Synthesis and activity of analogues of the isoleucyl tRNA synthetase inhibitor SB-203207

Article abstract of DOI:10.1016/S0968-0896(03)00237-2

Twenty two analogues of SB-203207 have been prepared by total synthesis, and evaluated as inhibitors of a range of tRNA synthetases. Changes to the bicyclic core, removing either the terminal amino substituent or the sulfonyl group from the side chain, and altering either the carbon skeleton or stereochemistry of the isoleucine residue, decreases the potency of inhibition of isoleucyl tRNA synthetase. Substituting the isoleucine residue with other amino acids produces inhibitors of the corresponding synthetases. In particular, a methionine derivative is 50-100 times more potent against methionyl tRNA synthetase than against any of the corresponding isoleucyl, leucyl, valyl, alanyl and prolyl synthetases.

Full text of DOI:10.1016/S0968-0896(03)00237-2

Bioorganic & Medicinal Chemistry 11 (2003) 2687–2694
Synthesis and Activity of Analogues of the Isoleucyl tRNA
Synthetase Inhibitor SB-203207
Curtis F. Crasto,a Andrew K. Forrest,b Tomislav Karoli,a Darren R. March,a
Lucy Mensah,b Peter J. O’Hanlon,b Michael R. Nairn,a Mark D. Oldham,a
Weimin Yue,a Martin G. Banwella,* and Christopher J. Eastona,*
aResearch School of Chemistry, Institute of Advanced Studies, Australian National University, Canberra, ACT 0200, Australia
bGlaxoSmithKline, New Frontiers Science Park, Harlow, CM19 5AW, UK
Received 8 January 2003; revised 31 March 2003; accepted 7 April 2003
Abstract—Twenty two analogues of SB-203207 have been prepared by total synthesis, and evaluated as inhibitors of a range of
tRNA synthetases. Changes to the bicyclic core, removing either the terminal amino substituent or the sulfonyl group from the side
chain, and altering either the carbon skeleton or stereochemistry of the isoleucine residue, decreases the potency of inhibition of
isoleucyl tRNA synthetase. Substituting the isoleucine residue with other amino acids produces inhibitors of the corresponding
synthetases. In particular, a methionine derivative is 50–100 times more potent against methionyl tRNA synthetase than against
any of the corresponding isoleucyl, leucyl, valyl, alanyl and prolyl synthetases.
# 2003 Elsevier Science Ltd. All rights reserved.
Introduction
In connection with a high-throughput screening pro-
gram aimed to identify new anti-infective agents,
SB-203207 (1a) was isolated from Streptomyces NCIMB
40513 and shown to inhibit isoleucyl tRNA synthetase
(IRS) from Staphylococcus aureus Oxford and rat liver,
with IC50 values of 1.7 and <2 nM, respectively.1,2
Since the yield of the inhibitor 1a from natural sources
was insufficient to allow for further testing and the pre-
paration of analogues, we obtained altemicidin (2)
through fermentation and used it to prepare a semi-
synthetic sample of 1a and several other compounds
with the same bicyclic core.3 In parallel studies, we
undertook the total synthesis of a more structurally
diverse but related series of compounds, in order to
establish comprehensive structure–activity relationships.
Accordingly, we recently reported the multi-component
assembly of the bicyclic alcohols (ꢀ)-3-(ꢀ)-5 and the
elaboration of (ꢀ)-3 to the diastereomeric analogues
8a(I) and 8a(II) of SB-203207 (1a).4 We now report the
synthesis of a further 22 analogues of 1a from (ꢀ)-3-
(ꢀ)-5, and the evaluation of the activity of these com-
pounds as inhibitors of a range of amino acyl tRNA
synthetases.
*Corresponding author. Tel.:+61-2-6125-8201; fax:+61-2-6125-8114;
e-mail: easton@rsc.anu.edu.au
0968-0896/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00237-2

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C. F. Crasto et al. / Bioorg. Med. Chem. 11 (2003) 2687–2694
Results and Discussion
The methods used to elaborate the alcohols (ꢀ)-3-(ꢀ)-5
are illustrated in Schemes 1–4. As shown in Scheme 1,
EDCI-mediated coupling of the protected amino acids
6a–i with ethyl 2-sulfamylacetate afforded 7a–i. Base
hydrolysis of these esters gave the corresponding car-
boxylic acids. The acids reacted with oxalyl chloride,
and the product acid chlorides were treated with the
bicyclic alcohol (ꢀ)-3 in the presence of triethylamine
and a catalytic amount of DMAP. Catalytic hydro-
genolysis of the products then gave 8a–i, respectively.
Similar methods were used to prepare 9a–e, 11a–c
(Scheme 2), 12a–c (Scheme 3) and 13a–c (Scheme 4),
except that the ethyl esters of glycine, b-alanine and
g-aminobutyric acid were used instead of ethyl 2-sulfa-
mylacetate to prepare 11a–c, and the alcohols (ꢀ)-4 and
(ꢀ)-5 were used instead of (ꢀ)-3 to prepare 12a–c and
13a–c, respectively. Compounds 8a–i, 9a–e, 11a–c, 12a–
c and 13a–c were each obtained as a ca. 1/1 mixture of
diastereomers, as illustrated. They were isolated as
either the hydrochloride or acetate salts, by freeze-dry-
ing from hydrochloric acid or acetic acid solutions,
respectively. Samples of the individual diastereomers
8a(I) and 8a(II) were also prepared using the separated
enantiomers of 3.
Compounds 8a–i, 9a–e, 11a–c, 12a–c and 13a–c were
evaluated as inhibitors of a range of tRNA synthetases
and the results, along with those obtained earlier for
SB-203207 (1a) and the semi-synthetic analogues 1b and
1c,3 are summarised in Table 1. The isoleucine deriva-
tive SB-203207 (1a) is a potent inhibitor of IRS but less
active against the corresponding leucyl and valyl syn-
thetases (LRS and VRS). Compound 14 is an enzyme-
bound intermediate in the reaction catalysed by IRS
and the other tRNA synthetases involve similar inter-
mediates, differing only in the amino acid side chains. It
is reasonable to assume that 1a inhibits IRS because it
resembles the enzyme’s intermediate 14. One of the rea-
sons for preparing 1b and 1c was because it therefore
seemed likely that replacing the isoleucine residue of
SB-203207 (1a) with, for example, leucine and valine,
would produce the corresponding analogues of the
intermediates of LRS and VRS, which would inhibit
those enzymes. This hypothesis was substantiated
through the selective inhibition of IRS, LRS and VRS
by 1a–c, respectively.3 Our earlier studies showed that
Scheme 2. (i) EDCI; ethyl glycinate, ethyl b-alaninate or ethyl
g-aminobutyrate, DMAP; (ii) NaOH, H2O; (iii) ClCOCOCl, Et3N;
(ꢀ)-3, Et3N, DMAP; (iv) 10% Pd/C, H2.
Scheme 1. (i) EDCI; H2NSO2CH2CO2Et, DMAP; (ii) NaOH, H2O;
(iii) ClCOCOCl, Et3N; (ꢀ)-3, Et3N, DMAP; (iv) 10% Pd/C, H2.

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2689
Table 1. Inhibition of tRNA synthetases by SB-203207 (1a) and
related compounds
Compd Synthetase IC50 values (mM) or percentage inhibition at
100 mM#
IRSa IRSb
LRSa VRSa ARSa MRSa PRSa KRSa
1a
1b
0.014 0.0042 1.55 0.126
0.91 0.068 0.016 0.29
1c
0.037 0.0029 2.3
0.03
6.35
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
10%
8a(I)
8a(II)
8b
8c
8d
8e
8f
8g
8h
8i
9a
9b
9c
9d
9e
11a
11b
11c
12a
12b
12c
13a
13b
13c
3.7
12.4
NI
NI
NI
0.57
13.5
NI
NI
NI
0.42
NI*
2.4
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
55% 11%
1.45 8%
38% 31% 13%
46% 17% 18%
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
17%
10%
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
4.9
NI
30% 68%
65%
Scheme 3. (i) EDCI; H2NSO2CH2CO2Et, DMAP; (ii) NaOH, H2O;
(iii) ClCOCOCl, Et3N; (ꢀ)-4, Et3N, DMAP; (iv) 10% Pd/C, H2.
26% 18%
31% 49%
NI
14%
38% 31% 13%
34% 46%
15% 13%
10% 33%
NI
24
NI
#Determined according to the method reported previously.5
*NI, no inhibition detected.
aFrom Staphylococcus aureus WCUH29.
bFrom rat liver.
their side chain amino acids. Except in the case of 8a,
they were only tested as ca. 1/1 mixtures of diaster-
eomers, and any activity probably derives mostly from
that stereoisomer most closely related to 1a–c and 8a(I).
Compounds 8a–i, 9a–e, 11a–c, 12a–c and 13a–c are
generally less active than 1a–c. The vinylogous ureas
8a–c, which are structurally the most similar to 1a–c,
are more active than the corresponding vinylogous car-
bamates 12a–c and their isomers 13a–c. Compounds
9a–c and 11a–c, which lack the terminal amino sub-
stituent and the sulfonyl group of the side chain of 1a–c,
respectively, show little or no inhibition. Changing
either the carbon framework or the stereochemistry at
either the a- or b-position of the isoleucine residue of 8a
results in the loss of enzyme inhibition with 8d, 8e, 8h
and 8i.
Scheme 4. (i) EDCI; H2NSO2CH2CO2Et, DMAP; (ii) NaOH, H2O;
(iii) ClCOCOCl, Et3N; (ꢀ)-5, Et3N, DMAP; (iv) 10% Pd/C, H2.
The correlation between the amino acid side chain and
the selectivity of tRNA synthetase inhibition is con-
firmed by the present study. Of 8a–c, 12a–c and 13a–c,
those having leucine residues in the side chain (8b, 12b
and 13b) inhibit LRS in preference to either IRS or
VRS. Those containing isoleucine (8a, 12a and 13a) are
generally more potent as inhibitors of IRS than of either
LRS or VRS. The valine derivatives 8c, 12c and 13c
show no significant inhibition of VRS but they are gen-
erally less potent than either 8a, 12a or 13a, or 8b, 12b
or 13b, as inhibitors of IRS and LRS, respectively.
substituting the isoleucine side chain of 1a with leucine
in 1b afforded a more potent inhibitor of LRS and a less
potent inhibitor of either IRS or VRS. The analogous
valine derivative 1c was more potent than either 1a or
1b as an inhibitor of VRS, although it was also a potent
IRS inhibitor.
For this reason the synthetic compounds 8a–i, 9a–e,
11a–c, 12a–c and 13a–c were evaluated as inhibitors of a
range of synthetases, including those corresponding to

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C. F. Crasto et al. / Bioorg. Med. Chem. 11 (2003) 2687–2694
120 A column was used, with the solvents indicated
[A=0.1% AcOH in H2O, B=0.1% AcOH in CH3CN/
water (9/1)], with detection at 254 or 300 nm) and a flow
rate of 3 mL minꢂ1, unless otherwise indicated. Ele-
mental analyses were performed by the Microanalytical
Laboratory, Research School of Chemistry, the Aus-
tralian National University.
The procedures used to prepare compound 8a from 6a
are representative of those used to obtain 8b–i, 9a–e,
11a–c, 12a–c and 13a–c.
The derivatives of lysine 9e, proline 9d and alanine 8f
were prepared and tested to further examine this
hypothesis. Consistent with this concept, the lysine
derivative 9e inhibits lysyl tRNA synthetase (KRS) but
not IRS, LRS or VRS, and the proline derivative 9d
inhibits the proline synthetase (PRS) but not IRS, LRS,
VRS or the alanine synthetase (ARS). The alanine deri-
vative 8f does not inhibit ARS but neither does it show
significant inhibition of IRS, LRS, VRS or PRS. Some-
what unexpectedly, the alanine and proline derivatives
8f and 9d also display inhibition of methionyl tRNA
synthetase (MRS). This implied that the methionine
derivative 8g would be a more potent MRS inhibitor.
When it was made and tested it was found that, of the
synthetic compounds 8a–i, 9a–e, 11a–c, 12a–c and 13a–
c, only 8a(I) is more potent than 8g against the corres-
ponding synthetases, and 8g is more selective. It is at
least 50–100 times more potent against MRS than
against either IRS, LRS, VRS, ARS or PRS.
Ethyl [[(2S,3S)-2-Benzyloxycarbonylamino-3-methyl-1-
oxopentyl]amino]sulfonyl]acetate (7a). To a solution of
DCC or EDCI (3 mmol) in DCM (50 mL) was added
(2S,3S)-N-benzyloxycarbonylisoleucine (6a) (2.9 mmol).
Ethyl 2-sulfamylacetate (3 mmol) and DMAP (8.85
mmol) were then added, and the mixture was heated at
reflux for 24 h. It was then diluted with EtOAc (150
mL), washed with 1 M HCl (3ꢁ50 mL) and brine (50
mL), dried over anhydrous Na2SO4 and concentrated
under reduced pressure. Column chromatography of the
residue on silica, eluting with hexanes/EtOAc/AcOH
(50/50/1, v/v/v), provided the title compound 7a, yield
94%; oil; HPLC Rt 7.7 min (gradient of solvents A/B,
20/80!0/100 over 30 min); 1H NMR (300 MHz,
CDCl3) d 10.11 (s, 1H), 7.36 (m, 5H), 5.47 (d, J=9.5
Hz, 1H), 5.18 and 5.12 (ABq, J=12.0 Hz, 2H), 4.39 (m,
1H), 4.43 and 4.34 (ABq, J=15.5 Hz, 2H), 4.19 (q,
J=7.0 Hz, 2H), 1.92 (m, 1H), 1.51 (m, 1H), 1.25 (t,
J=7.0 Hz, 3H), 1.13 (m, 1H), 0.99 (d, J=7.0 Hz, 3H),
0.90 (t, J=7.5 Hz, 3H); 13C NMR (75 MHz, CDCl3) d
171.8, 162.4, 156.9, 135.7, 128.5, 128.3, 128.0, 67.7, 62.6,
59.7, 56.0, 37.5, 24.2, 15.4, 13.8, 11.2; IR (neat) 3373,
3139, 2968, 2937, 1742, 1693, 1526, 1455, 1358, 1284,
1229, 1135, 1027, 909, 861, 734, 698 cmꢂ1; EI m/z 414
(M+, 1%), 369 (2), 325 (2), 307 (10), 250 (6), 221 (18),
220 (53), 177 (16), 176 (53), 107 (16), 100 (19), 92 (21),
91 (100), 65 (15); EI-HRMS m/z 414.1461. calcd for
C18H26N2O7S (M+) m/z 414.1461; Found: C, 51.54; H,
6.57; N, 6.88. calcd for C18H26N2O7S.0.3H2O: C, 51.49;
H, 6.39; N, 6.67%.
Thus, replacing the isoleucine residue of the IRS inhi-
bitor 8a(I) with methionine in 8g produced a selective
MRS inhibitor. In a similar manner, Creppy et al.,6,7
have reported that whereas ochratoxin A is an inhibitor
of a phenylalanyl tRNA synthetase, substituting the
phenylalanine residue of this compound with valine
afforded a VRS inhibitor. Consequently this approach
of amino acid substitution appears to be useful in the
search for new and selective synthetase inhibitors.
Experimental
Melting points were determined on a Kofler hot-stage
apparatus, equipped with a Reichert microscope, and
are uncorrected. NMR spectra were recorded on either
a Varian Inova 500 or a Varian Gemini 300 spectro-
meter, using the solvent systems specified. IR spectra
were recorded using neat liquids or KBr disks, on either
a Perkin–Elmer 1800 Fourier Transform Infrared spec-
(4aR,7S,7aS)- and (4aS,7R,7aR)-7-[[(2S,3S)-2-Amino-3-
methyl-1-oxopentyl]amino]sulfonyl]acetyloxy]-2,4a,5,6,7,7a-
hexahydro-2-methyl-1H-cyclopenta[c]pyridine-4-carbox-
amide (8a). A solution of the ethyl ester (7a) (0.52
mmol) in ethanol (5 mL) and NaOH (3 mL, 1 M aq)
was stirred for 1 h, before it was adjusted to pH 3 with
HCl (2 M, aq). The mixture was concentrated under
reduced pressure and the resulting suspension was
extracted with ethyl acetate (3ꢁ4 mL). The combined
organic extracts were dried (Na2SO4) and concentrated
under reduced pressure. The residual white solid was
stored in a dessicator overnight over P2O5, and then
used without further purification. The solid was added
to a mixture of triethylamine (0.44 mmol) in DCM,
maintained at 0 ꢃC. Then oxalyl chloride (0.46 mmol)
was added, and that mixture was stirred for 30 min. A
solution of the bicyclic alcohol (ꢀ)-3 (0.25 mmol), trie-
thylamine (0.22 mmol) and DMAP (3 mg) in DMF (1.5
mL) was then added dropwise to the mixture over 1
min. After stirring for 1 h, EtOH (2 mL) was added and
trophotometer or
a
Perkin–Elmer 683 spectro-
photometer. EIMS and HREIMS were recorded on
either a VG Micromass 7070F or an AEI MS-30 spec-
trometer, operating at an ionisation potential of 70 eV.
ESMS were obtained on a VG Quattro II mass spec-
trometer, operating with a cone voltage of 50 V.
LSIMS-HRMS were recorded by the Central Science
Laboratory, University of Hobart, using a Kratos Ana-
lytical Concept ISQ instrument, and m-nitrobenzyl
alcohol as the matrix. HPLC was carried out using
WatersTM 510HPLC pumps and the eluant was mon-
itored using a WatersTM 486 Tunable Absorbance
detector. A YMC-pack ODS-AQ, 250ꢁ10 mm, S-5 mm,

C. F. Crasto et al. / Bioorg. Med. Chem. 11 (2003) 2687–2694
2691
the mixture was allowed to warm to room temperature
before it was concentrated under reduced pressure. The
residue was diluted with water (4 mL) and extracted
with ethyl acetate (5ꢁ2 mL). The organic extracts were
concentrated under reduced pressure and the residue
was chromatographed on silica, eluting with AcOH/
EtOH/EtOAc (0.1/30/70, v/v/v), to yield a ca. 1/1 mix-
ture of diastereomers of the benzyloxycarbonyl deriva-
tive of 8a as a colourless solid (47%), mp 110–113 ꢃC;
HPLC Rt 11.1 min (gradient of solvents A/B, 30/70! 0/
1529, 1387, 1286, 1134, 1074, 845, 665 cmꢂ1; ESMS
(ꢂve) m/z 429 (MꢂH+); LSIMS-HRMS m/z 431.1954.
calcd for C18H31N4O6S (M+H+) m/z 431.1964.
(4aR,7S,7aS)- and (4aS,7R,7aR)-7-[[(S)-2-Amino-3-methyl-
1-oxobutyl]amino]sulfonyl]acetyloxy]-2,4a,5,6,7,7a-hexa-
hydro-2-methyl-1H-cyclopenta[c]pyridine-4-carboxamide
(8c). Isolated by freeze-drying from dilute aqueous
hydrochloric acid, mp 230 ꢃC (dec.); HPLC Rt 10.8 min
(gradient of solvents A/B, 100/0!10/90 over 20 min);
1H NMR (500 MHz, CD3OD) d 7.99 (s, 1H), 5.57 (m,
1H), 4.68 (m, 2H), 3.95 (m, 1H), 3.41 (m, 1H), 3.32 (s,
3H), 3.29 (m, 1H), 2.86 (m, 1H), 2.62 (m, 1H), 2.45 (m,
1H), 2.34 (m, 2H), 1.95 (m, 1H), 1.53 (m, 1H), 1.24 (d,
J=7.0 Hz, 3H), 1.15 and 1.14 (d and d, J=7.0 and 7.0
Hz, total 3H); 13C NMR (125 MHz, CD3OD) d 171.0,
170.0(3), 170.0(0), 163.7, 152.4, 92.6, 79.7, 60.0, 57.7,
45.3, 44.4, 38.6, 33.0, 31.2, 31.1, 30.2, 29.0, 28.9, 19.1,
19.0, 16.9; IR (KBr) 3430, 2982, 2921, 1737, 1627, 1463,
1356, 1282, 1193, 1128 cmꢂ1; ESMS (ꢂve) m/z 415
(MꢂH+); LSIMS-HRMS m/z 417.1800. calcd for
C17H29N4O6S (M+H+) m/z 417.1808.
1
100 over 10 min); H NMR (300 MHz, CD3OD) d 7.42
(m, 6H), 5.49 (m, 1H), 5.17 (m, 2H), 4.57 (m, 2H), 4.18
and 4.17 (d and d, J=7.0 and 7.0 Hz, total 1H), 3.16
(dd, J=5.5, 13.0 Hz, 1H), 3.08 and 3.07 (s and s, total
3H), 2.96 (m, 1H), 2.78 (m, 1H), 2.48 (m, 1H), 2.27 (m,
2H), 1.91 (m, 2H), 1.61 (m, 1H), 1.44 (m, 1H), 1.28 (m,
1H), 1.08 (d, J=6.5 Hz, 3H), 1.00 (t, J=7.0 Hz, 3H); IR
(neat) 3509, 3379, 3055, 2968, 2878, 1722, 1642, 1518,
1395, 1346, 1265, 1139, 1074, 896, 870, 737, 703 cmꢂ1
;
ESMS (+ve) m/z 587 (M+Na+), 603 (M+K+);
Found: C, 55.16; H, 6.47; N, 9.80. calcd for
C26H36N4O8S: C, 55.31; H, 6.43; N, 9.92%.
The benzyloxycarbonyl derivative of 8a was added to a
suspension of 10% Pd/C (5 mg) in MeOH (3 mL), and
the mixture was stirred under an atmosphere of hydro-
gen overnight at room temperature, before it was fil-
tered through Celite and the filtrate was concentrated
under reduced pressure. HPLC of the residue afforded a
ca. 1/1 mixture of diastereomers of the title compound
8a (84%) after freeze-drying from dilute aqueous
hydrochloric acid, mp 192 ꢃC (dec.); HPLC Rt 11.4 min
(gradient of solvents A/B, 100/0! 40/60 over 11 min);
1H NMR (300 MHz, CD3OD) d 7.92 (s, 1H), 5.46 (m,
1H), 4.59 (s, 2H), 3.93 (d, J=3.5 Hz, 1H), 3.33 (m, 1H),
3.24 (s, 3H), 3.20 (m, 1H), 2.78 (m, 1H), 2.52 (m, 1H),
2.24 (m, 2H), 2.06 (m, 1H), 1.85 (m, 1H), 1.48 (m, 2H),
1.23 (m, 1H), 1.12 (d, J=7.0 Hz, 3H), 0.99 (t, J=7.5
Hz, 3H); 13C NMR (75 MHz, CD3OD) d 170.9, 170.1,
170.0, 163.7, 152.5, 92.4, 79.7, 59.7, 57.8, 57.7, 45.4,
44.5, 38.5, 37.7, 37.6, 32.9, 30.1, 29.0, 28.9, 24.8, 24.7,
15.4, 11.8; IR (KBr) 3327, 2968, 1740, 1680, 1625, 1587,
1464, 1416, 1357, 1282, 1192, 1153, 1080, 899, 852, 509
(4aR,7S,7aS)- and (4aS,7R,7aR)-7-[[(2S,3R)-2-Amino-3-
methyl-1-oxopentyl]amino]sulfonyl]acetyloxy]-2,4a,5,6,7,7a-
hexahydro-2-methyl-1H-cyclopenta[c]pyridine-4-carbox-
amide (8d). Isolated by freeze-drying from dilute aqu-
eous hydrochloric acid, mp 190–210 ꢃC (dec.); HPLC Rt
11.9 min (gradient of solvents A/B, 100/0!0/100 over
1
20 min); H NMR (300 MHz, CD3OD) d 8.00 (s, 1H),
5.57 (m, 1H), 4.68 (s, 2H), 4.09 (m, 1H), 3.42 (m, 1H),
3.33 (s, 3H), 3.26 (m, 1H), 2.86 (m, 1H), 2.62 (m, 1H),
2.35 (m, 2H), 2.22 (m, 1H), 1.96 (m, 1H), 1.66 (m, 1H),
1.50 (m, 2H), 1.12 (t, J=7.5 Hz, 3H), 1.09 (d, J=7.0
Hz, 3H); 13C NMR (75 MHz, CD3OD) d 170.9, 170.3,
163.7, 152.4, 92.6, 79.7, 79.6, 58.9, 58.8, 57.6, 45.3, 44.5,
44.4, 38.6, 37.4, 37.3, 33.0, 30.2, 28.9, 27.1, 13.3, 12.0;
IR (KBr) 3410, 2967, 1739, 1679, 1625, 1587, 1463,
1356, 1283, 1158, 852 cmꢂ1; ESMS (ꢂve) m/z 429
(MꢂH+); LSIMS-HRMS m/z 431.1976. calcd for
C18H31N4O6S (M+H+) m/z 431.1964; Found: C, 39.89;
H, 6.51; N, 10.34. calcd for C18H30N4O6S.3HCl: C,
40.04; H, 6.16; N, 10.38%.
cmꢂ1
;
LSIMS-HRMS m/z 431.1980. calcd for
C18H31N4O6S (M+H+) m/z 431.1964; Found: C, 41.60;
H, 7.35; N, 10.63. calcd for C18H30N4O6S.3H2O.HCl:
C, 41.49; H, 7.16; N, 10.75%.
(4aR,7S,7aS)- and (4aS,7R,7aR)-7-[[(2R,3R)-2-Amino-3-
methyl-1-oxopentyl]amino]sulfonyl]acetyloxy]-2,4a,5,6,7,7a-
hexahydro-2-methyl-1H-cyclopenta[c]pyridine-4-carbox-
amide (8e). Isolated by freeze-drying from dilute aqu-
eous hydrochloric acid, mp 193–208 ꢃC (dec.); HPLC
Rt 11.4 min (gradient of solvents A/B, 100/0!0/100
(4aR,7S,7aS)- and (4aS,7R,7aR)-7-[[(S)-2-Amino-4-methyl-
1-oxopentyl]amino]sulfonyl]acetyloxy] - 2,4a,5,6,7,7a - hexa-
hydro-2-methyl-1H-cyclopenta[c]pyridine-4-carboxamide
(8b). Isolated by freeze-drying from acetic acid solu-
tion, mp 159–165 ꢃC; HPLC Rt 11.5 min (gradient of
solvents A/B, 100/0!0/100 over 20 min); 1H NMR
(500 MHz, CD3OD) d 7.42 (s, 1H), 5.48 (m, 1H), 4.31
(m, 2H), 3.72 (m, 1H), 3.26 (dd, J=13.0, 5.5 Hz, 1H),
3.11 (s, 3H), 2.98 (m, 1H), 2.80 (m, 1H), 2.50 (m, 1H),
2.28 (m, 2H), 1.91 (m, 3H), 1.71 (m, 1H), 1.45 (m, 1H),
1.08 (m, 6H); 13C NMR (125 MHz, CD3OD) d 176.8,
174.0, 166.0, 165.9, 146.0, 100.7(0), 100.6(8), 79.3(0),
79.2(7), 57.0, 55.5(2), 55.5(1), 44.9, 43.1, 41.9, 39.3, 34.5,
31.5(9), 31.5(6), 28.8(4), 28.7(9), 25.6, 23.3, 22.0; IR
(KBr) 3608, 3582, 3434, 3361, 3218, 2956, 1728, 1629,
1
over 20 min); H NMR (300 MHz, CD3OD) d 7.88 (s,
1H), 5.46 (m, 1H), 4.58 (s, 2H), 3.87 (d, J=4.5 Hz,
1H), 3.34 (m, 1H), 3.22 (s, 3H), 3.16 (m, 1H), 2.74 (m,
1H), 2.51 (m, 1H), 2.25 (m, 2H), 2.07 (m, 1H), 1.88 (m,
1H), 1.56 (m, 1H), 1.42 (m, 1H), 1.21 (m, 1H), 1.11 (d,
J=7.0 Hz, 3H), 0.99 (t, J=7.5 Hz, 3H); 13C NMR
(75 MHz, CD3OD) d 171.1, 170.1, 163.8, 152.2, 92.9,
79.7, 79.6, 59.9, 57.6, 45.3, 44.4, 38.6, 37.8, 37.7, 33.0,
30.2, 29.0, 24.7(4), 24.6(8), 15.5, 11.9; IR (KBr) 3430,
2975, 2926, 1740, 1626, 1463, 1356, 1281, 1156, 1118
cmꢂ1; ESMS (ꢂve) m/z 429 (MꢂH+); LSIMS-HRMS
m/z 431.1975. calcd for C18H31N4O6S (M+H+) m/z
431.1964.

2692
C. F. Crasto et al. / Bioorg. Med. Chem. 11 (2003) 2687–2694
(4aR,7S,7aS)- and (4aS,7R,7aR)-7-[[(S)-2-Amino-1-oxo-
propyl]amino]sulfonyl]acetyloxy]-2,4a,5,6,7,7a-hexahydro-
2-methyl-1H-cyclopenta[c]pyridine-4-carboxamide (8f).
Isolated by freeze-drying from acetic acid solution, mp
155–161 ꢃC; HPLC Rt 9.9 min (gradient of solvents A/
B, 100/0!60/40 over 13 min); 1H NMR (500 MHz,
CD3OD) d 7.42 (s, 1H), 5.48 (m, 1H), 4.32 (m, 2H), 3.77
(q, J=7.0 Hz, 1H), 3.26 (m, 1H), 3.11 (s, 3H), 2.98 (m,
1H), 2.79 (m, 1H), 2.50 (m, 1H), 2.28 (m, 2H), 1.93 (m,
1H), 1.57 (d, J=7.0 Hz, 3H), 1.45 (m, 1H); 13C NMR
(75 MHz, CD3OD) d 177.2, 174.3, 166.3, 146.3, 101.0,
79.6, 58.5, 53.1, 45.2, 43.4, 39.6, 34.9, 31.9, 29.1, 17.9;
IR (KBr) 3438, 3229, 2929, 1730, 1635, 1522, 1396,
1285, 1120, 765 cmꢂ1; ESMS (ꢂve) m/z 387 (MꢂH+);
LSIMS-HRMS m/z 389.1495. calcd for C15H25N4O6S
(M+H+) m/z 389.1494.
2H), 1.98 (m, 3H), 1.55 (m, 3H), 1.11 (t, J=7.5 Hz, 3H);
13C NMR (75 MHz, CD3OD) d 170.9, 170.6, 163.7,
152.4, 92.5, 79.7, 79.6, 57.6, 57.5, 55.0, 54.9, 45.4, 44.5,
44.4, 38.6, 34.0, 33.9, 33.0, 30.2, 29.0, 18.9, 14.0; IR
(KBr) 3418, 2965, 1738, 1678, 1625, 1464, 1355, 1282,
1152, 865 cmꢂ1; ESMS (ꢂve) m/z 415 (MꢂH+);
LSIMS-HRMS m/z 417.1788. calcd for C17H29N4O6S
(M+H+) m/z 417.1809.
(4aR,7S,7aS)- and (4aS,7R,7aR)-7-[[(S)-3-Methyl-1-
oxopentyl]amino]sulfonyl]acetyloxy] - 2,4a,5,6,7,7a - hexa-
hydro-2-methyl-1H-cyclopenta[c]pyridine-4-carboxamide
(9a). Mp 95–98 ꢃC; HPLC Rt 10.5 min (gradient of sol-
vents A/B, 55/45!0/100 over 20 min); 1H NMR
(300 MHz, CDCl3) d 8.62 (s, 1H), 7.36 (s, 1H), 5.69 (bs,
2H), 5.37 (m, 1H), 4.43 (ABq, J=17.0 Hz, 2H), 3.00 (s,
3H), 2.97 (m, 1H), 2.88 (m, 1H), 2.58 (m, 1H), 2.42 (m,
2H), 2.17 (m, 2H), 1.96 (m, 1H), 1.79 (m, 1H), 1.41 (m,
2H), 1.26 (m, 1H), 0.91 (d, J=7.5 Hz, 3H), 0.90 (t,
J=7.3 Hz, 3H); 13C NMR (75 MHz, CDCl3) d 173.5,
171.4, 162.5, 145.4, 98.4, 78.8, 56.4, 43.8, 43.5, 43.1,
37.8, 33.1, 31.8, 30.3, 29.4, 27.8, 19.2, 11.3; IR (neat)
3482, 3377, 3221, 2961, 2875, 1739, 1642, 1538, 1461,
(4aR,7S,7aS)- and (4aS,7R,7aR)-7-[[(S)-2-Amino-1-oxo-
5-thiahexyl]amino]sulfonyl]acetyloxy]-2,4a,5,6,7,7a-hexa-
hydro-2-methyl-1H-cyclopenta[c]pyridine-4-carboxamide
(8g). Isolated by freeze-drying from acetic acid solu-
tion, mp 135–142 ꢃC; HPLC Rt 11.5 min (gradient of
solvents A/B, 100/0! 0/100 over 20 min); 1H NMR
(300 MHz, CD3OD) d 7.32 (s, 1H), 5.39 (m, 1H), 4.24
(m, 2H), 3.78 and 3.75 (d and d, J=5.5 and 5.5 Hz,
total 1H), 3.16 (dd, J=13.0, 5.5 Hz, 1H), 3.01 (s, 3H),
2.88 (m, 1H), 2.66 (m, 3H), 2.41 (m, 1H), 2.19 (m, 3H),
2.11 (s, 3H), 2.06 (m, 1H), 1.82 (m, 1H), 1.37 (m, 1H);
13C NMR (75 MHz, CD3OD) d 176.1, 174.3, 166.3,
146.3, 101.0, 79.6, 57.8, 56.4, 45.2, 43.5, 39.6, 34.8(3),
34.8(0), 32.4, 31.9, 30.5, 29.2, 29.1, 15.3; IR (KBr) 3447,
2924, 1728, 1636, 1526, 1394, 1290, 1135, 1073, 848
cmꢂ1; ESMS (ꢂve) m/z 447 (MꢂH+); LSIMS-HRMS
m/z 449.1525. calcd for C17H29N4O6S2 (M+H+) m/z
449.1528.
1400, 1344, 1287, 1144, 1119, 1075, 909, 865, 755 cmꢂ1
;
ESMS (ꢂve) m/z 414 (MꢂH+); LSIMS-HRMS m/z
416.1847. calcd for C18H30N3O6S (M+H+) m/z
416.1855; Found: C, 51.62; H, 7.35; N, 10.06. calcd for
C18H29N3O6S: C, 52.03; H, 7.03; N, 10.11%.
(4aR,7S,7aS)- and (4aS,7R,7aR)-7-[[4-Methyl-1-oxo-
pentyl]amino]sulfonyl]acetyloxy]-2,4a,5,6,7,7a-hexahydro-
2-methyl-1H-cyclopenta[c]pyridine-4-carboxamide (9b).
Mp 115–117 ꢃC; HPLC Rt 9.7 min (gradient of solvents
1
A/B, 55/45!0/100 over 20 min); H NMR (300 MHz,
CD3OD) d 7.32 (s, 1H), 5.42 (m, 1H), 4.45 (m, 2H), 3.08
(m, 1H), 3.00 (s, 3H), 2.88 (m, 1H), 2.69 (m, 1H), 2.42
(m, 1H), 2.33 (m, 2H), 2.20 (m, 2H), 1.81 (m, 1H), 1.53
(m, 3H), 1.36 (m, 1H), 0.91 (d, J=6.5 Hz, 6H); 13C
NMR (75 MHz, CD3OD) d 175.4, 173.9, 164.0, 145.9,
100.8, 79.9, 57.0, 44.8, 43.1, 39.2, 35.3, 34.5, 31.6, 28.9,
28.8, 22.6; IR (KBr) 3369, 2952, 2868, 1734, 1638, 1542,
(4aR,7S,7aS)- and (4aS,7R,7aR)-7-[[(S)-2-Amino-1-oxo-
hexyl]amino]sulfonyl]acetyloxy]-2,4a,5,6,7,7a-hexahydro-
2-methyl-1H-cyclopenta[c]pyridine-4-carboxamide (8h).
Isolated by freeze-drying from dilute aqueous hydro-
chloric acid, mp 152–153 ꢃC; HPLC Rt 11.8 min (gra-
1
dient of solvents A/B, 100/0!0/100 over 20 min); H
1467, 1386, 1343, 1287, 1140, 1118, 1074, 873 cmꢂ1
;
NMR (300 MHz, CD3OD) d 7.32 (s, 1H), 5.38 (m, 1H),
4.23 (m, 2H), 3.58 (m, 1H), 3.17 (dd, J=5.5, 13.0 Hz,
1H), 3.01 (s, 3H), 2.88 (m, 1H), 2.70 (m, 1H), 2.40 (m,
1H), 2.18 (m, 2H), 1.84 (m, 3H), 1.39 (m, 5H), 0.94 (m,
3H); 13C NMR (75 MHz, CD3OD) d 176.6, 174.3,
166.1(8), 166.2(3), 146.3, 101.0, 79.6, 57.7, 57.3, 45.2,
43.4, 39.6, 34.9, 32.7, 31.9, 29.2, 28.4, 23.9, 14.5; IR
(KBr) 3439, 2957, 2926, 1729, 1638, 1539, 1291, 1118,
1074, 851 cmꢂ1; ESMS (ꢂve) m/z 429 (MꢂH+);
LSIMS-HRMS m/z 431.1964. calcd for C18H31N4O6S
(M+H+) m/z 431.1966.
ESMS (+ve) m/z 399 (MꢂNH3+H+), 416 (M+H+),
438 (M+Na+); LSIMS-HRMS m/z 416.1840. calcd for
C18H30N3O6S (M+H+) m/z 416.1855; Found: C, 52.19;
H, 6.96; N, 9.72. calcd for C18H29N3O6S: C, 52.03; H,
7.03; N, 10.11%.
(4aR,7S,7aS)- and (4aS,7R,7aR)-7-[[3-Methyl-1-oxo-
butyl]amino]sulfonyl]acetyloxy]-2,4a,5,6,7,7a-hexahydro-
2-methyl-1H-cyclopenta[c]pyridine-4-carboxamide (9c).
Mp 125–132 ꢃC; HPLC Rt 7.6 min (gradient of solvents
1
A/B, 55/45!0/100 over 20 min); H NMR (300 MHz,
CD3OD) d 7.32 (s, 1H), 5.42 (m, 1H), 4.47 (m, 2H), 3.08
(m, 1H), 3.00 (s, 3H), 2.88 (m, 1H), 2.71 (m, 1H), 2.40 (m,
1H), 2.18 (m, 5H), 1.82 (m, 1H), 1.38 (m, 1H), 0.98 (d,
J=6.5 Hz, 6H); 13C NMR (75 MHz, CD3OD) d 174.7,
173.9, 164.0, 146.0, 100.8, 79.9, 57.0, 46.2, 44.8, 43.1, 39.2,
34.5, 31.6, 28.9, 26.8, 22.7; IR (KBr) 3458, 3376, 3213,
2959, 2872, 1737, 1641, 1547, 1345, 1289, 1146, 1119, 1075,
904, 865 cmꢂ1; ESMS (+ve) m/z 385 (MꢂNH3+H+),
402 (M+H+); LSIMS-HRMS m/z 402.1686. calcd for
C17H28N3O6S (M+H+) m/z 402.1699.
(4aR,7S,7aS)- and (4aS,7R,7aR)-7-[[(S)-2-Amino-1-oxo-
pentyl]amino]sulfonyl]acetyloxy]-2,4a,5,6,7,7a-hexahydro-
2-methyl-1H-cyclopenta[c]pyridine-4-carboxamide (8i).
Isolated by freeze-drying from dilute aqueous hydro-
chloric acid, mp 190–210 ꢃC (dec.); HPLC Rt 11.3 min
(gradient of solvents A/B, 100/0!0/100 over 20 min);
1H NMR (300 MHz, CD3OD) d 8.01 (s, 1H), 5.57 (m,
1H), 4.67 (s, 2H), 4.11 (m, 1H), 3.41 (m, 1H), 3.33 (s,
3H), 3.26 (m, 1H), 2.86 (m, 1H), 2.62 (m, 1H), 2.34 (m,

C. F. Crasto et al. / Bioorg. Med. Chem. 11 (2003) 2687–2694
2693
(4aR,7S,7aS)- and (4aS,7R,7aR)-7-[[(S)-Pyrrolidin-2-oyl]-
amino]sulfonyl]acetyloxy] - 2,4a,5,6,7,7a - hexahydro - 2-
mp>150 ꢃC (dec.); HPLC Rt 15.6 min (gradient of sol-
vents A/B, 100/0!0/100 over 20 min); 1H NMR
(300 MHz, CD3OD) d7.31 (s, 1H), 5.35 (m, 1H), 3.55
(m, 1H), 3.45 (m, 2H), 3.00 (s, 3H), 2.98 (m, 1H), 2.89
(d, J=12.0 Hz, 1H), 2.71 (m, 1H), 2.59 (m, 2H), 2.38
(m, 1H), 2.18 (m, 2H), 1.77 (m, 2H), 1.53 (m, 1H), 1.36
(m, 1H), 1.18 (m, 1H), 0.95 (m, 6H); 13C NMR
(75 MHz, CD3OD) d 173.9, 172.9, 172.3, 145.9, 101.1,
78.3, 59.5, 45.0, 43.2, 43.1, 39.4, 38.8, 36.4, 34.7, 34.3,
31.6, 29.1, 25.6, 15.4, 11.8; IR (KBr) 3344, 3225, 3069,
2964, 2944, 2877, 1733, 1646, 1557, 1389, 1340, 1283,
1182, 1117, 1074, 769 cmꢂ1; ESMS (+ve) m/z 364
(MꢂNH3+H+), 381 (M+H+); LSIMS-HRMS m/z
381.2485. calcd for C19H33N4O4 (M+H+) m/z
381.2501.
methyl-1H-cyclopenta[c]pyridine-4-carboxamide
(9d).
Isolated by freeze-drying from acetic acid solution, mp
148–150 ꢃC; HPLC Rt 10.5 min (gradient of solvents A/
B, 100/0!0/100 over 20 min); 1H NMR (300 MHz,
CD3OD) d 7.42 (s, 1H), 5.49 (m, 1H), 4.32 (m, 2H), 4.16
(m, 1H), 3.49 (m, 1H), 3.37 (m, 1H), 3.29 (m, 1H), 3.11
(s, 3H), 2.98 (m, 2H), 2.79 (m, 1H), 2.45 (m, 2H), 2.29
(m, 3H), 2.10 (m, 1H), 1.90 (m, 1H), 1.45 (m, 1H); 13C
NMR (75 MHz, CD3OD) d 175.4, 174.0, 165.9, 146.1,
146.0, 100.7, 79.3(1), 79.2(5), 63.7, 47.3, 44.9, 44.8, 43.2,
43.1, 39.4, 39.3, 34.5(3), 34.4(7), 31.6(2), 31.5(6), 30.7,
29.0, 28.8, 24.9; IR (KBr) 3608, 3582, 3438, 3368, 2946,
1727, 1628, 1558, 1390, 1286, 1135, 1074, 845 cmꢂ1
;
ESMS (ꢂve) m/z 413 (MꢂH+); LSIMS-HRMS m/z
415.1628. calcd for C17H27N4O6S (M+H+) m/z
415.1651; Found: C, 46.66; H, 6.27; N, 12.54. calcd for
C17H26N4O6S.1.5H2O: C, 46.25; H, 6.62; N, 12.69%.
(4aR,7S,7aS)- and (4aS,7R,7aR)-7-[4-[(2S,3S)-2-Amino-
3-methyl-1-oxopentyl]amino]butanoyl]-2,4a,5,6,7,7a-hexa-
hydro-2-methyl-1H-cyclopenta[c]pyridine-4-carboxamide
(11c). Isolated by freeze-drying from acetic acid solu-
tion, mp>150 ꢃC (dec.); HPLC Rt 13.2 and 13.3 min
(gradient of solvents A/B, 100/0!0/100 over 20 min);
1H NMR (300 MHz, CD3OD) d 7.31 (s, 1H), 5.34 (m,
1H), 3.46 (m, 1H), 3.29 (m, 2H), 3.00 (s, 3H), 2.92 (m,
2H), 2.70 (m, 1H), 2.38 (m, 3H), 2.17 (m, 2H), 1.83 (m,
3H), 1.74 (m, 1H), 1.56 (m, 1H), 1.36 (m, 1H), 1.20 (m,
1H), 0.96 (m, 6H); 13C NMR (75 MHz, CD3OD) d
174.4, 173.9, 172.3, 145.9, 101.1, 78.2, 59.7, 45.1, 43.1,
39.7, 39.3, 38.9, 34.4, 32.3, 31.6, 29.2, 25.7, 25.6, 15.5,
11.8; IR (KBr) 3339, 3227, 3072, 2966, 2934, 2877, 1732,
1646, 1557, 1401, 1340, 1284, 1174, 1118, 1075, 880,
769, 651 cmꢂ1; ESMS (+ve) m/z 378 (MꢂNH3+H+),
395 (M+H+), 417 (M+Na+); LSIMS-HRMS m/z
395.2647. calcd for C20H35N4O4 (M+H+) m/z
395.2658.
(4aR,7S,7aS)- and (4aS,7R,7aR)-7-[[(S)-2,6-Diamino-1-
oxohexyl]amino]sulfonyl]acetyloxy] - 2,4a,5,6,7,7a - hexa-
hydro-2-methyl-1H-cyclopenta[c]pyridine-4-carboxamide
(9e). Isolated by freeze-drying from dilute aqueous
hydrochloric acid, mp 126–129 ꢃC; HPLC Rt 9.3 min
(gradient of solvents A/B, 100/0!50/50 over 10 min);
1H NMR (300 MHz, CD3OD) d 8.02 (s, 1H), 5.58 (m,
1H), 4.72 and 4.66 (ABq, J=14.5 Hz, 2H), 4.18 (m, 1H),
3.41 (m, 1H), 3.34 (s, 3H), 3.28 (m, 1H), 3.08 (t, J=7.5
Hz, 2H), 2.88 (m, 1H), 2.61 (m, 1H), 2.35 (m, 2H), 2.04
(m, 3H), 1.86 (m, 2H), 1.64 (m, 2H), 1.57 (m, 1H); 13C
NMR (75 MHz, CD3OD) d 171.2, 170.6, 164.1(2),
164.0(7), 152.8, 92.7, 80.0, 57.9, 57.8, 55.0, 45.7, 44.8,
40.5, 38.8, 33.3, 31.7, 31.6, 30.5, 29.3, 28.3, 22.9; IR
(KBr) 3608, 3582, 3350, 3235, 2943, 1728, 1630, 1549,
1401, 1286, 1133, 848 cmꢂ1; ESMS (ꢂve) m/z 444
(MꢂH+); LSIMS-HRMS m/z 446.2080. calcd for
C18H32N5O6S (M+H+) m/z 446.2073.
(4aR,7S,7aS)- and (4aS,7R,7aR)-7-[[(2S,3S)-2-Amino-3-
methyl-1-oxopentyl]amino]sulfonyl]acetyloxy]-2,4a,5,6,7,7a-
hexahydro-2-methyl-1H-cyclopenta[c]pyridine-4-carboxylic
acid methyl ester (12a). Isolated by freeze-drying from
acetic acid solution, mp 116–120 ꢃC; HPLC Rt 12.6 min
(gradient of solvents A/B, 60/40!0/100 over 20 min);
1H NMR (500 MHz, CD3OD) d 7.53 (s, 1H), 5.47 (m,
1H), 4.32 (m, 2H), 3.72 (s, 3H), 3.63 (m, 1H), 3.28 (dd,
J=13.0, 6.0 Hz, 1H), 3.12 (s, 3H), 3.00 (m, 1H), 2.84
(m, 1H), 2.47 (m, 1H), 2.24 (m, 2H), 2.10 (m, 1H), 1.91
(m, 1H), 1.73 (m, 1H), 1.44 (m, 1H), 1.35 (m, 1H), 1.14
(d, J=7.5 Hz, 3H), 1.06 (m, 3H); 13C NMR (125 MHz,
CD3OD) d 175.5, 175.4, 171.3, 165.9(2), 165.8(7), 148.4,
98.2(4), 98.2(3), 79.4(0), 79.3(9), 61.6, 61.5, 57.3, 51.0,
45.0(4), 45.0(3), 43.2, 39.0, 38.0, 34.7, 31.7, 28.9, 28.8,
25.5(0), 25.4(6), 15.4(8), 15.4(6), 12.2; IR (KBr) 3608,
3582, 3492, 3119, 2956, 1730, 1617, 1486, 1259, 1177,
(4aR,7S,7aS)- and (4aS,7R,7aR)-7-[[(2S,3S)-2-Amino-3-
methyl-1-oxopentyl]amino]acetyloxy]-2,4a,5,6,7,7a-hexa-
hydro-2-methyl-1H-cyclopenta[c]pyridine-4-carboxamide
(11a). Isolated by freeze-drying from acetic acid solu-
tion, mp>150 ꢃC (dec.); HPLC Rt 13.0 and 13.5 min
(gradient of solvents A/B, 100/0!0/100 over 20 min);
1H NMR (300 MHz, CD3OD) d 7.31 (s, 1H), 5.39 (m,
1H), 4.01 (m, 2H), 3.56 (d, J=5.5 Hz, 1H), 3.18 (m,
1H), 3.00 (s, 3H), 2.89 (m, 1H), 2.71 (m, 1H), 2.43 (m,
1H), 2.17 (m, 2H), 1.86 (m, 2H), 1.59 (m, 1H), 1.30 (m,
2H), 1.04 (d, J=7.0 Hz, 3H), 0.97 (m, 3H); 13C NMR
(75 MHz, CD3OD) d 174.0, 173.0, 172.9, 170.9(2),
170.8(6), 145.9, 101.1, 79.2, 59.7, 45.0, 43.1, 42.0, 39.5,
39.4, 38.9, 34.4(1), 34.3(6), 31.6, 29.1, 29.0, 25.5, 15.4,
11.8; IR (KBr) 3325, 3216, 2961, 2930, 1735, 1643, 1550,
1458, 1384, 1343, 1285, 1198, 1118, 1074, 1033, 768
cmꢂ1; ESMS (+ve) m/z 350 (MꢂNH3+H+) 367
(M+H+); LSIMS-HRMS m/z 367.2331. calcd for
C18H31N4O4 (M+H+) m/z 367.2345.
1129, 1076, 843 cmꢂ1
;
ESMS (+ve) m/z 414
(MꢂMeOH+H+), 446 (M+H+); LSIMS-HRMS m/z
446.1927. calcd for C19H32N3O7S (M+H+) m/z
446.1960.
(4aR,7S,7aS)- and (4aS,7R,7aR)-7-[[(S)-2-Amino-4-methyl-
1-oxopentyl]amino]sulfonyl]acetyloxy] - 2,4a,5,6,7,7a - hexa-
hydro-2-methyl-1H-cyclopenta[c]pyridine-4-carboxylic
acid methyl ester (12b). Isolated by freeze-drying from
acetic acid solution, mp 119–122 ꢃC; HPLC Rt 11.2 min
(4aR,7S,7aS)- and (4aS,7R,7aR)-7-[3-[(2S,3S)-2-Amino-3-
methyl-1-oxopentyl]amino]propanoyl]-2,4a,5,6,7,7a-hexa-
hydro-2-methyl-1H-cyclopenta[c]pyridine-4-carboxamide
(11b). Isolated by freeze-drying from acetic acid solution,

2694
C. F. Crasto et al. / Bioorg. Med. Chem. 11 (2003) 2687–2694
(gradient of solvents A/B, 80/20!0/100 over 20 min);
1H NMR (300 MHz, CD3OD) d 7.32 (s, 1H), 5.27 (m,
1H), 4.11 (m, 2H), 3.51 (s, 3H), 3.49 (m, 1H), 3.07 (dd,
J=13.0, 5.0 Hz, 1H), 2.92 (s, 3H), 2.80 (m, 1H), 2.63
(m, 1H), 2.26 (m, 1H), 2.04 (m, 2H), 1.74 (m, 3H), 1.53
(m, 1H), 1.23 (m, 1H), 0.89 (d, J=5.0 Hz, 3H), 0.86 (d,
J=6.5 Hz, 3H); 13C NMR (75 MHz, CD3OD) d 176.7,
171.3, 165.9(4), 165.8(9), 148.4, 98.2, 79.4, 56.9, 55.5,
51.0, 45.0, 43.2(3), 43.1(6), 42.0, 39.0, 34.7, 31.7, 28.9,
25.6, 23.3, 22.0; IR (KBr) 3604, 3582, 2954, 1731, 1666,
1615, 1438, 1410, 1287, 1260, 1177, 1134, 1075, 845
cmꢂ1; ESMS (ꢂve) m/z 444 (MꢂH+); LSIMS-HRMS
m/z 446.1973. calcd for C19H32N3O7S (M+H+) m/z
446.1960.
1H NMR (300 MHz, CD3OD) d 7.32 (s, 1H), 5.17 (m,
1H), 4.20 (m, 1H), 4.08 (m, 1H), 3.56 (m, 1H), 3.52 (s,
3H), 3.33 (m, 1H), 2.92 (s, 3H), 2.24 (m, 2H), 2.02 (m,
2H), 1.75 (m, 4H), 1.47 (m, 2H), 0.87 (br, 6H); 13C
NMR (75 MHz, CD3OD)
d 176.8, 171.3, 166.0,
147.6(2), 147.5(7), 92.5(2), 92.4(9), 78.3, 59.2, 57.1, 55.5,
51.2, 41.9(2), 41.8(7), 38.0, 28.1, 27.2(4), 27.1(7), 25.6,
23.4, 21.9, 17.8; IR (KBr) 3604, 3582, 2953, 1731, 1614,
1438, 1413, 1381, 1298, 1273, 1180, 1135, 1110, 845
cmꢂ1; ESMS (ꢂve) m/z 444 (MꢂH+); LSIMS-HRMS
m/z 446.1945. calcd for C19H32N3O7S (M+H+) m/z
446.1960.
(4aR,5S,7aR)- and (4aS,5R,7aS)-7-[[(S)-2-Amino-3-methyl-
1-oxobutyl]amino]sulfonyl]acetyloxy] - 4,4a,5,6,7,7a - hexa-
hydro-1-methyl-1H-cyclopenta[b]pyridine-3-carboxylic
acid methyl ester (13c). Isolated by freeze-drying from
dilute aqueous hydrochloric acid, mp 135–160 ꢃC;
HPLC Rt 14.0 min (gradient of solvents A/B, 100/0!0/
(4aR,7S,7aS)- and (4aS,7R,7aR)-7-[[(S)-2-Amino-3-
methyl-1-oxobutyl]amino]sulfonyl]acetyloxy]-2,4a,5,6,7,7a-
hexahydro-2-methyl-1H-cyclopenta[c]pyridine-4-carboxylic
acid methyl ester (12c). Isolated by freeze-drying from
dilute aqueous hydrochloric acid, mp 150–165 ꢃC;
HPLC Rt 14.1 min (gradient of solvents A/B, 100/0!0/
1
100 over 20 min); H NMR (300 MHz, CD3OD) d 7.44
(s, 1H), 5.35 (m, 1H), 4.58 (m, 2H), 4.04 and 4.00 (d and
d, J=4.0 and 4.0 Hz, total 1H), 3.77 (m, 1H), 3.64 (s,
3H), 3.46 (m, 1H), 3.03 (s, 3H), 2.36 (m, 2H), 2.16 (m,
2H), 1.93 (m, 2H), 1.58 (m, 1H), 1.15 and 1.14 (d and d,
J=7.0 and 7.0 Hz, total 3H), 1.03 (d, J=7.0 Hz, 3H);
13C NMR (75 MHz, CD3OD) d 171.3, 170.1, 163.8,
163.6, 147.8, 147.6, 92.4, 79.4(3), 79.3(6), 60.1, 59.2,
59.1, 57.5, 57.3, 51.2, 41.8, 38.2, 38.0, 31.2, 28.1, 27.4,
27.2, 19.0, 17.8, 16.7(2), 16.6(6); IR (KBr) 3608, 3582,
3400, 2964, 1737, 1612, 1462, 1355, 1275, 1150, 903, 856
cmꢂ1; ESMS (ꢂve) m/z 430 (MꢂH+); LSIMS-HRMS
m/z 432.1803. calcd for C18H30N3O7S (M+H+) m/z
432.1804.
1
100 over 20 min); H NMR (300 MHz, CD3OD) d 7.43
(s, 1H), 5.42 (m, 1H), 4.56 (s, 2H), 3.84 (m, 1H), 3.62 (s,
3H), 3.11 (m, 1H), 3.02 (s, 3H), 2.91 (m, 1H), 2.75 (m,
1H), 2.36 (m, 2H), 2.16 (m, 2H), 1.80 (m, 1H), 1.36 (m,
1H), 1.13 (d, J=7.0 Hz, 3H), 1.04 (d, J=7.0 Hz, 3H);
13C NMR (75 MHz, CD3OD) d 171.2, 170.0, 169.9,
163.9, 148.3, 98.4, 80.4(0), 80.3(5), 60.0, 57.4, 51.1, 45.0,
43.2(2), 43.1(6), 39.0, 34.6, 31.7, 31.2, 31.1, 28.9, 19.1(1),
19.0(6), 16.8; IR (KBr) 3604, 3582, 2920, 2848, 1733,
1615, 1460, 1356, 1302, 1273, 1147, 1126, 1075 cmꢂ1
;
ESMS (ꢂve) m/z 430 (MꢂH+); LSIMS-HRMS m/z
432.1784. calcd for C18H30N3O7S (M+H+) m/z
432.1804.
(4aR,5S,7aR)- and (4aS,5R,7aS)-7-[[(2S,3S)-2-Amino-3-
methyl-1-oxopentyl]amino]sulfonyl]acetyloxy]-4,4a,5,6,7,7a-
hexahydro-1-methyl-1H-cyclopenta[b]pyridine-3-carboxylic
acid methyl ester (13a). Isolated by freeze-drying from
acetic acid solution, mp 130–135 ꢃC; HPLC Rt 8.4 min
(gradient of solvents A/B, 80/20!0/100 over 20 min);
1H NMR (300 MHz, CD3OD) d 7.31 (s, 1H), 5.16 (m,
1H), 4.20 (m, 1H), 4.05 (m, 1H), 3.51 (s, 3H), 3.49 (m,
1H), 3.32 (m, 1H), 2.90 (s, 3H), 2.22 (m, 2H), 1.90 (m,
5H), 1.46 (m, 2H), 1.13 (m, 1H), 0.92 (d, J=7.0 Hz,
3H), 0.83 (m, 3H); 13C NMR (75 MHz, CD3OD) d
175.7, 171.6, 166.3, 147.8(9), 147.8(6), 92.9, 92.8,
78.6(4), 78.6(0), 61.8(3), 61.7(6), 59.5, 57.8, 51.4, 42.1,
38.4, 38.3, 28.3, 27.6, 27.5, 25.8, 25.7, 18.0, 15.8, 12.5;
IR (KBr) 3608, 3582, 3468, 2962, 1731, 1615, 1442,
Acknowledgements
We thank GlaxoSmithKline (Australia) Pty Ltd for
financial support and Dr. Brian Metcalf (formerly of
SmithKline Beecham, USA) for facilitating this colla-
boration.
References and Notes
1. Stefanska, A. L.; Cassels, R.; Ready, S. J.; Warr, S. R.
J. Antibiot. 2000, 53, 357.
2. Houge-Frydrych, C. S. V.; Gilpin, M. L.; Skett, P. W.;
Tyler, J. W. J. Antibiot. 2000, 53, 364.
3. Banwell, M. G.; Crasto, C. F.; Easton, C. J.; Forrest, A. K.;
Karoli, T.; March, D. R.; Mensah, L.; Nairn, M. R.; O’Han-
lon, P. J.; Oldham, M. D.; Yue, W. Bioorg. Med. Chem. Lett.
2000, 10, 2263.
4. Banwell, M. G.; Crasto, C. F.; Easton, C. J.; Karoli, T.;
March, D. R.; Nairn, M. R.; O’Hanlon, P. J.; Oldham, M. D.;
Willis, A. C.; Yue, W. Chem. Commun. 2001, 2210.
5. Pope, A. J.; Moore, K. J.; McVey, M.; Mensah, L.; Benson,
N.; Osbourne, N.; Broom, N.; Brown, M. J. B.; O’Hanlon,
P. J. J. Biol. Chem. 1998, 273, 31691.
6. Creppy, E. E.; Mayer, M.; Kern, D.; Schlegel, M.; Steyn,
P. S.; Vleggaar, R.; Dirheimer, G. Biochim. Biophys. Acta
1981, 656, 265.
7. Creppy, E. E.; Kern, D.; Steyn, P. S.; Vleggaar, R.; Roe-
schenthaler, R.; Dirheimer, G. Toxicol. Lett. 1983, 19, 217.
1415, 1383, 1325, 1271, 1180, 1131, 1075, 843, 762 cmꢂ1
;
ESMS (+ve) m/z 414 (MꢂMeOH+H+), 446
(M+H+), 468 (M+Na+); LSIMS-HRMS m/z
446.1954. calcd for C19H32N3O7S (M+H+) m/z
446.1960.
(4aR,5S,7aR)- and (4aS,5R,7aS)-7-[[(S)-2-Amino-4-methyl-
1-oxopentyl]amino]sulfonyl]acetyloxy] - 4,4a,5,6,7,7a - hexa-
hydro-1-methyl-1H-cyclopenta[b]pyridine-3-carboxylic
acid methyl ester (13b). Isolated by freeze-drying from
acetic acid solution, mp 129–131 ꢃC; HPLC Rt 11.2 min
(gradient of solvents A/B, 80/20!0/100 over 20 min);