Synthesis of analogs of (1,4)-3- and 5-imino oxazepane, thiazepane, and diazepane as inhibitors of nitric oxide synthases

Article abstract of DOI:10.1016/j.bmcl.2004.09.019

The preparation and SAR of a series of iNOS inhibitors leading to the most potent compound, incorporating a (1,4)-5-imino thiazepane (R = n-Pr) is disclosed. A series of 3- and 5-imino analogs from oxazepane, thiazepane, and diazepane was prepared and eva

Full text of DOI:10.1016/j.bmcl.2004.09.019

Bioorganic & Medicinal Chemistry Letters 14 (2004) 5907–5911
Synthesis of analogs of (1,4)-3- and 5-imino oxazepane,
thiazepane, and diazepane as inhibitors of nitric oxide synthasesq
K. Shankaran,a,* Karla L. Donnelly,a Shrenik K. Shah,a Charles G. Caldwell,a Ping Chen,a
William K. Hagmann,a Malcolm MacCoss,a John L. Humes,b Stephen G. Pacholok,b
Theresa M. Kelly,c Stephan K. Grantc and Kenny K. Wongc
aDepartment of Medicinal Chemistry, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA
bDepartment of Immunology and Inflammation Research, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA
cDepartment of Enzymology, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA
Received 14 July 2004; revised 8 September 2004; accepted 8 September 2004
Available online 30 September 2004
Abstract—A series of 3- and 5-imino analogs from oxazepane, thiazepane, and diazepane was prepared and evaluated as inhibitors
of human nitric oxide synthesis (NOS). The most potent iNOS inhibitor was the thiazepane analog 25 (IC50 = 0.19lM).
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
defense, is induced by inflammatory cytokines and
hence the name iNOS. Unwarranted production of
nitric oxide by iNOS has been associated with tissue
damage leading to inflammatory diseases such as arthri-
tis and inflammatory bowl disease. Thus selective inhibi-
tion of iNOS may offer possible treatments for such
diseases.
The isozymes of nitric oxide synthase (NOS) produce
nitric oxide (NO), a reactive inorganic radical gas, which
plays central role in physiological functions in various
mammalian tissues.2–5 All three NOS enzymes bring
about a five electron oxidation of L-arginine to NO
and L-citrulline, as shown in Figure 1. To date three iso-
forms of NOS enzymes have been identified. Of these,
two isoforms are constitutive and are implicated in the
cardiovascular and neuronal activities (eNOS and
nNOS). The third isoform, which is linked with host
Efforts toward NOS inhibitors have led to amino acids
as well as nonaminoacid based inhibitors, including ami-
noguanidines, isothioureas, amidines benzoxazolones,
and 2-amino-pyridines, respectively.6 Previously, pyrrol-
idin-2-imines7a,8 and piperidin-2-imines9 as inhibitors of
NOS displaying selectivity to varying degrees has been
reported (Fig. 2). In an obvious extension from the pyr-
rolidinyl and piperidinyl-2-imines, the analogous of 3-
and 5-imino derivatives of oxazepane, thiazepane, and
diazepane were prepared (Fig. 2).7b The synthesis and
SAR of these classes of inhibitors are the subject of this
report.
FAD, FMN, Ca/CaM
Tetrahydrobiopterin (H 4B)
NH
O
COOH
NH2
L-Arginine
COOH
NH2
H2N
N
H2N
N
H
O2, NOS
H
L-Citrulline
•NO
Nitric Oxide
1
Figure 1. Nitric oxide biosynthesis pathway.
R1
1
X
X
6
( )n
and
3
5
NH
3
R2
N
H
N
H
NH
N
H
NH
Keyword: NOS synthase.
q See Ref. 1.
X = O, S, NH
n = 1 and 2
*
Corresponding author. Tel.: +1 732 594 3979; fax: +1 732 594
3007; e-mail: kothandaraman_shankaran@merck.com
Figure 2.
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.09.019

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K. Shankaran et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5907–5911
2. Chemistry
involving the Dieckmann cyclization12 of 9 to afford
10 that was followed by its alkylation and the eventual
removal of allyloxycarbonyl moiety to furnish the tetra-
hydrothiopyran-4-one (11). Transformation of 11 to the
oxime followed by the Beckmann rearrangement of the
resultant oxime tosylate gave a mixture of two amides
(12), that could not be separated. The mixture of 12
was converted to the thioamides 13 and 14, at which
point they could be separated by flash chromatography.
The separated thioamides 13 and 14 were then trans-
formed individually to their respective amidines 15 and
16 in a sequences of reaction similar to the conversion
3 to 5 described in Scheme 1.
The preparation of unsubstituted 5-imino analogs of
oxazepane, thiazepane, and diazepane described herein
are shown in Scheme 1. In an identical sequence of reac-
tions both tetrahydropyran-4-one (X = O) and tetra-
hydrothiopyran-4-one (X = S) (1) were subjected to
oximation followed by tosylation to give the desired
tosylates (2). These tosylates (2) undergo smooth Beck-
mann rearrangement10 to afford the amides 3. The
amides 3 were transformed to the thioethers 4 in two
steps consisting of initial formation of the thioamide11
from 3 that was followed by S-alkylation in the subse-
quent step. The thioethers 4 on reflux with ammonium
chloride gave the amidines 5. The preparation of diaze-
pane 8 took a different route starting from the known
amide 6. Removal of the benzyl protecting group at
nitrogen on 6 followed by reprotection gave 7. The
transformation of 7 to the desired amidine 8 was accom-
plished in three steps that involve formation of imino-
ether, amidine, and then eventually N-Boc removal to
afford 8 as shown below.
The preparation of C-2 and C-7-methyl analogs of (1,4)-
5-imino thiazepanes 19 and 20 were carried out as in
Scheme 3. Oxidation of the b-ketoester 10 to the enone
17 was accomplished under active MnO2 conditions as
reported in the literature.13 The enone 17 underwent
smooth Cu-catalyzed 1,4-addition of MeLi followed by
Pd-mediated removal of the allyloxycarbonyl in subse-
quent step to give 18. The conversion of 18 to the de-
sired amidines 19 and 20 was analogous to Scheme 1.
The synthesis of C-3 and C-6 substituted analogs of
(1,4)-5-imino thiazepanes are shown in Scheme 2. The
bis-allylester 9 was transformed to 11 in three steps
The preparation of homochiral 3-(S)-propyl-(1,4)-5-
imino thiazepane 25 is shown in Scheme 4. The readily
available N-Boc alcohol 21 undergoes facile azirida-
tion14 under Mitsunobu protocol giving 22. The nucleo-
philic ring opening of 22 by b-mercapto propionic acid
gave the desired acid, that was subsequently deprotected
X
X
X
c
a, b
HN
3
O
O
NOTs
2
1 (X = O, S)
X
X
d, e
f
S
a
b, c
S
S
Me
HN
5
N
4
10
COOallyl
S
NH
SMe
O
O
Bn
N
Boc
N
H
N
17
18
g, h
e, f and i
Me
HN
6
Me
HN
7
HN
d
O
O
and
NH
8
HN
HN
NH
NH
Scheme 1. Reagents and conditions: (a) NH2OH, Py; (b) n-BuLi, TsCl,
THF, À78°C to rt; (c) dioxane, NEt3, rt; (d) Lawessonꢀs regent, Tol.,
90°C; (e) Me3OBF4, CH2Cl2; (f) NH4Cl, EtOH, reflux; (g) Pd(OH)2,
H2, EtOH, rt; (h) Boc2O, CH2Cl2, rt; (i) HCl, EtOAc, rt.
19
20
Scheme 3. Reagents and conditions: (a) active MnO2, CHCl3, rt; (b)
MeLi, CuI–Me2S, ether, À78°C to rt; (c) Pd(PPh3)4, THF, morpho-
line; (d) Scheme 1 and steps a–f.
S
S
S
COOallyl
a
b, c
g
S
Oallyl
OH
b, c
COOH
a
COOallyl
9
R
NBoc
O
O
n-Pr NH2
O
11
n-Pr
S
n-Pr NHBoc
10
22
23
S
21
S
d, e, and f
+
R
R
R
S
HN
HN
R
d
e
O
O
12
n-Pr
24
N
H
n-Pr
25
N
H
O
NH
S
S
S
S
h
+
and
R
R
1
HN
14
HN
HN
13
HN
S
S
OH
NH
S
NH
3
n-Pr
5
16
R = Me, Et, n-Pr, i-Bu
15
N
H
n-Pr NHBoc
NH
26
27
Scheme 2. Reagents and conditions: (a) NaH, ether, allyl alcohol; (b)
NaH, DMF, RI; (c) Pd(PPh3)4, THF, morpholine; (d) NH2OH, Py; (e)
n-BuLi, TsCl, THF, À78°C to rt; (f) dioxane, NEt3, rt; (g) Lawessonꢀs
regent, Tol., 90°C; (h) Scheme 1 and steps e and f.
Scheme 4. Reagents and conditions: (a) PPh3, THF, DIAD, rt; (b) b-
mercaptopropionic acid, Cs2CO3, DMF, 60°C; (c) EtOAc, HCl (g), rt;
(d) EDCÆHCl, DMF, NMM, rt; (e) Scheme 1 and steps d–f.

K. Shankaran et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5907–5911
5909
cles are shown in Table 1. In the (1,4)-5-imine class,
clearly the thiazepane analog 35 was relatively more po-
tent and also had a better selectivity than the oxazepane
analog 34, while the diazepane analog 8 was inactive
(IC50 > 50lM). The binding results for the (1,4)-3-imine
analogs 32 and 33 are also displayed in Table 1. The rel-
ative comparison of the oxazepane analogs 34 and 32
suggested that the later was significantly more potent
for the all three isoforms but neither displayed selectiv-
ity. Interestingly, the thiazepane analogs 35 and 33 were
both equipotent and, unlike their oxazepane counter-
parts displayed slightly better selectivity. Since the selec-
tivity data for both thiazepanes (33 and 35) appeared
better, the effects of substituents on (1,4)-5-imino thiaze-
pane (35) in order to enhance its potency and also to in-
crease the selectivity for the iNOS over the other
isoforms was explored. The assay results of these studies
were compared with 35 and are shown in Table 2.
O
O
a
c
O
COOMe
Br
NC
N
H
32
NH
N
H
30
O
28
b
c
S
S
H2N
N
H
N
H
O
NH
29
33
31
Scheme 5. Reagents and conditions: (a) RaNi, H2, 1000psi, 100°C;
(b) NaOEt, HSCH2COOEt, EtOH; (c) Scheme 1.
to the amino acid 23.15 Transformation of 23 to the lac-
tam 24 was accomplished using EDC, N-methylmorpho-
line in DMF. Conversion of 24 to the amidine 25 was
analogous to Scheme 1. A similar sequence of reactions
was in employment to transform 26 to the enantiomeric
3-(R)-propyl-(1,4)-5-imino thiazepane amidine 27.
The thiazepane analog 36 with methyl at C-2 resulted in
2-folds loss of potency for iNOS compared to 35 and
gain in the potency for other isoforms. The placement
of methyl at C-3 (37) led to a gain in some potency
for the all three isoforms. Analog 38 with a methyl at
C-6 resulted in a substantial loss versus all isoforms,
especially for the iNOS and eNOS, respectively. The
presence of a methyl substituent at C-7 (39) gave an ana-
log that was as potent as 35 or 36. Since the presence of
methyl at C-3 generated more potent analog (37), fur-
ther alterations were carried out at this position that
led to the preparation of additional analogs bearing lin-
ear and branched alkyls, as in 40–45. The C-3 hydroxy-
methyl and bicyclic analogs 40 and 41 were
unimpressive in that the former displayed no improve-
ment compared to 37 while the later lost all activity. A
2–3-fold gain in the iNOS potency (compared to 37)
was observed in the analogs 42–45. In general the gain
in the selectivity over eNOS was substantial for these
The preparation of isomeric analogs of (1,4)-3-imino
oxazepane (32) from 28 and the thiazepane (33) from
29 are shown in Scheme 5. This involved routine chem-
istry leading to the preparation of intermediate lactams
30 and 31 that was followed by steps given under
Scheme 1 to give 32 and 33.
3. Results and discussion
The hydrochloride salts of synthesized (1,4)-3- and 5-
imino analogs were evaluated for NOS binding activity
in an assay protocol that used recombinant versions of
the human enzymes.16 Results tabulated below describ-
ing the SAR for this class of inhibitors were compared
with N-iminoethyl lysine (L-NIL) and primarily centered
on the placement of substituents at all available position
of the heterocycles. The IC50 from the parent heterocy-
Table 1. NOS inhibition by (1,4)-3- and 5-imino oxazepane, thiazepane, and diazepanea
#
IC50 (lM)
Selectivity eNOS/iNOS
3.7
Structureb
L-NIL
iNOS
2.1
eNOS
7.9
nNOS
17.5
Selectivity eNOS/iNOS
8.3
—
H
N
8
>50
>50
ND
>50
ND
N
NH
H
O
S
32
33
34
0.16
0.45
10.5
25
2.8
8.8
2.1
0.16
2.8
1
N
H
NH
NH
1.2
2.3
0.7
N
H
O
11.8
8.7
N
H
NH
NH
S
35
1.4
13.4
9.5
4.3
3
N
H
a IC50s values are obtained from a 10 point titration using Sigma Plot, where each individual point is an average of duplicate determinations at that
concentration.
b The analogs in Table 1 were synthesized according to Schemes 1 and 5, ND = not determined, L-NIL = L-N-iminoethyl lysine.

5910
K. Shankaran et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5907–5911
Table 2. NOS inhibition by substituted (1,4)-5-imino thiazepanesa
#
IC50 (lM)
Structureb
iNOS
1.4
eNOS
13.4
Selectivity eNOS/iNOS
nNOS
4.3
Selectivity eNOS/iNOS
3.0
S
35
9.5
N
NH
S
H
Me
36
37
38
3.5
4.1
5.0
1.1
6.1
0.83
1.8
0.24
2.2
N
H
NH
NH
S
0.81
Me
S
N
H
Me
>50
>50
ND
>50
ND
N
H
NH
Me
S
39
40
3.0
5.0
6
1.6
5.3
1
0.3
2.4
N
H
NH
S
1.1
2.7
HO
N
H
NH
Me
NH
S
41
>50
>50
ND
>50
ND
N
H
S
42
43
0.31
0.29
1.1
50
3.7
0.59
2.8
2
Me
Me
Me
N
H
NH
S
172
10
N
H
NH
NH
S
Me
44
45
25
27
0.32
0.5
60
77
1.0
50
186
164
5.2
42
1.9
1.2
0.44
7.1
6
N
H
S
2.6
2.3
6
N
H
NH
Me
Me
S
0.19
1.2
N
H
H
NH
NH
S
Me
N
H
H
a IC50s values are obtained from a 10 point titration using Sigma Plot, where each individual point is an average of duplicate determinations at that
concentration.
b In Table 2 the analogs were synthesized according to Schemes 2 and 3 (compounds 35–39) and Scheme 4 (compounds 40–45 and including 25 and
27), ND = not determined.
analogs while the selectivity over the nNOS was modest.
As the n-propyl analog 43 was among the best, it was
probed further to broaden the scope of these
observations.
selectivity for neither compound matched that of the
racemic analog 43 or other analogs displayed in Table 2.
In conclusion, limited SAR studies described herein
established the (1,4)-5-imino thiazepanes as potent and
selective iNOS inhibitors. We have also shown that
iNOS potency can be improved by the placement of n-
propyl substituent at C-3 of thiazepane and that the
analog 25 was more potent and 27 was more selective.
Our observations described herein is similar to an anal-
ogous studies conducted previously on these systems by
Mooremann et al.7b Nevertheless studies described here-
in when compared with the earlier observations suggest
Since 43 was racemic, the chiral synthesis of enantiomers
25 and 27 were undertaken to see if an additional po-
tency/selectivity are to be gained. Table 2 reveals that
S-isomer (25) displayed 6-fold more potency for the
iNOS compared to the (R)-isomer 27.
Paradoxically, the selectivity (over eNOS) data for the
25 was modest in comparison to 27. Interestingly, the

K. Shankaran et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5907–5911
5911
that the pyrrolidin-2-imine6h or piperidin-2-imine6h,9
were significantly more potent and had a more balanced
inhibitory profile than the analogs described herein.
P.; Durette, P.; Esser, C. K.; Lanza, T. J.; Kopka, I. E.;
Guthikonda, R.; Shah, S. K.; Green, B. G.; Humes, J. L.;
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References and notes
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15. The crude acid 23 was taken as is to the next step to afford
lactam 24 whose purification was facile.
16. The NOS inhibitory activity of the compound was
determined by comparing the conversion of 3H-L-arginine
3
to H-citrulline in the presence of inhibitor with control.
The assay mixture containing 1lM of 3H-L-arginine,
cofactors and the inhibitor or aqueous DMSO (control)
was incubated for 30min at room temperature. The
reaction was quenched by adding a slurry of Dowex
50W-K8 resin to complex and remove the unreacted
substrate. The concentration of the 3H-L-citrulline product
in the supernatant fluid was determined on a scintillation
counter. Under the assay conditions the production of L-
citrulline was linear with time for the duration of the
experiment.