3,4-Dihydropyrimido(1,2-a)indol-10(2H)-ones as potent non-peptidic inhibitors of caspase-3

Article abstract of DOI:10.1016/j.bmc.2009.09.036

Cysteine-dependant aspartyl protease (caspase) activation has been implicated as a part of the signal transduction pathway leading to apoptosis. It has been postulated that caspase-3 inhibition could attenuate cell damage after an ischemic event and thereby providing for a novel neuroprotective treatment for stroke. As part of a program to develop a small molecule inhibitor of caspase-3, a novel series of 3,4-dihydropyrimido(1,2-a)indol-10(2H)-ones (pyrimidoindolones) was identified. The synthesis, biological evaluation and structure-activity relationships of the pyrimidoindolones are described.

Full text of DOI:10.1016/j.bmc.2009.09.036

Bioorganic & Medicinal Chemistry 17 (2009) 7755–7768
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
3,4-Dihydropyrimido(1,2-a)indol-10(2H)-ones as potent non-peptidic
inhibitors of caspase-3
a
a
a
a
a
Lisa M. Havran a, , Dan C. Chong , Wayne E. Childers , Paul J. Dollings , Arlene Dietrich , Boyd L. Harrison ,
Vasilios Marathias a, Gregory Tawa a, Ann Aulabaugh b, Rebecca Cowling b, Bhupesh Kapoor b, Weixin Xu c,
Lidia Mosyak c, Franklin Moy c, Wah-Tung Hum c, Andrew Wood d, Albert J. Robichaud a
*
a Chemical Sciences, Wyeth Research, CN 8000, Princeton, NJ 08543, USA
b Screening Sciences, Wyeth Research, 401 N. Middletown Road, Pearl River, NY 10965, USA
c Chemical Sciences, Wyeth Research, 200 CambridgePark Drive, Cambridge, MA 02140, USA
d Discovery Neuroscience, Wyeth Research, CN 8000, Princeton, NJ 08543, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 15 July 2009
Revised 17 September 2009
Accepted 18 September 2009
Available online 24 September 2009
Cysteine-dependant aspartyl protease (caspase) activation has been implicated as a part of the signal
transduction pathway leading to apoptosis. It has been postulated that caspase-3 inhibition could atten-
uate cell damage after an ischemic event and thereby providing for a novel neuroprotective treatment for
stroke. As part of a program to develop a small molecule inhibitor of caspase-3, a novel series of 3,4-
dihydropyrimido(1,2-a)indol-10(2H)-ones (pyrimidoindolones) was identified. The synthesis, biological
evaluation and structure–activity relationships of the pyrimidoindolones are described.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Caspase-3
Stroke
Apoptosis
Pyrimidoindolone
1. Introduction
inhibitors is an electrophilic group that can form a reversible or
irreversible bond with the active site cysteine leading to the inacti-
Cysteine-dependant aspartyl proteases (caspases) have been
implicated in the signal transduction cascade leading to apoptosis.
The 11 known human caspases can be divided into three sub-types
based on their structure and function.1,2 Group I caspases (1, 4, 5
and 14) are primarily involved in inflammation. Group II caspases
(6, 8, 9 and 10) are primary involved in apoptosis as upstream reg-
ulators of the Group III caspases (3 and 7). The Group III caspases are
effector caspases that, once activated, stimulate a signalling path-
way that ultimately leads to the death of the cell. Over the past dec-
ade, evidence has emerged that caspase inhibition can provide for
tissue protection and reduce infarct volume in rodent models of
ischemia.3–8 While these studies have been carried out using pri-
marily peptide-based broad spectrum caspase inhibitors, more re-
cently, there has been a focus on the identification of selective,
non-peptidic caspase inhibitors. Multiple groups have reported
their efforts toward selective, small molecule caspase-3 inhibi-
tors9–14 and one of these compounds has been shown to reduce tis-
sue damage in an isolated rabbit heart model of ischemia
injury.15,16 A key structural feature of almost all known caspase
vation of the enzyme. In an effort to identify potent, selective cas-
pase-3 inhibitors as potential therapeutic treatments for ischemic
stroke, we undertook an extensive structure–activity based optimi-
zation of several key leads identified from our compound collection.
PhO
O
O
S
O
N
N
O
O
N
N
1
2
RO
N
O
O
S
10
8
O
N
N
3
R = Ph (3) Caspase-3 IC50 = 7 nM
R = CH3 (4) Caspase-3 IC50 = 6 nM
* Corresponding author. Tel.: +1 732 274 4164; fax: +1 732 274 4505.
E-mail address: havranl@wyeth.com (L.M. Havran).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.09.036

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L. M. Havran et al. / Bioorg. Med. Chem. 17 (2009) 7755–7768
Through a high throughput screen of our compound library we
identified 1 as a singleton hit for caspase-3 inhibition. Modification
in our exploratory chemistry group and recognition of the possible
similarity to a previously reported series of caspase-3 inhibitors (2)9
allowed us to identify a lead series of novel 3,4-dihydropyrimi-
do(1,2-a)indol-10(2H)-ones (pyrimidoindolones) (3 and 4) as po-
tent and selective inhibitors of caspase-3.17 An X-ray crystal
structure of 3 bound to human caspase-3 (Fig. 1)18 was obtained
and revealed that a covalent bond forms between the electrophilic
10-position ketone of 3 and the nucleophilic active site cysteine of
the activated caspase-3 in the enzyme–inhibitor complex. Using
this information, we began to optimize this series of compounds.
Herein, we describe the synthesis and structure–activity relation-
ships of this novel series of pyrimidoindolones.
BnO
N
O
O
-O
O
S
O
S
Na+
c
a, b
O
O
3
O
O
N
N
H
H
5
6
BnO
BnO
N
O
S
O
N
O
O
O
d
e, f
S
O
O
O
O
O
N
CN
N
H
8
7
BnO
N
TsO
N
2. Chemistry
O
O
i, j
O
O
g, h
O
S
O
S
A general approach to the preparation of phenoxy analogs is
shown in Scheme 1. 5-Chlorosulfonylisatin was prepared as previ-
ously reported19 by treating sodium 5-isatin sulfonate 5 with phos-
phorus oxychloride in sulfolane at 60 °C. The resulting sulfonyl
chloride was reacted with (S)-2-(benzyloxymethyl)-pyrrolidine
(13) in the presence of Hunig’s base to give sulfonamide 6. The
3-position carbonyl was protected as a ketal and subsequently
alkylated with 3-chloro-2,2-dimethyl-propionitrile20 to give nitrile
8. Hydrogenation with Raney nickel followed by heating of the
resultant primary amine in a sealed tube yielded the protected
pyrimidoindolone 9. The benzyl protecting group was removed un-
der phase transfer hydrogenolysis conditions and a tosylate group
was installed to afford the common intermediate 10, through
which the first key analogs were prepared. Reaction of 10 with
substituted phenols or other heterocyclic phenols followed by
deprotection of the ketal gave target analogs 11a–n. Tosylate inter-
mediate 10 could also be used to prepare amino analogs 14a–d as
shown in Scheme 2 by heating with various amines in THF fol-
lowed by the usual ketal deprotection.
O
O
N
N
N
N
9
10
RO
HO
BnO
O
O
N
S
O
k, l
NBoc
N
NH
13
N
12
11a-n
Scheme 1. Reagents and conditions: (a) POCl3, sulfolane, 60 °C, 3 h, 66%; (b) 13,
DIPEA, CH2Cl2, rt, 1 h, 78%; (c) 1,3-propanediol, pTsOH, PhH, reflux, 14 h, 90%; (d) 3-
chloro-2,2-dimethyl-propionitrile, KOt-Bu, DMSO, 132 °C, 21 h, 99%; (e) H2, Ra/Ni,
2 M NH3 in EtOH/THF, rt, 22 h; (f) 135 °C (sealed tube), 2 M NH3 in EtOH, 18 h, 60%
(two steps); (g) cyclohexadiene, 10% Pd/C, EtOH, reflux, 2 d, 72%; (h) pTsCl, DIPEA,
DMAP, CH2Cl2, rt, 2 d, 97%; (i) NaH, ROH, THF/DMF (1/1), 100 °C, o/n; (j)
methanesulfonic acid, CH2Cl2, 50 °C, o/n, 16–70% (two steps); (k) NaH, BnBr, THF,
rt, 17 h, 86%; (l) TFA, CH2Cl2, 0 °C, 1 h, 92%.
In an effort to investigate the substitution of the sulfonyl moiety
for a carbonyl, the construction of the corresponding amide deriv-
atives of 3 and 4 was undertaken (Scheme 3). A similar approach as
above was taken to build up the pyrimidoindolone core starting
with commercially available 5-bromoisatin (15). An exocyclic vinyl
group was then introduced using a palladium-mediated Stille cou-
pling. Ozonolysis followed by saponification of the resultant ester
yielded carboxylic acid 18, which was then routinely coupled with
(S)-2-(methoxymethyl)-pyrrolidine or (S)-2-(phenoxymethyl)-pyr-
rolidine.9 Deprotection of the ketal, as before, provided the desired
amides 19a–b.
The corresponding reverse sulfonamide analogs were prepared
starting from 5-nitroisatin (20) as shown in Scheme 4. Once again,
the 3-carbonyl was protected and the nitrogen was alkylated with
3-chloro-2,2-dimethyl-propionitrile. Hydrogenation under Raney
Figure 1. X-ray co-crystal of 3 with human caspase-3 (left) and with the residues within 5 Å illustrated (right).

L. M. Havran et al. / Bioorg. Med. Chem. 17 (2009) 7755–7768
7757
nickel catalysis transformed the nitrile to the primary amine with
concomitant reduction of the 5-nitro group. Heating in a sealed
tube in 2 M NH3 in EtOH provided the protected pyrimidoindolone
21. Compound 21 could be treated with a variety of sulfonyl chlo-
rides to give the corresponding sulfonamides. The target reverse
sulfonamides 22a–h were obtained following the usual ketal
deprotection.
Spiroannulated pyrimidoindolone derivatives (25a–f) were pre-
pared as shown in Scheme 5 by methods very similar to those de-
scribed previously. The key step was the alkylation of 23 with the
appropriate 2,2-spiroalkyl-3-chloro-propanenitriles (26a–c).20
Once synthesized, all compounds were tested in a fluorescence
based recombinant human caspase-3 assay for their ability to inhi-
bit substrate (AcDEVD-AFC) cleavage by caspase-3 using a proce-
dure that has been described previously.21–23 Data are shown in
Tables 1–5.
NR1R2
TsO
3. Results and discussion
O
S
O
O
N
O
N
Early in our caspase program, our SAR studies examined the sul-
fonamide region of the molecule with the goals of improving bind-
ing affinity and modifying the physicochemical properties of the
lead compound. As our program became more advanced, we iden-
tified an issue with the aqueous stability of the pyrimidoindolone
ring system itself and then focused on improvement of this moiety.
These efforts are detailed below.
O
a, b
S
O
N
O
N
N
N
10
14a-d
Scheme 2. Reagents and conditions: (a) NHR1R2, THF, 100 °C, 2 d; (b) methane-
sulfonic acid, CH2Cl2, rt, o/n, 36–76% (two steps).
Initially, our SAR studies focused on the substitution at the
phenoxy substituent of the pyrrolidine. Overall, multiple groups
were generally well tolerated as seen in Table 1. From this exami-
nation, the optimal aryl groups identified were phenyl (3,
IC50 = 7 nM), or the 4-substituted derivatives; 4-F-phenyl (11a,
IC50 = 10.77 nM) and 4-CH3O-phenyl (11b, IC50 = 13.83 nM). How-
ever, bulky groups at the 4-position, such as t-butyl (11e), resulted
in a 10-fold reduction in potency from the parent compound 3.
Disubstituted analogs were prepared (entries 11f–g), but, unfortu-
nately, were 2–6-less potent as compared to the corresponding
4-monosubstituted analogs (e.g., 11a vs 11f, 11b vs 11g). In an
attempt to improve the physical chemical properties, the 2- and
3-pyridyl (11h–n) analogs were prepared and were shown to be
very potent with the advantage of increased solubility.24
O
O
O
Br
Br
e
a-d
f, g
O
N
N
N
H
16
15
O
O
O
O
h, i
O
HO
N
N
N
N
18
17
Replacement of the phenoxy moiety with simple amines was
also examined (Table 2) with the goal of improving solubility.
These changes were also well tolerated with morpholino (14a) or
N-carboxyethyl-piperazine (14b) proving to be the optimal substit-
uents for enzyme inhibition. Similar to the pyridyl analogs, these
amino-based analogs demonstrated improved physicochemical
OR
N
O
O
N
N
19a-b
Scheme 3. Reagents and conditions: (a) 1,3-propanediol, pTsOH, PhH, reflux, 14 h,
85%; (b) 3-chloro-2,2-dimethyl-propionitrile, KOt-Bu, DMSO, 135 °C, 21 h, 82%; (c)
H2, Ra/Ni, 2 M NH3 in EtOH/THF, 22 h; (d) 135 °C (sealed tube), 2 M NH3 in EtOH, 18,
85% (two steps); (e) vinyl Sn(nBu)3, Pd(PPh3)4, dioxane, 100 °C, 6 h, 79%; (f) O3, 2.5 N
NaOH, MeOH, ꢀ60 °C, 15 m, 54%; (g) 1 N NaOH, H2O, THF/EtOH, reflux, 1 h, 50%; (h)
(S)-2-(methoxymethyl)-pyrrolidine or (S)-2-(phenoxymethyl)-pyrrolidine, DCC,
HOBT, TEA, CH2Cl2, rt, o/n; (i) methanesulfonic acid, CH2Cl2, rt, o/n, 20–26% (two
steps).
RO
RO
O
O
O
N
a-c
d
O
N
O
S
5
S
O
O
O
O
CN
O
N
CN
N
H
(
)
n
Cl
23a R = CH3
23b R = Ph
(
)
n
26a n = 1
26b n = 2
26c n = 3
R = CH3
R = Ph
24a n = 1 24b n = 1
O
O
H2N
O
24d n = 2
24f n = 3
O2N
24c n = 2
24e n = 3
a-d
N
O
N
RO
N
H
21
20
O
N
e-g
O
S
O
N
O
N
H
N
O
N
R
S
e, f
N
O
(
)
n
25a-f
22a-h
Scheme 5. Reagents and conditions: (a) POCl3, sulfolane, 60 °C, 3 h, 66%; (b) (S)-2-
(methoxymethyl)-pyrrolidine or (S)-2-(phenoxymethyl)-pyrrolidine, DIPEA, CH2Cl2,
rt, 1 h, 62–93%; (c) 1,3-propanediol, pTsOH, PhH, reflux, 14 h, 45–62%; (d) 26a–c,
KOt-Bu, DMSO, 185 °C, 21 h, 72–92%; (e) H2, Ra/Ni, 2 M NH3 in EtOH/THF, 22 h; (f)
135 °C (sealed tube), 2 M NH3 in EtOH, 18 h, 64–87% (two steps); (g) methanesulf-
onic acid, CH2Cl2, rt, 14 h, 48–68%.
Scheme 4. Reagents and conditions: (a) 1,3-propanediol, pTsOH, PhH, reflux, 14 h,
96%; (b) 3-chloro-2,2-dimethyl-propionitrile, KOt-Bu, DMSO, 180 °C, 21 h, 90%; (c)
H2, Ra/Ni, 2 M NH3 in EtOH/THF, 22 h; (d) 135 °C (sealed tube), 2 M NH3 in EtOH,
18 h, 67% (two steps); (e) R-PhSO2Cl, Et3N, CH2Cl2, rt, 1 h; (f) methanesulfonic acid,
CH2Cl2, rt, 15 h, 15–58% (two steps).

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L. M. Havran et al. / Bioorg. Med. Chem. 17 (2009) 7755–7768
Table 1
Phenoxy analogs
RO
O
O
N
S
O
N
N
Compound
R
IC50 (nM)
c log D @ pH 7.4
Solubility @ pH 7.4 (lg/mL)
3
Phenyl
4-F-phenyl
4-OCH3-phenyl
4-Cl phenyl
4-Ac phenyl
4-t-Bu phenyl
4-F-3-CH3 phenyl
4-OCH3-2-Cl phenyl
2-Pyridyl
5-Cl 2-pyridyl
6-CH3 2-pyridyl
3-Pyridyl
5-Cl 3-pyridyl
2-CH3 3-pyridyl
5-CO2CH3 3-pyridyl
7
4.84
5.06
4.91
5.58
4.43
6.91
5.57
5.65
3.47
4.21
3.99
3.47
4.21
3.99
3.56
11
0
17
5
39
1
1
11a
11b
11c
11d
11e
11f
11g
11h
11i
11j
11k
11l
11m
11n
10.77 0.49
13.83 1.42
37
30
81
23
82
3
3
6
2
6
7
7.81 0.55
10.24 0.52
12.57 0.61
2.40 0.13
4.43 0.51
7.18 0.59
10.14 0.72
13
28
55
>100
30
66
>100
Table 2
Table 4
Amino analogs
Reverse sulfonamide analogs
NR1R2
O
H
O
S
N
R
N
O
O
O
N
N
S
O
N
N
Compound
R
IC50 (nM)
364 23
22a
22b
22c
22d
22e
22f
H
2-F
Compound NR1R2
IC50 (nM)
c log D @
pH 7.4
Solubility
@ pH 7.4
g/mL)
148
191
6
9
3-CF3
3-OCH3
4-Cl
4-OCH3
4-OCF3
4-F
350 58
1338 92
1503 159
2524 95
2394 140
(
l
14a
14b
14c
14d
4.99 1.30 1.56
14.32 1.17 0.82
43.63 2.68 1.44
34
45
47
55
HN
N
O
22g
22h
N
CO2CH2CH3
HN
H2N
N
Table 5
Spirocyclic analogs
59
8
1.98
RO
N
O
O
S
O
N
(
Table 3
Amide analogs
N
OR
N
)
n
O
O
Compound
R
n
IC50 (nM)
Stability:
% Parent @ 24 h
N
N
25a
25b
25c
25d
25e
25f
CH3
Ph
CH3
Ph
CH3
Ph
1
1
2
2
3
3
3.30 0.28
1.29 0.51
14.83 1.68
2.32 0.32
13.61 0.24
24.63 4.96
74
69
87
89
71
72
Compound
R
% Inhibition
19a
19b
Ph
CH3
25% @ 50
lM
10% @ 1 lM
properties (solubility,24 lower calculated log D25) than the parent
compound, 3.
After determining that the phenoxy region was amenable to a
variety of changes, we examined the role of the sulfonamide

L. M. Havran et al. / Bioorg. Med. Chem. 17 (2009) 7755–7768
7759
moiety. The amide analogs of 3 (19a) or 4 (19b) (Table 3) were con-
siderably less potent than the corresponding sulfonamides, thus
limiting the breadth of this substitution, and further attempts to
alter this moiety were halted.
3.1. Stability studies of compound 3
As work focused on the pyrimidoindolone series, the apparent
stability of these compounds was questioned as key experiments
showed the potency of several analogs substantially decreased
over time under standard aqueous conditions.26 Extensive NMR
studies determined that the conversion (shown in Scheme 6) was
occurring under neutral conditions and that as much as 30% of 3
rearranged after 24 h (Fig. 2). Under these conditions, it is hypoth-
esized that the parent structure 3 undergoes the addition of two
molecules of water to form the dihydrate intermediate 27. Subse-
quent expulsion of a water molecule and opening to the nine-
membered keto-amide structure 28 completes the transformation.
It was initially postulated that we could improve the stability of
this class of molecules by modifying the electrophilicity of the car-
bonyl group of the isatin. However, this carbonyl is key to the
inhibitory activity and must remain sufficiently electrophilic in or-
der for the active site cysteine to form a covalent bond leading to
inactivation of the enzyme. As observed with most caspase inhib-
itors, the formation of a covalent bond between an electrophilic
group and the active site cysteine plays the primary role in the
compound’s ability to inhibit caspase-3. In exploring the electro-
philic nature of this key moiety, we turned to an examination of
the energetic characteristics of the carbonyl. The reactivity of the
carbonyl in parent compound 3 is dependent on its LUMO energy.
The lower the LUMO energy, the easier it is for nucleophiles such as
water and cysteine sulfur to share electrons with the carbonyl car-
bon, thereby resulting in chemical reaction. The calculated LUMO
of the 10-carbonyl group of the parent compound 3 is ꢀ30 kcal/
mol. By proposing a series of reverse sulfonamides, we strove to re-
tain the non-covalent interactions between the sulfonamide por-
tion of the molecule and the enzyme while reducing the
electrophilicity of the 10-carbonyl. The hope was that by modulat-
ing the electrophilicity of the ketone, the new entity would be
resistant to intermolecular attack by water and subsequent ring
expansion, but could still form a bond with the active site cysteine.
Molecular modelling calculations determined the calculated LUMO
of the ketone in the reverse sulfonamide 22a to be ꢀ22 kcal/mol, a
change of 8 kcal/mol. Modelling analysis of an overlay of com-
pounds 3 and 22a (Fig. 3) shows that the sulfonyl groups of both
molecules can form similar hydrogen bond interactions with
Arg-207. This study also suggests that the reverse sulfonamide
can make additional favourable interactions in the binding pocket
Figure 2. The time course NMR study illustrating the conversion of 3 to 28 via the
intermediate 27. Shown is the aromatic region of the proton NMR spectra with the
integration values (in parenthesis) for each resonance in DMSO and for the first
time interval of the time course study. The arrows indicate resonances arising from
the formation of 28. Resonances from other trace byproducts can also be observed.
OPh
OPh
O
HO
O
O
N
OH
OH
N
S
S
O
2 H2O
10
O
NH
N
N
N
3
27
OPh
O
O
O
N
S
NH
O
- H2O
HN
28
Scheme 6. Conversion of 3 to 28 under standard aqueous conditions.
Figure 3. Molecular modelling overlay of compound 3 (magenta) and 22a (violet).

7760
L. M. Havran et al. / Bioorg. Med. Chem. 17 (2009) 7755–7768
such as an edge to face
of 22a and Phe-256 and a hydrogen bond between the sulfonamide
NH with the guanidine group of Arg-207.
p
–
p
interaction27 between the phenyl ring
studies. Furthermore, these analogs maintained low nanomolar
capsase-3 inhibitory activity. Somewhat surprisingly, the next ring
size increment, the spirocyclohexyl analogs (25e or 25f), while
maintaining potency, did not improve stability over earlier leads.
Attempts to determine the reason for the improved stability
associated with the spirocyclopentyl analogs by molecular model-
ling calculations of the energy differences between the parents, the
hydrated species and the open nine-membered degradation prod-
ucts have not yet led to definitive conclusions. However, we are
continuing studies to order to understand this effect. Nevertheless,
with the results of the SAR program and our ability to favourably
impart stability to the ring system, this pyridoindolone lead series
shows excellent promise as caspase-3 inhibitors.
Based on this modelling analysis, a series of reverse sulfona-
mides 22a–h (Table 4) was prepared and evaluated. Although
these compounds were not as potent as some of our earlier leads,
we were gratified to see that we could dramatically reduce the
electrophilicity of the carbonyl and maintain a reasonable level
of caspase-3 inhibition. This study also revealed a clear preference
for ortho or meta substitution on the aryl ring of the reverse sulfon-
amide. The most potent compounds from this series were the 2-F
(22b, IC50 = 148 nM) and the 3-CF3 (22c, IC50 = 191 nM) analogs.
para-Substituted compounds 22e–h showed a dramatic reduction
in activity. It seems likely that substitution at the 4-position of
the phenyl ring cannot be easily accommodated in the active site,
leading to an unfavourable interaction and the observed reduction
in binding affinity as compared to ortho or meta substitution.
Selected members of the reverse sulfonamide series were chosen
for NMR stability studies, however, these compounds showed no
improvement in ring stability over the earlier lead 3. This work
indicated that despite dramatically reducing the electrophilicity
of the 10-carbonyl group, hydration in the reversed sulfonamide
series is still a facile event which ultimately leads to nine-member
ring formation.
Based on the disappointing results for the reverse sulfonamides,
we explored a different approach to improve the stability of the
pyrimidoindolones (Scheme 7). NMR studies on early program
leads that were unsubstituted at the 3-position, such as 29, indi-
cated that these compounds were extremely unstable in aqueous
media, with none of the parent structure remaining after 24 h.
Introduction of a gem-dimethyl group imparted improved stability
at 24 h that we observed for compound 3.17 In an attempt to in-
crease stability of the ring system by substitution at this position
and to avoid metabolic stability liabilities, we chose to tie the
methyl groups together to afford the spiroannulated structure 25.
A series of spiroannulated analogs (25a–f) investigating the effects
of ring size were prepared and are shown in Table 5.
4. Conclusion
In summary, we have described the synthesis and examined the
structure–activity relationships for a series of pyrimidoindolone
capsase-3 inhibitors. We determined that the phenoxy region
was amenable to variety of substituents, which can be used to
modify the physicochemical properties of the molecules. Addition-
ally, the stability of this series under aqueous biological assay con-
ditions was improved by the preparation of spiropentacyclic
analogs 25c and 25d while maintaining low nanomolar caspase-3
inhibitory activity.
5. Experimental
5.1. General methods: chemistry
Melting points were determined on a Thomas–Hoover capillary
or an Electrothermal melting point apparatus and are uncorrected.
1H NMR spectra were recorded on a Varian Unity Plus 400 or a
Varian 500 Unity INOVA spectrometer. The chemical shifts (d) are
reported in parts per million (ppm) downfield from zero relative
to the residual DMSO signal (2.49 ppm). Coupling constants are
reported in hertz (Hz). Mass spectra were recorded on a Hewlett–
Packard 5995A, a Finnigan Trace MS or a Micromass LCT spectrom-
eter. C,H,N combustion analyses were determined on either a
Perkin–Elmer 2400 analyzer or were performed by Robertson
Microlit (Madison, NJ). Unless otherwise noted, reagents were
obtained from commercial sources and were used without further
purification. Chromatographic purifications were performed by
Spirocyclobutyl analogs with either methoxy-methyl pyrroli-
dine (25a) or phenoxy-methyl pyrrolidine (25b) sulfonamide sub-
stituents were some of the most potent compounds prepared with
IC50 values of 3.30 and 1.29 nM, respectively. Unfortunately, they
showed a stability profile similar to that of 3 with 69–74% of the
parent structure remaining after 24 h. However, the spirocyclopen-
tyl analogs with either methoxy (25c) or phenoxy (25d) substitu-
tion showed an improved stability profile with 87–89% of the
parent structure remaining after 24 h in the NMR aqueous stability
flash chromatography using Baker 40-lm silica gel.
5.1.1. (S)-2-(Benzyloxymethyl)-pyrrolidine (13)
Step 1: To a slurry of sodium hydride (60%) (2.18 g, 54.50 mmol,
1.1 equiv) in THF (100 mL) was added (S)-1-(tert-butoxycarbonyl)-
2-pyrrolidinemethanol (10 g, 49.68 mmol, 1 equiv) in THF
(100 mL) and the mixture was stirred at room temperature for
1 h. Benzyl bromide (8.86 mL, 74.5 mmol, 1.5 equiv) was added
and the reaction was stirred at room temperature overnight. It
was then poured into brine and extracted with ethyl acetate. The
combined organics were washed with brine, dried over magnesium
sulfate and concentrated. The crude residue purified by column
chromatography using ethyl acetate/hexanes (10/90) as an eluent
to give (S)-tert-butyl 2-(benzyloxymethyl) pyrrolidine-1-carboxyl-
ate as a yellow oil (12.41 g, 86%). 1H NMR (400 MHz, DMSO-d6) d
ppm 7.32–7.21 (m, 5H) 4.44 (s, 2H) 3.82–3.71 (m, 1H) 3.49–3.39
(m, 1H) 3.33–3.23 (m, 1H) 3.20–3.1 (m, 2H) 1.90–1.63 (m, 4H)
1.23–1.40 (m, 9H).
OPh
OCH3
O
O
N
O
O
N
S
S
O
N
O
N
N
N
3
3
29
3
OR
O
O
N
S
O
N
(
N
R = Ph or CH3
n = 1-3
Step 2: A solution of (S)-tert-butyl 2-(benzyloxymethyl) pyrroli-
dine-1-carboxylate (5.57 g, 19.11 mmol, 1 equiv) in CH2Cl2 (15 mL)
was cooled to 0 °C and TFA (15 mL) was added. The reaction was
stirred at room temperature for 1 h and then poured carefully into
)
n
25a-f
Scheme 7. Spiroannulated analogs.

L. M. Havran et al. / Bioorg. Med. Chem. 17 (2009) 7755–7768
7761
1 N NaOH. The pH was adjusted to basic with 2.5 N NaOH and it was
extracted with CH2Cl2. The combined organics dried over sodium
sulfate and concentrated to afford the title compound as yellow
oil (3.35 g, 92%), which was used crude in the following reaction.
1H NMR (400 MHz, DMSO-d6) d ppm 7.35–7.21 (m, 5H) 4.45 (s,
2H) 3.30–3.20 (m, 2H) 3.18–3.10 (m, 1H) 2.78–2.65 (m, 2H) 2.33
(br s, 1H) 1.73–1.66 (m, 1H) 1.61–1.52 (m, 2H) 1.34–1.27 (m, 1H).
26.6 mmol, 1 equiv) all at one time under a dry N2 atmosphere.
After stirring 20 min, 3-chloro-2,2-dimethylpropionitrile20 (9.38
g, 79.8 mmol, 3 equiv) was added drop-wise and the reaction
was heated at 132 °C for 21 h. After cooling in an ice bath, the reac-
tion mixture was poured into H2O and extracted with Et2O. The
combined organic extracts were dried over Na2SO4, filtered and
concentrated. The crude product was purified on Biotage KP silica
gel using a step gradient of CH2Cl2/CH3OH/NH4OH (99.25/0.5/0.25
to 96/2/1) to give the title compound as a yellow solid (14.21 g,
99%yield). 1H NMR (500 MHz, DMSO-d6) d ppm 7.89 (dd, J = 8.5,
1.8 Hz, 1H) 7.68 (d, J = 1.8 Hz, 1H) 7.53 (d, J = 8.2 Hz, 1H) 7.40–
7.23 (m, 5H) 4.73 (m, 2H) 4.50 (s, 2H) 4.01–3.91 (m, 4H) 3.75–
3.65 (m, 1H) 3.57 (dd, J = 9.5, 4.0 Hz, 1H) 3.42 (dd, J = 9.3, 7.8 Hz,
1H) 3.29 (m, 1H) 3.09–2.99 (m, 1H) 2.33–2.18 (m, 1H) 1.83–1.73
(m, 2H) 1.68 (m, 1H) 1.56–1.45 (m, 2H) 1.38 (s, 6H).
5.1.2. General Procedure A—amidation—50-({(2S)-2-
[(benzyloxy)methyl]pyrrolidin-1-yl}sulfonyl)-1H-indole-2,3-
dione (6)
Step 1: A mixture of isatin-5-sulfonic acid sodium salt dihydrate
(5) (10.00 g, 35.1 mmol, 1 equiv) and phosphorous oxychloride
(18.5 mL, 198 mmol, 5.6 equiv) in tetramethylene sulfone (50 mL)
was heated at 60 °C for 3 h under a dry N2 atmosphere. The reac-
tion was cooled in an ice bath to 0 °C and water was cautiously
added drop-wise, keeping the internal temperature below 6 °C.
The resulting green solid was collected by filtration and was
washed well with water. The solid was dissolved in ethyl acetate
(200 mL) and washed again with water (3 ꢁ 50 mL), dried over
magnesium sulfate, filtered and concentrated. The crude product
was recrystallized from ethyl acetate/hexanes with hot filtration
to give 2,3-dioxo-2,3-dihydro-1H-indole-5-sulfonyl chloride
(5.81 g, 66%). 1H NMR (400 MHz, DMSO-d6) d ppm 11.09 (s, 1H)
7.77 (dd, J = 8.1, 1.7 Hz, 1H) 7.55 (d, J = 1.6 Hz, 1H) 6.84 (d,
J = 8.1 Hz, 1H).
5.1.5. General Procedure D—reduction/cyclization—80-({(2S)-2-
[(benzyloxy)methyl]pyrrolidin-1-yl}sulfonyl)-30,30-dimethyl-
30,40-dihydro-20H-spiro[1,3-dioxane-2,100-pyrimido[1,2-
a]indole] (9)
A mixture of 8 (3.00 g, 5.56 mmol, 1 equiv), wet Raney nickel
(3.18 g) in 2 M NH3 in EtOH (180 mL) and THF (30 mL) was hydro-
genated at 54 psi for 22 h. After the reaction was filtered through
Celite, the filtrate was poured into a sealed tube and heated to
132 °C for 18 h. After cooling to room temperature, the reaction
was concentrated and purified on Biotage KP silica gel eluting with
CH2Cl2/CH3OH/NH4OH (98.5/1/0.5) to give the title compound as a
white solid (1.77 g, 60%). 1H NMR (400 MHz, DMSO-d6) d ppm 7.77
(dd, J = 8.3, 1.9 Hz, 1H) 7.56 (d, J = 1.8 Hz, 1H) 7.44–7.13 (m, 5H)
6.91 (d, J = 8.2 Hz, 1H) 5.04 (m, 2H) 4.50 (s, 2H) 3.85–3.75 (m,
2H) 3.69–3.60 (m, 1H) 3.57 (dd, J = 9.3, 3.7 Hz, 1H) 3.40 (m, 1H)
3.28–3.18 (m, 5H) 3.05–2.90 (m, 1H) 2.17 (m, 1H) 1.82–1.68 (m,
2H) 1.57 (m, 1H) 1.53–1.42 (m, 2H) 0.95 (s, 6H).
Step 2: To a suspension of 2,3-dioxo-2,3-dihydro-1H-indole-5-
sulfonyl chloride (9.33 g, 38.0 mmol, 1 equiv) in a 1:1 mixture of
CHCl3/THF (410 mL) was added drop-wise a solution of (S)-2-(ben-
zyloxymethyl)-pyrrolidine (8.00 g, 41.8 mmol, 1.1 equiv) and N,N-
diisopropylethylamine (12.2 mL, 70.0 mmol, 1.8 equiv) in CHCl3
(63 mL) over 1.25 h with cooling in an ice bath under a dry N2
atmosphere. The reaction was complete (by TLC) after stirring for
1 h at room temperature. The reaction was concentrated and puri-
fied on silica gel eluting with 50/50 pet ether/EtOAc to give the title
5.1.6. {(2S)-1-[(30,30-Dimethyl-30,40-dihydro-20H-spiro[1,3-
dioxane-2,100-pyrimido[1,2-a]indol]-80-yl)sulfonyl]pyrrolidin-
2-yl}methyl 4-methylbenzenesulfonate (10)
compound as
a
bright orange solid (11.8 g, 77%). 1H NMR
(400 MHz, DMSO-d6) d ppm 11.40 (br s, 1H) 7.98 (dd, J = 8.5,
2.0 Hz, 1H) 7.73 (d, J = 1.7 Hz, 1H) 7.36–7.24 (m, 5H) 7.04 (d,
J = 8.2 Hz, 1H) 4.48 (s, 2H) 3.71–3.67 (m, 1H) 3.58–3.53 (m, 1H)
3.42–3.38 (m, 1H) 3.30–3.25 (m, 1H) 3.08–3.02 (m, 1H) 1.80–
1.71 (m, 2H) 1.55–1.47 (m, 2H).
Step 1: A mixture of 9 (0.250 g, 0.480 mmol, 1 equiv) and 10%
Pd/C (0.250 g) in EtOH (10 mL) was degassed for 20 min, then 1,4
cyclohexadiene (5 mL) was added and the mixture was refluxed
for 2 days. The reaction was filtered through Celite and the filtrate
was concentrated. The crude product was purified on Biotage KP
silica gel eluting with acetone/hexane (30/70) to give {(2S)-1-
[(30,30-dimethyl-30,40-dihydro-20H-spiro[1,3-dioxane-2,100-pyrimi-
do[1,2-a]indol]-80-yl)sulfonyl]pyrrolidin-2-yl}methanol as a white
foam (0.150 g, 72%). 1H NMR (500 MHz, DMSO-d6) d ppm 7.77
(dd, J = 8.2, 1.8 Hz, 1H) 7.57 (d, J = 1.8 Hz, 1H) 6.94 (d, J = 8.2 Hz,
1H) 5.10–4.99 (m, 2H) 4.83 (t, J = 5.8 Hz, 1H) 3.84–3.76 (m, 2H)
3.59–3.51 (m, 1H) 3.48–3.40 (m, 1H) 3.31–3.22 (m, 6H) 3.00–
2.91 (m, 1H) 2.25–2.12 (m, 1H) 1.83–1.68 (m, 2H) 1.59 (m, 1H)
1.53–1.42 (m, 1H) 1.42–1.32 (m, 1H) 0.96 (s, 6H). MS: (ES+) m/z
436.1 [M+H].
5.1.3. General Procedure B—ketalization—preparation of 50-
({(2S)-2-[(Benzyloxy)methyl]pyrrolidin-1-yl}sulfonyl)spiro[1,3-
dioxane-2,30-indol]-20(10H)-one (7)
A suspension of 6 (11.8 g, 29.4 mmol, 1 equiv), p-toluenesul-
fonic acid monohydrate (2.24 g, 11.8 mmol, 0.4 equiv) and 1,3-pro-
panediol (8.63 mL, 119.4 mmol, 4 equiv) in benzene (527 mL) was
refluxed for 14 h with a Dean Stark Trap. After cooling to room
temperature, the reaction was washed with satd aq NaHCO3
(3ꢁ), water (3ꢁ) and brine (3ꢁ), dried over Na2SO4, filtered and
concentrated. The crude product was purified on silica gel eluting
with 60/40 pet ether/EtOAc to give the title compound as a yellow
foam (12.25 g, 90%). 1H NMR (500 MHz, DMSO-d6) d ppm 10.88 (s,
1H) 7.77–7.72 (m, 1H) 7.57 (d, J = 1.9 Hz, 1H) 7.43 (d, J = 7.9 Hz, 1H)
7.33–7.25 (m, 5H) 4.74–4.67 (m, 2H) 4.47 (s, 2H) 3.96–3.87 (m, 2H)
3.67–3.60 (m, 1H) 3.55–3.52 (m, 1H) 3.40–3.30 (m, 1H) 3.27–3.20
(m, 1H) 3.00–2.90 (m, 1H) 2.25–2.10 (m, 1H) 1.80–1.70 (m, 2H)
1.65–1.57 (m, 1H) 1.50–1.40 (m, 2H). MS: (ES+) m/z 459.1 [M+H].
Step 2: A solution of {(2S)-1-[(30,30-dimethyl-30,40-dihydro-20H-
spiro[1,3-dioxane-2,100-pyrimido[1,2-a]indol]-80-yl)sulfonyl]pyrr-
olidin-2-yl}methanol (0.100 g, 0.23 mmol, 1 equiv), p-toluenesul-
fonyl chloride (0.070 g, 0.34 mmol, 1.5 equiv), N,N-diisopro
pylethylamine (0.100 mL, 0.58 mmol, 2.5 equiv) and 4-(dimethyl-
amino)pyridine (0.020 g, 0.07 mmol, 0.3 equiv) in CH2Cl2 (5 mL)
was stirred at rt for 2 days. The reaction was poured into brine
and extracted with EtOAc. The combined organic extracts were
dried over Na2SO4, filtered and concentrated to give the title com-
pound as a white foam (0.132 g, 97%). 1H NMR (400 MHz, DMSO-
d6) d ppm 7.81 (d, J = 8.3 Hz, 2H) 7.71 (dd, J = 8.4, 2.0 Hz, 1H)
7.54 (d, J = 2.0 Hz, 1H) 7.50 (d, J = 8.3 Hz, 2H) 6.92 (d, J = 8.4 Hz,
1H) 5.03 (m, 2H) 4.16–3.97 (m, 2H) 3.85–3.75 (m, 2H) 3.71–3.60
(m, 1H) 3.31 (s, 2H) 3.27–3.12 (m, 3H) 2.99–2.86 (m, 1H) 2.43 (s,
5.1.4. General Procedure C—alkylation—preparation of 3-[50-
({(2S)-2-[(Benzyloxy)methyl]pyrrolidin-1-yl}sulfonyl)-20-oxo
spiro[1,3-dioxane-2,30-indol]-10(20H)-yl]-2,2-dimethyl
propanenitrile (8)
To a solution of potassium t-butoxide (3.58 g, 31.9 mmol,
1.2 equiv) in anhydrous DMSO (72 mL) was added 7 (12.20 g,

7762
L. M. Havran et al. / Bioorg. Med. Chem. 17 (2009) 7755–7768
3H) 2.25–2.08 (m, 1H) 1.78–1.36 (m, 5H) 0.95 (s, 6H). MS: (ES+) m/
z 590.3 [M+H].
MS: (ES+) m/z 496.1 [M+H]. HRMS calcd for C26H29N3O5S:
496.1901; found: 496.1902.
5.1.7. 8-({(2S)-2-[(4-Methoxyphenoxy)methyl]pyrrolidin-1-yl}
sulfonyl)-3,3-dimethyl-3,4-dihydropyrimido[1,2-a]indol-10
(2H)-one (11b)
5.1.11. 8-({(2S)-2-[(4-tert-Butylphenoxy)methyl]pyrrolidin-1-
yl}sulfonyl)-3,3-dimethyl-3,4-dihydropyrimido[1,2-a]indol-10
(2H)-one (11e)
To
a
solution of 4-methoxy-phenol (0.104 g 0.838 mmol,
The title compound was prepared as yellow foam in 40% yield
from 10 and 4-t-butylphenol according to the procedure for 11b.
1H NMR (500 MHz, DMSO-d6) d ppm 8.07 (dd, J = 8.4, 2.0 Hz, 1H)
7.83 (d, J = 1.8 Hz, 1H) 7.30 (d, J = 8.8 Hz, 2H) 7.20 (d, J = 8.5 Hz,
1H) 6.86 (d, J = 8.8 Hz, 2H) 4.08 (dd, J = 9.6, 3.5 Hz, 1H) 3.93 (m,
1H) 3.86 (m, 1H) 3.46 (s, 2H) 3.43–3.38 (m, 3H) 3.17–3.05 (m,
1H) 1.82 (m, 2H) 1.70–1.52 (m, 2H) 1.25 (s, 9H) 0.98 (s, 6H). MS:
(ESꢀ) m/z 508.2 [MꢀH]. HRMS calcd for C28H35N3O4S [M+H]:
510.2421; found: 510.2420.
1.4 equiv) in THF (5 mL) was added NaH (60% dispersion in mineral
oil) (0.048 g, 1.200 mmol, 2 equiv) and the reaction was stirred at
rt for 1 h. Compound 10 (0.352 g, 0.59 mmol, 1 equiv) in 1/1 THF
/DMF (6 mL) was then added and the reaction heated overnight
at 100 °C. The reaction was quenched with water and extracted
with EtOAc. The combined organic extracts were dried over
Na2SO4, filtered, concentrated and purified on silica gel eluting
with acetone/hexane (30/70) to give the ketal intermediate as a
white foam (0.258 g, 80%). 0.149 g of the resulting intermediate
was dissolved in CH2Cl2 (5 mL), methanesulfonic acid (5 mL) was
added and the solution was stirred at 50 °C overnight. The reaction
mixture was poured onto ice, basified to pH 11 with ammonium
hydroxide and extracted with EtOAc. The combined organic ex-
tracts were dried over Na2SO4, filtered, concentrated and purified
on silica gel eluting with acetone/hexane (30/70) to give the title
5.1.12. 8-({(2S)-2-[(4-Fluoro-3-methylphenoxy)methyl]
pyrrolidin-1-yl}sulfonyl)-3,3-dimethyl-3,4-dihydropyrimido
[1,2-a]indol-10(2H)-one (11f)
The title compound was prepared as light brown foam in 70%
yield from 10 and 4-fluoro, 3-methylphenol according to the pro-
cedure for 11b. 1H NMR (400 MHz, DMSO-d6) d ppm 8.06 (dd,
J = 8.5, 2.0 Hz, 1H) 7.82 (d, J = 2.0 Hz, 1H) 7.18 (d, J = 8.5 Hz, 1H)
7.03 (dd, J = 9.1, 9.1 Hz, 1H) 6.85 (dd, J = 6.1, 3.0 Hz, 1H) 6.79–
6.64 (m, 1H) 4.00–4.12 (m, 1H) 3.97–3.92 (m, 2H) 3.47 (s, 2H)
3.44–3.35 (m, 3H) 3.21–3.08 (m, 1H) 2.20 (s, 3H) 1.99–1.76 (m,
2H) 1.75–1.51 (m, 2H) 0.99 (s, 6H). MS: (ES+) m/z 486.2 [M+H].
HRMS calcd for C25H28FN3O4S [M+H]: 486.1857; found: 486.1855.
compound as
a
light yellow foam (0.064 g, 48%). 1H NMR
(500 MHz, DMSO-d6) d ppm 8.05 (dd, J = 8.5, 1.8 Hz, 1H) 7.81 (d,
J = 1.8 Hz, 1H) 7.17 (d, J = 8.5 Hz, 1H) 6.84 (s, 4H) 4.02 (dd, J = 9.3,
2.9 Hz, 1H) 3.95–3.78 (m, 2H) 3.68 (s, 3H) 3.45 (s, 2H) 3.42–3.33
(m, 3H) 3.19–3.04 (m, 1H) 1.90–1.80 (m, 2H) 1.73–1.46 (m, 2H)
0.97 (s, 6H). MS: (ES+) m/z 484.1 [M+H]. HRMS calcd for
C25H29N3O5S [M+H]: 484.1900; found: 484.1898.
5.1.13. 8-({(2S)-2-[(2-Chloro-4-methoxyphenoxy)methyl]
pyrrolidin-1-yl}sulfonyl)-3,3-dimethyl-3,4-dihydropyrimido
[1,2-a]indol-10(2H)-one (11g)
5.1.8. 8-({(2S)-2-[(4-Fluorophenoxy)methyl]pyrrolidin-1-yl}
sulfonyl)-3,3-dimethyl-3,4-dihydropyrimido[1,2-a]indol-10
(2H)-one (11a)
The title compound was prepared as light brown foam in 33%
yield from 10 and 2-chloro 4-methoxyphenol according to the pro-
cedure for 11b. 1H NMR (500 MHz, DMSO-d6) d ppm 8.04 (dd,
J = 8.5, 1.8 Hz, 1H) 7.80 (d, J = 1.8 Hz, 1H) 7.14 (d, J = 8.5 Hz, 1H)
7.04 (d, J = 9.0 Hz, 1H) 6.99 (d, J = 3.0 Hz, 1H) 6.85 (dd, J = 9.0,
2.9 Hz, 1H) 4.12–4.04 (m, 1H) 4.03–3.96 (m, 1H) 3.96–3.85 (m,
1H) 3.72 (s, 3H) 3.46 (s, 2H) 3.44–3.35 (m, 3H) 3.23–3.13 (m, 1H)
2.10–1.83 (m, 2H) 1.80–1.54 (m, 2H) 0.98 (s, 3H) 0.97 (s, 3H).
MS: (ES+) m/z 518.1 [M+H]. HRMS calcd for C25H28ClN3O5S
[M+H]: 518.1511; found: 518.1507.
The title compound was prepared as yellow foam in 55% yield
from 10 and 4-fluorophenol according to the procedure for 11b.
1H NMR (400 MHz, DMSO-d6) d ppm 8.05 (dd, J = 8.5, 2.0 Hz, 1H)
7.81 (d, J = 2.0 Hz, 1H) 7.17 (d, J = 8.5 Hz, 1H) 7.14–7.04 (m, 2H)
6.98–6.85 (m, 2H) 4.05 (dd, J = 9.4, 3.2 Hz, 1H) 3.98–3.91 (m, 1H)
3.90–3.81 (m, 1H) 3.45 (s, 2H) 3.40 (s, 2H) 3.39–3.33 (m, 1H)
3.18–3.07 (m, 1H) 1.95–1.75 (m, 2H) 1.73–1.50 (m, 2H) 0.98 (s,
6H).MS: (ES+) m/z 472.2 [M+H]. HRMS calcd for C24H26FN3O4S
[M+H]: 472.1700; found: 472.1697.
5.1.9. 8-({(2S)-2-[(4-Chlorophenoxy)methyl]pyrrolidin-1-yl}
sulfonyl)-3,3-dimethyl-3,4-dihydropyrimido[1,2-a]indol-10
(2H)-one (11c)
5.1.14. 3,3-Dimethyl-8-({(2S)-2-[(pyridin-2-yloxy)methyl]
pyrrolidin-1-yl}sulfonyl)-3,4-dihydropyrimido[1,2-a]indol-
10(2H)-one (11h)
The title compound was prepared as brown foam in 52% yield
from 10 and 4-chlorophenol according to the procedure for 11b.
1H NMR (500 MHz, DMSO-d6) d ppm 8.06 (dd, J = 8.5, 1.6 Hz, 1H)
7.82 (d, J = 1.5 Hz, 1H) 7.31 (d, J = 8.8 Hz, 2H) 7.17 (d, J = 8.5 Hz,
1H) 6.94 (d, J = 8.8 Hz, 2H) 4.06 (dd, J = 9.6, 3.5 Hz, 1H) 3.97 (dd,
J = 7.0, 2.4 Hz, 1H) 3.93–3.81 (m, 1H) 3.46 (s, 2H) 3.40 (s, 2H)
3.39–3.34 (m, 1H) 3.21–3.09 (m, 1H) 1.98–1.76 (m, 2H) 1.75–
1.52 (m, 2H) 0.98 (s, 6H). MS: (ES+) m/z 488.1 [M+H]. HRMS calcd
for C24H26ClN3O4S [M+H]: 488.1405; found: 488.1408.
The title compound was prepared as yellow foam in 20% yield
from 10 and 2-pyridinol according to the procedure for 11b. 1H
NMR (500 MHz, DMSO-d6) d ppm 8.17 (m, 1H) 8.06 (dd, J = 8.6,
2.0 Hz, 1H) 7.84 (d, J = 1.7, 1H) 7.69 (m, 1H) 7.18 (d, J = 8.5, 1H)
6.97 (m, 1H) 6.77 (d, J = 8.4 Hz, 1H) 4.42 (dd, J = 10.7, 4.0 Hz, 1H)
4.18 (dd, J = 10.7, 7.4 Hz, 1H) 4.00–3.82 (m, 1H) 3.46 (s, 2H) 3.43
(s, 2H) 3.42–3.34 (m, 1H) 3.21–3.10 (m, 1H) 1.92–1.75 (m, 2H)
1.71–1.47 (m, 2H) 0.98 (s, 6H). MS: (ESꢀ) m/z 453.2 [MꢀH]. HRMS
calcd for C23H26N4O4S [M+H]: 455.1745; found: 455.1748.
5.1.10. 8-({(2S)-2-[(4-Acetylphenoxy)methyl]pyrrolidin-1-yl}
sulfonyl)-3,3-dimethyl-3,4-dihydropyrimido[1,2-a]indol-
10(2H)-one (11d)
5.1.15. 8-[((2S)-2-{[(5-Chloropyridin-2-yl)oxy]methyl}
pyrrolidin-1-yl)sulfonyl]-3,3-dimethyl-3,4-dihydropyrimido
[1,2-a]indol-10(2H)-one (11i)
The title compound was prepared as yellow foam in 46% yield
from 10 and 4-acetylphenol according to the procedure for 11b.
1H NMR (500 MHz, DMSO-d6) d ppm 8.06 (dd, J = 8.5, 1.8 Hz, 1H)
7.90 (d, J = 8.8 Hz, 2H) 7.81 (d, J = 1.8 Hz, 1H) 7.16 (d, J = 8.5 Hz,
1H) 7.01 (d, J = 8.8 Hz, 2H) 4.28–4.02 (m, 2H) 3.99–3.82 (m, 1H)
3.44 (s, 2H) 3.42–3.34 (m, 3H) 3.21–3.10 (m, 1H) 2.50 (s, 3H)
2.01–1.78 (m, 2H) 1.77–1.52 (m, 2H) 0.99 (s, 3H) 0.98 (s, 3H).
The title compound was prepared as a yellow foam in 16% yield
from 10 and 5-chloro-2-pyridinol according to the procedure for
11b. 1H NMR (400 MHz, DMSO-d6) d ppm 8.21 (d, J = 2.7 Hz, 1H)
8.05 (dd, J = 8.5, 2.0 Hz, 1H) 7.83 (d, J = 1.9 Hz, 1H) 7.79 (dd,
J = 8.8, 2.7 Hz, 1H) 7.17 (d, J = 8.5 Hz, 1H) 6.84 (d, J = 8.84 Hz, 1H)
4.39 (dd, J = 10.7, 4.1 Hz, 1H) 4.21 (dd, J = 10.7, 7.1 Hz, 1H) 3.93
(m, 1H) 3.46 (s, 2H) 3.40 (s, 2H) 3.37 (m, 1H) 3.17 (m, 1H) 1.83

L. M. Havran et al. / Bioorg. Med. Chem. 17 (2009) 7755–7768
7763
(m, 2H) 1.68 (m, 1H) 1.57 (m, 1H) 0.97 (s, 6H). MS: (ES+) m/z 489.2
[M+H]. HRMS calcd for C23H25ClN4O4S [M+H]: 489.1358; found:
489.1358.
J = 8.5 Hz, 1H) 4.32–4.08 (m, 2H) 3.98–3.91 (m, 1H) 3.89 (s, 3H)
3.45 (s, 2H) 3.43–3.36 (m, 3H) 3.22–3.09 (m, 1H) 2.02–1.43 (m,
4H) 0.97 (s, 6H). MS: (ES+) m/z 513.2 [M+H]. HRMS calcd for
C25H28N4O6S [M+H]: 513.1802; found: 513.1799.
5.1.16. 3,3-Dimethyl-8-[((2S)-2-{[(6-methylpyridin-2-yl)
oxy]methyl}pyrrolidin-1-yl)sulfonyl]-3,4-dihydropyrimido[1,2-
a]indol-10(2H)-one (11j)
5.1.21. 8-({(2S)-2-[(Cyclohexylamino)methyl]pyrrolidin-1-yl}
sulfonyl)-3,3-dimethyl-3,4-dihydropyrimido[1,2-a]indol-10
(2H)-one (14d)
The title compound was prepared as yellow foam in 32% yield
from 10 and 6-methyl-2-pyridinol according to the procedure for
11b. 1H NMR (400 MHz, DMSO-d6) d ppm 8.06 (dd, J = 8.5, 2.0 Hz,
1H) 7.83 (d, J = 2.0 Hz, 1H) 7.55 (m, 1H) 7.18 (d, J = 8.5 Hz, 1H)
6.82 (d, J = 7.1 Hz, 1H) 6.55 (d, J = 8.3 Hz, 1H) 4.40 (dd, J = 10.5,
3.9 Hz, 1H) 4.14 (dd, J = 10.5, 7.6 Hz, 1H) 4.03–3.85 (m, 1H) 3.47
(s, 2H) 3.45–3.36 (m, 3H) 3.22–3.10 (m, 1H) 2.40 (s, 3H) 1.97–
1.74 (m, 2H) 1.73–1.49 (m, 2H) 0.99 (s, 6H). MS: (ES+) m/z 469.2
[M+H]. HRMS calcd for C24H28N4O4S [M+H]: 469.1904; found:
469.1902.
To a solution of 10 (0.10 g, 0.17 mmol, 1 equiv) in THF (2 mL)
was added cyclohexylamine (0.034 g, 0.34 mmol, 2 equiv) and
the reaction was stirred at 70 °C overnight. Additional cyclohexyl-
amine (0.088 g, 0.85 mmol, 5 equiv) was added to the reaction and
stirred at 100 °C for 2 days. The reaction was quenched with water
and extracted with EtOAc. The combined organic extracts were
dried over Na2SO4, filtered, concentrated and purified on Biotage
Si 12+M cartridge silica gel eluting with acetone/hexane (35/65).
The resulting product was dissolved in CH2Cl2 (2 mL), methane-
sulfonic acid (2 mL) was added and the solution was stirred at rt
overnight. The reaction mixture was poured onto ice, basified to
pH 11 with ammonium hydroxide and extracted with EtOAc
(3ꢁ). The combined organic extracts were dried over Na2SO4, fil-
tered, concentrated and purified on Biotage Si 12+M cartridge silica
gel eluting with acetone/hexane (35/65) to give the title compound
as a yellow foam (0.036 g, 46%). 1H NMR (500 MHz, DMSO-d6) d
ppm 8.02 (d, J = 8.5 Hz, 1H) 7.80 (s, 1H) 7.20 (d, J = 8.5 Hz, 1H)
3.58–3.50 (m, 1H) 3.46 (s, 2H) 3.42 (s, 2H) 3.27–3.21 (m, 1H)
3.13–3.03 (m, 1H) 2.81–2.69 (m, 1H) 2.53 (m, 1H) 2.35 (m, 1H)
1.85–1.59 (m, 6H) 1.58–1.40 (m, 3H) 1.28–1.00 (m, 5H) 0.98 (s,
6H) NH proton not distinctively observed. MS: (ESI+) m/z 459.3
[M+H]. HRMS calcd for C24H34N4O3S [M+H]: 459.2424; found:
459.2423.
5.1.17. 3,3-Dimethyl-8-({(2S)-2-[(pyridin-3-yloxy)methyl]
pyrrolidin-1-yl}sulfonyl)-3,4-dihydropyrimido[1,2-a]indol-10
(2H)-one (11k)
The title compound was prepared as yellow foam in 39% yield
from 10 and 3-pyridinol according to the procedure for 11b. 1H
NMR (500 MHz, DMSO-d6) d ppm 8.26 (d, J = 2.7 Hz, 1H) 8.16 (dd,
J = 4.5, 1.3 Hz, 1H) 8.06 (dd, J = 8.5, 1.9 Hz, 1H) 7.82 (dd,
J = 1.8 Hz, 1H) 7.39 (ddd, J = 8.4, 2.9, 1.4 Hz, 1H) 7.32 (dd, J = 8.4,
4.5 Hz, 1H) 7.17 (d, J = 8.5 Hz, 1H) 4.14 (dd, J = 9.8, 4.7 Hz, 1H)
4.06 (dd, J = 9.8, 6.9 Hz, 1H) 3.92 (m, 1H) 3.46 (s, 2H) 3.40 (s, 2H)
3.37 (m, 1H) 3.14 (m, 1H) 1.92–1.77 (m, 2H) 1.66 (m, 1H) 1.55
(m, 1H) 0.96 (s, 6H). MS: (ESꢀ) m/z 453.2 [MꢀH]. HRMS calcd for
C23H26N4O4S [M+H]: 455.1745; found: 455.1748.
5.1.18. 8-[((2S)-2-{[(5-Chloropyridin-3-yl)oxy]methyl}
pyrrolidin-1-yl)sulfonyl]-3,3-dimethyl-3,4-dihydropyrimido
[1,2-a]indol-10(2H)-one (11l)
5.1.22. 3,3-Dimethyl-8-{[(2S)-2-(morpholin-4-ylmethyl)
pyrrolidin-1-yl]sulfonyl}-3,4-dihydropyrimido[1,2-a]indol-10
(2H)-one (14a)
The title compound was prepared as yellow foam in 62% yield
from 10 and 5-chloro-3-pyridinol according to the procedure for
11b. 1H NMR (400 MHz, DMSO-d6) d ppm 8.22 (dd, J = 11.7,
2.2 Hz, 2H) 8.06 (dd, J = 8.5, 2.0 Hz, 1H) 7.82 (d, J = 1.7 Hz, 1H)
7.59 (m, 1H) 7.17 (d, J = 8.5 Hz, 1H) 4.25–4.04 (m, 2H) 4.01–3.86
(m, 1H) 3.46 (s, 2H) 3.43–3.33 (m, 3H) 3.21–3.11 (m, 1H) 1.98–
1.78 (m, 2H) 1.76–1.54 (m, 2H) 0.98 (s, 6H). MS: (ES+) m/z 489.1
[M+H]. HRMS calcd for C23H25ClN4O4S [M+H]: 489.1358; found:
489.1357.
The title compound was prepared as yellow foam in 65% yield
from 10 and morpholine according to the procedure for 14d. 1H
NMR (400 MHz, DMSO-d6) d ppm 8.04 (dd, J = 8.5, 1.9 Hz, 1H)
7.81 (d, J = 1.8 Hz, 1H) 7.18 (d, J = 8.5 Hz, 1H) 3.76–3.65 (m, 1H)
3.54 (m, 4H) 3.45 (s, 2H) 3.42 (s, 2H) 3.28–3.22 (m, 1H) 3.10–
2.97 (m, 1H) 2.47–2.41 (m, 3H) 2.40–2.27 (m, 3H) 1.85–1.68 (m,
2H) 1.59–1.42 (m, 2H) 0.98 (s, 6H). MS: (ES+) m/z 447.2 [M+H].
HRMS calcd for C22H30N4O4S [M+H]: 447.2061; found: 447.2062.
5.1.23. Ethyl 4-({(2S)-1-[(3,3-dimethyl-10-oxo-2,3,4,10-tetra-
hydropyrimido[1,2-a]indol-8-yl)sulfonyl]pyrrolidin-2-yl}
methyl)piperazine-1-carboxylate (14b)
5.1.19. 3,3-Dimethyl-8-[((2S)-2-{[(2-methylpyridin-3-yl)oxy]
methyl}pyrrolidin-1-yl)sulfonyl]-3,4-dihydropyrimido[1,2-a]
indol-10(2H)-one (11m)
The title compound was prepared as yellow foam in 36% yield
from 10 and ethyl piperazine-1-carboxylate according to the pro-
cedure for 14d. 1H NMR (400 MHz, DMSO-d6) d ppm 8.05 (dd,
J = 8.4, 2.0 Hz, 1H) 7.82 (d, J = 2.0 Hz, 1H) 7.18 (d, J = 8.5 Hz, 1H)
4.02 (q, J = 7.1 Hz, 2H) 3.71 (m, 1H) 3.46 (s, 2H) 3.42 (s, 2H) 3.32
(m, 4H) 3.28–3.23 (m, 1H) 3.10–3.00 (m, 1H) 2.47–2.41 (m, 3H)
2.39–2.29 (m, 3H) 1.83–1.70 (m, 2H) 1.60–1.45 (m, 2H) 1.17 (t,
J = 7.2 Hz, 3H) 0.98 (s, 6H). MS: (ES+) m/z 518.2 [M+H]. HRMS calcd
for C25H35N5O5S [M+H]: 518.2432; found: 518.2430.
The title compound was prepared as yellow foam in 50% yield
from 10 and 2-methyl-3-pyridinol according to the procedure for
11b. 1H NMR (400 MHz, DMSO-d6) d ppm 8.05 (dd, J = 8.5, 2.0 Hz,
1H) 7.98 (dd, J = 4.8, 1.1 Hz, 1H) 7.81 (d, J = 2.0 Hz, 1H) 7.29 (dd,
J = 8.3, 1.2 Hz, 1H) 7.20–7.09 (m, 2H) 4.13–4.06 (m, 1H) 4.06–
3.91 (m, 2H) 3.46 (s, 2H) 3.43–3.35 (m, 3H) 3.24–3.13 (m, 1H)
2.31 (s, 3H) 2.01–1.83 (m, 2H) 1.72 (m, 1H) 1.60 (m, 1H) 0.98 (s,
6H). MS: (ES+) m/z 468.2 [M+H]. HRMS calcd for C24H28N4O4S
[M+H]: 469.1904; found: 469.1903.
5.1.24. 3,3-Dimethyl-8-({(2S)-2-[(4-methylpiperazin-1-yl)
methyl]pyrrolidin-1-yl}sulfonyl)-3,4-dihydropyrimido[1,2-a]
indol-10(2H)-one (14c)
The title compound was prepared as yellow foam in 76% yield
from 10 and n-methyl piperazine according to the procedure for
14d. 1H NMR (400 MHz, DMSO-d6) d ppm 8.04 (dd, J = 8.5, 2.0 Hz,
1H) 7.81 (d, J = 2.0 Hz, 1H) 7.19 (d, J = 8.5 Hz, 1H) 3.77–3.59 (m,
1H) 3.46 (s, 2H) 3.42 (s, 2H) 3.28–3.20 (m, 1H) 3.11–2.97 (m, 1H)
2.47–2.17 (m, 10H) 2.13 (s, 3H) 1.84–1.63 (m, 2H) 1.51–1.43 (m,
5.1.20. Methyl 5-({(2S)-1-[(3,3-dimethyl-10-oxo-2,3,4,10-tetra-
hydropyrimido[1,2-a]indol-8-yl)sulfonyl]pyrrolidin-2-yl}
methoxy)nicotinate (11n)
The title compound was prepared as yellow foam in 40% yield
from 10 and 5-hydroxynicotinic acid methyl ester according to
the procedure for 11b. 1H NMR (400 MHz, DMSO-d6) d ppm 8.68
(d, J = 1.7 Hz, 1H) 8.49 (d, J = 2.9 Hz, 1H) 8.06 (dd, J = 8.5, 2.0 Hz,
1H) 7.84 (d, J = 2.0 Hz, 1H) 7.77 (dd, J = 2.9, 1.7 Hz, 1H) 7.14 (d,

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L. M. Havran et al. / Bioorg. Med. Chem. 17 (2009) 7755–7768
2H) 0.97 (s, 6H). MS: (ES+) m/z 460.2 [M+H]. HRMS calcd for
C23H33N5O3S [M+H]: 460.2377; found: 460.2373.
EtOAc (3ꢁ). The combined organic extracts were dried over
Na2SO4, filtered and concentrated to give the title compound as
white solid (0.097 g, 50%). 1H NMR (400 MHz, DMSO-d6) d ppm
12.23 (br s, 1H) 7.91 (dd, J = 8.3, 1.7 Hz, 1H) 7.79 (d, J = 1.7 Hz,
1H) 6.81 (d, J = 8.3 Hz, 1H) 5.04 (m, 2H) 3.78 (m, 2H) 3.25 (s, 2H)
3.23 (s, 2H) 2.25–2.04 (m, 1H) 1.59 (m, 1H) 0.95 (s, 6H). MS:
(ES+) m/z 317.3 [M+H].
5.1.25. 80-Bromo-30,30-dimethyl-30,40-dihydro-20H-spiro[1,3-
dioxane-2,100-pyrimido[1,2-a]indole] (16)
Step 1: 50-Bromospiro[1,3-dioxane-2,30-indol]-20(10H)-one was
synthesized from 5-bromo isatin in 85% yield as a white solid
according to General Procedure B. 1H NMR (500 MHz, DMSO-d6)
d ppm 10.58 (br s, 1H) 7.52–7.39 (m, 2H) 6.77 (d, J = 8.2 Hz, 1H)
4.76–4.64 (m, 2H) 3.95–3.86 (m, 2H) 2.20–2.05 (m, 1H) 1.70–
1.58 (m, 1H). MS: (ES) m/z 282.0 [MꢀH].
5.1.28. 3,3-Dimethyl-8-{[(2S)-2-(phenoxymethyl)pyrrolidin-1-
yl]carbonyl}-3,4-dihydropyrimido[1,2-a]indol-10(2H)-one
(19a)
Step
2:
3-(50-Bromo-20-oxospiro[1,3-dioxane-2,30-indol]-
To a solution of 18 (0.090 g, 0.284 mmol, 1 equiv) in CH2Cl2
(7 mL) was added (S)-2-(phenoxymethyl)-pyrrolidine9 (0.75 g,
0.423 mmol, 1.5 equiv), DCC (0.082 g, 0.397 mmol, 1.4 equiv), HOBT
(0.038 g, 0.284 mmol, 1 equiv) and Et3N (0.045 mL, 0.323 mmol,
1.1 equiv) and the solution was stirred overnight at rt. The reaction
was filtered through 1 cm of silica gel washing with EtOAc. The fil-
trate was dried over Na2SO4, filtered, concentrated and purified on
silica gel eluting with acetone/hexane (30/70). The resulting white
foam was dissolved in CH2Cl2 (1.5 mL), methanesulfonic acid
(1 mL) was added and the solution was stirred at rt overnight. The
reaction was poured onto ice, basified to pH 11 with ammonium
hydroxide and extracted with EtOAc (3ꢁ). The combined organic ex-
tracts were dried over Na2SO4, filtered, concentrated and purified on
Biotage Si 12+M cartridge silica gel eluting with acetone/hexane (35/
65) to give the title compound as a yellow foam (0.021 g, 20%). 1H
NMR (400 MHz, DMSO-d6) d ppm 7.74 (dd, J = 8.3, 1.2 Hz, 1H) 7.60
(d, J = 1.1 Hz, 1H) 7.26 (m, 2H) 7.01 (d, J = 8.3 Hz, 1H) 6.98–6.77
(m, 3H) 4.41 (m, 1H) 4.30–3.94 (m, 2H) 3.58–3.48 (m, 1H) 3.43 (m,
3H) 3.37 (s, 2H) 2.17–1.61 (m, 4H) 0.97 (s, 6H). (Resonance broaden-
ing due to amide rotamers). MS: (ES+) m/z 418.3 [M+H] HRMS calcd
for C25H27N3O3 [M+H]: 418.2125; found: 418.2125.
10(20H)-yl)-2,2-dimethylpropanenitrile was synthesized from 50-
bromospiro[1,3-dioxane-2,30-indol]-20(10H)-one in 82% yield as a
white solid according to General Procedure C. 1H NMR (500 MHz,
DMSO-d6) d ppm 7.61 (dd, J = 8.5, 2.1 Hz, 1H) 7.52 (d, J = 2.1 Hz,
1H) 7.31 (d, J = 8.5 Hz, 1H) 4.76–4.59 (m, 2H) 3.99–3.91 (m, 2H)
3.87 (s, 2H) 2.25–2.08 (m, 1H) 1.73–1.63 (m, 1H) 1.36 (s, 6H).
MS: (EI) m/z 364.0 [M+].
Step 3: The title compound was synthesized from 3-(50-bromo-
20-oxospiro[1,3-dioxane-2,30-indol]-10(20H)-yl)-2,2-dimethylpro-
panenitrile in 85% yield as a light brown solid according to General
Procedure D. 1H NMR (500 MHz, DMSO-d6) d ppm 7.46 (dd, J = 8.4,
2.0 Hz, 1H) 7.37 (d, J = 1.8 Hz, 1H) 6.71 (d, J = 8.5 Hz, 1H) 5.01 (m,
2H) 3.76 (m, 2H) 3.21 (s, 2H) 3.18 (s, 2H) 2.23–1.98 (m, 1H) 1.57
(m, 1H) 0.93 (s, 6H). MS: (EI) m/z 350 [MꢀH].
5.1.26. 30,30-Dimethyl-80-vinyl-30,40-dihydro-20H-spiro[1,3-
dioxane-2,100-pyrimido[1,2-a]indole] (17)
To the stirred solution of 16 (2.576 g, 7.33 mmol, 1 equiv) in
dioxane (70 mL) was added tributyl(vinyl) tin (3.490 g,
11.00 mmol, 1.5 equiv) and the solution was degassed with N2
for 15 min. Pd(PPh3)4 (0.507 g, 0.439 mmol, 0.06 equiv) was added
and the mixture was stirred at 100 °C for 6 h. The reaction was
diluted with EtOAc, washed with brine and concentrated. The
resulting residue was dissolved in EtOAc and stirred with 1 M aq
potassium fluoride solution (20 mL) overnight. The reaction was
filtered and the filtrate was washed with brine. The organics were
dried over Na2SO4, concentrated and purified on a silica gel column
eluting with acetone/hexane (10/90) to give the title compound as
white solid (1.720 g, 79%). 1H NMR (400 MHz, DMSO-d6) d ppm
7.37–7.32 (m, 2H) 6.67–6.59 (m, 2H) 7.63 (d, J = 17.5 Hz, 1H)
5.05–5.00 (m, 3H) 3.75–3.71 (m, 2H) 3.21 (s, 2H) 3.18 (s, 2H)
2.15–2.05 (m, 1H) 1.55–1.50 (m, 1H) 0.91 (s, 6H).
5.1.29. 8-{[(2S)-2-(Methoxymethyl)pyrrolidin-1-yl]carbonyl}-
3,3-dimethyl-3,4-dihydropyrimido[1,2-a]indol-10(2H)-one
(19b)
The title compound was prepared as an orange foam in 26%
yield from 18 and (S)-2-(methoxymethyl)-pyrrolidine according
to the procedure for 17a. 1H NMR (400 MHz, DMSO-d6) d ppm
7.76 (dd, J = 8.2, 1.6 Hz, 1H) 7.61 (d, J = 1.5 Hz, 1H) 7.04 (d,
J = 8.3 Hz, 1H) 4.26–4.11 (m, 1H) 3.51–3.39 (m, 4H) 3.37–3.30 (s,
2H) 3.29–3.15 (m, 5H) 2.04–1.57 (m, 4H) 0.97 (s, 6H) (Resonance
broadening due to amide rotamers). MS: (ES+) m/z 356.2 [M+H].
HRMS calcd for C20H25N3O3 [M+H]: 356.1969; found: 356.1967.
5.1.27. 30,30-Dimethyl-30,40-dihydro-20H-spiro[1,3-dioxane-
2,100-pyrimido[1,2-a]indole]-80-carboxylic acid (18)
5.1.30. 30,30-Dimethyl-30,40-dihydro-20H-spiro[1,3-dioxane-
2,100-pyrimido[1,2-a]indol]-80amine (21)
Step 1: To a solution of 17 (0.687 g, 2.303 mmol, 1 equiv) in
CH2Cl2 (15 mL), was added 2.5 N NaOH (7.5 mL) and MeOH
(7.5 mL) and the solution was cooled to ꢀ60 °C. A stream of O3
was bubbled into the reaction for 15 min until TLC indicated start-
ing material was consumed. H2O (20 mL) was then added to the
reaction and it was extracted with EtOAc. The combined organics
were dried over Na2SO4, filtered, concentrated and purified on a
silica gel column eluting with acetone/hexane (10/90) to give
methyl 30,30-dimethyl-30,40-dihydro-20H-spiro[1,3-dioxane-2,100-
pyrimido[1,2-a]indole]-80-carboxylate as a white solid (0.410 g,
54%). 1H NMR (400 MHz, DMSO-d6) d ppm 7.94 (dd, J = 8.3,
1.7 Hz, 1H) 7.80 (d, J = 1.5 Hz, 1H) 6.85 (d, J = 8.5 Hz, 1H) 5.04 (m,
2H) 3.81 (s, 3H) 3.80–3.75 (m, 2H) 3.26 (s, 2H) 3.24 (s, 2H) 2.23–
2.10 (m, 1H) 1.59 (m, 1H) 0.95 (s, 6H). MS: (ES+) m/z 331.2 [M+H].
Step 2: To a solution of methyl 30,30-dimethyl-30,40-dihydro-
20H-spiro[1,3-dioxane-2,100-pyrimido[1,2-a]indole]-80-carboxylate
(0.200 g, 0.610 mmol, 1 equiv) in 1/1 THF /EtOH (8 mL) was added
1 N NaOH (2 mL) and H2O (2 mL) and the reaction was refluxed for
1 h. The reaction was quenched with 1 N HCl and extracted with
Step 1: 50-Nitrospiro[1,3-dioxane-2,30-indol]-20(10H)-one was
synthesized from 5-nitro isatin in 96% yield according to General
Procedure B. 1H NMR (400 MHz, DMSO-d6) d ppm 11.17 (s, 1H)
8.26 (dd, J = 8.7, 2.4 Hz, 1H) 8.08 (d, J = 2.6 Hz, 1H) 7.02 (d,
J = 8.6 Hz, 1H) 4.72 (m, 2H) 3.95 (m, 2H) 2.31–2.10 (m, 1H) 1.76–
1.59 (m, 1H).
Step 2: 3-(50-Nitro-20-oxospiro[1,3-dioxane-2,30-indol]-10(20H)-
yl)-2,2-dimethylpropanenitrile was prepared in 90% yield from
50-nitrospiro[1,3-dioxane-2,30-indol]-20(10H)-one and 3-chloro-
2,2-dimethylpropionitrile20 following General Procedure C. 1H
NMR (400Mz, DMSO-d6): d ppm 8.35 (dd, J = 8.8, 2.4 Hz, 1H) 8.10
(d, J = 2.3 Hz, 1H) 7.57 (d, J = 8.8 Hz, 1H) 4.72–4.65 (m, 2H) 3.97
(m, 2H) 3.95 (s, 2H) 2.23 (m, 1H) 1.69 (m, 1H) 1.36 (s, 6H).
Step 3: The title compound was prepared in 67% yield from 3-
(50-nitro-20-oxospiro[1,3-dioxane-2,30-indol]-10(20H)-yl)-2,2-dim-
ethylpropanenitrile following General Procedure D. 1H NMR
(400Mz, DMSO-d6): d ppm 6.62 (d, J = 2.2 Hz, 1H) 6.45 (dd,
J = 8.1, 2.3 Hz, 1H) 6.37 (d, J = 8.1 Hz, 1H) 5.05 (m, 2H) 4.63 (s,

L. M. Havran et al. / Bioorg. Med. Chem. 17 (2009) 7755–7768
7765
2H) 3.68 (m, 2H) 3.08 (s, 2H) 3.06 (s, 2H) 2.04 (m, 1H) 1.52 (m, 1H)
5.1.36. N-(3,3-Dimethyl-10-oxo-2,3,4,10-tetrahydropyrimido
0.89 (s, 6H).
[1,2-a]indol-8-yl)-4-methoxybenzenesulfonamide (22f)
The title compound was prepared as a yellow foam in 29% yield
from 21 and 4-methoxy benzenesulfonyl chloride according to the
procedure for 22a. 1H NMR (400Mz, DMSO-d6): 1H 1H NMR
(400 MHz, DMSO-d6) d ppm 9.99 (s, 1H) 7.59 (d, J = 8.9 Hz,, 2H)
7.27 (dd, J = 8.5, 2.2 Hz, 1H) 7.14 (d, J = 2.2 Hz, 1H) 7.05 (d,
J = 9.0 Hz, 2H) 6.91 (d, J = 8.5 Hz, 1H) 3.78 (s, 3H) 3.37 (s, 2H)
3.25 (s, 2H) 0.93 (s, 6H). MS: (APPI) m/z 398 [MꢀH]. HRMS calcd
for C20H21N3O4S [M+H]: 400.1325; found: 400.1325.
5.1.31. N-(3,3-Dimethyl-10-oxo-2,3,4,10-tetrahydropyrimido
[1,2-a]indol-8-yl)benzenesulfonamide (22a)
To a solution of 21 (0.060 g, 0.21 mmol, 1 equiv) in CH2Cl2
(2 mL) was added Et3N (0.060 mL, 0.43 mmol, 2.05 equiv) and ben-
zenesulfonyl chloride (0.030 mL, 0.23 mmol, 1.07 equiv). The reac-
tion was stirred at rt 1 h and concentrated. The crude residue
purified by column chromatography using acetone/hexanes (30/
70) as an eluent. The resulting white solid was dissolved in CH2Cl2
(4 mL) and methanesulfonic acid (2 mL) was added. The reaction
was stirred at rt 15 h. and then poured into brine. It was basified
to pH 10, saturated with solid NaCl and extracted with EtOAc.
The combined organics were dried over Na2SO4 and concentrated.
The crude residue purified by column chromatography using ace-
tone/hexanes (40/60) as an eluent to give the title compound as
a yellow foam (0.032 g, 58%). 1H NMR (500Mz, DMSO-d6): d ppm
10.14 (br s, 1H) 7.70 (m, 2H) 7.61 (m, 1H) 7.55 (m, 2H) 7.13 (dd,
J = 8.6, 2.2 Hz, 1H) 7.27 (d, J = 2.1 Hz, 1H) 6.91 (d, J = 8.5 Hz, 1H)
3.36 (s, 2H) 3.25 (s, 2H) 0.92 (s, 6H). MS: (ESꢀ) m/z 368 [MꢀH].
HRMS calcd for C19H19N3O3S [M+H]: 370.1220; found: 370.1217.
5.1.37. N-(3,3-Dimethyl-10-oxo-2,3,4,10-tetrahydropyrimido
[1,2-a]indol-8-yl)-4-(trifluoromethoxy)benzenesulfonamide
(22g)
The title compound was prepared as a yellow foam in 17% yield
from 21 and 4-trifluoromethoxy benzenesulfonyl chloride accord-
ing to the procedure for 22a. 1H NMR (400 MHz, DMSO-d6) d ppm
10.26 (br s, 1H) 7.82 (d, J = 8.8 Hz, 2H) 7.55 (d, J = 8.8 Hz, 2H) 7.27
(dd, J = 8.5, 2.2 Hz, 1H) 7.14 (d, J = 2.0 Hz, 1H) 6.93 (d, J = 8.5 Hz,
1H) 3.37 (s, 2H) 3.26 (s, 2H) 0.93 (s, 6H). MS: (APPI) m/z 452
[MꢀH]. HRMS calcd for C20H18F3N3O4S [M+H]: 454.1043; found:
454.1048.
5.1.38. N-(3,3-Dimethyl-10-oxo-2,3,4,10-tetrahydropyrimido
[1,2-a]indol-8-yl)-4-fluorobenzenesulfonamide (22h)
5.1.32. N-(3,3-Dimethyl-10-oxo-2,3,4,10-tetrahydropyrimido
[1,2-a]indol-8-yl)-2-fluorobenzenesulfonamide (22b)
The title compound was prepared as a yellow foam in 17% yield
from 21 and 4-fluoro benzenesulfonyl chloride according to the
procedure for 22a. 1H NMR (400 MHz, DMSO-d6) d ppm 10.15 (br
s, 1H) 7.75 (m, 2H) 7.46–7.33 (m, 2H) 7.26 (dd, J = 8.7, 2.1 Hz,
1H) 7.14 (d, J = 2.2 Hz, 1H) 6.91 (d, J = 8.5 Hz, 1H) 3.37 (s, 2H)
3.26 (s, 2H) 0.93 (s, 6H). MS: (APPI) m/z 386 [MꢀH]. HRMS calcd
for C19H18FN3O3S [M+H]: 388.1126; found: 388.1120.
The title compound was prepared as a yellow foam in 17% yield
from 21 and 2-fluorobenzenesulfonyl chloride according to the
procedure for 22a. 1H NMR (400 MHz, DMSO-d6) d ppm 10.45 (br
s, 1H) 7.74 (m, 1H) 7.71–7.61 (m, 1H) 7.41 (m, 1H) 7.37–7.27 (m,
2H) 7.17 (d, J = 2.0 Hz, 1H) 6.91 (d, J = 8.5 Hz, 1H) 3.36 (s, 2H)
3.25 (s, 2H) 0.92 (s, 6H). MS: (APPI) m/z 386 [MꢀH]. HRMS calcd
for C19H18FN3O3S [M+H]: 388.1125; found: 388.1120.
5.1.39. 50-{[(2S)-2-(Methoxymethyl)pyrrolidin-1-yl]sulfonyl}
spiro[1,3-dioxane-2,30-indol]-20(10H)-one (23a)
5.1.33. N-(3,3-Dimethyl-10-oxo-2,3,4,10-tetrahydropyrimido
[1,2-a]indol-8-yl)-3-(trifluoromethyl)benzenesulfonamide
(22c)
The title compound was prepared as a white solid in 61% yield
from isatinsulfonic acid sodium salt hydrate and (S)-2-(methoxy-
methyl)-pyrrolidine according to General Procedures A and B. 1H
NMR (500 MHz, DMSO-d6) d ppm 10.93 (s, 1H) 7.80 (dd, J = 8.2,
1.7 Hz, 1H) 7.61 (d, J = 1.5 Hz, 1H) 7.01 (d, J = 8.2 Hz, 1H) 4.74 (m,
2H) 3.93 (m, 2H) 3.63–3.55 (m, 1H) 3.44 (dd, J = 9.4, 3.7 Hz, 1H)
3.28 (s, 2H) 3.26 (s, 3H) 3.04–2.94 (m, 1H) 2.22 (m, 1H) 1.83–
1.59 (m, 3H) 1.52–1.40 (m, 2H). MS: (ESI) m/z 383 [M+H]. Mp:
127–128 °C.
The title compound was prepared as a yellow foam in 15% yield
from 21 and 3-trifluoromethyl benzenesulfonyl chloride according
to the procedure for 22a. 1H NMR (400 MHz, DMSO-d6) d ppm
10.29 (br s, 1H) 8.03 (d, J = 7.6 Hz, 1H) 7.98–7.93 (m, 2H) 7.81
(dd, J = 8.5, 2.2 Hz, 1H) 7.27 (dd, J = 8.4, 2.1 Hz, 1H) 7.11 (d,
J = 2.2 Hz, 1H) 6.94 (d, J = 8.6 Hz, 1H) 3.38 (s, 2H) 3.27 (s, 2H)
0.94 (s, 6H). MS: (APPI) m/z 436 [MꢀH]. HRMS calcd for
C20H18F3N3O3S [M+H]: 438.1094; found: 438.1087.
5.1.40. 50-{[(2S)-2-(Phenoxymethyl)pyrrolidin-1-yl]sulfonyl}
spiro[1,3-dioxane-2,30-indol]-20(10H)-one (23b)
5.1.34. N-(3,3-Dimethyl-10-oxo-2,3,4,10-tetrahydropyrimido
[1,2-a]indol-8-yl)-3-methoxybenzenesulfonamide (22d)
The title compound was prepared as a yellow foam in 51% yield
from 21 and 3-methoxy benzenesulfonyl chloride according to the
procedure for 22a. 1H NMR (400 MHz, DMSO-d6) d ppm 10.12 (br s,
1H) 7.45 (m, 1H) 7.28 (dd, J = 8.5, 2.2 Hz, 1H) 7.26–7.11 (m, 4H)
6.91 (d, J = 8.5 Hz, 1H) 3.75 (s, 3H) 3.37 (s, 2H) 3.25 (s, 2H) 0.92
(s, 6H). MS: (APPI) m/z 398 [MꢀH]. HRMS calcd for C20H21N3O4S
[M+H]: 400.1325; found: 400.1328.
The title compound was prepared as a white solid in 58% yield
from isatinsulfonic acid sodium salt hydrate and (S)-2-(phenoxy-
methyl)-pyrrolidine9 according to General Procedures A and B. 1H
NMR (400 MHz, DMSO-d6) d ppm 10.94 (br s, 1H) 7.83 (dd,
J = 8.3, 2.0 Hz, 1H) 7.65 (d, J = 1.7 Hz, 1H) 7.26–7.34 (m, 2H) 7.01
(d, J = 8.3 Hz, 1H) 6.98–6.91 (m, 3H) 4.74 (m, 2H) 4.10 (dd, J = 9.5,
3.7 Hz, 1H) 3.98–3.88 (m, 2H) 3.82 (dd, J = 7.3, 3.9 Hz, 1H) 3.36
(dd, J = 6.8, 3.4 Hz, 2H) 3.08–2.98 (m, 1H) 2.28–2.13 (m, 1H)
1.94–1.77 (m, 2H) 1.70–1.46 (m, 3H). MS: (ESI) m/z 445 [M+H].
Mp: 201–203 °C.
5.1.35. 4-Chloro-N-(3,3-dimethyl-10-oxo-2,3,4,10-tetrahydro
pyrimido[1,2-a]indol-8-yl)benzenesulfonamide (22e)
5.1.41. 1-{[50-{[(2S)-2-(Methoxymethyl)pyrrolidin-1-yl] sul
fonyl}-20-oxospiro[1,3-dioxane-2,30-indol]-10(20H)-yl] methyl}
cyclobutanecarbonitrile (24a)
The title compound was prepared as a white solid in 96% yield
from 23a and 1-chloromethyl-cyclobutanecarbonitrile20 according
to General Procedure C. 1H NMR (300 MHz, DMSO-d6) d ppm 7.91
(dd, J = 8.4, 2.0 Hz, 1H) 7.67 (d, J = 1.8 Hz, 1H) 7.51 (d, J = 8.4 Hz,
1H) 4.73 (dd, J = 11.4, 9.7 Hz, 2H) 4.15 (s, 2H) 3.96 (dd, J = 11.5,
The title compound was prepared as a yellow solid in 17% yield
from 21 and 4-chloro benzenesulfonyl chloride according to the
procedure for 22a. 1H NMR (400 MHz, DMSO-d6) d ppm 10.21 (br
s, 1H) 7.69 (d, J = 8.8 Hz, 2H) 7.63 (d, J = 8.8 Hz, 2H) 7.26 (dd,
J = 8.5, 2.4 Hz, 1H) 7.14 (d, J = 2.2 Hz, 1H) 6.92 (d, J = 8.5 Hz, 1H)
3.37 (s, 2H) 3.26 (s, 2H) 0.93 (s, 6H). MS: (APPI) m/z 402 [MꢀH].
HRMS calcd for C19H18ClN3O3S [M+H]: 404.0830; found:
404.0829. Mp: 161.1–162.4 °C.

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L. M. Havran et al. / Bioorg. Med. Chem. 17 (2009) 7755–7768
3.7 Hz, 2H) 3.65 (m, 1H) 3.50–3.40 (m, 1H) 3.33–3.28 (m, 2H) 3.25
(s, 3H) 3.03 (m, 1H) 2.43–2.33 (m, 4H) 2.32–2.20 (m, 1H) 2.13–1.97
(m, 2H) 1.83–1.62 (m, 3H) 1.56–1.39 (m, 2H). MS: (ES+) m/z 476.2
[M+H]. Mp: 130.0–131.1 °C.
3.42–3.34 (m, 1H) 3.09 (m, 1H) 2.33–2.17 (m, 1H) 1.95–1.79 (m,
4H) 1.77–1.30 (m, 11H). MS: (ES+) m/z 566.2 [M+H].
5.1.47. 80-{[(2S)-2-(Methoxymethyl)pyrrolidin-1-yl]sulfonyl}
spiro[cyclopentane-1,30-pyrimido[1,2-a]indol]-100(20H)-one
(25c)
5.1.42. 1-{[20-Oxo-50-{[(2S)-2-(phenoxymethyl)pyrrolidin-1-yl]
sulfonyl}spiro[1,3-dioxane-2,30-indol]-10(20H)-yl]methyl}
cyclobutanecarbonitrile (24b)
Step 1: 80-{[(2S)-2-(methoxymethyl)pyrrolidin-1-yl]sulfonyl}-
20H-dispiro[cyclopentane-1,30-pyrimido[1,2-a]indole-100,200-[1,3]
dioxane] was prepared as a white solid in 91% yield from 24c
according to General Procedure D. 1H NMR (400 MHz, DMSO-d6)
d ppm 7.79 (dd, J = 8.3, 1.8 Hz, 1H) 7.56 (d, J = 1.8 Hz, 1H) 6.95 (d,
J = 8.3 Hz, 1H) 4.96–5.10 (m, 2H) 3.73–3.84 (m, 2H) 3.58 (m, 1H)
3.46 (dd, J = 9.4, 3.8 Hz, 1H) 3.40 (s, 2H) 3.34 (s, 2H) 3.28–3.32
(m, 2H) 3.27 (s, 3H) 2.90–3.05 (m, 1H) 2.18 (m, 1H) 1.53–1.81
(m, 7H) 1.34–1.50 (m, 6H). MS: (ES+) m/z 476.2 [M+H].
The title compound was prepared as a white foam in 80% yield
from 23b and 1-chloromethyl-cyclobutanecarbonitrile20 according
to General Procedure C. 1H NMR (400 MHz, DMSO-d6) d ppm 7.94
(dd, J = 8.5, 2.0 Hz, 1H) 7.70 (d, J = 2.0 Hz, 1H) 7.51 (d, J = 8.5 Hz,
1H) 7.34–7.23 (m, 2H) 7.00–6.88 (m, 3H) 4.72 (m, 2H) 4.15 (s,
2H) 4.10 (dd, J = 9.6, 3.5 Hz, 1H) 4.00–3.91 (m, 3H) 3.90–3.80 (m,
1H) 3.42–3.33 (m, 1H) 3.08 (m, 1H) 2.43–2.33 (m, 4H) 2.31–2.17
(m, 1H) 2.11–2.01 (m, 2H) 1.94–1.79 (m, 2H) 1.73–1.45 (m, 3H).
MS: (ES+) m/z 538.2 [M+H].
Step 2: To a solution of 80-{[(2S)-2-(methoxymethyl)pyrrolidin-
1-yl]sulfonyl}-20H-dispiro[cyclopentane-1,30-pyrimido[1,2-a]in-
dole-100,200-[1,3]dioxane] (0.220 g, 0.46 mmol, 1 equiv) in CH2Cl2
(12 mL) was added methanesulfonic acid (4 mL). The reaction
was stirred at rt 14 h. and poured into brine. It was basified to
pH 13 with NH4OH and extracted with EtOAc. The combined
organics were dried over sodium sulfate and concentrated. The
crude residue purified by column chromatography using acetone/
hexane (25/75) as an eluent to give the title compound as a yellow
solid (0.112 g, 58%). 1H NMR (400 MHz, DMSO-d6) d ppm 8.04 (dd,
J = 8.5, 2.0 Hz, 1H) 7.80 (d, J = 2.0 Hz, 1H) 7.22 (d, J = 8.5 Hz, 1H)
3.66 (m, 1H) 3.57 (s, 2H) 3.50 (s, 2H) 3.45 (dd, J = 9.4, 3.8 Hz, 1H)
3.37–3.28 (m, 2H) 3.27 (s, 3H) 3.06 (m, 1H) 1.83–1.60 (m, 6H)
1.58–1.32 (m, 6H). Anal. Calcd for C21H27N3O4S: C, 60.41; H, 6.52;
N, 10.06. Found: C, 60.24; H, 6.42; N, 9.98. MS: (ESꢀ) m/z 416
[MꢀH]. HRMS calcd for C21H27N3O4S [M+H]: 418.1795; found:
418.1794. Mp: 171.2–171.9 °C.
5.1.43. 1-{[50-{[(2S)-2-(Methoxymethyl)pyrrolidin-1-yl] sulfo-
nyl}-20-oxospiro[1,3-dioxane-2,30-indol]-10(20H)-yl] methyl}
cyclopentanecarbonitrile (24c)
The title compound was prepared as a white solid in 77% yield
from 23a and 1-chloromethyl-cyclopentanecarbonitrile20 accord-
ing to General Procedure C. 1H NMR (400 MHz, DMSO-d6) d ppm
7.91 (dd, J = 8.3, 2.0 Hz, 1H) 7.67 (d, J = 2.0 Hz, 1H) 7.54 (d,
J = 8.3 Hz, 1H) 4.81–4.63 (m, 2H) 4.02 (s, 2H) 4.00–3.91 (m, 2H)
3.70–3.58 (m, 1H) 3.44 (dd, J = 9.4, 3.8 Hz, 1H) 3.34–3.28 (m, 2H)
3.26 (s, 3H) 3.09–2.98 (m, 1H) 2.36–2.18 (m, 1H) 2.05–1.95 (m,
2H) 1.92–1.83 (m, 2H) 1.83–1.64 (m, 7H) 1.54–1.39 (m, 2H). MS:
(ES+) m/z 490.2 [M+H]. Mp: 70.3–72.1 °C.
5.1.44. 1-{[20-Oxo-50-{[(2S)-2-(phenoxymethyl)pyrrolidin-1-yl]
sulfonyl}spiro[1,3-dioxane-2,30-indol]-10(20H)-yl]methyl}
cyclopentanecarbonitrile (24d)
5.1.48. 80-{[(2S)-2-(Methoxymethyl)pyrrolidin-1-yl]sulfonyl}
spiro[cyclobutane-1,30-pyrimido[1,2-a]indol]-100(20H)-one
(25a)
The title compound was prepared as a white solid in 79% yield
(2 steps) from 23b and 1-chloromethyl-cyclopentanecarbonitrile20
according to General Procedure C. 1H NMR (400 MHz, DMSO-d6) d
ppm 7.94 (dd, J = 8.5, 2.0 Hz, 1H) 7.70 (d, J = 2.0 Hz, 1H) 7.54 (d,
J = 8.5 Hz, 1H) 7.35–7.23 (m, 2H) 6.99–6.88 (m, 3H) 4.73 (m, 2H)
4.10 (dd, J = 9.5, 3.7 Hz, 1H) 4.01 (s, 2H) 3.99–3.92 (m, 3H) 3.86
(m, 1H) 3.44–3.33 (m, 2H) 3.16–3.02 (m, 1H) 2.34–2.15 (m, 1H)
2.06–1.95 (m, 1H) 1.93–1.65 (m, 8H) 1.65–1.45 (m, 2H) 1.28–
1.18 (m, 1H). MS: (ES+) m/z 552.22 [M+H]. Mp: 82.2–83.9 °C.
The title compound was prepared as a yellow solid in 54% yield
(two steps) from 24a according to the procedure for 25c. 1H NMR
(400 MHz, DMSO-d6) d ppm 8.06 (dd, J = 8.5, 1.9 Hz, 1H) 7.79 (d,
J = 1.7 Hz, 1H) 7.24 (d, J = 8.5 Hz, 1H) 3.73 (s, 2H) 3.70 (s, 2H)
3.61–3.69 (m, 1H) 3.45 (dd, J = 9.4, 3.8 Hz, 1H) 3.34–3.28 (m, 2H)
3.27 (s, 3H) 3.06 (m, 1H) 2.06–1.92 (m, 2H) 1.91–1.81 (m, 4H)
1.81–1.65 (m, 2H) 1.50 (m, 2H). MS: (ESꢀ) m/z 402 [MꢀH]. HRMS
calcd for C20H25N3O4S [M+H]: 404.1639; found: 404.1640. Mp:
146.8–147.4 °C.
5.1.45. 1-{[50-{[(2S)-2-(Methoxymethyl)pyrrolidin-1-yl]
sulfonyl}-20-oxospiro[1,3-dioxane-2,30-indol]-10(20H)-yl]
methyl}cyclohexanecarbonitrile (24e)
5.1.49. 80-{[(2S)-2-(Phenoxymethyl)pyrrolidin-1-yl]sulfonyl}
spiro[cyclobutane-1,30-pyrimido[1,2-a]indol]-100(20H)-one
(25b)
The title compound was prepared as a white foam in 76% yield
from 23a and 1-chloromethyl-cyclohexanecarbonitrile20 according
to General Procedure C. 1H NMR (400 MHz, DMSO-d6) d ppm 7.89
(dd, J = 8.4, 1.9 Hz, 1H) 7.67 (d, J = 2.0 Hz, 1H) 7.54 (d, J = 8.5 Hz,
1H) 4.81–4.60 (m, 2H) 4.01–3.95 (m, 2H) 3.94 (s, 2H)
3.69–3.60 (m, 1H) 3.44 (dd, J = 9.3, 3.9 Hz, 1H) 3.34–3.29 (m, 2H)
3.26 (s, 3H) 3.10–2.96 (m, 1H) 2.36–2.16 (m, 1H) 1.94–1.84 (m,
2H) 1.81–1.60 (m, 7H) 1.57–1.30 (m, 6H). MS: (ES+) m/z 504.2
[M+H].
The title compound was prepared as a light yellow foam in 29%
yield (two steps) from 24b according to the procedure for 25c. 1H
NMR (400 MHz, DMSO-d6) d ppm 8.08 (dd, J = 8.5, 2.0 Hz, 1H) 7.81
(d, J = 2.0 Hz, 1H) 7.31–7.24 (m, 2H) 7.22 (d, J = 8.5 Hz, 1H) 6.98–
6.87 (m, 3H) 4.08 (dd, J = 9.8, 3.4 Hz, 1H) 4.00–3.93 (m, 1H) 3.89
(m, 1H) 3.73 (s, 2H) 3.69 (s, 2H) 3.37 (m, 1H) 3.14 (m, 1H) 2.05–
1.92 (m, 2H) 1.93–1.78 (m, 6H) 1.73–1.64 (m, 1H) 1.63–1.52 (m,
1H). MS: (ESꢀ) m/z 464 [MꢀH]. HRMS calcd for C25H27N3O4S
[M+H]: 466.1795; found: 466.1797.
5.1.46. 1-{[20-Oxo-50-{[(2S)-2-(phenoxymethyl)pyrrolidin-1-yl]
sulfonyl}spiro[1,3-dioxane-2,30-indol]-10(20H)-yl]methyl}
cyclohexanecarbonitrile (24f)
The title compound was prepared as a white foam in 94% yield
from 23b and 1-chloromethyl-cyclohexanecarbonitrile20 according
to General Procedure C. 1H NMR (400 MHz, DMSO-d6) d ppm 7.92
(dd, J = 8.4, 1.8 Hz, 1H) 7.70 (d, J = 1.7 Hz, 1H) 7.54 (d, J = 8.5 Hz,
1H) 7.34–7.25 (m, 2H) 6.98–6.91 (m, 3H) 4.72 (m, 2H) 4.10 (dd,
J = 9.5, 3.7 Hz, 1H) 4.00–3.94 (m, 2H) 3.94 (s, 3H) 3.87 (m, 1H)
5.1.50. 80-{[(2S)-2-(Phenoxymethyl)pyrrolidin-1-yl]sulfonyl}
spiro[cyclopentane-1,30-pyrimido[1,2-a]indol]-100(20H)-one
(25d)
The title compound was prepared as a yellow solid in 29% yield
(two steps) from 24d according to the procedure for 25c. 1H NMR
(400 MHz, DMSO-d6) d ppm 8.06 (dd, J = 8.5, 2.0 Hz, 1H) 7.82 (d,
J = 2.0 Hz, 1H) 7.32–7.24 (m, 2H) 7.21 (d, J = 8.5 Hz, 1H) 6.97–

L. M. Havran et al. / Bioorg. Med. Chem. 17 (2009) 7755–7768
7767
6.87 (m, 3H) 4.08 (dd, J = 9.5, 3.4 Hz, 1H) 4.00–3.92 (m, 1H) 3.89
(m, 1H) 3.57 (s, 2H) 3.49 (s, 2H) 3.37 (m, 1H) 3.14 (m 1H) 1.97–
1.76 (m, 2H) 1.73–1.54 (m, 6H) 1.50–1.32 (m, 4H). Anal. Calcd for
C26H29N3O4S: C, 65.11; H, 6.09; N, 8.76. Found: C, 64.85; H, 5.98;
N, 8.60. MS: (ESꢀ) m/z 478 [MꢀH]. HRMS calcd for C26H29N3O4S
[M+H]: 480.1952. Found: 480.1956. Mp: 142.9.8–144.5 °C.
marin (Ac-DEVD-AFC purchased from Biomol). The assays are con-
ducted at pH 7.2 in a buffered system containing 20 mM PIPES,
100 mM NaCl, 1 mM EDTA, 0.1% CHAPS, 10% sucrose and 5 mM
L-cysteine. The final concentration of the substrate is 25 lM. Enzy-
mic cleavage between the aspartate and the AFC fluorophore liber-
ates 7-amino-4-trifluoromethyl coumarin which is detected using
an excitation wavelength of 400 nm and an emission wavelength
of 505 nm in a SpectraMax GeminiXS plate reader operated at
room temperature. A steady state rate of substrate cleavage is ob-
tained for analysis.
5.1.51. 80-{[(2S)-2-(Methoxymethyl)pyrrolidin-1-yl]sulfonyl}
spiro[cyclohexane-1,30-pyrimido[1,2-a]indol]-100(20H)-one
(25e)
The title compound was prepared as a light yellow foam in 41%
yield (two steps) from 24e according to the procedure for 25c. 1H
NMR (400 MHz, DMSO-d6) d ppm 8.04 (dd, J = 8.5, 1.9 Hz, 1H)
7.79 (d, J = 1.8 Hz, 1H) 7.31 (d, J = 8.5 Hz, 1H) 3.67 (m, 1H) 3.53
(s, 4H) 3.45 (dd, J = 9.3, 3.9 Hz, 1H) 3.34–3.28 (m, 2H) 3.27 (s,
3H) 3.07 (m, 1H) 1.82–1.64 (m, 2H) 1.58–1.42 (m, 7H) 1.41–1.22
(m, 5H). Anal. Calcd for C22H29N3O4S: C, 60.32; H, 6.69; N, 9.55.
Found: C, 60.69; H, 6.84; N, 9.52. MS: (ESꢀ) m/z 430 [MꢀH]. HRMS
calcd for C22H29N3O4S [M+H]: 432.1952; found: 432.1950.
For IC50 determination, typically 11 concentrations ranging
from 20
lM to 20 nM were freshly prepared by serial dilution with
assay buffer containing no cysteine with 80
l
L of 31.25 M sub-
l
strate added to the assay well. Once substrate and inhibitor were
added to the assay plate, the reaction was initiated by addition
of 10 lL of 2.5 nM enzyme, prepared in assay buffer containing
50 mM Cysteine, to the assay mixture (final concentration
0.25 nM). After the reaction was initiated with the addition of en-
zyme, AFC production was monitored continuously for 90 min by
exciting at 400 nm and measuring the emission at 505 nm every
42 s. The progress curves generated were fitted by computer to
5.1.52. 80-{[(2S)-2-(Phenoxymethyl)pyrrolidinyl]sulfonyl}spiro
[cyclohexane-1,30-pyrimido[1,2-a]indol]-100(20H)-one (25f)
The title compound was prepared as a light yellow foam in 31%
yield (two steps) from 24f according to the procedure for 25c. 1H
NMR (400 MHz, DMSO-d6) d ppm 8.06 (dd, J = 8.5, 2.0 Hz, 1H)
7.81 (d, J = 2.0 Hz, 1H) 7.29 (m, 3H) 6.99–6.85 (m, 3H) 4.08 (dd,
J = 9.4, 3.3 Hz, 1H) 3.99–3.93 (m, 1H) 3.89 (m, 1H) 3.53 (s, 2H)
3.52 (s, 2H) 3.38 (m, 1H) 3.14 (m, 1H) 1.96–1.79 (m, 2H) 1.73–
1.65 (m, 1H) 1.64–1.55 (m, 1H) 1.52–1.42 (m, 5H) 1.41–1.25 (m,
5H). MS: (ESꢀ) m/z 492 [MꢀH]. HRMS calcd for C27H31N3O4S
[M+H]: 494.2108; found: 494.2112.
n
n
n
*
Eq. 1 to generate an IC50 value. Eq. 1: y = Bmax (1 ꢀ (x /(K + x ))),
where Bmax is rate in the absence of inhibitor.
5.4. Modelling
Starting with the X-ray co-crystal structure of compound 3
bound to human caspase-3 a molecular mechanics optimization
of the whole structure was performed using Molecular Operating
Environment (MOE)28 and the MMFF94 potential.29 Using this
optimized complex compound 1 was then stripped down to its
maximum common substructure with compound 22a (pyrimidoin-
dolone fused heterocycle with attached gem-dimethyl). Then com-
pound 22a was grown into the binding pocket one torsion group at
a time. After each added torsion group a full rotor search was per-
formed to set the optimal torsion angle. Once compound 22a was
completely grown into the pocket a full minimization of 22a and
all caspase-3 residues within 5 Å was performed. The optimized
structures of compounds 3 and 22a bound to caspase-3 were then
used to analyze the interactions between the compounds and var-
ious residues in the caspase-3 binding site. Compounds 3 and 22a
were then removed from the caspase-3 binding pocket and opti-
mized to their nearest local minima using the MMFF94 potential.
The LUMO energies of compounds 3 and 22a were then calculated
in the PM3 approximation using MOPAC.30 The SYBYL31 interface
was used to perform these computations.
5.2. Preparation of active caspase-3
Caspase-3 was expressed intracellularly in Escherichia coli with
a c-terminal His tag. Fermentation was performed at 25 °C in a B.
Braun Biotech Biostat C 10 litre bioreactor vessel. The culture
was collected in 1 L bottles and centrifuged in Komspin KA-
7.1000 rotors at approximately 8000 RCF (Relative Centrifugal
Force). The cell pellets were re-suspended in 20 mM Tris pH 8.0,
500 mM NaCl and 5 mM imidazole. The cell suspension was dis-
rupted by passing 5 times through a microfluidizer Model 110Y
(Microfluidics Corp, Newton, Mass). After centrifugation (13 kg,
30 min at 4C), the supernatant was applied to a column of
Nickle-NTA agarose. The caspase-3 was eluted with a gradient of
5–150 mM imidazole in the above buffer. Fractions containing cas-
pase-3 were pooled and concentrated with a Millipore Ultrafree fil-
tration device. The concentrated caspase-3 solution was loaded
unto a TSK gel G3000sw column (Tosoh Bioseph LLC), equilibrated
with a buffer of 20 mM PIPES pH 7.2, 100 mM NaCl, 1 mM EDTA
and 5 mM Cysteine. Fractions containing caspase-3 were pooled
and concentrated. Sometimes CHAPS was added to 0.1% and
sucrose to 10% into the protein sample. The caspase-3 obtained
with this method shows two subunits of 17 and 13 kD on reduced
SDS–PAGE and aliquots stored at ꢀ80 °C.
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