N
H
>16
>16
>8
>16
N
H
Cl
O
O
>16
N
H
CF3
>8
N
H
Cl
Cl
Cl
O
8g
8h
8i
>100
0.29
0.25
1.6
>32
>16
>8
>32
N
H
O
O
O
O
O
>16
N
H
>8
N
H
8j
>16
>128
>16
>16
1
>16
Ph
N
H
N
8k
8l
0.30
>100
>100
0.06
0.20
0.14
>128
N
H
>16
N
H
N
N
N
O
O
8m
8n
8o
8p
32
N
H
2
S
N
H
O
O
S
0.5
1
N
H
N
>16
>16
S
N
H
(continued on next page)

F. Arhin et al. / Bioorg. Med. Chem. 14 (2006) 5812–5832
5817
Table 2 (continued)
Compound
R1
iPr
iPr
iPr
iPr
iPr
iPr
iPr
R2
IC50 (lM),
S. aureus RNAP
MIC (lg/mL), S. aureus
ATCC 13709
MIC (lg/mL),
S. aureus RN4220
O
8q
8r
8s
8t
0.49
>100
>100
5
1
>128
S
N
H
O
O
S
>8
>4
>8
>4
N
H
CO2Me
S
N
H
CO2Me
N
O
O
>64
>8
>64
>8
N
H
O
O
8u
8v
8w
8x
8y
9
>100
>100
6
Ph
N
N
>8
>8
O
O
O
>16
1
>16
4
N
O
0.16
0.25
>100
>100
O
O
S
N
H
S
2
>128
>32
>128
N
H
O
iPr
iPr
>32
>128
O
Ph
O
10
S NHPh
O
The activity of 6c against the Rifampicin-resistant
strains would be indicative of a mechanism of action
or a binding site which is different from Rifampicin.
H N
X
N
S
NH
O
Figure 2.
There was no activity of 6c outside the Staphylococcus
genus among the Gram-positives in the panel (S. pyoge-
nes, S. pneumoniae, E. faecalis, and Bacillus subtilis, not
shown). Likewise, 6c lacked activity against the Gram-
negative bacteria (E. coli, P. aeruginosa, Salmonella
typhimurium, Haemophilus influenzae) and the yeast
(Candida albicans, and Candida parapsilosis) in the panel
(not shown).
one lipophilic arm did not impact the inhibitory activity
at all. This tends to indicate that the analogs as made are
unable to fill the space occupied by the 8-membered ring
in 6c in the binding site, benefiting only from the inter-
actions made by the ester and the urea functionalities.
This binding site obviously has a fairly hydrophobic sur-
face associated with it.
The impact of serum on the antibacterial activity was
established in the ATCC 13709 strain of S. aureus.
The MIC values of 6c shift from 0.5 to 128 lg/mL when
50% mouse or human serum is used in the growth medi-
um. If 4% human serum albumin is used the MIC shifts
to 16 lg/mL. This detrimental effect of serum and serum
albumin is also observed with 6j (128 lg/mL with 50%
serum, 32 lg/mL with 4% albumin), 8n (>128 lg/mL
with 50% serum, 32 lg/mL with 4% albumin), 8x
(128 lg/mL with 50% serum, 32 lg/mL with 4% albu-
min), and 12b (>128 lg/mL with 50% serum, 16 lg/mL
with 4% albumin).
4. In vitro characterization of activity
Compound 6c was selected for further characterization
in light of its antibacterial activity. This later was deter-
mined against a panel of microbes.
Staphylococcus spp. including S. epidermidis and S. hyi-
cus were eradicated with MIC values of 0.25–1 lg/mL.
The spectrum of activity against S. aureus is summarized
in Table 4.

5818
F. Arhin et al. / Bioorg. Med. Chem. 14 (2006) 5812–5832
Table 3. Inhibitory activities against the RNAP enzyme and antibac-
terial activities of compounds 12a–k
140
120
100
80
CO2iPr
R1
NH
R2
S
NHPh
O
12(a-k)
DNA
RNA
Protein
60
Compound
IC50 (lM) MIC (lg/mL) MIC
ATCC 13709 (lg/mL)
RN4220
R1
R2
40
20
12a
12b
12c
12d
12e
0.12
0.05
0.14
>64
>64
0
0.001
0.01
0.1
1
10
100
concentration (mg/ml)
0.5
0.5
Figure 3. Effect of 6c on macromolecular biosynthesis.
>8
>16
>32
>8
>16
>32
S. aureus.18 Figure 3 shows that 6c inhibits the synthesis
of both RNA and protein, but not of DNA. This is rem-
iniscent of the mechanism of action of Rifampicin, and
likely reflects the immediate cessation of protein synthe-
sis upon inhibition of transcription due to the tight cou-
pling of transcription and translation in bacteria.
17
EtO2C
O
1.6
2.5
The time-kill curves (Fig. 4) of 6c provide another inter-
esting insight into the activity of this compound: an ini-
tial dip in the number of viable bacteria is followed by
normal growth in the RN4220 strain of S. aureus. In
the ATCC 13709 strain, the same effect is observed, ex-
cept at high multiples of the MIC, where a bacteriostatic
effect is seen. This provides a basis for the fact that the
compounds in this class are either clearly antibacterial
or without effect. Indeed, if the bacteria recover from
the initial dip, they will be able to grow normally and
reach saturation at the endpoint of the MIC assay
(18–24 h). Only the most potent compounds, for which
the initial killing is more profound and sustained, will
result in a measurable MIC.
12f
128
128
N
Me
12g
12h
12i
8.6
2.5
4
>128
>128
>16
>128
>128
>16
O
O
12j
5
>16
>16
This phenomenon is in fact reflected in the in vitro fre-
quencies of resistance, measured at 4· MIC, which are
3.2 · 10ꢀ5 against RN4220 and 6.8 · 10ꢀ8 against
ATCC 13709. These are indeed high for RN4220 (the
measured frequency of resistance of RN4220 to Rifa-
mycins is ꢁ10ꢀ7), and again display the same strain-
dependence.
O
O
12k
9
>128
>128
Table 4. In vitro susceptibility of drug- or multidrug-resistant or efflux
pump mutants of Staphylococcus aureus to compound 6c
Resistant category
n
MIC (lg/mL)
MIC or MIC range MIC50 MIC90
0.5 (11 strains)
5. In vivo evaluation
Compound 6c does not display any cytotoxicity against
the HeLa cell line (MTS assay) or human primary hepa-
tocytes (ATP assay), nor is it mutagenic in the Ames test
(not shown). It is therefore not surprising that it is well
tolerated in vivo, with 100% survival of mice that re-
ceived up to 2· 100 mg/kg of body weight po in
10%DMSO:90% peanut oil, or 2· 100 mg/kg ip or
50 mg/kg iv both in 15:15:70 solutol:PEG400:PBS.
Mupirocin-resistant 12 >128 (1 strain)
Rifampicin-resistant
MRSA
0.5
0.25
0.5
na
0.5
0.5
1
9
14 0.25–2
<0.125–1
VISA ATCC 700699
K2068-MDR
1199B-NorA
1
1
1
0.25
0.25
1
na
na
na
na
na
The mechanism of antibacterial activity was also con-
firmed through the impact of 6c on the synthesis of mac-
romolecules at the cellular level in the RN4220 strain of
Not unexpectedly given its large serum shift, 6c failed to
protect mice from lethal S. aureus ATCC 13709
infection at 72 h postinfection in the mouse peritonitis

F. Arhin et al. / Bioorg. Med. Chem. 14 (2006) 5812–5832
Time-kill of RN4220
5819
Time-kill of ATCC13709
A
B
1.E+10
1.E+09
1.E+08
1.E+07
1.E+06
1.E+05
1.E+04
1.E+03
1.E+10
1.E+09
1.E+08
1.E+07
1.E+06
1.E+05
1.E+04
1.E+03
No compound
2xMIC
4xMIC
No compound
4xMIC
8xMIC
16xMIC
8xMIC
0
6
12
18
24
0
6
12
18
24
Time (hours)
Time (hours)
Figure 4. Time-kill curves of 6c against the ATCC 13709 (A) and the RN4220 (B) strains of Staphylococcus aureus.
Table 5. Pharmacokinetic parameters for 6c
resistance. Additional studies focusing on the 8-mem-
bered macrocyclic portion of the structure may open
up a larger chemical space to ultimately circumvent
these liabilities.
Dose (mg/kg)
Route
tmax (h)
25
50
po
iv
—
0.50
Cmax (lg/mL)
AUC (h mg/mL)
t1/2 (h)
—
36.6
2.09
6.18
4.46
This study has outlined a strategy to identify useful
chemical tools with which to probe the bacterial RNA
polymerase. The reverse chemical genetics approach de-
scribed here started with the desired protein target (the
RNAP holoenzyme of S. aureus), screened for small
molecules that affect its activity, then asked whether
the small molecule causes a phenotypic change in
S. aureus. This approach is analogous to reverse genet-
ics, in which a gene is deliberately mutated or knocked
out in order to study the resulting phenotype. Current
small molecule- or natural product inhibitors of bacte-
rial RNAP are restricted to the Rifamycins, which act
on the b (polymerase) subunit, and recently described
synthetic compounds which apparently inhibit the inter-
action between the core and the sigma (promoter speci-
ficity) subunit.7b Additional compounds are required to
better understand the enzymology of transcription. The
well-defined mechanism of action of compound 6c in
inhibiting transcription in growing S. aureus cells
establishes a chemical platform with which to further
probe the workings of a multicomponent enzyme, the
bacterial RNAP.
0.52
0.31
11.3
Vss (L/kg)
Cl (mL/min/kg)
Bioavailability (%)
—
—
—
8.4
model when used iv at up to 50 mg/kg. Statistically
significant efficacy (60% survival) was seen only when
compound 6c was administered ip at 2· 100 mg/kg.
Pharmacokinetic parameters for plasma concentrations
were measured with a 15:15:70 solutol:PEG400:PBS
formulation (Table 5).
6. Conclusion
The modifications of the substituents of compound 1
have led to derivatives with potent inhibitory activity to-
ward the S. aureus RNAP holoenzyme and antibacterial
activity against Staphylococcus spp. More extensive
studies on the activity of one of these congeners, com-
pound 6c, provided a more complete picture in terms
of mechanism of action. This inhibitor clearly inhibits
transcription in S. aureus as judged by its rapid and pro-
found impact on both RNA and protein synthesis as is
seen with the Rifamycins. However, the compound
maintains its excellent antibacterial activity against
Rifampicin-resistant strains of S. aureus, suggesting an
alternate mechanism of action or binding site on the
RNAP holoenzyme compared to the Rifamycins’ bind-
ing site on the b subunit of the RNAP core.
7. Experimental
7.1. General
1H NMR spectra were recorded on a Varian MercuryTM
400 spectrometer. The reported chemical shifts (in parts
per million) are referenced using the signals assigned to
the residual non-deuterated solvents. Mass spectral
analyses were performed on an Agilent mass spectrom-
eter under electron spray ionization (ESI). Reactions
were monitored by TLC on Silica gel Gel 60 F254
(0.25 mm, Merck). Column chromatography was per-
formed on Silica gel Gel 60 (70–230 mesh). All reactions
were carried out under an argon atmosphere with anhy-
drous solvents, unless otherwise noted. All chemicals,
unless otherwise stated, were obtained from commercial
sources.
The transient activity of 6c as judged by time-kill exper-
iments with S. aureus RN4220 and the elevated frequen-
cy of resistance of this strain to 6c, however, remain
significant hurdles to further development of these com-
pounds as antibacterial agents.
The very stringent structural requirements needed to
confer antibacterial activity do not bode well for
tackling the issues associated with serum binding and

5820
F. Arhin et al. / Bioorg. Med. Chem. 14 (2006) 5812–5832
7.2. Chemistry
2H), 1.90–1.98 (m, 2H), 2.22–2.32 (m, 5H), 2.59–2.66
(m, 2H), 3.45 (s, 2H), 4.86–4.90 (m, 1H).
7.2.1. Cyanoacetate esters (2a–m). Ethyl 2-cyanoacetate
(2a), methyl 2-cyanoacetate (2b), isopropyl 2-cyanoace-
tate (2c), tert-butyl 2-cyanoacetate (2d), isobutyl 2-cya-
noacetate (2e), and allyl 2-cyanoacetate (2g) were
commercially available. Others were prepared as follows:
7.2.1.7. (R)-tert-Butyl 3-(2-cyanoacetoyloxy)pyrroli-
dine-1-carboxylate (2m). To a solution of (R)-pyrroli-
din-3-ol (0.78 g, 8.9 mmol) in THF (18 mL) at 0 ꢁC was
added Boc anhydride (1.94 g, 8.9 mmol). The solution
was stirred for 48 h at room temperature, then concentrat-
ed to dryness. Purification by flash chromatography on
silica gel, using 50% EtOAc/hexanes as eluent, provided
(R)-tert-butyl 3-hydroxypyrrolidine-1-carboxylate as a
Cyanoacetates 2f,h–k were prepared via the Fischer ester-
ification procedure as follows: to a solution of cyanoace-
tic acid (2.0 g, 23.5 mmol) and alcohol (35.3 mmol) in
benzene (50 mL) were added a few drops of concd
H2SO4. The mixture was stirred at reflux for 2.5 h, using
a Dean–Stark condenser to remove water. The reaction
mixture was concentrated to half its initial volume under
reduced pressure, diluted with EtOAc, washed with satu-
rated NaHCO3 solution, water, and brine. The organic
layer was dried over Na2SO4, filtered, and concentrated
under reduced pressure. The crude product was purified
by flash chromatography on silica gel.
1
colorless oil (1.66 g, 99% yield). H NMR (400 MHz,
CDCl3) d 1.46 (s, 9H), 1.92 (br s, 1H), 1.94–2.04 (m,
2H), 3.30–3.52 (m, 4H), 4.44–4.47 (m, 1H). (R)-tert-Butyl
3-hydroxypyrrolidine-1-carboxylate (1.0 g, 5.34 mmol)
was coupled with cyanoacetic acid using the general pro-
cedure above. Cyanoacetate 2m was obtained as a white
1
solid (858 mg, 79% yield). H NMR (400 MHz, CDCl3)
d 1.46 (s, 9H), 2.10 (br s, 2H), 3.39–3.61 (m, 6H), 5.37
(br s, 1H) broad signals due to carbamate isomerism.
7.2.1.1. Benzyl 2-cyanoacetate (2f). Chromatographic
eluent: 50% CH2Cl2 in hexanes. Yield: 93% (colorless
oil). H NMR (400 MHz, CDCl3) d 3.49 (s, 2H), 5.24
(s, 2H), 7.36–7.40 (m, 5H).
7.2.2. a-Cyanoketones (3a,b). To a solution of cyanoace-
tic acid (1 g, 11.8 mmol) in THF (100 mL), cooled in a
dry ice/acetone bath, was added a 1.6 M solution of n-
BuLi in THF (29 mL, 47.0 mmol). The resulting solu-
tion was warmed to room temperature over 1 h, stirred
for 30 min at this temperature, and cooled back in a dry
ice/acetone bath. The acid chloride (11.8 mmol) was
added dropwise and the resulting mixture was stirred
for 30 min at the same temperature, warmed to room
temperature, and stirred for 1 h at this temperature. A
20% aqueous solution of hydrochloric acid (50 mL)
was then added and the resulting mixture was vigorously
stirred for 2h. Et2O was added to the mixture, and the
organics were collected. The aqueous layer was extract-
ed twice more with Et2O, the combined organic layers
were washed with brine, dried over MgSO4, and concen-
trated under reduced pressure.
1
7.2.1.2. Cyclohexyl 2-cyanoacetate (2h). Chromato-
graphic eluent: 10% EtOAc in hexanes Yield: quantita-
tive (colorless oil). 1H NMR (400 MHz, CDCl3) d
1.26–1.57 (m, 6H), 1.71–1.79 (m, 2H), 1.84–1.90 (m,
2H), 3.43 (s, 2H), 4.83–4.90 (m, 1H).
7.2.1.3. Cyclopentyl 2-cyanoacetate (73). Chromato-
graphic eluent: 20% EtOAc in hexanes. Yield: quantita-
tive (colorless oil). 1H NMR (400 MHz, CDCl3) d 1.59–
1.65 (m, 2H), 1.71–1.79 (m, 4H), 1.86–1.93 (m, 2H), 3.41
(s, 2H), 5.24–5.28 (m, 1H).
7.2.1.4. ( )-Tetrahydrofuran-3-yl 2-cyanoacetate (2j).
Chromatographic eluent: 50% EtOAc in hexanes. Yield:
60% (colorless oil). 1H NMR (400 MHz, CDCl3) d 2.03–
2.10 (m, 1H), 2.18–2.28 (m, 1H), 3.47 (s, 2H), 5.84–5.97
(m, 4H), 5.37–5.41 (m, 1H).
7.2.2.1. 4-Methyl-3-oxopentanenitrile (3a). The crude
product was purified by flash chromatography on silica
gel using 5–20% EtOAc in hexanes (linear gradient) as
the eluent. Yield: 72% (pale yellow oil). 1H NMR
(400 MHz, CDCl3) d 1.19 (d, J = 6.9 Hz, 6H), 2.83 (m,
1H), 3.53 (s, 2H).
7.2.1.5. ( )-1-Methoxypropan-2-yl 2-cyanoacetate
(2k). Chromatographic eluent: 40% EtOAc in hexanes.
1
Yield: 85% (colorless oil). H NMR (400 MHz, CDCl3)
7.2.2.2. 5-Methyl-3-oxohexanenitrile (3b). The prod-
uct was purified by distillation under reduced pressure
(bp = 150 ꢁC/20 mmHg). Yield: 72% (colorless liquid).
1H NMR (400 MHz, CDCl3) d 0.96 (d, J = 6.9 Hz,
6H), 2.83 (m, 1H), 2.50 (d, J = 6.9 Hz, 2H), 3.44 (s, 2H).
d 1.28 (d, J = 6.5 Hz, 3H), 3.37 (s, 3H), 3.43–3.49 (m,
2H), 3.47 (s, 2H), 5.15–5.21 (m, 1H).
Cyanoacetates 2l,m were prepared via DCC coupling as
follows: to a solution of DCC (1.29 g, 6.25 mmol) in
MeCN (6 mL) was added dropwise a solution of cyano-
acetic acid (483 mg, 5.68 mmol) and alcohol
(7.10 mmol) in MeCN (6 mL). After stirring for 3.5 h,
the suspension was filtered through Celite. The filtrate
was concentrated and the crude product was purified
by flash chromatography on silica gel.
7.2.3. 2-Cyano-N-isopropylacetamide (4). To isopropyl-
amine (1.85 g, 31.2 mmol) was added slowly ethyl cya-
noacetate (1.5 mL, 12.5 mmol). The mixture was
stirred for 18 h and concentrated to dryness, yielding 4
as a yellow solid (1.97 g, quantitative). 1H NMR
(400 MHz, CDCl3) d 1.20 (d, J = 6.6 Hz, 6H), 3.34 (s,
2H), 4.08 (sept, J = 6.8 Hz, 1H), 5.92 (br s, 1H).
7.2.1.6. 1-Methylpiperidin-4-yl 2-cyanoacetate (2l).
Chromatographic eluent: gradient of 30–40% EtOAc
in hexanes. Yield: 52% (yellow oil which slowly solidi-
fied). 1H NMR (400 MHz, CDCl3) d 1.73–1.82 (m,
7.2.4. 4,5,6,7,8,9-Hexahydrocycloocta[b]thiophen-2-amines
(5). To
a
solution of cyclooctanone (500 mg,
3.96 mmol) in solvent (4 mL) in a pressure tube were

F. Arhin et al. / Bioorg. Med. Chem. 14 (2006) 5812–5832
5821
added sulfur (127 mg, 3.96 mmol), 2-cyanoacetate ester
(3.96 mmol), and amine base (5.54 mmol). The tube was
capped and the mixture was stirred at 70 ꢁC for 18 h, after
which the reaction mixture was brought to room
temperature. The resulting solution was diluted with
EtOAc and washed with H2O twice and with brine twice.
The organic layer was dried over MgSO4, filtered, and
concentrated under reduced pressure. The crude product
was purified by flash chromatography on silica gel.
d 1.27–1.32 (m, 2H), 1.41–1.47 (m, 2H), 1.53–1.59 (m,
4H), 2.58–2.61 (m, 2H), 2.79–2.83 (m, 2H), 5.27 (s,
2H), 5.93 (br s, 2H), 7.30–7.42 (m, 5H).
7.2.4.7. Allyl 2-amino-4,5,6,7,8,9-hexahydrocyclooc-
ta[b]thiophene-3-carboxylate (5g). Using DMF as sol-
vent, morpholine as the amine base, and cyanoacetate
2g. Chromatographic eluent: 50% CH2Cl2 in hexanes.
1
Yield: 42% (yellow oil). H NMR (400 MHz, CDCl3)
d 1.28–1.34 (m, 2H), 1.43–1.49 (m, 2H), 1.56–1.65 (m,
4H), 2.59–2.62 (m, 2H), 2.82–2.85 (m, 2H), 4.74 (dt,
J = 5.7 Hz, 1.3, 2H), 5.25 (dq, J = 10.5, 1.3 Hz, 1H),
5.36 (dq, J = 17.2, 1.5 Hz, 1H), 5.93 (br s, 2H), 5.96–
6.05 (m, 1H).
7.2.4.1. Ethyl 2-amino-4,5,6,7,8,9-hexahydrocyclooc-
ta[b]thiophene-3-carboxylate (5a). Using ethanol as the
solvent, morpholine as the amine base, and cyanoacetate
2a. Chromatographic eluent: 50% CH2Cl2 in hexanes.
1
Yield: 68% (yellow oil). H NMR (400 MHz, CDCl3)
d 1.28–1.34 (m, 2H), 1.35 (t, J = 7.1 Hz, 3H), 1.43–1.49
(m, 2H), 1.54–1.66 (m, 4H), 2.59–2.62 (m, 2H), 2.81–
2.84 (m, 2H), 4.28 (q, J = 7.1 Hz, 2H), 5.91 (br s, 2H).
7.2.4.8. Cyclohexyl 2-amino-4,5,6,7,8,9-hexahydrocy-
cloocta[b]thiophene-3-carboxylate (5h). Using DMF as
solvent, morpholine as the amine base, and cyanoacetate
2h. Chromatographic eluent: 50% CH2Cl2 in hexanes.
1
7.2.4.2. Methyl 2-amino-4,5,6,7,8,9-hexahydrocyclo-
octa[b]thiophene-3-carboxylate (5b). Using methanol
as the solvent, morpholine as the amine base, and
cyanoacetate 2b. Chromatographic eluent: 50% CH2Cl2
in hexanes. Yield: 38% (yellow solid). 1H NMR
(400 MHz, CDCl3) d 1.27–1.33 (m, 2H), 1.43–1.48 (m,
2H), 1.54–1.64 (m, 4H), 2.59–2.62 (m, 2H), 2.79–2.82
(m, 2H), 3.80 (s, 3H), 5.90 (br s, 2H).
Yield: 33% (yellow oil). H NMR (400 MHz, CDCl3)
d 1.29–1.34 (m, 3H), 1.41–1.48 (m, 4H), 1.52–1.59 (m,
5H), 1.62–1.66 (m, 2H), 1.71–1.77 (m, 2H), 1.89–1.95
(m, 2H), 2.59–2.62 (m, 2H), 2.83–2.86 (m, 2H), 4.97
(quint, J = 3.9 Hz, 1H), 5.93 (br s, 2H).
7.2.4.9. Cyclopentyl 2-amino-4,5,6,7,8,9-hexahydrocy-
cloocta[b]thiophene-3-carboxylate (5i). Using DMF as
solvent, morpholine as the amine base, and cyanoacetate
2i. Chromatographic eluent: 50% CH2Cl2 in hexanes.
Yield: ca. 44% (yellow oil). The compound was insepa-
rable from cyclooctanone and was used as such in the
next stage. 1H NMR (400 MHz, CDCl3) d 1.28–1.34
(m, 2H), 1.42–1.48 (m, 2H), 1.52–1.67 (m, 6H), 1.74–
1.91 (m, 6H), 2.59–2.62 (m, 2H), 2.78–2.81 (m, 2H),
5.34–5.38 (m, 1H), 5.95 (br s, 2H).
7.2.4.3. Isopropyl 2-amino-4,5,6,7,8,9-hexahydrocyclo-
octa[b]thiophene-3-carboxylate (5c). Using i-PrOH as the
solvent, morpholine as the amine base, and cyanoacetate
2c. Chromatographic eluent: 50% CH2Cl2 in hexanes.
Yield: 63% (pale yellow oil, which solidified over time).
1H NMR (400 MHz, CDCl3) d 1.32 (d, J = 6.2 Hz, 6H),
1.43–1.49 (m, 6H), 1.53–1.66 (m, 2H), 2.59–2.62 (m,
2H), 2.81–2.84 (m, 2H), 5.18 (sept, J = 6.2 Hz, 1H),
5.92 (br s, 2H). Mass calculated for C14H21NO2S: 267,
found: 268.0 (M+H)+.
7.2.4.10. ( )-Tetrahydrofuran-3-yl 2-amino-4,5,6,7,8,
9-hexahydrocycloocta[b]thiophene-3-carboxylate (5j).
Using DMF as solvent, morpholine as the amine base,
and cyanoacetate 2j. Chromatographic eluent:
80–100% CH2Cl2 in hexanes then 10% EtOAc in
CH2Cl2. Yield: 40% (dark yellow oil). 1H NMR
(400 MHz, CDCl3) d 1.28–1.34 (m, 2H), 1.43–1.48 (m,
2H), 1.54–1.65 (m, 4H), 2.09–2.26 (m, 2H), 2.59–2.62
(m, 2H), 2.79–2.82 (m, 2H), 3.90–3.95 (m, 3H), 4.00
(dd, J = 10.4, 4.6 Hz, 1H), 5.47–5.51 (m, 1H), 5.97 (br
s, 2H).
7.2.4.4. tert-Butyl 2-amino-4,5,6,7,8,9-hexahydrocy-
cloocta[b]thiophene-3-carboxylate (5d). Using EtOH as
the solvent, morpholine as the amine base, and cyanoac-
etate 2d. Chromatographic eluent: 50% CH2Cl2 in hex-
anes. Yield: 44% (yellow oil). 1H NMR (400 MHz,
CDCl3) d 1.28–1.34 (m, 2H), 1.43–1.48 (m, 2H), 1.55
(s, 9H), 1.52–1.64 (m, 4H), 2.59–2.62 (m, 2H), 2.79–
2.82 (m, 2H), 5.88 (br s, 2H).
7.2.4.5. Isobutyl 2-amino-4,5,6,7,8,9-hexahydrocyclo-
octa[b]thiophene-3-carboxylate (5e). Using DMF as sol-
vent, morpholine as the amine base, and cyanoacetate
2e. Chromatographic eluent: 50% CH2Cl2 in hexanes.
7.2.4.11. ( )-1-Methoxypropan-2-yl 2-amino-4,5,6,7,
8,9-hexahydrocycloocta[b]thiophene-3-carboxylate (5k).
Using DMF as solvent, morpholine as the amine base,
and cyanoacetate 2k. Chromatographic eluent: CH2Cl2.
Yield: 43% (dark orange oil). 1H NMR (400 MHz,
CDCl3) d 1.27–1.34 (m, 2H), 1.32 (d, J = 6.4 Hz, 3H),
1.43–1.48 (m, 2H), 1.53–1.66 (m, 4H), 2.59–2.62 (m,
2H), 2.81–2.84 (m, 2H), 3.37 (s, 3H), 3.45 (dd, J = 10.2
Hz, 4.3, 1H), 3.53 (dd, J = 10.2 Hz, 6.0, 1H), 5.20–5.26
(m, 1H), 5.89 (br s, 2H).
1
Yield: 41% (yellow oil). H NMR (400 MHz, CDCl3)
d 1.00 (d, J = 6.7 Hz, 6H), 1.31–1.35 (m, 2H), 1.43–
1.48 (m, 2H), 1.55–1.66 (m, 4H), 2.03 (sept, J = 6.7
Hz, 1H), 2.60–2.62 (m, 2H), 2.83–2.86 (m, 2H), 4.02
(d, J = 6.4 Hz, 2H), 5.96 (br s, 2H).
7.2.4.6. Benzyl 2-amino-4,5,6,7,8,9-hexahydrocyclooc-
ta[b]thiophene-3-carboxylate (5f). Using DMF as sol-
vent, morpholine as the amine base, and cyanoacetate
2f. Chromatographic eluent: 50% CH2Cl2 in hexanes.
7.2.4.12. 1-Methylpiperidin-4-yl 2-amino-4,5,6,7,8,9-
hexahydrocycloocta[b]thiophene-3-carboxylate (5l). Using
DMF as solvent, morpholine as the amine base, and cya-
noacetate 2l. Chromatographic eluent: 0–5% MeOH in
1
Yield: 37% (yellow oil). H NMR (400 MHz, CDCl3)

5822
F. Arhin et al. / Bioorg. Med. Chem. 14 (2006) 5812–5832
1
CH2Cl2. Yield: 23% (orange gum). H NMR (400 MHz,
CDCl3) d 1.28–1.35 (m, 2H), 1.42–1.47 (m, 2H), 1.54–
1.64 (m, 4H), 1.78–1.88 (m, 2H), 1.98–2.06 (m, 2H), 2.31
(s, 3H), 2.28–2.38 (m, 2H), 2.59–2.70 (m, 4H), 2.82–2.85
(m, 2H), 4.99–5.03 (m, 1H), 6.00 (br s, 2H).
(92 mg, 91% yield). 1H NMR (400 MHz, CDCl3) d
1.22 (dd, J = 6.5, 1.0 Hz, 6H), 1.36–1.42 (m, 2H),
1.44–1.50 (m, 2H), 1.56–1.68 (m, 4H), 2.61–2.64 (m,
2H), 2.70–2.74 (m, 2H), 4.20 (sept, J = 6.4 Hz, 1H),
5.68 (br s, 3H).
7.2.4.13. (R)-N-Boc-pyrrolidin-3-yl 2-amino-4,5,6,7,8,
9-hexahydrocycloocta[b]thiophene-3-carboxylate (5m).
Using DMF as solvent, morpholine as the amine base,
and cyanoacetate 2m. Chromatographic eluent: 0–5%
EtOAc in CH2Cl2. Yield: 47% (orange foam). 1H
NMR (400 MHz, CDCl3) d 1.27–1.33 (m, 2H), 1.40–
1.48 (m, 2H), 1.45 (s, 9H), 2.09–2.15 (m, 2H), 2.58–
2.61 (m, 2H), 2.73–2.78 (m, 2H), 3.42–3.67 (m, 4H),
5.47 (br s, 1H), 5.99 (br s, 2H).
7.2.5. 3-Substituted 4,5,6,7,8,9-hexahydro-2-(3-phenyl-
ureido)cycloocta[b]thiophenes. Three procedures were
used:
Procedure A. To a solution of 3-substituted 2-amino-
4,5,6,7,8,9-hexahydrocycloocta[b]thiophene (2.98 mmol)
in pyridine (6 mL) was added phenylisocyanate (747 lL,
6.85 mmol). The mixture was heated in a pressure tube
at 65 ꢁC for 18 h, after which the reaction mixture was
brought to room temperature and concentrated under
reduced pressure. The crude material was stirred in
EtOAc, undissolved diphenyl urea was removed by fil-
tration, and the filtrate was concentrated and purified
by SiO2 chromatography.
7.2.4.14. 1-(2-Amino-4,5,6,7,8,9-hexahydrocycloocta-
[b]thiophen-3-yl)-2-methylpropan-1-one
(5n).
Using
MeOH as solvent, triethylamine as the amine base,
and a-cyanoketone 3a. Chromatographic eluent: 40–
70% CH2Cl2 in hexanes. The fractions containing the
product were used in the next step as such.
Procedure B. To a solution of 3-substituted 2-amino-
4,5,6,7,8,9-hexahydrocycloocta[b]thiophene
(100 mg,
7.2.4.15. 1-(2-Amino-4,5,6,7,8,9-hexahydrocycloocta-
[b]thiophen-3-yl)-3-methylbutan-1-one
MeOH as solvent, triethylamine as the amine base,
and a-cyanoketone 3b. Chromatographic eluent: 40–
70% CH2Cl2 in hexanes. The fractions containing the
product were used in the next step as such.
0.29 mmol) in CHCl3 (1.5 mL) were added phenylisocy-
anate (63 lL, 0.58 mmol) and a catalytic amount of
DMAP. The mixture was heated in a pressure tube at
70 ꢁC for 18 h, after which the reaction mixture was
brought to room temperature and concentrated under
reduced pressure. The crude material was purified by
flash chromatography on silica gel.
(5o).
Using
7.2.4.16.
2-Amino-4,5,6,7,8,9-hexahydrocycloocta-
[b]thiophene-3-carbonitrile (5p). Using EtOH as solvent,
morpholine as the amine base, and malononitrile. Chro-
matographic eluent: 0–40% EtOAc in hexanes. Yield:
88% (yellow solid). 1H NMR (400 MHz, CDCl3) d
1.29–1.38 (m, 4H), 1.46–1.57 (m, 4H), 2.48 (t,
J = 6.1 Hz, 2H), 2.56 (t. J = 6.3 Hz, 2H) 6.88 (s, 2H).
Procedure C. To a stirred solution of 3-substituted
2-amino-4,5,6,7,8,9-hexahydrocycloocta[b]thiophene
(0.757 mmol) in CHCl3 were added phenylisocyanate
(82 lL, 0.757 mmol) and DMAP (ꢁ10 mg). The result-
ing solution was stirred at reflux for 12 h before anoth-
er portion of phenylisocyanate (82 lL, 0.757 mmol)
was added. After another 12 h at reflux, the solution
was cooled to room temperature, diluted with CH2Cl2
and H2O. The organics were collected and the aqueous
layer was extracted twice more with CH2Cl2. The com-
bined organic layers were washed with brine, dried
over MgSO4, and concentrated under reduced pressure.
The crude product was purified by two consecutive col-
umn chromatographies on silica gel.
7.2.4.17. N-Isopropyl 2-amino-4,5,6,7,8,9-hexahydro-
cycloocta[b]thiophene-3-carboxamide (5q). To a solution
of cyclooctanone (300 mg, 2.38 mmol) and cyanoaceta-
mide 4 (300 mg, 2.38 mmol) in benzene (10 mL) were
added ammonium acetate (37 mg, 0.48 mmol) and acetic
acid (109 lL, 1.90 mmol). The mixture was stirred under
˚
reflux, with 4 A molecular sieves and a Dean–Stark con-
denser, for 48 h, after which it was diluted with EtOAc,
washed with H2O twice and with brine, dried (MgSO4),
filtered, and concentrated. The crude product was puri-
fied by column chromatography on silica gel using 10%
EtOAc/hexanes as eluent. 2-Cyano-2-cyclooctylidene-N-
isopropylacetamide was obtained as an off-white solid
(90 mg, 16% yield). 1H NMR (400 MHz, CDCl3) d
1.20 (d, J = 6.5 Hz, 6H), 1.34–1.40 (m, 2H), 1.47–1.56
(m, 4H), 1.82–1.92 (m, 4H), 2.65–2.68 (m, 2H), 2.94–
2.97 (m, 2H), 4.09 (sept, J = 6.5 Hz, 1H), 5.92 (br s,
1H). To a solution of 2-cyano-2-cyclooctylidene-N-iso-
propylacetamide (88 mg, 0.38 mmol) in EtOH (1 mL)
were added sulfur powder (14 mg, 0.45 mmol) and mor-
pholine (26 lL, 0.30 mmol). The mixture was stirred at
70 ꢁC for 18 h in a pressure tube. After concentration
to dryness, the crude product was purified by column
chromatography on silica gel using 20% EtOAc/CH2Cl2
as eluent, providing compound 5q as a beige solid
7.2.5.1. Ethyl 4,5,6,7,8,9-hexahydro-2-(3-phenylure-
ido)cycloocta[b]thiophene-3-carboxylate (1). Procedure
A used. Chromatographic eluent: 50–100% CH2Cl2 in
1
hexanes. Yield: 85% (white foam). H NMR (400 MHz,
CDCl3) d 1.25–1.30 (m, 2H), 1.35 (t, J = 7.1 Hz, 3H),
1.44–1.49 (m, 2H), 1.59–1.65 (m, 4H), 2.70–2.73 (m,
2H), 2.86–2.89 (m, 2H), 4.29 (q, J = 7.1 Hz, 2H), 6.76
(s, 1H), 7.14 (t, J = 7.4 Hz, 1H), 7.35 (t, J = 8.3 Hz,
2H), 7.44 (d, J = 8.4 Hz, 2H), 10.93 (s, 1H). Mass calcu-
lated for C20H24N2O3S: 372, found: 373.1 (M+H)+.
7.2.5.2. Methyl 4,5,6,7,8,9-hexahydro-2-(3-phenylure-
ido)cycloocta[b]thiophene-3-carboxylate (6b). Procedure
A used. Chromatographic eluent: 50% CH2Cl2 in hex-
anes. Yield: 40% (white foam). 1H NMR (400 MHz,
CDCl3) d 1.25–1.30 (m, 2H), 1.44–1.49 (m, 2H), 1.59–
1.65 (m, 4H), 2.69–2.72 (m, 2H), 2.85–2.88 (m, 2H),

F. Arhin et al. / Bioorg. Med. Chem. 14 (2006) 5812–5832
5823
3.81 (s, 3H), 6.83 (s, 1H), 7.14–7.17 (m, 1H), 7.34–7.38
(m, 2H), 7.42–7.45 (m, 2H), 10.84 (s, 1H). Mass calculat-
ed for C19H22N2O3S: 358, found: 359.1 (M+H)+.
hexanes. Yield: 78% (white foam). 1H NMR
(400 MHz, CDCl3) d 1.27–1.31 (m, 2H), 1.35–1.49 (m,
5H), 1.52–1.68 (m, 7H), 1.72–1.80 (m, 2H), 1.89–1.95
(m, 2H), 2.70–2.73 (m, 2H), 2.89–2.92 (m, 2H), 4.94–
4.99 (m, 1H), 6.78 (s, 1H), 7.10–7.14 (m, 1H), 7.31–
7.37 (m, 2H), 7.42–7.45 (m, 2H), 11.05 (s, 1H). Mass cal-
culated for C24H30N2O3S: 426, found: 427.2 (M+H)+.
7.2.5.3. Isopropyl 4,5,6,7,8,9-hexahydro-2-(3-phenyl-
ureido)cycloocta[b]thiophene-3-carboxylate (6c). Proce-
dure
A
used. Chromatographic eluent: 50–60%
CH2Cl2 in hexanes. Yield: 89% (white foam). 1H
NMR (400 MHz, CDCl3) d 1.24–1.30 (m, 2H), 1.32 (d,
J = 6.2 Hz, 6H), 1.43–1.49 (m, 2H), 1.58–1.66 (m, 4H),
2.69–2.72 (m, 2H), 2.86–2.89 (m, 2H), 5.16 (sept,
J = 6.2 Hz, 1H), 7.03 (s, 1H), 7.12 (t, J = 7.4 Hz, 1H),
7.34 (t, J = 7.5 Hz, 2H), 7.45 (d, J = 7.5 Hz, 2H), 10.99
(s, 1H). Mass calculated for C21H26N2O3S: 386, found:
387.1 (M+H)+.
7.2.5.9. Cyclopentyl 4,5,6,7,8,9-hexahydro-2-(3-pheny-
lureido)cycloocta[b]thiophene-3-carboxylate (6i). Proce-
dure B used. Chromatographic eluent: 50–60% CH2Cl2
in hexanes. Yield: 79% (white foam). 1H NMR
(400 MHz, CDCl3) d 1.25–1.31 (m, 2H), 1.43–1.48 (m,
2H), 1.59–1.70 (m, 6H), 1.75–1.92 (m, 6H), 2.69–2.72
(m, 2H), 2.84–2.87 (m, 2H), 5.35–5.38 (m, 1H), 6.78 (s,
1H), 7.10–7.14 (m, 1H), 7.32–7.36 (m, 2H), 7.43–7.46
(m, 2H), 11.05 (s, 1H). Mass calculated for
C23H28N2O3S: 412, found: 413.1 (M+H)+.
7.2.5.4. tert-Butyl 4,5,6,7,8,9-hexahydro-2-(3-phenyl-
ureido)cycloocta[b]thiophene-3-carboxylate (6d). Proce-
dure A used. Chromatographic eluent: 50% CH2Cl2 in
1
hexanes. Yield: 68% (white foam). H NMR (400 MHz,
7.2.5.10. ( )-Tetrahydrofuran-3-yl 4,5,6,7,8,9-hexahy-
dro-2-(3-phenylureido)cycloocta[b]thiophene-3-carboxyl-
ate (6j). Procedure A used. Chromatographic eluent: 5%
EtOAc in CH2Cl2/hexanes (1:1). Yield: 37% (white
foam). 1H NMR (400 MHz, CDCl3) d 1.25–1.31 (m,
2H), 1.43–1.48 (m, 2H), 1.59–1.66 (m, 4H), 2.11–2.27
(m, 2H), 2.69–2.72 (m, 2H), 2.85–2.88 (m, 2H), 3.92–
4.00 (m, 4H), 5.48–5.51 (m, 1H), 6.94 (s, 1H), 7.11–
7.15 (m, 1H), 7.32–7.36 (m, 2H), 7.43–7.46 (m, 2H),
10.82 (s, 1H). Mass calculated for C22H26N2O4S: 414,
found: 415.2 (M+H)+.
CDCl3) d 1.26–1.31 (m, 2 H), 1.43–1.49 (m, 2H), 1.56 (s,
9H), 1.59–1.65 (m, 4H), 2.69–2.72 (m, 2H), 2.85–2.88
(m, 2H), 6.81 (s, 1H), 7.09–7.13 (m, 1H), 7.31–7.35 (m,
2H), 7.44–7.46 (m, 2H), 11.06 (s, 1H). Mass calculated
for C22H28N2O3S: 400, found: 401.1 (M+H)+.
7.2.5.5. Isobutyl 4,5,6,7,8,9-hexahydro-2-(3-phenylure-
ido)cycloocta[b]thiophene-3-carboxylate (6e). Procedure
A used. Chromatographic eluent: 50–60% CH2Cl2 in
hexanes. Yield: 78% (white foam). 1H NMR
(400 MHz, CDCl3) d (d, J = 6.7 Hz, 6H), 1.26–1.32 (m,
2H), 1.43–1.48 (m, 2H), 1.59–1.67 (m, 4H), 2.03 (sept,
J = 6.7 Hz, 1H), 2.70–2.73 (m, 2H), 2.89–2.92 (m, 2H),
4.02 (d, J = 6.5 Hz, 2H), 6.91 (s, 1H), 7.11–7.15 (m,
1H), 7.35 (t, J = 7.5 Hz, 2H), 7.44 (d, J = 7.7 Hz, 2H),
11.00 (s, 1H). Mass calculated for C22H28N2O3S: 400,
found: 401.1 (M+H)+.
7.2.5.11. ( )-1-Methoxypropan-2-yl 4,5,6,7,8,9-hexa-
hydro-2-(3-phenylureido)cycloocta[b]thiophene-3-carbox-
ylate (6k). Procedure B used. Chromatographic eluent
(two columns used): CH2Cl2 (first column) then 25%
Et2O in hexanes (second column). Yield: 25% (white
1
foam). H NMR (400 MHz, CDCl3) d 1.27–1.33 (m,
2H), 1.37 (d, J = 6.4 Hz, 3H), 1.43–1.49 (m, 2H), 1.59–
1.69 (m, 4H), 2.71–2.74 (m, 2H), 2.87–2.90 (m, 2H),
3.45 (s, 3H), 3.55 (dd, J = 10.9, 3.7 Hz, 1H), 3.65 (dd,
J = 10.9 Hz, 7.0, 1H), 5.23–5.27 (m, 1H), 6.89 (s, 1H),
7.07–7.12 (m, 1H), 7.30–7.34 (m, 2H), 7.43–7.46 (m,
2H), 10.61 (s, 1H). Mass calculated for C22H28N2O4S:
416, found: 417.1 (M+H)+.
7.2.5.6. Benzyl 4,5,6,7,8,9-hexahydro-2-(3-phenylure-
ido)cycloocta[b]thiophene-3-carboxylate (6f). Procedure
A used. Chromatographic eluent: 70% CH2Cl2 in hex-
anes. Yield: 46% (white foam). 1H NMR (400 MHz,
CDCl3) d 1.21–1.28 (m, 2H), 1.40–1.46 (m, 2H), 1.51–
1.63 (m, 4H), 2.69–2.72 (m, 2H), 2.84–2.87 (m, 2H),
5.28 (s, 2H), 6.66 (s, 1H), 7.11–7.15 (m, 1H), 7.30–7.43
(m, 9H), 10.86 (s, 1H). Mass calculated for
C25H26N2O3S: 434, found: 435.1 (M+H)+.
7.2.5.12. 1-Methylpiperidin-4-yl 4,5,6,7,8,9-hexahy-
dro-2-(3-phenylureido)cycloocta[b]thiophene-3-carboxyl-
ate (6l). Procedure B used. Chromatographic eluent: 0–
5% MeOH in CH2Cl2. Yield: 62% (beige foam). 1H
NMR (400 MHz, CDCl3) d 1.25–1.32 (m, 2H), 1.43–
1.49 (m, 2H), 1.59–1.66 (m, 4H), 1.80–1.89 (m, 2H),
1.97–2.05 (m, 2H), 2.31 (s, 3H), 2.33–2.38 (m, 2H),
2.62–2.69 (m, 2H), 2.70–2.73 (m, 2H), 2.89–2.92 (m,
2H), 4.98–5.02 (m, 1H), 6.91 (s, 1H), 7.11–7.15 (m,
1H), 7.32–7.37 (m, 2H), 7.43–7.46 (m, 2H), 11.00 (s,
1H). Mass calculated for C24H31N3O3S: 441, found:
442.2 (M+H)+.
7.2.5.7. Allyl 4,5,6,7,8,9-hexahydro-2-(3-phenylure-
ido)cycloocta[b]thiophene-3-carboxylate (6g). Procedure
A used. Chromatographic eluent: 50–60% CH2Cl2 in
hexanes. Yield: 60% (white foam). 1H NMR
(400 MHz, CDCl3) d1.25–1.30 (m, 2H), 1.44–1.49 (m,
2H), 1.59–1.66 (m, 4H), 2.70–2.73 (m, 2H), 2.87–2.90
(m, 2H), 4.73 (dt, J = 5.7 Hz, 1.3, 2H), 5.26–5.29 (m,
1H), 5.33–5.39 (m, 1H), 5.93–6.03 (m, 1H), 6.80 (s,
1H), 7.12–7.16 (m, 1H), 7.33–7.38 (m, 2H), 7.42–7.45
(m, 2H), 10.89 (s, 1H). Mass calculated for
C21H24N2O3S: 384, found: 385.0 (M+H)+.
7.2.5.13. (R)-1-Methylpyrrolidin-3-yl 4,5,6,7,8,9-hexa-
hydro-2-(3-phenylureido)cycloocta[b]thiophene-3-carbox-
ylate (6m). Procedure B was used, with 5m and
phenylisocyanate. The crude material was purified by
flash chromatography on silica gel using a gradient of
7.2.5.8. Cyclohexyl 4,5,6,7,8,9-hexahydro-2-(3-phenyl-
ureido)cycloocta[b]thiophene-3-carboxylate (6h). Proce-
dure A. Chromatographic eluent: 50% CH2Cl2 in

5824
F. Arhin et al. / Bioorg. Med. Chem. 14 (2006) 5812–5832
0–5%
EtOAc/hexanes.
(R)-N-Boc-pyrrolidin-3-yl
(m, 4H), 2.19 (m, 1H), 2.67–2.72 (m, 4H), 2.87 (br t,
4,5,6,7,8,9-hexahydro-2-(3-phenylureido)cycloocta[b]thi-
ophene-3-carboxylate was obtained as a pale yellow
foam in 50% yield. 1H NMR (400 MHz, CDCl3) d
1.24–1.29 (m, 2H), 1.46 (s, 11H), 1.57–1.69 (m, 4H),
2.16 (br s, 2H), 2.69–2.71 (m, 2H), 2.80–2.83 (m,
2H), 3.46–3.61, 3.75–3.79 (2m, 4H), 5.41–5.47 (m, 1H),
7.09–7.14 (m, 1H), 7.18 (br s, 1H), 7.32–7.36 (m, 2H),
7.47–7.48 (m, 2H), 10.63, 10.75 (2s, 1H), rotamers due
to carbamate isomerism. Mass calculated for
C27H35N3O5S: 513, found: 414.1 (MꢀBoc+H)+. It was
then treated with a solution of TFA/CH2Cl2 (40%, v/v)
and stirred during 40 minutes and concentrated to dry-
ness. After two co-evaporations with benzene, the
TFA salt was obtained as a beige solid in quantitative
J = 6.2 Hz, 2H), 7.11 (dt, J = 8.5, 1.5 Hz, 1H), 7.33
(m, 2H), 7.47 (m, 2H), 7.83 (br s, 1H), 12.42 (br s,
1H). Mass calculated for C22H28N2O2S: 384, found:
385.2 (M+H)+.
7.2.5.16.
1-(3-Cyano-4,5,6,7,8,9-hexahydrocyclooc-
ta[b]thiophen-2-yl)-3-phenylurea (6p). A solution of 5p
(0.91 g, 4.4 mmol) and phenylisocyanate (0.97 mL,
8.8 mmol) in DCM (10 mL) was stirred at room tem-
perature for 20 hr. The reaction was quenched by the
addition of water. After stirring the mixture at room
temperature for 1 h, the precipitate was collected by
filtration and the crude product was recrystallized
from EtOAc/hexanes, yielding 750 mg (52%) of 6p as
a white solid. 1H NMR (400 MHz, CDCl3) d 1.38–
1.46 (m, 4H), 1.62–1.75 (m, 4H), 2.70–2.74 (m, 4H),
7.10–7.14 (m, 1H), 7.35 (dd, J = 8.6, 7.5 Hz, 2H),
7.47 (dd, J = 8.7, 1.1 Hz, 2H), 7.54 (s, 1H), 8.57 (s,
1H). Mass calculated for C18H19N3OS: 325, found:
326.1 (M+H)+.
1
yield. H NMR (400 MHz, DMSO-d6) d 1.21–1.29 (m,
2H), 1.40–1.46 (m, 2H), 1.53–1.65 (m, 4H), 2.20–2.27
(m, 2H), 2.69–2.72 (m, 2H), 2.80–2.87 (m, 2H), 3.22–
3.28 (m, 1H), 3.39–3.47 (m, 2H), 3.54–3.60 (m, 1H),
5.51–5.55 (m, 1H), 7.00–7.05 (m, 1H), 7.30–7.33 (m,
2H), 7.48–7.50 (m, 2H), 9.04 (br s, 1H), 9.13 (br s,
1H), 10.24 (s, 1H), 10.66 (s, 1H). Mass calculated for
C22H27N3O5S: 413, found: 414.1 (M+H)+. The TFA salt
(121 mg, 0.23 mmol) was dissolved in a solution of for-
mic acid (1.0 mL) and formaldehyde (1.0 mL), and stir-
red under reflux for 30 min, after which it was
concentrated to dryness. The residue was poured into
sat. NaHCO3 solution and extracted three times with a
mixture of CHCl3/i-PrOH (4:1), the combined organic
layers were dried (MgSO4), filtered, and concentrated.
Crude 6m was purified by flash chromatography, 0–3%
MeOH/CH2Cl2, then reverse phase on C18, 20–100%
MeCN/H2O, followed by prep. TLC, 10% MeOH/
EtOAc. Compound 6m was obtained as a white foam
(10 mg, 10% yield). 1H NMR (400 MHz, CDCl3) d
1.25–1.32 (m, 2H), 1.42–1.48 (m, 2H), 1.58–1.67 (m,
4H), 1.97–2.05 (m, 1H), 2.30–2.39 (m, 1H), 2.46–2.52
(m, 1H), 2.48 (s, 3H), 2.70–2.73 (m, 2H), 2.79–2.95 (m,
5H), 5.40–5.45 (m, 1H), 7.08–7.12 (m, 2H), 7.33 (t,
J = 7.5 Hz, 2H), 7.45 (d, J = 7.5 Hz, 2H), 10.62 (s,
1H). Mass calculated for C23H29N3O3S: 427, found:
428.2 (M+H)+.
7.2.5.17. N-Isopropyl 4,5,6,7,8,9-hexahydro-2-(3-
phenylureido)cycloocta[b]thiophene-3-carboxamide (6q).
Procedure B was used, with 5q and phenylisocyanate.
The crude material was purified by flash chromatogra-
phy on silica gel using a gradient of 0–2% EtOAc/
CH2Cl2, followed by HPLC prep. on XTerra Prep
RP18 (10· 300 mm), gradient of MeCN in water (con-
taining 0.05% TFA) 20–75% (20 min) then isocratic
75% (15 min). Compound 6q was obtained as a white
1
solid (34 mg, 27% yield). H NMR (400 MHz, CDCl3)
d 1.25 (d, J = 6.5 Hz, 6H), 1.37–1.43 (m, 2H), 1.46–
1.52 (m, 2H), 1.62–1.71 (m, 4H), 2.73–2.76 (m, 2H),
2.78–2.81 (m, 2H), 4.21 (sept, J = 6.6 Hz, 1H), 5.91 (d,
J = 6.6 Hz, 1H), 6.98 (s, 1H), 7.04–7.094 (m, 1H),
7.27–7.32 (m, 2H), 7.42–7.44 (m, 2H), 11.41 (s, 1H).
Mass calculated for C21H27N3O2S: 385, found: 386.1
(M+H)+.
7.2.6. 4,5,6,7,8,9-Hexahydro-2-(3-phenylureido)cyclooc-
ta[b]thiophene-3-carboxylic acid (6r). tert-Butyl ester 6d
(55 mg, 0.14 mmol) was stirred for 1 h in a solution of
TFA/CH2Cl2 (40%, v/v, 4 mL), after which the reaction
mixture was concentrated under reduced pressure, to af-
ford 35 mg (74%) of 6r as a white solid. 1H NMR
(400 MHz, DMSO-d6) d 1.18–1.25 (m, 2H), 1.38–1.45
(m, 2H), 1.51–1.61 (m, 4H), 2.67–2.71 (m, 2H), 2.84–
2.89 (m, 2H), 6.99–7.03 (m, 1H), 7.28–7.32 (m, 2H),
7.48–7.50 (m, 2H), 10.15 (s, 1H), 10.72 (s, 1H), 12.81
(br s, 1H). Mass calculated for C18H20N2O3S: 344,
found: 345.1 (M+H)+.
7.2.5.14.
1-(4,5,6,7,8,9-Hexahydro-3-(isobutyryl)-
cycloocta[b]thiophen-2-yl)-3-phenylurea (6n). Procedure
C used. Chromatographic eluent (two columns used):
40–70% CH2Cl2 in hexanes (first column), 5–20%
EtOAc in hexanes (second column). Yield: 82% (white
solid). 1H NMR (400 MHz, CDCl3)
d 1.20 (d,
J = 6.7 Hz, 6H), 1.33 (m, 2H), 1.50 (m, 2H), 1.62–1.72
(m, 4H), 2.75 (br t, J = 5.9 Hz, 2H), 2.88 (br t,
J = 8.7 Hz, 2H), 3.36 (m, 1H), 6.76 (br s, 1H), 7.12
(dt, J = 7.4, 0.8 Hz, 1H), 7.35 (m, 2H), 7.46 (m, 2H),
12.42 (br s, 1H). Mass calculated for C11H26N2O2S:
370, found: 371.5 (M+H)+.
7.2.7. Ethyl 4,5,6,7,8,9-hexahydro-2-(3-phenylthioure-
ido)cycloocta[b]thiophene-3-carboxylate (7). Prepared as
for 1 but substituting phenyl isothiocyanate for pheny-
lisocyanate. The reaction mixture was diluted in EtOAc,
washed with 5% HCl solution twice, saturated NaHCO3
solution, and satd NaCl solution. The organic layer was
dried over Na2SO4, filtered, and concentrated under re-
duced pressure. The crude product was purified by flash
chromatography on silica gel, using 50% CH2Cl2 in hex-
anes as eluent. Compound 7 was obtained in 76% yield,
7.2.5.15. 1-(3-(3-Methylbutanoyl)-4,5,6,7,8,9-hexahy-
drocycloocta[b]thiophen-2-yl)-3-phenylurea (6o). Proce-
dure C used. Chromatographic eluent (two columns
used): 40–70% CH2Cl2 in hexanes (first column), 5–
20% EtOAc in hexanes (second column). Yield: 88%
1
(white foam). H NMR (400 MHz, CDCl3) d 0.93 (d,
J = 6.7 Hz, 6H), 1.32 (m, 2H), 1.48 (m, 2H), 1.59–1.69

F. Arhin et al. / Bioorg. Med. Chem. 14 (2006) 5812–5832
5825
1
as a white solid. H NMR (400 MHz, CDCl3) d 1.22–
2.69–2.72 (m, 2H), 2.85–2.88 (m, 2H), 3.81 (s, 3H),
5.15 (sept, J = 6.2 Hz, 1H), 6.58 (s, 1H), 6.89–6.91 (m,
2H), 7.31–7.34 (m, 2H), 10.89 (s, 1H). Mass calculated
for C22H28N2O4S: 416, found: 417.1 (M+H)+.
1.28 (m, 2H), 1.25 (t, J = 7.1 Hz, 3H), 1.43–1.48 (m,
2H), 1.57–1.67 (m, 4H), 2.72–2.75 (m, 2H), 2.83–2.86
(m, 2H), 7.13 (q, J = 7.1 Hz, 2H), 7.34 (d, J = 7.2 Hz,
2H), 7.39 (d, J = 7.4 Hz, 1H), 7.48 (t, J = 7.9 Hz, 2H),
7.74 (s, 1H), 12.23 (s, 1H). Mass calculated for
C20H24N2O2S2: 388, found: 389.1 (M+H)+.
7.2.8.4.
Isopropyl
2-(3-(3-chlorophenyl)ureido)-
4,5,6,7,8,9-hexahydrocycloocta[b]thiophene-3-carboxyl-
ate (8d). Procedure A was used with 5c and 3-chloroph-
enylisocyanate. Chromatographic eluent: 50–60%
CH2Cl2 in hexanes. This was followed by trituration in
hexanes. Yield: 38% (white solid). 1H NMR
(400 MHz, CDCl3) d 1.27–1.30 (m, 2H), 1.36 (d,
J = 6.3 Hz, 6H), 1.44–1.48 (m, 2H), 1.60–1.65 (m, 4H),
2.71–2.73 (m, 2H), 2.87–2.90 (m, 2H), 5.19 (sept,
J = 6.3 Hz, 1H), 6.87 (s, 1H), 7.06–7.09 (m, 1H), 7.24–
7.26 (m, 2H), 7.61–7.62 (m, 1H), 11.06 (s, 1H). Mass cal-
culated for C21H25ClN2O3S: 420, found: 421.0 (M+H)+.
7.2.8. Susbstituted 4,5,6,7,8,9-hexahydro-2-ureidocyclo-
octa[b]thiophene-3-carboxylates (8a–y). Two procedures
were used:
Procedure A. To a solution of 5c/5j (2.98 mmol) in pyri-
dine (6 mL) was added an isocyanate (6.85 mmol). The
mixture was heated in a pressure tube at 65 ꢁC for 18 h,
after which the reaction mixture was brought to room
temperature and concentrated under reduced pressure.
The crude material was purified by SiO2 chromatography.
7.2.8.5. Isopropyl 2-(3-(3-(trifluoromethyl)phenyl)ure-
ido)-4,5,6,7,8,9-hexahydrocycloocta[b]thiophene-3-car-
boxylate (8e). Procedure A was used with 5c and a,a,a-
trifluoro-m-tolylisocyanate. Chromatographic eluent:
Procedure B. To a solution of 5c/5j (0.37 mmol) in
CH2Cl2 (7 mL) was added dropwise a solution of tri-
phosgene (110 mg, 0.37) in CH2Cl2 (1 mL), followed
by dropwise addition of triethylamine (80 lL,
0.59 mmol) at 0 ꢁC. After stirring for 2 h at room tem-
perature, the appropriate amine (3.0 mmol) was added
and the reaction mixture was stirred for 1 h after which
H2O was added. The resulting solution was extracted
three times with CH2Cl2. The combined organic layers
were washed with brine, dried over Na2SO4, and con-
centrated. The crude product was purified by flash chro-
matography on silica gel.
1
80% CH2Cl2 in hexanes. Yield: 36% (white solid). H
NMR (400 MHz, CDCl3) d 1.25–1.28 (m, 2H), 1.35–
1.36 (d, J = 6.3 Hz, 6H), 1.44–1.49 (m, 2H), 1.59–1.67
(m, 4H), 2.71–2.73 (m, 2H), 2.87–2.90 (m, 2H), 5.15–
5.22 (m, 1H), 6.96 (s, 1H), 7.34–7.36 (d, J = 7.6 Hz,
1H), 7.42–7.46 (t, J = 8.2 Hz, 1H), 7.59–7.61 (d,
J = 8.4 Hz, 1H), 7.82 (s, 1H), 11.12 (s, 1H). Mass calcu-
lated for C22H25F3N2O3S: 454, found: 455.1 (M+H)+.
7.2.8.6. Isopropyl 2-(3-(2-chlorophenyl)ureido)-4,5,
6,7,8,9-hexahydrocycloocta[b]thiophene-3-carboxylate
(8f). Procedure A was used with 5c and 2-chloropheny-
lisocyanate. Chromatographic eluent: 50–60% CH2Cl2
in hexanes. Yield: 45% (yellow foam). 1H NMR
(400 MHz, CDCl3) d 1.27–1.30 (m, 2H), 1.37–1.38 (d,
J = 6.3 Hz, 6H), 1.45–1.49 (m, 2H), 1.60–1.66 (m, 4H),
2.71–2.74 (m, 2H), 2.88–2.89 (m, 2H), 5.22 (m, 1H),
7.00–7.05 (m, 2H), 7.26–7.30 (m, 1H), 7.36–7.38 (d,
J = 8.4 Hz, 1H), 8.21–8.23 (d, J = 8.4 Hz, 1H), 11.11
(s, 1H). Mass calculated for C21H25ClN2O3S: 420,
found: 421.0 (M+H)+.
7.2.8.1. Isopropyl 4,5,6,7,8,9-hexahydro-2-(3-p-tolyl-
ureido)cycloocta[b]thiophene-3-carboxylate (8a). Proce-
dure A was used with 5c and p-tolylisocyanate.
Chromatographic eluent: 50–60% CH2Cl2 in hexanes.
Yield: 54% (light pink foam). 1H NMR (400 MHz,
CDCl3) d 1.25–1.31 (m, 2H), 1.33 (d, J = 6.3 Hz, 6H),
1.44–1.48 (m, 2H), 1.59–1.65 (m, 4H), 2.33 (s, 3H),
2.70–2.73 (m, 2H), 2.86–2.89 (m, 2H), 5.18 (sept,
J = 6.2 Hz, 1H), 6.59 (s, 1H), 7.14–7.17 (m, 2H), 7.30–
7.33 (m, 2H), 10.96 (s, 1H). Mass calculated for
C22H28N2O3S: 400, found: 401.1 (M+H)+.
7.2.8.2. Isopropyl 2-(3-(4-chlorophenyl)ureido)-4,5,
6,7,8,9-hexahydrocycloocta[b]thiophene-3-carboxylate
(8b). Procedure A was used with 5c and 4-chloropheny-
lisocyanate. Chromatographic eluent: 50% CH2Cl2 in
7.2.8.7. Isopropyl 2-(3-(2,6-dichlorophenyl)ureido)-4,5,
6,7,8,9-hexahydrocycloocta[b]thiophene-3-carboxylate
(8g). Procedure A was used with 5c and 2,6-dichloroph-
enylisocyanate. Chromatographic eluent: 50–60%
CH2Cl2 in hexanes. Yield: 31% (white foam). 1H
NMR (400 MHz, CDCl3) d 1.27–1.28 (m, 2H), 1.31–
1.32 (d, J = 6.3 Hz, 6H), 1.45 (m, 2H), 1.56–1.62 (m,
4H), 2.68–2.70 (m, 2H), 2.85–2.88 (m, 2H), 5.15 (m,
1H), 6.47 (s, 1H), 7.17–7.23 (m, 1H), 7.42 (d,
J = 5.6 Hz, 2H), 10.97 (s, 1H). Mass calculated for
C21H24Cl2N2O3S: 455, found: 456.1 (M+H)+.
1
hexanes. Yield: 57% (light pink foam). H NMR (400
MHz, CDCl3)
d
1.25–1.30 (m, 2H), 1.35 (d,
J = 6.3 Hz, 6H), 1.44–1.48 (m, 2H), 1.60–1.65 (m, 4H),
2.70–2.73 (m, 2H), 2.86–2.89 (m, 2H), 5.19 (sept,
J = 6.3 Hz, 1H), 6.77 (s, 1H), 7.28–7.30 (m, 2H), 7.39–
7.42 (m, 2H), 11.05 (s, 1H). Mass calculated for
C21H25ClN2O3S: 420, found: 421.1 (M+H)+.
7.2.8.3. Isopropyl 4,5,6,7,8,9-hexahydro-2-(3-(4-meth-
oxyphenyl)ureido)cycloocta[b]thiophene-3-carboxylate
(8c). Procedure A was used with 5c and 4-methoxyphe-
nylisocyanate. Chromatographic eluent: 75–80%
CH2Cl2 in hexanes. Yield: 77% (beige foam). H NMR
(400 MHz, CDCl3) d 1.25–1.30 (m, 2H), 1.31 (d,
J = 6.3 Hz, 6H), 1.44–1.47 (m, 2H), 1.58–1.64 (m, 4H),
7.2.8.8. Isopropyl 2-(3-cyclohexylureido)-4,5,6,7,8,9-
hexahydrocycloocta[b]thiophene-3-carboxylate (8h). Pro-
cedure A was used with 5c and cyclohexylisocyanate.
Chromatographic eluent: 80% CH2Cl2 in hexanes.
Yield: 15% (colorless oil). H NMR (400 MHz, CDCl3)
d 1.12–1.44 (m, 16H), 1.60–1.61 (m, 4H), 1.70–1.73 (m,
2H), 1.97–1.99 (m, 2H), 2.67–2.70 (m, 2H), 2.84–2.87
1
1

5826
F. Arhin et al. / Bioorg. Med. Chem. 14 (2006) 5812–5832
(m, 2H), 3.64 (m, 1H), 4.70 (d, J = 8.0 Hz, 1H), 5.14–
5.21 (m, 1H), 10.67 (s, 1H). Mass calculated for
C21H32N2O3S: 392, found: 393.1 (M+H)+.
Yield: 33% (colorless oil). 1H NMR (400 MHz,
CDCl3) d 1.25–1.31 (m, 8H), 1.44–1.46 (m, 2H),
1.58–1.63 (m, 4H), 2.68–2.71 (m, 2H), 2.84–2.88 (m,
2H), 5.13–5.30 (m, 1H), 6.80 (s, 1H), 6.90–6.92 (m,
1H), 7.01–7.03 (m, 1H), 7.26 (br s, 1H), 11.02 (s,
1H). Mass calculated for C19H24N2O3S2: 392, found:
393.1 (M+H)+.
7.2.8.9. Isopropyl 2-(3-cyclopentylureido)-4,5,6,7,8,9-
hexahydrocycloocta[b]thiophene-3-carboxylate (8i). Pro-
cedure A was used with 5c and cyclopentylisocyanate.
Chromatographic eluent: 80% CH2Cl2 in hexanes. Yield:
17% (colorless oil). 1H NMR (400 MHz, CDCl3) d 1.26–
1.46 (m, 12H), 1.60–1.69 (m, 8H), 2.00–2.04 (m, 2H),
2.67–2.70 (m, 2H), 2.84–2.87 (m, 2H), 4.09 (m, 1H), 4.80
(m, 1H), 5.16–5.19 (m, 1H), 10.71 (s, 1H). Mass calculated
for C20H30N2O3S: 378, found: 379.2 (M+H)+.
7.2.8.15. Isopropyl 4,5,6,7,8,9-hexahydro-2-(3-(thio-
phen-3-yl)ureido)cycloocta[b]thiophene-3-carboxylate (8o).
Procedure A was used with 5c and 3-thienylisocyanate.
Chromatographic eluent: 5% MeOH in CH2Cl2. Yield:
28% (colorless oil). 1H NMR (400 MHz, CDCl3) d
1.23–1.30 (m, 8H), 1.42–1.47 (m, 2H), 1.56–1.63 (m,
4H), 2.66–2.69 (m, 2H), 2.84–2.87 (m, 2H), 5.09–5.14
(m, 1H), 7.02 (d, J = 5.1 Hz, 1H), 7.22–7.26 (m, 1H),
7.35 (s, 1H), 7.93 (s, 1H), 10.94 (s, 1H). Mass calculated
for C19H24N2O3S2: 392, found: 393.1 (M+H)+.
7.2.8.10. Isopropyl 2-(3-benzylureido)-4,5,6,7,8,9-
hexahydrocycloocta[b]thiophene-3-carboxylate (8j). Pro-
cedure A was used with 5c and benzylisocyanate. Chro-
matographic eluent: 80% CH2Cl2 in hexanes. Yield: 28%
1
(colorless oil). H NMR (400 MHz, CDCl3) d 1.24–1.27
(m, 2H), 1.29–1.31 (d, J = 6.3 Hz, 6H), 1.42–1.47 (m,
2H), 1.56–1.64 (m, 4H), 2.66–2.68 (m, 2H), 2.84–2.87
(m, 2H), 4.47 (d, J = 5.6 Hz, 2H), 5.04–5.11 (m, 1H),
5.58 (s, 1H), 7.24–7.33 (m, 5H), 10.80 (s, 1H). Mass cal-
culated for C22H28N2O3S: 400, found: 401.2 (M+H)+.
7.2.8.16. Isopropyl 4,5,6,7,8,9-hexahydro-2-(3-(thia-
zol-2-yl)ureido)cycloocta[b]thiophene-3-carboxylate (8p).
Procedure B was used with 5c and 2-aminothiazole.
Chromatographic eluent: 5% MeOH in CH2Cl2. Yield:
44% (colorless oil). 1H NMR (400 MHz, CDCl3) d
1.25–1.30 (m, 2H), 1.36–1.38 (d, J = 6.3 Hz, 6H), 1.46–
1.48 (m, 2H), 1.62–1.67 (m, 4H), 2.72–2.75 (m, 2H),
2.88–2.92 (m, 2H), 5.21–5.28 (m, 1H), 6.93 (d,
J = 3.7 Hz, 1H), 7.78 (d, J = 3.7 Hz, 1H), 11.35 (s,
1H). Mass calculated for C18H23N3O3S2: 393, found:
394.1 (M+H)+.
7.2.8.11. Isopropyl 4,5,6,7,8,9-hexahydro-2-(3-(pyri-
din-4-yl)ureido)cycloocta[b]thiophene-3-carboxylate (8k).
Procedure B was used with 5c and 4-aminopyridine.
Chromatographic eluent: 10% hexanes in CH2Cl2. Yield:
30% (white powder). 1H NMR (400 MHz, CDCl3) d 1.25–
1.30 (m, 2H), 1.32–1.34 (d, J = 6.3 Hz, 6H), 1.44–1.47 (m,
2H), 1.62–1.66 (m, 4H), 2.71–2.74 (m, 2H), 2.85–2.89 (m,
2H), 5.11–5.17 (m, 1H), 7.47 (d, J = 6.1 Hz, 2H), 8.34 (s,
1H), 8.47 (s, 2H), 11.16 (s, 1H). Mass calculated for
C20H25N3O3S2: 387, found: 388.1 (M+H)+.
7.2.8.17. Isopropyl 4,5,6,7,8,9-hexahydro-2-(3-(5-
methylthiophen-2-yl)ureido)cycloocta[b]thiophene-3-car-
boxylate (8q). To a solution of 5-methylthiophene-2-car-
boxylic acid (71 mg, 0.50 mmol) in benzene (50 mL) was
added dropwise diphenyl azidophosphate (110 lL,
0.50 mmol), followed by the dropwise addition of trieth-
ylamine (110 lL, 0.75 mmol) at room temperature.
After stirring for 3 h at reflux, the amine 5c (134 mg,
0.50 mmol) was added and the reaction mixture was stir-
red and refluxed overnight. The resulting solution was
evaporated to dryness and the crude product was puri-
fied by flash chromatography on silica gel using 10%
hexanes in CH2Cl2. Yield 55% (dark powder). 1H
NMR (400 MHz, CDCl3) d 1.26–1.29 (m, 8H), 1.42–
1.47 (m, 2H), 1.57–1.64 (m, 4H), 2.44 (s, 3H), 2.67–
2.70 (m, 2H), 2.84–2.87 (m, 2H), 5.10–5.30 (m, 1H),
6.56 (s, 1H), 6.65 (s, 1H), 7.26 (s, 1H), 10.94 (s, 1H).
Mass calculated for C20H26N2O3S2: 406, found: 407.1
(M+H)+.
7.2.8.12. Isopropyl 4,5,6,7,8,9-hexahydro-2-(3-(pyri-
din-2-yl)ureido)cycloocta[b]thiophene-3-carboxylate (8l).
Procedure B was used with 5c and 2-aminopyridine.
Chromatographic eluent: 10% hexanes in CH2Cl2.
Yield: 34% (white powder). 1H NMR (400 MHz,
CDCl3) d 1.24–1.31 (m, 2H), 1.32–1.34 (d, J = 6.3 Hz,
6H), 1.43–1.47 (m, 2H), 1.61–1.67 (m, 4H), 2.71–2.74
(m, 2H), 2.85–2.89 (m, 2H), 5.25–5.33 (m, 1H), 7.01–
7.10 (m, 2H), 7.67–7.69 (m, 1H), 8.43 (s, 1H), 11.16 (s,
1H). Mass calculated for C20H25N3O3S2: 387, found:
388.1 (M+H)+.
7.2.8.13. Isopropyl 4,5,6,7,8,9-hexahydro-2-(3-(pyra-
zin-2-yl)ureido)cycloocta[b]thiophene-3-carboxylate (8m).
Procedure B was used with 5c and 2-aminopyrazine.
Chromatographic eluent: 10% hexanes in CH2Cl2. Yield:
30% (white powder). 1H NMR (400 MHz, CDCl3) d 1.25–
1.31 (m, 2H), 1.37–1.39 (d, J = 6.3 Hz, 6H), 1.45–1.51 (m,
2H), 1.63–1.69 (m, 4H), 2.75–2.79 (m, 2H), 2.90–2.95 (m,
2H), 5.28–5.34 (m, 1H), 8.27 (s, 1H), 8.37 (s, 1H), 8.64 (s,
1H), 9.21 (s, 1H), 13.32 (s, 1H). Mass calculated for
C19H24N4O3S: 388, found: 389.1 (M+H)+.
7.2.8.18.
Isopropyl
4,5,6,7,8,9-hexahydro-2-(3-
(3-(methylcarboxy)thiophen-2-yl)ureido)cycloocta[b]thio-
phene-3-carboxylate (8r). Procedure B was used with 5c
and methyl 5-aminothiophene-2-carboxylate. Chroma-
tographic eluent: 10% hexanes in CH2Cl2. Yield: 27%
1
(white powder). H NMR (400 MHz, CDCl3) d 1.26–
1.31 (m, 4H), 1.37–1.38 (d, J = 6.3 Hz, 6H), 1.43–1.49
(m, 2H), 1.60–1.67 (m, 4H), 2.71–2.74 (m, 2H), 2.88–
2.91 (m, 2H), 3.88 (s, 3H), 5.20–5.30 (m, 1H), 6.68–
6.69 (d, J = 6.3 Hz, 1H), 7.16–7.17 (d, J = 6.3 Hz, 1H),
10.66 (s, 1H), 11.24 (s, 1H). Mass calculated for
C21H26N2O5S2: 450, found: 451.1 (M+H)+.
7.2.8.14. Isopropyl 4,5,6,7,8,9-hexahydro-2-(3-(thio-
phen-2-yl)ureido)cycloocta[b]thiophene-3-carboxylate (8n).
Procedure A was used with 5c and 2-thienylisocya-
nate. Chromatographic eluent: 5% MeOH in CH2Cl2.

F. Arhin et al. / Bioorg. Med. Chem. 14 (2006) 5812–5832
5827
7.2.8.19.
Isopropyl
4,5,6,7,8,9-hexahydro-2-(3-
in CH2Cl2 as eluent, then reverse phase chromatography
on Biotage (C18, 25+M), using a gradient of MeCN in
H2O could not separate the two products. The mixture
obtained after those separation attempts was treated
with a solution of 5%, v/v Et3N in MeOH, at 65 ꢁC dur-
ing 5 h, then concentrated. Flash chromatography on
silica gel using a gradient of 0–5% EtOAc in CH2Cl2
as eluent, then recrystallization in Et2O/CH2Cl2 finally
provided 8x as a beige solid (29 mg, 16% yield). 1H
NMR (400 MHz, CDCl3) d 1.25–1.30 (m, 2H), 1.42–
1.48 (m, 2H), 1.57–1.63 (m, 4H), 2.08–2.24 (m, 2H),
2.68–2.71 (m, 2H), 2.83–2.86 (m, 2H), 3.87–3.98 (m,
4H), 5.44–5.48 (m, 1H), 6.76–6.78 (m, 1H), 6.88–6.91
(m, 1H), 7.01–7.02 (m, 1H), 7.63 (br s, 1H), 10.86 (s,
1H). Mass calculated for C20H24N2O4S2: 420, found:
421.0 (M+H)+.
(2-(methylcarboxy)thiophen-3-yl)ureido)cycloocta[b]thio-
phene-3-carboxylate (8s). Procedure A was used with 5c
and 3-thienylisocyanate. Chromatographic eluent: 10%
hexanes in CH2Cl2. Yield: 21% (dark powder). 1H
NMR (400 MHz, CDCl3) d 1.23–1.27 (m, 8H), 1.44–
1.47 (m, 2H), 1.56–1.63 (m, 4H), 2.66–2.69 (m, 2H),
2.84–2.89 (m, 2H), 5.09–5.14 (m, 1H), 7.22–7.24 (d,
J = 6.3 Hz, 1H), 7.22–7.24 (m, 1H), 7.34–7.36 (d,
J = 6.3 Hz, 1H), 7.93 (s, 1H), 10.94 (s, 1H). Mass calcu-
lated for C19H24N2O3S2: 392, found: 393.1 (M+H)+.
7.2.8.20. Isopropyl 4,5,6,7,8,9-hexahydro-2-(3-(3,5-
dimethylisoxazol-4-yl)ureido)cycloocta[b]thiophene-3-car-
boxylate (8t). Procedure A was used with 5c and
3,5-dimethylisoxazol-4-ylisocyanate. Chromatographic
eluent: 10% MeOH in CH2Cl2. Yield: 15% (colorless
1
oil). H NMR (400 MHz, CDCl3) d 1.28–1.30 (m, 8H),
7.2.8.25. ( )-Tetrahydrofuran-3-yl 4,5,6,7,8,9-hexahy-
dro-2-(3-(thiophen-3-yl)ureido)cycloocta[b]thiophene-3-
1.45 (m, 2H), 1.61 (m, 4H), 2.24 (s, 3H), 2.39 (s, 3H),
2.67–2.70 (m, 2H), 2.84–2.87 (m, 2H), 5.11–5.14 (m,
1H), 6.25 (s, 1H), 10.93 (s, 1H). Mass calculated for
C20H27N3O4S: 405, found: 406.2 (M+H)+.
carboxylate (8y). To
a solution of 5j (133 mg,
0.45 mmol), in EtOAc (2.3 mL), was added 3-thienyliso-
cyanate (113 mg, 0.90 mmol). The mixture was stirred at
room temperature during 18 h. After concentrating to
dryness, crude 8y was purified by flash chromatography
on silica gel using a gradient of 0–5% EtOAc in CH2Cl2
as eluent, followed by triturations: first in EtO2 (dithie-
nylurea insoluble), then in hexanes (product insoluble),
providing 8y as an off-white solid (113 mg, 70% yield).
1H NMR (400 MHz, CDCl3) d 1.26–1.31 (m, 2H),
1.43–1.49 (m, 2H), 1.59–1.66 (m, 4H), 2.11–2.25 (m,
2H), 2.69–2.72 (m, 2H), 2.81–2.87 (m, 2H), 3.91–4.00
(m, 4H), 5.47–5.50 (m, 1H), 7.01 (dd, J = 5.2 Hz, 1.4
Hz, 1H), 7.14 (br s, 1H), 7.26–7.28 (m, 1H), 7.36–7.37
(m, 1H), 10.82 (s, 1H). Mass calculated for
C20H24N2O4S2: 420, found: 421.0 (M+H)+.
7.2.8.21. Isopropyl 4,5,6,7,8,9-hexahydro-2-(3-methyl-
3-phenylureido)cycloocta[b]thiophene-3-carboxylate (8u).
Procedure B was used with 5c and N-methylanilinium tri-
fluoroacetate. Chromatographic eluent: 10% hexanes in
CH2Cl2. Yield: 19% (white powder). 1H NMR
(400 MHz, CDCl3) d 1.17–1.19 (d, J = 6.3 Hz, 6H),
1.21–1.24 (m, 2H), 1.41–1.44 (m, 2H), 1.60–1.67 (m,
4H), 2.66–2.69 (m, 2H), 2.79–2.82 (m, 2H), 3.37 (s, 3H),
4.97–5.00 (m, 1H), 7.31 (d, J = 6.3 Hz, 2H), 7.39 (t,
J = 6.3 Hz, 1H), 7.46–7.50 (t, 2H), 10.64 (s, 1H). Mass cal-
culated for C22H28N2O3S: 400, found: 401.1 (M+H)+.
7.2.8.22. Isopropyl 4,5,6,7,8,9-hexahydro-2-(piperi-
dine-1-carboxamido)cycloocta[b]thiophene-3-carboxylate
(8v). Procedure B was used with 5c and piperidine.
Chromatographic eluent: 10% hexanes in CH2Cl2.
Yield: 35% (white powder). 1H NMR (400 MHz,
CDCl3) d 1.19–1.25 (m, 2H), 1.38 (d, J = 6.3 Hz, 6H),
1.40–1.45 (m, 2H), 1.57–1.68 (m, 10H), 2.65–2.68 (m,
2H), 2.82–2.88 (m, 2H), 3.47 (s, 4H), 5.17–5.23 (m,
1H), 11.18 (s, 1H). Mass calculated for C20H30N2O3S:
378, found: 379.2 (M+H)+.
7.2.9. Benzyl 3-(isopropoxycarbonyl)-4,5,6,7,8,9-hexahy-
drocycloocta[b]thiophen-2-ylcarbamate (9). To a solution
of 5c (100 mg, 0.375 mmol) in pyridine (0.8 mL) was add-
ed benzylchloroformate (120 lL, 0.86 mmol). The mix-
ture was heated in a pressure tube at 65 ꢁC for 18 h,
after which the reaction mixture was brought to room
temperature and concentrated under reduced pressure.
The crude material was purified by SiO2 chromatography,
using 10% hexanes in CH2Cl2 as the eluent to furnish
141 mg of product 9 (0.35 mmol, 47% yield) as a colorless
1
7.2.8.23. Isopropyl 4,5,6,7,8,9-hexahydro-2-(morpho-
line-4-carboxamido)cycloocta[b]thiophene-3-carboxylate
(8w). Procedure B was used with 5c and morpholine.
Chromatographic eluent: 10% hexanes in CH2Cl2.
Yield: 32% (white powder). 1H NMR (400 MHz,
CDCl3) d 1.25–1.27 (m, 2H), 1.35 (d, J = 6.3 Hz, 6H),
1.43–1.46 (m, 2H), 1.59–1.64 (m, 4H), 2.69–2.72 (m,
2H), 2.86–2.89 (m, 2H), 3.52–3.55 (m, 4H), 3.73–3.76
(m, 4H), 5.18–5.21 (m, 1H), 11.28 (s, 1H). Mass calculat-
ed for C19H28N2O4S: 380, found: 381.1 (M+H)+.
oil. H NMR (400 MHz, CDCl3) d 1.24–1.32 (m, 8H),
1.44 (m, 2H), 1.60 (m, 4H), 2.70 (m, 2H), 2.86 (m, 2H),
5.11–5.22 (m, 3H), 7.21–7.42 (m, 5H), 10.63 (s, 1H). Mass
calculated for C22H27NO4S: 401, found: 402.1 (M+H)+.
7.2.10. 1-(3-(isopropoxycarbonyl)-4,5,6,7,8,9-hexahydro-
cycloocta[b]thiophen-2-yl)-3-phenyl sulfuric diamide (10).
To a solution of 5c (257 mg, 0.96 mmol) in CH2Cl2 were
added phenylsulfamoyl chloride (460 mg, 2.4 mmol) and
triethylamine (0.33 mL, 2.4 mmol). The mixture was re-
fluxed for 18 h, after which the reaction mixture was
brought to room temperature and concentrated under
reduced pressure. The crude material was purified by
SiO2 chromatography, using 50/47/3 CH2Cl2–hexanes–
methanol as the eluent to furnish 114 mg of product
7.2.8.24. ( )-Tetrahydrofuran-3-yl 4,5,6,7,8,9-hexahy-
dro-2-(3-(thiophen-2-yl)ureido)cycloocta[b]thiophene-3-
carboxylate (8x). Procedure B was used, but a mixture of
desired product and product of the addition of two iso-
cyanates was obtained. Purification by flash chromato-
graphy on silica gel using a gradient of 0–5% EtOAc
1
10 (0.27 mmol, 36% yield) as a colorless oil. H NMR
(400 MHz, CDCl3) d 1.24–1.31 (m, 8H), 1.43–1.48 (m,

5828
F. Arhin et al. / Bioorg. Med. Chem. 14 (2006) 5812–5832
2H), 1.55–1.64 (m, 4H), 2.69–2.72 (m, 2H), 2.78–2.81
(m, 2H), 4.99–5.06 (m, 1H), 6.93 (s, 1H), 7.06–7.10 (m,
3H), 7.22–7.26 (m, 2H), 10.49 (s, 1H). Mass calculated
for C20H26N2O4S2: 422, found: 423.1 (M+H)+.
(dt, J = 7.2, 1.0 Hz, 3H), 1.29–1.32 (m, 6H), 1.74–1.84
(m, 1H), 2.14–2.20 (m, 1H), 2.60–2.79 (m, 4H), 2.90–
2.97 (m, 1H), 4.16 (q, J = 7.2 Hz, 2H), 5.15 (d sept,
J = 6.3, 1.0 Hz, 1H), 5.95 (br s, 2H).
7.2.11. Substituted isopropyl 2-aminothiophene-3-carb-
oxylates (11). To a solution of ketone (3.96 mmol) in
solvent (4 mL) were added sulfur (150 mg, 4.68 mmol
of elemental S), isopropyl cyanoacetate (500 lL,
3.96 mmol), and base (5.54 mmol). The mixture was stir-
red at the indicated temperature for 18 h. The mixture
was concentrated under reduced pressure and purified
as indicated.
7.2.11.5. Isopropyl 2-amino-5,7-dihydro-4H-thie-
no[2,3-c]pyran-3-carboxylate (11e). Prepared from tetra-
hydropyran-4-one using morpholine in isopropanol at
70 ꢁC. The product was purified by flash chromatogra-
phy on silica gel using a gradient of 20–25% EtOAc in
hexanes containing 1% Et3N as eluent. Yield: 82%
1
(white solid). H NMR (400 MHz, CDCl3) d 1.31 (d,
J = 6.3 Hz, 6H), 2.82 (m, 2H), 3.91 (t, J = 5.7 Hz, 2H),
4.56 (t, J = 2.1 Hz, 2H), 5.16 (sept, J = 6.3 Hz, 1H),
5.98 (br s, 2H).
7.2.11.1. Isopropyl 2-amino-5,6,7,8-tetrahydro-4H-
cyclohepta[b]thiophene-3-carboxylate (11a). Prepared
from cycloheptanone using morpholine in isopropanol
at 70 ꢁC. The product was purified by flash chroma-
tography on silica gel, using 50% CH2Cl2 in hexanes
as eluent. Yield: 78% (yellow oil). 1H NMR
(400 MHz, CDCl3) d 1.33 (d, J = 6.3 Hz, 6H), 1.58–
1.67 (m, 4H), 1.78–1.84 (m, 2H), 2.56–2.59 (m, 2H),
2.96–2.98 (m, 2H), 5.18 (sept, J = 6.1 Hz, 1H), 5.74
(br s, 2H).
7.2.11.6. Isopropyl 2-amino-8-methyl-8-aza-thieno[2,3-
c]bicyclo[3.2.1]octane-3-carboxylate (11f). Prepared from
tropinone using morpholine in DMF at room tempera-
ture. The residue slowly crystallized, and the resulting
crystals were washed with cold CH2Cl2. Yield: 17%
1
(light brown solid). H NMR (400 MHz, DMSO-d6) d
1.25–1.28 (m, 6H), 1.77 (m, 1H), 2.01 (m, 1H), 2.28
(m, 2H), 2.61 (2 s, 3H), 3.06 (m, 1H), 3.78 (m, 1H),
3.92 (br s, 1H), 4.45 (br m, 1H), 5.00 (sept, J = 6.2 Hz,
1H), 7.42 (s, 2H).
7.2.11.2. Isopropyl 2-amino-5,6,7,8,9,10-hexahydro-
4H-cyclonona[b]thiophene-3-carboxylate (11b). Prepared
from cyclononanone using morpholine in isopropanol
at 70 ꢁC. The product was purified by flash chromatog-
raphy on silica gel, using a gradient of 20–50% CH2Cl2
in hexanes as eluent. This was obtained as mixture of
product and cyclononanone, 2:1 ratio, which was used
7.2.11.7. Isopropyl 2-amino-4-cyclohexylthiophene-3-
carboxylate (11g). Prepared from cyclohexyl methyl ke-
tone using diethylamine in pyridine at 80 ꢁC. The prod-
uct was purified by flash chromatography on silica gel
using a gradient of 5% EtOAc in hexanes as eluent.
1
1
directly for the next step. H NMR (400 MHz, CDCl3)
Yield: 24%. H NMR (400 MHz, CDCl3) d 1.16–1.42
d 1.26–1.30 (m, 2H), 1.32 (d, J = 6.2, 6H), 1.37–1.41
(m, 2H), 1.44–1.48 (m, 4H), 1.54–1.61 (m, 4H), 2.62–
2.65 (m, 2H), 2.79–2.82 (m, 2H), 5.18 (sept, J = 6.3,
1H), 5.96 (br s, 2H).
(m, 5H), 1.33 (d, J = 6.3 Hz, 6H), 1.72–1.97 (m, 5H),
3.05 (m, 1H), 5.19 (sept, J = 6.3 Hz, 1H), 5.85 (s, 1H),
6.10 (br s, 2H).
7.2.11.8. Isopropyl 2-amino-4-cyclopentylthiophene-3-
carboxylate (11h). Prepared from cyclopentyl methyl ke-
tone using diethylamine in pyridine at 80 ꢁC. The prod-
uct was purified by flash chromatography on silica gel
using 8% EtOAc in hexanes as eluent. Yield: 28%.
1H NMR (400 MHz, CDCl3) d 1.33 (d, J = 6.3 Hz,
6H), 1.43–1.53 (m, 2H), 1.58–1.75 (m, 4H), 2.04 (m,
2H), 3.43 (quintet, J = 7.43 1H), 5.20 (sept, J = 6.3 Hz,
1H), 5.89 (s, 1H), 6.05 (br s, 2H).
7.2.11.3. Isopropyl 2-amino-4,5,6,7,8,9,10,11-octahy-
drocyclodeca[b]thiophene-3-carboxylate (11c). Prepared
from cyclodecanone using morpholine in isopropanol
at 90 ꢁC for four days. The product was purified by flash
chromatography on silica gel, using 70% CH2Cl2 in hex-
anes as eluent, followed by another flash chromatogra-
phy on silica gel, using 50% CH2Cl2 in hexanes as
eluent, providing a mixture of desired compound and
starting cyclodecanone, the latter was removed on Ku-
gelrohr apparatus (80 ꢁC, hi-vacuum), and pure 11c
was obtained as a dark yellow oil (207 mg, 21% yield).
1H NMR (400 MHz, CDCl3) d 1.12–1.17 (m, 2H), 1.32
(d, J = 6.3 Hz, 6H), 1.41–1.44 (m, 4H), 1.57 (br s, 2H),
1.62–1.72 (m, 4H), 2.77–2.84 (m, 4H), 5.18 (sept,
J = 6.3 Hz, 1H), 6.00 (br s, 2H).
7.2.11.9. Isopropyl 2-amino-4-butyl-5-propylthioph-
ene-3-carboxylate (11i). Prepared from nonanone using
morpholine in isopropanol at 70 ꢁC. The product was
purified by flash chromatography on silica gel using a
gradient of 20–25% EtOAc in hexanes containing 1%
Et3N as eluent. Yield: 45% (yellow oil). 1H NMR
(400 MHz, CDCl3) d 0.92 (t, J = 7.0 Hz, 3H), 0.95 (t,
J = 7.4 Hz, 3H), 1.32 (d, J = 6.4 Hz, 6H), 1.32–1.58
(m, 6H), 2.52 (t, J = 7.6 Hz, 2H), 2.63 (t, J = 7.8 Hz,
2H), 5.20 (sept, J = 6.2 Hz, 1H), 5.96 (br s, 2H).
7.2.11.4. 6-Ethyl 3-isopropyl 2-amino-4,5,6,7-tetra-
hydrobenzo[b]thiophene-3,6-dicarboxylate (11d). Pre-
pared from ethyl 4-oxocyclohexane carboxylate using
morpholine in DMF at 70 ꢁC. Crude mixture was dilut-
ed with EtOAc, washed with 2· H2O, 2· brine, dried
(Na2SO4), filtered, and concentrated. The product was
purified by flash chromatography on silica gel using a
gradient of 80–100% CH2Cl2 in hexanes as eluent. Yield:
7.2.11.10. 2-Ethyl 4-isopropyl 5-amino-3-propylthioph-
ene-2,4-dicarboxylate (11j). Prepared from ethyl 3-oxo-
hexanoate using morpholine in isopropanol at 70 ꢁC.
The product was purified by flash chromatography on
silica gel using a gradient of 20–25% EtOAc in hexanes
1
89% (white solid). H NMR (400 MHz, CDCl3) d 1.27

F. Arhin et al. / Bioorg. Med. Chem. 14 (2006) 5812–5832
5829
containing 1% Et3N as eluent. Yield: 41% (yellow oil).
1H NMR (400 MHz, CDCl3) d 0.98 (t, J = 7.2 Hz,
3H), 1.31 (t, J = 7.2 Hz, 3H), 1.33 (d, J = 6.4 Hz, 6H),
1.48–1.57 (m, 2H), 3.19 (m, 2H), 4.24 (q, J = 7.1 Hz,
2H), 5.18 (sept, J = 6.3 Hz, 1H), 5.96 (br s, 2H).
2H), 1.34 (d, J = 6.3 Hz, 6H), 1.44–1.47 (m, 4H), 1.61–
1.67 (m, 4H), 2.73–2.76 (m, 2H), 2.85–2.88 (m, 2H),
5.19 (sept, J = 6.1 Hz, 1H), 6.67 (s, 1H), 7.10–7.14 (m,
1H), 7.31–7.36 (m, 2H), 7.43–7.45 (m, 2H), 11.08 (s,
1H). Mass calculated for C22H28N2O3S: 400, found:
401.1 (M+H)+.
7.2.11.11. 2-Ethyl 4-isopropyl 5-amino-3-methylthio-
phene-2,4-dicarboxylate (11k). Prepared from ethyl ace-
toacetate using morpholine in isopropanol at 70 ꢁC.
The product was purified by flash chromatography on
silica gel using 20% EtOAc in hexanes containing 1%
Et3N as eluent. Yield: 71% (white solid). 1H NMR
(400 MHz, CDCl3) d 1.31 (t, J = 7.2 Hz, 3H) overlap-
ping with 1.33 (d, J = 6.0 Hz, 6H), 2.68 (s, 3H), 4.24
(q, J = 7.1 Hz, 2H), 5.18 (sept, J = 6.3 Hz, 1H), 6.59
(br s, 2H).
7.2.12.3. Isopropyl 4,5,6,7,8,9,10,11-octahydro-2-(3-
phenylureido)cyclodeca[b]thiophene-3-carboxylate (12c).
Procedure C. Heated at 80 ꢁC during 72 h. The crude
product was purified by flash chromatography on silica
gel using CH2Cl2 as eluent, then reverse phase chroma-
tography on Biotage (C18, 12+M), using a gradient of
MeCN in H2O. Yield: 77%. 1H NMR (400 MHz,
CDCl3) d 1.07–1.13 (m, 2H), 1.30–1.36 (m, 2H), 1.34
(dd, J = 6.3 Hz, 0.8 Hz, 6H), 1.43–1.45 (m, 4H), 1.62–
1.69 (m, 2H), 1.77–1.84 (m, 2H), 2.87–2.90 (m, 4H),
5.18 (d sept, J = 6.3 Hz, 0.7 Hz, 1H), 6.85 (s, 1H),
7.10–7.15 (m, 1H), 7.31–7.36 (m, 2H), 7.44–7.47 (m,
2H), 11.14 (s, 1H). Mass calculated for C23H30N2O3S:
414, found: 415.2 (M+H)+.
7.2.12. Substituted isopropyl 2-(phenylureido)thiophene-
3-carboxylates (12a–k). Three procedures were used:
Procedure A. To a solution of isopropyl 2-aminothioph-
ene-3-carboxylate (2.98 mmol) in pyridine (6 mL) was
added phenylisocyanate (747 lL, 6.85 mmol). The mix-
ture was heated in a pressure tube at 65 ꢁC for 18 h, after
which the reaction mixture was brought to room
temperature and concentrated under reduced pressure.
7.2.12.4. 6-Ethyl 3-isopropyl 4,5,6,7-tetrahydro-2-(3-
phenylureido)benzo[b]thiophene-3,6-dicarboxylate (12d).
Procedure B. The crude product was purified by flash
chromatography on silica gel using a gradient of 80–
100% CH2Cl2 in hexanes as eluent, then another flash
chromatography on silica gel using a gradient of 0–5%
Et2O in CH2Cl2/hexanes (1:1) as eluent. Yield: 34%.
1H NMR (400 MHz, CDCl3) d 1.28 (t, J = 7.2 Hz,
3H), 1.32 (dd, J = 6.2, 4.2 Hz, 6H), 1.80–1.88 (m, 1H),
2.18–2.23 (m, 1H), 2.68–2.75 (m, 2H), 2.86–2.89 (m,
2H), 2.94–3.00 (m, 1H), 4.18 (q, J = 7.2 Hz, 2H), 5.15
(sept, J = 6.2 Hz, 1H), 6.88 (s, 1H), 7.11–7.15 (m, 1H),
7.32–7.36 (m, 2H), 7.42–7.46 (m, 2H), 10.92 (s, 1H).
Mass calculated for C22H26N2O5S: 430, found: 431.1
(M+H)+.
Procedure B. To a solution of isopropyl 2-aminothioph-
ene-3-carboxylate (100 mg, 0.29 mmol) in CHCl3
(1.5 mL) were added phenylisocyanate (63 lL,
0.58 mmol) and a catalytic amount of DMAP. The mix-
ture was heated in a pressure tube at 70 ꢁC for 18 h, after
which the reaction mixture was brought to room tem-
perature and concentrated under reduced pressure.
The crude material was purified by flash chromatogra-
phy on silica gel.
Procedure C. To a solution of isopropyl 2-aminothioph-
ene-3-carboxylate (470 mg, 1.76 mmol) in CHCl3 (5 mL)
were added phenylisocyanate (0.38 mL, 3.52 mmol) and
triethylamine (0.29 mL, 2.11 mmol), and the mixture
was heated at 70 ꢁC for 5 h, after which the reaction
mixture was brought to room temperature and concen-
trated under reduced pressure
7.2.12.5. Isopropyl 5,7-dihydro-2-(3-phenylureido)-4H-
thieno[2,3-c]pyran-3-carboxylate (12e). Procedure A. The
crude product was purified by flash chromatography on
silica gel using a gradient of 50–100% CH2Cl2 in hexanes
as eluent, followed by prep HPLC on XTerra PrepRP18
column (gradient of 20–90% MeCN in water containing
0.005% TFA, over 25 min). Yield: 15% (white solid). 1H
NMR (400 MHz, CDCl3) d 1.33 (d, J = 6.3, 6H), 2.86 (t,
J = 5.6 Hz, 2H), 3.94 (t, J = 5.6 Hz, 2H), 4.68 (t,
J = 1.8 Hz, 2H), 5.16 (sept, J = 6.3 Hz, 1H), 6.95 (br s,
1H), 7.14 (t, J = 7.4 Hz, 1H), 7.35 (t, J = 8.0 Hz, 2H),
7.44 (d, J = 7.5 Hz, 2H), 10.87 (s, 1H). Mass calculated
for C18H20N2O4S: 360, found: 361.0 (M+H)+.
7.2.12.1. Isopropyl 5,6,7,8-tetrahydro-2-(3-phenylure-
ido)-4H-cyclohepta[b]thiophene-3-carboxylate (12a). Pro-
cedure A. The product was purified by flash
chromatography on silica gel, using a gradient of 50–
80% CH2Cl2 in hexanes as eluent. Yield: 66% (white
foam). 1H NMR (400 MHz, CDCl3) d 1.33 (d,
J = 6.3 Hz, 6H), 1.60–1.69 (m, 4H), 1.81–1.86 (m, 2H),
2.68–2.71 (m, 2H), 2.99–3.02 (m, 2H), 5.16 (sept,
J = 6.2 Hz, 1H), 6.72 (s, 1H), 7.10–7.14 (m, 1H), 7.32–
7.36 (m, 2H), 7.42–7.44 (m, 2H), 10.83 (s, 1H). Mass cal-
culated for C20H24N2O3S: 372, found: 373.1 (M+H)+.
7.2.12.6. Isopropyl 8-methyl-8-aza-2-(3-phenylure-
ido)thieno[2,3-c]bicyclo[3.2.1]octane-3-carboxylate, TFA
salt (12f). Procedure A. The crude product was purified
by flash chromatography on silica gel using a gradient of
50–100% CH2Cl2 in hexanes as eluent, followed by prep
HPLC on XTerra PrepRP18 column (gradient of 20–
90% MeCN in water containing 0.005% TFA, over
7.2.12.2. Isopropyl 5,6,7,8,9,10-hexahydro-2-(3-phenyl-
ureido)-4H-cyclonona[b]thiophene-3-carboxylate (12b).
Procedure A. The crude product was purified by flash
chromatography on silica gel, using a gradient of 50–
60% CH2Cl2 in hexanes as eluent. Yield: 45% (white
1
25 min). Yield: 27% (TFA salt, fluffy white sold). H
NMR (400 MHz, DMSO-d6) d 1.35–1.40 (m, 6H),
1.98–2.03 (m, 1H), 2.31–2.58 (m, 3H), 2.85 and 2.90
(2s, 3H), 3.07 (dd, J = 7.7 Hz, 18.0, 1H), 3.47 (dd,
1
foam). H NMR (400 MHz, CDCl3) d 1.23–1.28 (m,

5830
F. Arhin et al. / Bioorg. Med. Chem. 14 (2006) 5812–5832
J = 4.9 Hz, 18.0, 1H), 4.16–4.22 (m, 1H), 4.75–4.82 (m,
1H), 5.20–5.26 (m, 1H), 7.06 (t, J = 7.4 Hz, 1H), 7.30
(t, J = 7.2 Hz, 2H), 7.47 (d, J = 8.6 Hz, 2H), 9.92 (s,
1H), 10.83 and 10.87 (2s, 1H). Mass calculated for
C21H25N3O3S: 399, found: 400.2 (M+H)+.
3H), 4.28 (q, J = 7.2 Hz, 2H), 5.17 (sept, J = 6.3 Hz,
1H), 7.15 (t, J = 7.4 Hz, 1H), 7.35 (t, J = 8.0 Hz, 2H),
7.47 (d, J = 8.0 Hz, 2H), 11.25 (s, 1H). Mass calculated
for C19H22N2O5S: 390, found: 391.0 (M+H)+.
7.3. Biology
7.2.12.7. Isopropyl 4-cyclohexyl-2-(3-phenylureido)thio-
phene-3-carboxylate (12g). Procedure C. The crude
product was purified by flash chromatography on silica
gel using 8% EtOAc in hexanes as eluent. Yield: 75%. 1H
NMR (400 MHz, CDCl3) d 1.21–1.41 (m, 5H), 1.33 (d,
J = 6.3 Hz, 6H), 1.72–1.97 (m, 5H), 3.05 (m, 1H), 5.19
(sept, J = 6.3 Hz, 1H), 6.32 (s, 1H), 7.04 (s, 1H), 7.12
(t, J = 7.23 Hz, 1H), 7.33 (t, J = 8.21 Hz, 2H), 7.45 (d,
J = 8.41 Hz, 2H), 11.11 (s, 1H). Mass calculated for
C21H26N2O3S: 386, found: 387.1 (M+H)+.
7.3.1. Determination of inhibitory activity toward the
RNA polymerase enzyme. In vitro transcription assays
used 12.5 nM of S. aureus sigma factor and 12.5 nM
of S. aureus core RNA polymerase in a total volume
of 25 lL containing 40 mM Tris–acetate, pH 7.9,
100 mM NaCl, 5 mM MgCl2, 1 mM DTT, 0.1 mg/mL
BSA, 150 lM each of ATP, GTP, CTP, 30 lM UTP,
200,000–300,000 cpm of a-[32P] UTP (3000 Ci/mmol),
1 U RNasin (Promega), and 40 ng of pTMSM template
DNA (a vector derived by modification of pTOO2119).
To measure 50% inhibitory concentrations (IC50s), com-
pounds were prepared in serial dilution typically ranging
from 1 nM to 100 lM and added to the reaction mix-
ture. The final concentration of DMSO in the reaction
mixture was 2%. Rifampicin was used as a positive con-
trol and DMSO alone was used as a negative control.
The reaction mixture was incubated for 1 h at 37 ꢁC in
a 96-well PCR-plate (BD-FalconTM). Samples were trans-
ferred to a 96-well MultiscreenTM GF-B plate (Millipore)
and subjected to a 10% TCA precipitation step for 1 h at
4 ꢁC in the presence of 10 lg of single strand DNA as a
carrier in a total volume of 180 lL. After filtration, the
wells were washed three times (225 lL per wash) with
10% TCA, two times (225 lL per wash) with 95% etha-
nol. Liquid scintillation cocktail (75 lL) was then added
and the plates were counted for 15 s in a TriluxTM
Microbeta counter (Perkin-Elmer-Packard).
7.2.12.8.
Isopropyl
4-cyclopentyl-2-(3-phenylure-
ido)thiophene-3-carboxylate (12h). Procedure C. The
crude product was purified by flash chromatography
on silica gel using 5% EtOAc in hexanes as eluent. Yield:
75%. 1H NMR (400 MHz, CDCl3) d 1.33 (d, J = 6.3 Hz,
6H), 1.49–1.76 (m, 6H), 2.0 (m, 2H), 3.47 (quint,
J = 7.43 1H), 5.17 (sept, J = 6.3 Hz, 1H), 6.37 (s, 1H),
7.04 (br s, 1H), 7.12 (t, J = 7.23 Hz, 1H), 7.33 (t,
J = 8.41 Hz, 2H), 7.45 (d, J = 8.41 Hz, 2H), 11.10 (s,
1H). Mass calculated for C20H24N2O3S: 372, found:
373.1 (M+H)+.
7.2.12.9. Isopropyl 4-butyl-2-(3-phenylureido)-5-pro-
pylthiophene-3-carboxylate (12i). Procedure A. The
crude product was purified by flash chromatography
on silica gel using a gradient of 50–80% CH2Cl2 in hex-
anes as eluent. Yield: 72% (light yellow oil which slowly
solidified). 1H NMR (400 MHz, CDCl3) d 0.93 (t,
J = 7.1 Hz, 3H), 0.96 (t, J = 7.3 Hz, 3H), 1.33 (d,
J = 6.3 Hz, 6H), 1.35–1.49 (m, 4H), 1.62 (q,
J = 7.5 Hz, 2H), 2.60 (t, J = 7.6 Hz, 2H), 2.68 (dd,
J = 6.8 Hz, 8.6, 2H), 5.19 (sept, J = 6.3 Hz, 1H), 7.03
(br s, 1H), 7.11 (t, J = 7.4 Hz, 1H), 7.33 (t, J = 8.0 Hz,
2H), 7.45 (d, J = 7.5 Hz, 2H), 11.06 (s, 1H). Mass calcu-
lated for C22H30N2O3: 402, found: 403.2 (M+H)+.
7.3.2. Determination of cell based activities. The antimi-
crobial activity of the compounds was tested against S.
aureus strains RN4220 and ATCC 13709. Minimum
inhibitory concentration (MIC) testing was performed
by the microdilution method according to the guidelines
set by the Clinical and Laboratory Standards Institute
(formerly the National Committee for Clinical Labora-
tory Standards).20
7.2.12.10. 2-Ethyl 4-isopropyl 5-(3-phenylureido)-3-
propylthiophene-2,4-dicarboxylate (12j). Procedure A.
The crude product was purified by flash chromatogra-
phy on silica gel using 15% EtOAc in hexanes as eluent.
Yield: 38% (light yellow gum). 1H NMR (400 MHz,
CDCl3) d 0.98 (t, J = 7.3 Hz, 3H), 1.32 (t, J = 7.1 Hz,
3H), 1.33 (d, J = 6.3 Hz, 6H), 1.50–1.60 (m, 2H), 3.24
(m, 2H), 4.27 (q, J = 7.1 Hz, 2H), 5.17 (sept,
J = 6.3 Hz, 1H), 7.14 (t, J = 7.4 Hz, 1H), 7.34 (t,
J = 8.0 Hz, 2H), 7.43 (br s, 1H), 7.47 (d, J = 7.6 Hz,
2H), 11.31 (s, 1H). Mass calculated for C21H26N2O5S:
418, found: 419.1 (M+H)+.
The cytotoxicities of compounds 1 and 6c were deter-
mined by the MTS and the ATP assays as reported.
The mutagenicity of compound 6c against S. typhimuri-
um was determined as described.21
For resistance frequency determination, overnight cul-
tures of S. aureus strains RN4220 and ATCC 13709 in
TSB were diluted or concentrated appropriately in saline
and plated on TSA without any compound or containing
compound 6c at two- and fourfold the MIC. Plates were
incubated at 37 ꢁC for 48 h. Mutation frequencies were
calculated as the ratio of the number of resistant colonies
at 48 h to the number of CFU inoculated. To purify,
selected colonies were streaked once on TSA plates con-
taining the selected concentration of compound.
7.2.12.11. 2-Ethyl 4-isopropyl 5-(3-phenylureido)-3-
methylthiophene-2,4-dicarboxylate (12k). Procedure A.
The crude product was purified by flash chromatogra-
phy on silica gel using a gradient of 50% CH2Cl2 in hex-
anes to neat CH2Cl2 as eluent. Yield: 49% (light yellow
gum). 1H NMR (400 MHz, CDCl3) d 1.33 (t, J = 7.2 Hz,
3H) overlapping with 1.35 (d, J = 6.0 Hz, 6H), 2.73 (s,
Time-kill curves were generated by growing S. aureus
strains RN4220 and ATCC 13709 to mid-logarithmic
phase at 37 ꢁC with vigorous shaking. Compound 6c

F. Arhin et al. / Bioorg. Med. Chem. 14 (2006) 5812–5832
5831
was added at the indicated folds over MIC and the incu-
bation was continued. To assess the number of surviving
bacteria, samples were taken immediately prior to drug
exposure and at the indicated time points thereafter.
Samples were diluted in saline and were plated onto cat-
ion-adjusted Mueller–Hinton agar.
7.3.4.3. Determination of 6c in plasma. Female CD-1
mice were administered with 6c intravenously (a single bo-
lus dose of 25 mg/kg of body weight), or orally (1 dose of
50 mg/kg of body weight). Animals were sacrificed by
CO2 inhalation at specified time points after dosing.
Blood samples were collected by cardiac puncture and
transferred in BD vacutainer tubes (green cap) for plasma
isolation. The plasma components were obtained by cen-
trifugation of these samples and they were stored at
ꢀ80 ꢁC until analysis. Plasma samples (100 lL) were
mixed with 900 lL of acetonitrile. The mixture was vor-
texed for 30 min and centrifuged for 15 min at 10,000g.
The supernatant was evaporated to dryness under a nitro-
gen stream and the dried residue was reconstituted in
200 lL water–acetonitrile 1/4. The amount of compound
6c was evaluated from a 8-point calibration curve by
injecting 20 lL of the reconstituted sample into a LC ana-
lyzer (Agilent 1100 LC). The analysis was done on a Zor-
baxTM SB-C18 column (2.1· 30 mm, 3.5 lL) using
ammonium acetate (10 mM, pH 6.8) and acetonitrile as
eluents, in isocratic conditions (65% acetonitrile, run time
8 min) at a flow rate of 0.3 mL/min. The diode array
detector was set at 254 nm.
7.3.3. Effect of 6c on macromolecular synthesis. The syn-
thesis of DNA, RNA, and proteins was evaluated by
the incubation of radiolabeled macromolecule precur-
sors into bacteria followed by trichloroacetic acid
(TCA) precipitation of radiolabeled macromolecules.
S. aureus RN4220 was grown overnight at 37 ꢁC in
CAMHB. The overnight culture was diluted 1:100 in
fresh CAMHB and grown to OD565 = 0.3. Broth was
removed from the bacteria by centrifugation for
15 min at 3500g. The S. aureus pellet was washed twice
in synthetic medium comprising M6322 supplemented
with 0.5% casamino acids, 3 lg/mL thymine, 6.25 ng/
mL biotin, 3.1 lg/mL sodium pantothenate, and
1.55 lg/mL nicotinic acid. The pellet was resuspended
in the supplemented synthetic medium and diluted to
OD565 = 0.3. Macromolecular synthesis was measured
by pre-incubating cells with different amounts of com-
pound 6c for 5 min, followed by labeling 100 lL of
cells with 10 lL of 3H-thymidine (0.01 mCi/mL),
References and notes
3
10 lL of H-uridine (0.01 mCi/mL), and 4 lL of 35S-
methionine (0.05 mCi/mL) for 10 min at 37 ꢁC. The
labeling reactions were stopped by adding 10 lL of
1.23 mM cold label (thymidine, uridine or methionine)
containing 0.2% sodium azide. The reaction mixtures
were precipitated with 110 lL of cold 10% TCA for
30 min on ice and filtered through a 96-well multi-
screen plate (MAFBNOB10, Millipore Corporation,
Bedford, MA, USA). The samples were washed
sequentially with 200 lL of cold 5% TCA, 1% TCA,
and 95% ethanol, and dried. After addition of 100 lL
of scintillation fluid, the samples were counted in a li-
quid scintillation counter (Trilux 1450TM Microbeta
counter, Perkin-Elmer Biosciences, Turku, Finland).
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