Synthesis and biological evaluation of novel macrocyclic bis-7-azaindolylmaleimides as potent and highly selective glycogen synthase kinase-3β (GSK-3β) inhibitors

Article abstract of DOI:10.1016/j.bmc.2003.09.047

Palladium catalyzed cross-coupling reactions were used to synthesize two key intermediates 3 and 5 that resulted in the synthesis of novel series of macrocyclic bis-7-azaindolylmaleimides. Among the three series of macrocycles, the oxygen atom and thiophe

Full text of DOI:10.1016/j.bmc.2003.09.047

Bioorganic & MedicinalChemistry 12 (2004) 1239–1255
Synthesis and biological evaluation of novel macrocyclic
bis-7-azaindolylmaleimides as potent and highly selective
glycogen synthase kinase-3ꢀ (GSK-3ꢀ) inhibitors§
Lan Shen, Catherine Prouty, Bruce R. Conway, Lori Westover, Jun Z. Xu,
Richard A. Look, Xin Chen, Mary Pat Beavers, Jerry Roberts, William V. Murray,
Keith T. Demarest and Gee-Hong Kuo*
Drug Discovery Division, Johnson and Johnson Pharmaceutical Research and Development, L.L.C., 1000 Route 202,
PO Box300, Raritan, NJ 08869, USA
Received 2 June 2003; accepted 11 September 2003
Abstract—Palladium catalyzed cross-coupling reactions were used to synthesize two key intermediates 3 and 5 that resulted in the
synthesis of novel series of macrocyclic bis-7-azaindolylmaleimides. Among the three series of macrocycles, the oxygen atom and
thiophene containing linkers yielded molecules with higher inhibitory potency at GSK-3b (Ki=0.011–0.079 mM) while the nitrogen
atom containing linkers yielded molecules with lower potency (Ki=0.150–>1 mM). Compound 33 and 36 displayed 1–2 orders of
magnitude selectivity at GSK-3b against CDK2, PKCbII, Rsk3 and little or no inhibitions to the other 62 protein kinases. Com-
pound 46 was at least 100-fold more selective towards GSK-3b than PKCbII, and it had little or no activity against a panel of 65
protein kinases, almost behaved as a GSK-3b ‘specific inhibitor’. All three compounds showed good potency in GS assay. Molec-
ular docking studies were conducted in an attempt to rationalize the GSK-3b selectivity of azaindolylmaleimides. The high selec-
tivity, inhibitory potency and cellular activities of these non-crown-ether typed molecules may provide them as a valuable
pharmacological tools in elucidating the complex roles of GSK-3b in cell signaling pathways and the potential usage for the treat-
ment of elevated level of GSK-3b involved diseases.
# 2003 Elsevier Ltd. All rights reserved.
1. Introduction
receptor substrate-1 (IRS-1) and GS, the two key tar-
gets in the insulin signaling pathway.6 Suppression of
these targets may limit most insulin-mediated biological
responses. In addition, elevated GSK-3 activity was
found in diabetic tissues, reinforcing GSK-3 as a prom-
ising therapeutic target for insulin resistance and Type 2
diabetes. Tau is a known substrate of GSK-3 in vivo.
Tau hyperphosphorylation has been postulated to pro-
mote microtubule disassembly, an early event in the
progression of Alzheimer’s disease.7 Inhibition of GSK-
3 has also been shown to attenuate apoptotic signals.8
Therefore, GSK-3 inhibitors may be usefulfor the
treatment of Alzheimer’s disease and protection
against cell death.9 Lithium ions and valproic acid
have been used as mood stabilizers for the chronic
treatment of patients with bipolar disorder. Recently,
these compounds have been shown to be GSK-3
inhibitors.10 Finally, studies on fibroblasts from the
GSK-3b knockout mice indicate that inhibition of
GSK-3 may be usefulin treating inflammatory dis-
orders or diseases through the negative regulation of
NFkB activity.11
Glycogen synthase kinase-3 (GSK-3) is a serine/threo-
nine protein kinase that was first identified over 20 years
ago because of its ability to phosphorylate and inhibit
glycogen synthase (GS),1 the rate-limiting enzyme of
glycogen biosynthesis.2 Mammalian GSK-3 exists as
two isoforms, GSK-3a and GSK-3b, sharing 98%
homology in their catalytic domain.3Both isoforms are
ubiquitously expressed in cells and tissues, and have
similar biochemical properties.3 Today, it is known that
GSK-3 is involved in diverse cellular processes and
might have multiple substrates.4,5 For example, GSK-3
phosphorylates and inhibits the functioning of insulin
Keywords: Specific inhibitor; Kinase; Azaindolemaleimide; Cross–
x11dummy;coupling.
§Supplementary data associated with this article can be found, in the
online version, at doi:10.1016/j.bmc.2003.09.047.
* Corresponding author. Tel.: +1-908-704-4330; fax: +1-908-203-
8109; e-mail: gkuo@prdus.jnj.com
0968-0896/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2003.09.047

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L. Shen et al. / Bioorg. Med. Chem. 12 (2004) 1239–1255
Scheme 1.
Since Chiron disclosed purines12 (Scheme 1) are capable
of exhibiting GSK-3 inhibitory activities, many chemi-
The first one, N-methyl-bis-7-azaindolylmaleimide 3,
was synthesized in two steps from the known 3-iodo-7-
azaindole derivative 1.20 The tributyltin analogue of 2
was reported to synthesize from 1 in 19% yield.20 We
found out that if trimethyltin chloride was mixed with
the iodide 1 at ꢀ78 ꢁC before the addition of n-butyl-
lithium, the yield of 2 was increased to 55–60%. A pal-
ladium-catalyzed cross-coupling reaction21 of 2 with
2,3-dichloro -N-methylmaleimide gave the desired cou-
pling product 3.22 Later, it was found out that some of
the base-sensitive molecules were decomposed during
the base hydrolysis and ammonolysis conditions
required for the transformation of N-methylmaleimides
to N-H maleimides. Therefore, we prepared the N-2,4-
13,14
calseries
(pyrazines, pyrimidines, heterocyclic-pyr-
imidones, amine pyrazoles, bisindole-maleimides,
hymenialdisine, paullones, indirubines and anilino-mal-
eimides) have been developed as ATP competitive GSK-
3 inhibitors while thiadiazolidinones15 were reported to
be the first ATP non-competitive GSK-3 inhibitors.
However, except the anilino-maleimides16,17 were
demonstrated as selective GSK-3 inhibitors over 24
protein kinases, the majority of ATP competitive GSK-
3 inhibitors all showed significant activities at other
protein kinases. To minimize the potentialside effects, it
is always desirable to have the clinical agent targeting
the enzyme specifically.
dimethoxybenzyl-bis-7-zazindolylmaleimide
5
that
could be deprotected under acidic condition. Treatment
of 3,4-dichloro-furan-2,5-dione with 2,4-dimethoxy-
benzylamine in refluxing acetic acid gave dichloride 4.
A palladium-catalyzed cross-coupling reaction of 2 with
4 gave the desired N-2,4-dimethoxybenzylprotected
maleimide 5.
In an attempt to identify a potent and selective protein
kinase C gamma (PKCg) inhibitors for the treatment of
chronic pain, we unexpectedly discovered that poly-
oxygenated-macrocyclic-maleimides (Scheme 1) are
potent and highly selective GSK-3b inhibitors if both A
and B are nitrogen atoms and n=2 or 3 (n=2,
IC50=0.034 mM; n=3, IC50=0.048 mM).18 However,
these compounds are less attractive due to the cytotoxi-
city concern of its crown-ether typed structures.19 This
article describes our continued efforts to the identifica-
tion of severalnon-crown-ether typed macrocyclic bis-7-
azaindolylmaleimides as potent and highly selective
GSK-3b inhibitors.
The synthesis of the symmetricalnitrogen containing
macrocyclic maleimides 10 and 12 were shown in
Scheme 3. Alkylation of N-methylmaleimide 3 with the
bismesylate 7, which in turn was prepared from carba-
mate diol 6,23 in the presence of Cs2CO3 in DMF24 gave
the cyclic maleimide 8. Removalof the Boc protecting
group, following by hydrolysis and ammonolysis24 gave
the N-H cyclic maleimide 10. Ethylation of 9 gave 11.
Hydrolysis and ammonolysis of 11 provided the N-
ethylated cyclic maleimide 12. The synthesis of unsym-
metrical nitrogen containing macrocyclic maleimides 19
and 21 were shown in Scheme 4. Reaction of d-valer-
olactone and 4-amino-1-butanol gave the diol 13. Bor-
ane reduction followed by N-Boc protection, and
mesylation gave the required bismesylate 16. Alkylation
2. Chemistry
The synthesis of macrocyclic bis-7-azaindolylmaleimides
described in Table 1 resulted from bis-alkylations of var-
ious linkers with the upper maleimides. Two key N-pro-
tected maleimides were used in these studies (Scheme 2).

L. Shen et al. / Bioorg. Med. Chem. 12 (2004) 1239–1255
1241
Table 1. Inhibition constants at GSK-3ba
Compd
R
GSK-3ꢀ KiꢂSEM (ꢁM)
10
12
H
H
–(CH2)5NH(CH2)5—
–(CH2)5NH(CH2)4—
0.318ꢂ0.048
0.541ꢂ0.201
19
21
H
H
31% inhib. @ 1 mM
44% inhib. @ 1 mM
28
33
36
41
46
45
H
0.079ꢂ0.001
0.058ꢂ0.005
H
H
0.011ꢂ0.002
H
H
0.150ꢂ0.009
0.036ꢂ0.001
Protected
0% inhib. @ 1 mM
a
Assay details were described in the Experimental.
b SEM, standard error mean.
Scheme 2. (i) Me3SnCl, ꢀ78 ꢁC, n-BuLi; (ii) 2,3-dichloro-N-methylmaleimide, PdCl2(PPh3)2, LiCl, toluene, 100 ꢁC; (iii) HOAc, reflux; (iv) 2,
PdCl2(PPh3)2, LiCl, toluene, 100 ꢁC.

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L. Shen et al. / Bioorg. Med. Chem. 12 (2004) 1239–1255
Scheme 3. (i) MsCl, pyridine; (ii) 3, Cs2CO3, DMF, 100 ꢁC; (iii) TFA; (iv) (a) KOH, EtOH, 80 ꢁC; 10% citric acid; (b) NH4OAc, 140 ꢁC; (v) C2H5I,
THF, 70 ꢁC.
ꢁ
.
Scheme 4. (i) Xylene; (ii) (a) BH3 SMe2, THF; (b) HCl, Et2O; (iii) Et3N, CH3OH, Boc2O; (iv) MsCl, pyridine; (v) 5, Cs2CO3, DMF, 70 C;
(vi) TFA; (vii) CH3SO3H; (viii) C2H5I, THF.
of 5 with 16 gave 17. Treatment of 17 with tri-
fluoroacetic acid removed the N-Boc protecting group
selectively to give 18. As desired, removalof the N-2,4-
dimethoxybenzylprotecting group was accompislhed
with the treatment of methanesulfonic acid at 20 ꢁC to
provide 19 in quantitative yield.25 Ethylation of 18 gave
20. Acid treatment of 20 provided the unsymmetrical N-
ethylated cyclic maleimide 21.
Deprotection and mesylation yielded 31. Alkylation of 5
with 31 gave 32, followed by acid promoted deprotec-
tion to give the symmetricalmaelimide 33 (Scheme 6).
Reduction of 31 gave the alcohol 34. Alkylation of 5 with
34, following by deprotection of 35 gave the hydroxyl
containing macrocyclic maleimide 36 (Scheme 7).
Bis-alkylation of 2,6-lutidine with 3-bromo-propoxy-
tert-butyldimethylsilanes gave 37. Deprotection fol-
lowed by mesylation gave bismesylate 39. Alkylation of
3 with 39, following by hydrolysis and ammonolysis
yielded the pyridine containing macrocyclic maleimide
41 (Scheme 8). In a similar way, bis-alkylation of thio-
phene gave 42. Deprotection, mesylation and alkyl-
ation of 44 with 5, followed by deprotection to give the
thiophene containing macrocyclic maleimide 46
(Scheme 9).
The synthesis of unsymmetricalcarbonylgroup contain-
ing macrocyclic maleimide 28 was shown in Scheme 5.
The carbonylgroup of 22 was protected as cyclic ketyl
23, followed by reduction to give the diol 24. Deprotec-
tion and mesylation yielded the bismesylate 26. Alkyl-
ation of 3 with 26 gave 27, followed by hydrolysis and
ammonolysis to provide the maleimide 28. Similarly,
reduction of the cyclic ketyl 29 gave the diol 30.

L. Shen et al. / Bioorg. Med. Chem. 12 (2004) 1239–1255
1243
Scheme 5. (i) EtOH, HCl(concd.); (ii) CH(OEt) , TsOH, HO(CH2)2OH; (iii) LiAlH4; (iv) H2SO4, acetone; (v) MsCl, Et3N; (vi) 3, Cs2CO3, DMF,
3
100 ꢁC; (vii) (a) KOH, EtOH, 80 ꢁC; 10% citric acid; (b) NH4OAc, 140 ꢁC.
Scheme 6. (i) CH(OEt)3, TsOH, HO(CH2)2OH; (ii) LiAlH4; (iii) MsCl, Et3N; (iv) 5, Cs2CO3, DMF, 70 ꢁC; (v) CH3SO3H.
Scheme 7. (i) NaBH4, EtOH, THF; (ii) 5, Cs2CO3, DMF, 80 ꢁC; (iii) CH3SO3H.
3. Results and discussion
lower potency (Ki=0.318 mM). It was thought that the
reduced potency might be due to the unfavorable extra
hydrogen bond donor –NH moiety presented in the linker.
Compound 12 was then synthesized with the –NH moiety
replaced with –NC2H5 moiety. However, no improve-
ment was observed (Ki=0.541 mM for 12 vs 0.318 mM
for 10). Since it was evident that conformationally
In the polyoxygenated macrocyclic maleimide series, the
19-membered macrocycle (n=2) gave the best potency
at GSK-3b. Thus, we first prepared compound 10 (the
nitrogen containing linker, Table 1) with the same ring
size; however, this replacement resulted in ꢃ10-fold

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L. Shen et al. / Bioorg. Med. Chem. 12 (2004) 1239–1255
Scheme 8. (i) n-BuLi, THF; (ii) TBAF; (iii) MsCl, Et3N; (iv) 3, Cs2CO3, DMF, 100 ꢁC; (v) (a) KOH, EtOH, 80 ꢁC; 10% citric acid; (b) NH4OAc,
140 ꢁC.
Scheme 9. (i) n-BuLi, THF; (ii) TBAF; (iii) MsCl, Et3N; (iv) 5, Cs2CO3, DMF, 90 ꢁC; (v) CH3SO3H.
constrained analogues may exhibit better binding affin-
ities,26 we next examined the potency of the smaller
macrocycles 19 and 21. With disappointment, both 18-
membered macrocycles containing either –NH or –NC2H5
in the linker displayed even poorer potency (for 19, 31%
inhibition at 1 mM; for 21, 44% inhibition at 1 mM). It
was hypothesized that the incorporation of a nitrogen
atom in the bottom linker may not be desirable to
achieve high inhibitory potency at GSK-3b.
it appears that altering the functional groups in the lin-
ker has a greater impact on the GSK-3b inhibitory
potency than altering the ring sizes does, at least
between the 17- to 19-membered macrocycles range.
The totalaboislhed potency of 2,4-dimethoxybenzy-l
protected maleimide 45 (0% inhibition at 1 mM) sug-
gested that the maleimide NH seems to be critical for
the binding.
We then went back to re-visit the oxygen atom con-
taining linkers but to place the oxygen atom out of the
chain instead. Interestingly, the carbonyl group con-
taining 19-membered macrocycle 28 (Ki=0.079 mM) did
display approximately 4-fold greater inhibitory potency
when compared to the nitrogen containing 19-mem-
bered macrocycle 10 (Ki=0.318 mM). We continued to
prepare a smaller analogue, the 17-membered macro-
cycle 33. The conformationally more constrained 33
displayed modest improvement in potency (Ki=0.058
mM) over 28. Replacement of the carbonyl group of 33
with a hydroxylgroup gave 36 (Ki=0.011 mM), which
exhibited another 5-fold increase in potency relative to
33. The high potency of 36 may partially contributed
from the hydrogen bond donor capability of –OH
group. Replacing the hydroxyl group in 36 with a pyri-
dine ring gave 41. When comparing macrocycle 41 to
macrocycle 12 (which has a comparable ring size), the
aromatic nitrogen ring was more favorable than the
tert-alkyl amine moiety for the inhibitory activity (for
41, Ki=0.150 mM). Replacement of the hydroxyl group
in 36 with a thiophene ring and shortening two carbon
atoms gave macrocycle 46. Compound 46 displayed
good potency (Ki=0.036 mM) although this compound
was not as potent as the hydroxylanaol gue 36. Overall,
4. Kinase selectivity
Recently, literature reports have described potent
cyclin-dependent kinase (CDK) inhibitors that could
also inhibit GSK-3b.27 The bisindole-maleimide deriva-
tives (Scheme 1) were identified as dualPKC/GSK-3
inhibitors,28 and have been reported to be competitive
inhibitors of ATP binding presumably by interacting at
the ATP binding site.29 Our compounds, in common
with most of the other protein kinase inhibitors, also
inhibited GSK-3b in an ATP competitive manner.30
Therefore, it is criticalto evaulate our compounds
against a panelof kinases inculding CDK and PKC
isoenzymes in order to determine the selectivity. In
addition, many reported specific inhibitors of protein
kinases were found to be non-specific when re-examined
against a large panel of protein kinases by Cohen.31
Therefore, we submitted compounds 33, 36 and 46 to
UBI (Upstate Biotech Inc.) for broad screening against
a panelof 66 protein kinases. In the presence of 100 mM
ATP, compound 33 inhibited GSK-3b kinase activity by
96% at a concentration of 10 mM. Compounds 36 or 46
inhibited GSK-3b kinase activity by 99% at 10 mM
(Table 2). Interestingly, compounds 33 and 36 only
showed very weak inhibitory activities at the other 63

L. Shen et al. / Bioorg. Med. Chem. 12 (2004) 1239–1255
1245
(or 62 for 36) kinases, and moderate inhibitions at
CDK2/cyclin A and PKCbII (and Rsk3 for 36). The Ki
value of these compounds at CDK2, PKCbII and Rsk3
assays were shown in Table 3. Compound 33 exhibited
ꢃ20-fold selectivity at GSK-3b versus CDK2/cyclin A
and PKCbII whereas compound 36 exhibited ꢃ60-fold
selectivity against the three low selective kinases.
Remarkably, compound 46 exhibited very low activity
against all of the other 65 kinases, and had 97-fold
selectivity against the poorly selective one, therefore
representing a ‘specific inhibitor’ of GSK-3b.
Table 2. Activities at various protein kinasesa
Protein Kinase
Activity (% of control)
36
33
46
GSK3b (h)
AMPK (r)
Blk (m)
4
98
77
1
100
85
91
80
62
43
93
86
91
103
101
62
1
108
93
92
70
93
80
100
109
103
123
81
CAMKII (r)
CAMKIV (h)
CDK1/cyclinB (h)
CDK2/cyclinA
CDK5/p35 (h)
CDK6/cyclin D3
CDK7/cyclinH
CHK1 (h)
CHK2 (h)
CK1 (y)
89
78
61
50
98
85
86
104
101
78
5. Cellular activity
84
Among the multiple cellular processes in which GSK-3
has been implicated, the ability to phosphorylate and
inhibit GS was the first regulation process being dis-
covered and is perhaps the most thoroughly studied.1
Therefore, the cell-based assay examining GS activation
represents a direct functionalassay to measure the ce-l
lular activity of GSK-3 inhibitors. Compounds 33, 36,
46 and LiClwere tested for the abiilty to increase GS
activity in human embryonic kidney (HEK293) cells. As
described above, LiClis a known inhibitor of GSK-3 b.
LiClincreases GS activity in isoal ted rat adipocytes and
hepatocytes,32 and produces intracellular effects similar
to that achieved by GSK-3 inhibition.33,34 Compound
33 (EC50=1.26 mM), compound 36 (EC50=0.33 mM)
and compound 46 (EC50=1.25 mM) all exhibited much
CK2 (h)
CSK
Fes (h)
Fyn (h)
FGFR3
IGF-1R
IKK a
IKK b
IR
JNK1a1 (h)
JNK2a2 (h)
JNK3 (r)
116
93
101
107
78
104
111
106
92
97
106
72
86
73
85
101
114
100
100
91
103
147
64
99
99
98
88
99
97
106
71
77
37
99
77
112
80
97
103
64
99
116
122
97
92
99
56
77
77
92
102
89
96
68
72
93
100
111
103
106
113
77
77
87
95
Lck (h)
Lyn
MAPK1 (h)
MAPK2 (h)
MAPK2 (m)
MAPKAP-K2 (h)
MEK1 (h)
MKK4 (m)
MKK6 (h)
MKK7b (h)
MSK1 (h)
PAK2 (h)
PDGFR a
PDGFR b
PDK1 (h)
PKA (b)
98
105
105
101
92
112
123
101
92
106
133
63
103
106
95
94
96
113
106
83
70
28
92
71
85
86
96
98
92
79
99
38
121
105
96
92
91
103
85
97
95
125
greater potency than LiCl(EC
>3000 mM) in the
50
HEK293 cells (Table 4).
90
101
113
70
112
111
89
94
91
112
104
68
84
56
102
77
97
87
91
104
98
72
85
74
114
95
113
99
100
97
96
124
82
6. Molecular docking studies
Compounds 33, 36 and 46 are very selective at GSK-3b,
demonstrating only moderate or poor activity for other
Ser/Thr kinases (Tables 2 and 3). In an attempt to
rationalize this high selectivity, the molecular docking
studies of 46 were conducted, based on the X-ray struc-
ture of GSK-3b.35 Compound 46 was docked into the
ATP binding site of GSK-3b. The binding mode derived
from the best docking pose is illustrated in Fig. 1.
According to this model, compound 46 may form three
H-bonds with GSK-3b when binding into the ATP-
binding site. The first H-bond may form between the
backbone carbonyloxygen of ASP133 to the imide NH.
The second H-bond is generated between the backbone
nitrogen of VAL135 to the imide carbonyloxygen. The
third H-bond may form between the side-chain guani-
dine group of ARG141 to the azaindole nitrogen atom.
The first two H-bonds that are formed with the back-
bone atoms are identicalto those observed when staur-
osporine (Scheme 1) binds to various kinases, such as
CDK2,36 LcK37 and so on. Meanwhile, the H-bond
with ARG141 turns out to be unique to GSK-3b and
provides an interesting modelto expal in the high GSK-
3b selectivity observed for our 7-azaindolemaleimides.
PKA (h)
PKBa (h)
PKBb (h)
PKCa (h)
PKCbII (h)
PKCg (h)
PKCe (h)
PKCy
PRAK (h)
c-RAF (h)
ROCK-II (h)
ROCK-II (r)
Rsk1 (r)
100
98
99
97
93
81
84
56
119
105
96
94
108
98
Rsk2 (h)
Rsk3 (h)
SAPK2a (h)
SAPK2b (h)
SAPK3 (h)
SAPK4 (h)
p70S6K (h)
SGK (h)
cSRC (h)
Syk
Yes (h)
94
86
87
114
ZAP-70 (h)
123
(a) Protein kinase were assayed with 10 mM of 33, 36 and 46 in the
presence of 100 mM ATP. Activities were given as the mean percentage
of that in controlincubations (averages of dupilcate determinations).
Assay details were described in the Experimental.
The aligned sequences of several kinases selected from
the UBI panelis shown in Fig. 2. This alignment was
adopted from the Protein Kinase Resource (PKR)

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L. Shen et al. / Bioorg. Med. Chem. 12 (2004) 1239–1255
Table 3. KiꢂSEM (mM) and Ki ratio at selected kinasea
Compd
KiꢂSEM (mM)
Ki ratio
GSK-3b
CDK2/cyclin A
PKCbII
RsK3
CDK2/GSK-3b
PKCbII/GSK-3b
RsK3/GSK-3b
33
36
46
0.058ꢂ0.005
0.011ꢂ0.002
0.036ꢂ0.001
1.233ꢂ0.057
0.682ꢂ0.022
NT
1.050ꢂ0.024
0.710ꢂ0.025
3.488ꢂ0.067
NTb
0.789ꢂ0.097
NT
21
62
—
18
65
97
—
72
—
a
Assay details were described in the Experimental. b Not tested due to less than 50% inhibition.
Table 4. Glycogen synthase activity in HEK293 cellsa
Compd
EC50ꢂSEM (mM)
33
36
46
LiCl
1.26ꢂ0.16
0.33ꢂ0.05
1.25ꢂ0.44
>3000
(a) Assay details were described in the Experimental.
database,38 which includes hundreds of kinases whose
sequences have been aligned by Hanks and Quinn.39 It
clearly demonstrates that the positive-charged ARG141
is quite unique to GSK-3b, with many of the other
kinases having a negative-charged residue at the same
alignment position. Conceivably, the replacement of
ARG141 by a negative-charged residue will not only
disrupt the H-bonding with the azaindole nitrogen,
but also introduce an unfavorable repellent interac-
tion with the lone-pair electrons on that nitrogen.
This unique binding site may explain why the bis-7-
azaindole 46 exhibit high selectivity at GSK-3b versus
other kinases.
Figure 2. Sequence alignment of some kinases selected from PKR
database, to demonstrate the uniqueness of ARG141 at GSK-3b. The
positive-charged residues are colored in blue, while the negative-
charged residues are colored in red.
There are also a few kinases with a neutral or even
positive-charged residue at the alignment position cor-
responding to ARG141 at GSK-3b, as shown in Fig. 2.
For example, CDK2 bears a positive-charged LYS86
at that position. However, a close inspection of the X-
ray structure of CDK2 and staurosporine complex
(1aq1.pdb)36 reveals that the positive-charged side chain
of LYS86 is actually oriented into a different space, and
instead, the negative-charged side chain of ASP83 is the
closest residue to the 7-position of the indole ring of
staurosporine. Therefore, we speculate that the selectiv-
ity difference of the 7-azaindolemaleimides observed
may contribute significantly from the different interac-
tion strengths with ARG141 at GSK-3b or the spatially
equivalent residues at other kinases.
The lower GSK-3b potency observed for molecules that
contain an aliphatic nitrogen-atom linker (compounds
10, 12, 19 and 21) may partially explained as the penalty
paid from an unfavorable desolvation with water during
the binding processes. A full discussion of the structure
activity relationships between these compounds requires
a carefulconsideration of severalfactors inculding H-
bonding, solvation/desolvation effects, van der Waals
forces and electrostatic interactions, the subject of
future computationalstudies.
7. Conclusion
Palladium-catalyzed cross-coupling reactions were
used to synthesize two key intermediates 3 and 5 that
resulted in the synthesis of novel series of macrocyclic
Figure 1. Illustration of the binding mode of compound 46 in the
ATP-binding site of GSK-3b, suggested by the molecular docking
studies. Compound 46 and its H-bonding partners (ASP133, VAL135,
and ARG141) are represented in the stick model. The protein struc-
ture is described in the ribbon model. The key H-bonds between
compound 46 and GSK-3b are indicated by the pink dotted lines,
together with the bond lengths in the unit of angstrom. (Atom color
scheme: hydrogen in white; oxygen in red; nitrogen in blue; carbon on
compound 46 in green; and carbon on GSK-3b in light blue.).
bis-7-azaindolylmaleimides.
N-2,4-Dimethoxybenzyl-
maleimide 5 could be deprotected under acidic condition
is particularly useful for the synthesis of molecules that
may not survived the base-hydrolysis/ammonolysis reac-
tion conditions. Among the three series of macrocycles,

L. Shen et al. / Bioorg. Med. Chem. 12 (2004) 1239–1255
1247
1
the oxygen atom and thiophene containing linkers
yielded molecules with higher inhibitory potency at
GSK-3b (Ki=0.011–0.079 mM) while the nitrogen atom
containing linkers yielded molecules with lower potency
(Ki=0.150–>1 mM). The ring-size of the macrocycles
seems to have less impact on the potency. Compound
33 and 36 displayed 1–2 orders of magnitude selec-
tivity at GSK-3b against CDK2, PKCbII, Rsk3 and
little or no inhibitions versus 62 other protein kinases.
Compound 46 displayed a 100-fold greater selectivity at
GSK-3b versus a panelof 65 protein kinases, and
behaved as a GSK-3b ‘specific inhibitor’. All three
compounds showed good potency in GS assay.
Molecular docking studies were conducted in an
attempt to rationalize the GSK-3b selectivity of bis-7-
azaindolylmaleimides. The high selectivity, inhibitory
potency and cellular activities of these non-crown-
ether typed molecules suggested that these compounds
may prove to be valuable pharmacological tools in
elucidating the complex roles of GSK-3b in cell
signaling pathways and the potential usage for the
treatment of elevated level of GSK-3b involved
diseases.
oil: H NMR (300 MHz, CDCl3) d 8.45 (d, J=4.9 Hz,
1H), 7.77 (d, J=7.6 Hz, 1H), 7.48 (s, 1H), 7.13 (dd,
J=7.7, 4.8 Hz, 1H), 1.65 (s, 9H), 0.36 (m, 9H); MS (ES)
m/z: 405 (M+Na).
8.1.3. 1-Methyl-3,4-bis-(1H-pyrrolo[2,3-b]pyridine-3-yl)-
pyrrole - 2,5-dione (3). A mixture of 2 (185 mg, 0.486
mmol), 2,3-dichloro-N-methylmaleimide (29 mg, 0.162
mmol), PdCl2(PPh3)2 (5.4 mg, 0.0077 mmol) and LiCl
(32 mg, 0.77 mmol) in anhydrous toluene (2 mL) was
stirred at 95 ꢁC overnight. The solvent was removed
under reduced pressure. The product was purified by
column chromatography (SiO2) to give 23 mg (41%) of
1
3 as an orange-red solid: H NMR (300 MHz, DMSO-
d6) d 12.35 (s, 2H), 8.12 (brd, J=3.9 Hz, 2H), 7.92 (s,
2H), 7.08 (d, J=7.7 Hz, 2H), 6.73 (m, 2H), 3.06 (s, 3H);
MS (ES) m/z: 344 (M+H+). Anal. calcd for
C19H13N5O2 2.2TFA 0.5H2O: C, 46.59; H, 2.71; N,
11.61. Found: C, 46.25; H, 2.41; N, 11.34.
.
.
8.1.4. 3,4-Dichloro-1-(2,4-dimethoxy-benzyl)-pyrrole-2,5-
dione (4). A mixture of 2,3-dichloromaleic anhydride
(1.02 g, 6.10 mmol), 2,4-dimethoxybenzylic amine (1.02
g, 6.10 mmol) in glacial acetic acid (18 mL) was heated
to 80 C for 5 h. After it was cooled to 20 ꢁC, the mixture
was concentrated under reduced pressure, and diluted
with CH2Cl2 (50 mL). The mixture was sequentially
washed with water (15 mL), 2 M aqueous Na2CO3 (15
mL), water (15 mL) and brine (15 mL). After the com-
bined organic phases were concentrated, the residue was
filtered through a short pad of SiO2, eluting with
CH2Cl2 to give 4 (1.42 g, 74%) as a light brown solid:
1H NMR (300 MHz, CDCl3) d 7.20 (d, J=8.7 Hz, 1H),
6.44 (d, J=2.3 Hz, 1H), 6.42 (s, 1H), 4.72 (s, 2H), 3.79
(s, 3H), 3.78 (s, 3H).
8. Experimental
8.1. Chemistry
1
8.1.1. General information. H NMR spectra were mea-
sured on a Bruker AC-300 (300 MHz) spectrometer using
tetramethylsilane as an internal standard. Elemental ana-
lyses were obtained by Quantitative Technologies Inc.
(Whitehouse, NJ, USA), and the results were within
0.4% of the calculated values unless otherwise men-
tioned. Melting points were determined in open capil-
lary tubes with a Thomas-Hoover apparatus and were
uncorrected. The opticalrotations were measured at
25 ꢁC with an AutopolIII poalrimeter. Eelctrospray
mass spectra (MS-ES) were recorded on a Hewlett
Packard 59987A spectrometer. High resolution mass
spectra (HRMS) were obtained on a Micromass Auto-
spec. E. spectrometer. The term ‘DMAP’ refers to
dimethylaminopyridine, ‘TFA’ refers to trifluoroacetic
acid, ‘NMP’ to 1-methyl-2-pyrrolidinone, ‘DPPF’ to 1,
10-bis(diphenylphosphino)ferrocene, ‘Pd2(dba)3’ to tris
(dibenzylideneacetone)dipalladium(0)-chloroform
8.1.5. 1-(2,4-Dimethoxy-benzyl)-3,4-bis-(1H-pyrrolo[2,3-
b]pyridin-3-yl)-pyrrole-2,5-dione (5). A mixture of 2 (500
mg, 1.31 mmol), 4 (180 mg, 0.57 mmol), PdCl2(PPh3)2
(80 mg, 0.11 mmol) and LiCl (240 mg, 8.6 mmol) in
toluene (9.0 mL) was heated at 100 ꢁC for 20 h. After
the solvent was removed under reduced pressure, the
residue was dry-loaded on silica gel, eluting with
EtOAc/hexane to give 5 (160 mg, 58%) as an orange red
1
solid: H NMR (300 MHz, DMSO-d6) d 12.30 (s, 2H),
8.12 (d, J=4.6 Hz, 2H), 7.93 (d, J=2.8 Hz, 2H), 7.08
(m, 3H), 6.73 (dd, J=8.0, 4.7 Hz, 2H), 6.58 (d, J=2.1
Hz, 1H), 6.48 (d, J=8.4 Hz, 1H), 4.68 (s, 2H), 3.82 (s,
3H), 3.74 (s, 3H); MS (ES) m/z: 480 (M+H+). Anal.
calcd for C27H21N5O4 0.3H2O: C, 66.88; H, 4.49; N,
14.44. Found: C, 66.51; H, 4.23; N, 14.61.
adduct and ‘DPPA’ refers to diphenylphosphoryl azide.
.
8.1.2. 3-Trimethylstannanyl-pyrrolo[2,3-b]pyridine-1-car-
boxylic acid tert-butyl Ester (2). To a THF solution (15
mL) of 7-aza-1-(tert-butyloxycarbonyl)-3-iodoindole 1
(1.82 g, 5.3 mmol) at ꢀ78 ꢁC was added trimethyltin
chloride (26.5 mL, 1 M in THF, 26.5 mmol) under
nitrogen. After 10 min, n-BuLi (10 mL, 1.6 M in hexane,
16 mmol) was added dropwise at ꢀ78 ꢁC and the reac-
tion mixture was allowed to warm up to 20 ꢁC over-
night. Water (4 mL) was added and the solvent was
removed under reduced pressure. The residue was dilu-
ted with hexane (250 mL) and the organic layer was
washed with water, dried (Na2SO4) and concentrated.
The product was purified by column chromatography
(SiO2) to give 1.198 g (60%) of organostannane 2 as an
8.1.6. Methanesulfonic acid 5-[tert-butoxycarbonyl-(5-
methanesulfonyloxy-pentyl)-amino]-pentyl ester (7). To a
solution of carbamate diol 6 (1.06 g, 3.66 mmol,23) in
CH2Cl2 (13 mL) was added pyridine (1.2 mL, 14.6
mmol) and MsCl (1.1 mL, 14.6 mmol) at 0 ꢁC. The
mixture was stirred at 20 ꢁC for 1.5 h. It was diluted
with diethyl ether (10 mL), and washed sequentially
with cold aqueous HCl (5%), NaOH (5%), water and
brine. The organic solution was dried (MgSO4), filtered
and concentrated. The product was purified by column
chromatograph on silica gel (eluting with hexane/

1248
L. Shen et al. / Bioorg. Med. Chem. 12 (2004) 1239–1255
1
EtOAc) to give 7 as a colorless oil (1.20 g, 74%): H
NMR (400 MHz, CDCl3) d 4.23 (t, J=6.4 Hz, 4H), 3.16
(s, br, 4H), 3.01 (s, 6H), 1.78 (m, 4H), 1.55 (m, 4H), 1.45
(s, 9H), 1.40 (m, 4H); MS (ES) m/z: 468 (M+Na).
CD3OD) d 8.27 (dd, J=4.7, 1.5 Hz, 2H), 7.65 (s, 2H),
7.55 (dd, J=8.0, 1.5 Hz, 2H), 7.01 (dd, J=8.0, 4.8 Hz,
2H), 4.32 (t, J=5.9 Hz, 4H), 2.23 (t, J=6.3 Hz, 4H),
1.81 (t, J=5.9 Hz, 4H), 1.40 (m, 4H), 0.94 (t, J=7.5 Hz,
4H); MS (ES) m/z: 483 (M+H+); FAB-HRMS
(M+H+). calcd 483.2508, found 483.2513.
8.1.7. 7,8,9,10,13,14,15,16,24,25-Decahydro-24-methyl-
23,25 - dioxo-6H,23H- 5,26:17,22 - dimethenodipyrido[2,3 -
n:30,20 - t]pyrrolo[3,4 - q][1,7,13]triazacycloheneicosine - 11
(12H)-carboxylic acid, 1,1-dimethylethyl ester (8). A
mixture of 3 (41 mg, 0.12 mmol) and Cs2CO3 (190 mg,
0.58 mmol) in DMF (20 mL) was heated to 100 ꢁC. A
DMF solution (5 mL) of the bismesylate 7 (77 mg, 0.17
mmol) was added via syringe pump over 1.5 h. After the
addition was complete, the reaction mixture was stirred
at 20 ꢁC for 21 h. The mixture was quenched with aqu-
eous ammonium chloride (30 mL), and extracted with
CH2Cl2 (2 ꢄ 30 mL). The combined organic layers was
washed with water (3 ꢄ 20 mL) and brine (15 mL). It
was then dried (Na2SO4) and concentrated. The product
was purified by column chromatography on silica gel
eluting with hexane/EtOAc to give 8 (36 mg, 50%) as an
8.1.10. 11-Ethyl-7,8,9,10,11,12,13,14,15,16-decahydro-24-
methyl-6H,23H-5,26:17,22-dimethenodipyrido[2,3-n:30,20 -
t]pyrrolo[3,4 -q][1,7,13]triazacycloheneicosine-23,25(24H)-
dione (11). A mixture of 9 (14 mg, 0.028 mmol) and
iodoethane (0.004 mL, 0.063 mmol) in THF (1.0 mL)
was heated to reflux for 2 days. The reaction mixture
was concentrated and the product was purified by col-
umn chromatography eluting with MeOH/CH2Cl2/
NH4OH to give 11 (12 mg, 75%) as an orange solid: 1H
NMR (400 MHz, CD3OD) d 8.27 (dd, J=4.7, 1.6 Hz,
2H), 7.64 (s, 2H), 7.62 (dd, J=8.0, 1.6 Hz, 2H), 7.03
(dd, J=8.0, 4.7 Hz, 2H), 4.31 (m, 4H), 3.14 (s, 3H), 2.44
(m, 2H), 2.11 (m, 4H), 1.84 (m, 4H), 1.25 (m, 4H), 0.98
(m, 7H); MS (ES) m/z: 525 (M+H+).
1
orange solid: H NMR (300 MHz, CD3OD) d 8.29 (dd,
J=4.7, 1.5 Hz, 2H), 7.66 (s, br, 2H), 7.58 (s, 2H), 7.05
(dd, J=8.0, 4.7 Hz, 2H), 4.30 (t, J=6.5 Hz, 4H), 3.15 (s,
3H), 2.73 (s, br, 4H), 1.75 (t, J=6.6 Hz, 4H), 1.42 (s,
9H), 1.34 (m, 4H), 1.03 (m, 4H); MS (ES) m/z: 597
(M+H+).
8.1.11. 11-Ethyl - 7,8,9,10,11,12,13,14,15,16 - decahydro -
6H,23H-5,26:17,22-dimethenodipyrido[2,3-n:30,20 -t][pyr-
rolo[3,4
- q][1,7,13]triazacycloheneicosine-23,25(24H)-
dione (12). Compound 11 (12 mg, 0.023 mmol) was
transformed into 12 (6 mg, 50%) using the same proce-
dure described for covert 9 to 10. Compound 12 was
isolated as an orange solid: 1H NMR (400 MHz,
CD3OD) d 8.27 (dd, J=4.7, 1.4 Hz, 2H), 7.62 (s, 2H),
7.60 (dd, J=7.7, 1.5 Hz, 2H), 7.03 (dd, J=8.0, 4.7 Hz,
2H), 4.30 (m, 4H), 2.44 (q, J=7.1 Hz, 2H), 2.11 (m,
4H), 1.83 (m, 4H), 1.26 (m, 4H), 0.98 (m, 7H); MS (ES)
m/z: 511 (M+H+); FAB-HRMS (M+H+). calcd
511.2821, found 511.2840.
8.1.8. 7,8,9,10,11,12,13,14,15,16 - Decahydro - 24 - methyl -
6H,23H - 5,26:17,22 - dimethenodipyrido[2,3-n:30,20 - t][pyr-
rolo[3,4-q][1,7,13]triazacycloheneicosine-23,25(24H)-dione
(9). To a solution of 8 (13 mg, 0.022 mmol) in CH2Cl2
(1.0 mL) was added TFA (0.2 mL). After the mixture
was stirred at 20 ꢁC for 1 h, solvent and excess TFA
were removed under reduced pressure. Ammonium
hydroxide was carefully added, and orange solids were
precipitated out. The solids were collected by filtration
and washed with water to give 9 (10 mg, 100%) after
8.1.12. (4-Hydroxy-butyl)-(5-hydroxy-pentyl)-carbamic
acid tert-butyl ester (15). A mixture of d-valerolactone
(1.7 mL, 18.3 mmol), 4-amino-1-butanol (1.7 mL, 18.3
mmol) in m-xylene (50 mL) was heated to 120 ꢁC for 20
h. After it was cooled to 20 ꢁC, the lower layer was
separated from the upper xylene layer, and concentrated
under reduced pressure to give crude 13 (3.50 g, 99%).
A solution of crude 13 (1.91 g, 10.1 mmol) in THF (50
mL) was heated to reflux. Borane dimethylsulfide com-
plex (20 mL, 2 M in THF, 40.0 mmol) was added drop-
wise via addition funnel. After the addition was
completed, the mixture was refluxed for another h. It
was cooled to 20 ꢁC and quenched with MeOH (4.0
mL). Hydrogen chloride (12 mL, 1 M in Et2O, 12.0
mmol) was added, and after the mixture was stirred at
20 ꢁC for 10 min, it was concentrated under reduced
pressure to give crude diolsatl 14. The crude 14 was
then mixed with MeOH (40 mL), Et3N (5.7 mL, 40.4
mmol) and Boc2O (2.7 g, 12.1 mmol). After the mixture
was refluxed for 3 h, it was cooled to 20 ꢁC, con-
centrated and taken up in CH2Cl2 (40 mL). It was
quickly washed with cold 1 N HCl, dried (Na2SO4) and
concentrated. The product was purified by column
chromatography eluting with EtOAc to give 15 (1.90 g,
1
drying under vacuum: H NMR (300 MHz, CD3OD) d
8.27 (dd, J=4.7, 1.4 Hz, 2H), 7.66 (s, 2H), 7.56 (dd,
J=8.0, 1.4 Hz, 2H), 7.02 (dd, J=8.0, 4.8 Hz, 2H), 4.33
(t, J=5.9 Hz, 4H), 3.14 (s, 3H), 2.26 (t, J=6.5 Hz, 4H),
1.84 (m, 4H), 1.40 (m, 4H), 0.96 (m, 4H); MS (ES) m/z:
497 (M+H+).
8.1.9. 7,8,9,10,11,12,13,14,15,16 - decahydro - (6H,23H -
5,26:17,22 - dimethenodipyrido[2,3 - n:30,20 - t]pyrrolo[3,4 -
q][1,7,13]triazacycloheneicosine-23,25(24H)-dione (10). A
mixture of 9 (10 mg, 0.020 mmol) and KOH (198 mg,
3.53 mmol) in ethanol (2.0 mL) was heated under reflux
for 18 h. It was cooled to 20 ꢁC and concentrated under
reduced pressure. The residue was dissolved in water
(3.0 mL) and acidified with 10% citric acid (pH 4). The
mixture was stirred at 20 ꢁC for 10 min and con-
centrated. The resulting residue was mixed with ammo-
nium acetate solids (2.4 g, 31.2 mmol), and heated to
140 ꢁC for 3 h. The mixture was cooled to 20 ꢁC, diluted
with water (3.0 mL), basified with 20% aqueous NaOH
till pH 10, and extracted with EtOAc (3 ꢄ 25 mL). The
combined organic layers was dried (Na2SO4) and con-
centrated. The product was purified by column chro-
matography (eluting with MeOH/CH2Cl2) to give 10 (4
mg, 42%) as an orange solid: 1H NMR (300 MHz,
1
70%) as a colorless oil: H NMR (300 MHz, CDCl3) d
3.67 (m, 4H), 3.18 (m, 4H), 1.60 (m, 10H), 1.45 (s, 9H);
MS (ES) m/z: 298 (M+Na).

L. Shen et al. / Bioorg. Med. Chem. 12 (2004) 1239–1255
1249
8.1.13. Methanesulfonic acid 5-[tert-butoxycarbonyl-(4-
methanesulfonyloxy-butyl)-amino]-pentyl ester (16). A
solution of 15 (1.90 g, 6.91 mmol) in CH2Cl2 (20 mL)
was cooled in an ice bath, pyridine (2.2 mL, 27.6 mmol)
was added, followed by MsCl (2.1 mL, 27.6 mmol). The
mixture was stirred at 20 ꢁC for 1.5 h. It was diluted
with Et2O (15 mL), and washed with cold 5% HCl and
5% NaOH. The organic phase was dried (Na2SO4) and
concentrated. The product was purified by column
chromatography on silica gel (eluting with hexane/
(19). To a solution of 18 (5 mg, 0.008 mmol) in CH2Cl2
(1.0 mL) was added methanesulfonic acid (0.5 mL).
After the mixture was stirred at 20 ꢁC for 6 h, ammo-
nium hydroxide was added carefully to basify the mix-
ture. It was extracted with EtOAc (2 ꢄ 10 mL), and the
organic layers were combined, washed with water (5
mL), brine (5 mL), dried (Na2SO4) and concentrated.
The product was purified by column chromatography
on silica gel (eluting with MeOH/CH2Cl2/NH4OH) to
give 19 (5 mg, 100%) as an orange solid: 1H NMR
(300 MHz, CDCl3) d 8.35 (m, 2H), 7.96 (d, J=7.9 Hz,
1H), 7.55 (s, 1H), 7.53 (d, J=8.1 Hz, 1H), 7.42 (s, 1H),
7.09 (dd, J=8.0, 4.7 Hz, 1H), 6.97 (dd, J=8.0, 4.7 Hz,
1H), 4.33 (t, J=6.0 Hz, 2H), 4.22 (t, J=6.6 Hz, 2H),
2.45 (t, J=6.4 Hz, 2H), 2.32 (t, J=6.3 Hz, 2H), 1.87 (m,
2H), 1.73 (m, 2H), 1.35 (m, 2H), 1.25 (m, 2H), 1.13 (m,
2H); MS (ES) m/z: 469 (M+H+).
1
EtOAc) to give 16 (2.40 g, 82%) as colorless oil: H
NMR (400 MHz, CDCl3) d 4.24 (m, 4H), 3.19 (m, 4H),
3.01 (s, 3H), 3.00 (s, 3H), 1.75 (m, 4H), 1.64 (m, 2H),
1.56 (m, 2H), 1.45 (s, 9H), 1.41 (m, 2H); MS (ES) m/z:
454 (M+Na).
8.1.14. 23-[(2,4-Dimethoxyphenyl)methyl]-6,7,8,9,12,13,
14,15,23,24-decahydro-22,24-dioxo-5,25:16,21-dimetheno-
22H - dipyrido[2,3- m:30,20 -s]pyrrolo[3,4 - p][1,6,12]triaza-
cycloeicosine-10(11H)-carboxylic acid, 1,1-dimethylethyl
ester (17). A mixture of 5 (38 mg, 0.079 mmol) and
Cs2CO3 (300 mg, 0.92 mmol) in DMF (12 mL) was
heated to 70 ꢁC. A DMF solution (2 mL) of bismesylate
16 (60 mg, 0.14 mmol) was added via syringe pump over
1 h. After the addition was complete, it was stirred at
70 ꢁC for 22 h. The mixture was cooled to 20 ꢁC,
quenched with saturated aqueous ammonium chloride
(30 mL), and diluted with EtOAc (50 mL). The organic
phase was separated, washed with water (3 ꢄ 20 mL)
and brine (15 mL). It was then dried (Na2SO4) and
concentrated. The crude product was chromatographed
on silica gel column (eluting with hexane/EtOAc) to
give 17 (27 mg, 48%) as an orange solid: 1H NMR
(400 MHz, CDCl3) d 8.40 (dd, J=4.8, 1.5 Hz, 2H), 8.29
(m, 2H), 7.78 (s, 1H), 7.18 (dd, J=8.0, 4.7 Hz, 2H), 7.10
(s, 1H), 6.85 (m, 1H), 6.46 (s, 1H), 6.43 (d, J=2.4 Hz,
1H), 4.85 (s, 2H), 4.44 (m, 2H), 4.14 (m, 2H), 3.86 (s,
3H), 3.78 (s, 3H), 3.18 (m, 2H), 2.90 (m, 2H), 2.56 (m,
2H), 1.90 (m, 2H), 1.64 (m, 2H), 1.39 (s, 9H), 1.13 (m,
2H), 0.74 (m, 2H); MS (ES) m/z: 719 (M+H+).
8.1.17. 23 - [(2,4 - Dimethoxyphenyl)methyl] - 10 - ethyl -
6,7,8,9,10,11,12,13,14,15 - decahydro - 5,25:16,21 - dime-
theno - 22H - dipyrido[2,3 - m:30,20 - s]pyrrolo[3,4 - p]
[1,6,12]triazacycloeicosine-22,24(23H)-dione (20).
A
mixture of 18 (17 mg, 0.027 mmol), and iodoethane (5
mL, 0.062 mmol) in THF (0.8 mL) was refluxed for 2
days. It was cooled and concentrated under reduced
pressure. The product was purified by column chroma-
tography (eluting with MeOH/CH2Cl2) to give 20 (6
mg, 35%) as an orange solid: 1H NMR (400 MHz,
CD3OD) d 8.32 (dd, J=4.7, 1.5 Hz, 1H), 8.25 (m, 2H),
7.85 (s, 1H), 7.32 (s, 1H), 7.27 (d, J=7.8 Hz, 1H), 7.22
(dd, J=8.0, 4.8 Hz, 1H), 7.14 (d, J=8.4 Hz, 1H), 6.93
(dd, J=8.0, 4.8 Hz, 1H), 6.54 (d, J=2.3 Hz, 1H), 6.46
(dd, J=8.4, 2.3 Hz, 1H), 4.79 (s, 2H), 4.45 (m, 2H), 4.15
(m, 2H), 3.84 (s, 3H), 3.77 (s, 3H), 2.83 (m, 4H), 2.27
(m, 2H), 1.99 (m, 2H), 1.65 (t, J=6.4 Hz, 2H), 1.27 (m,
4H), 1.15 (m, 2H), 0.88 (t, J=7.3 Hz, 3H); MS (ES) m/
z: 647 (M+H+).
8.1.18. 10 - Ethyl - 6,7,8,9,10,11,12,13,14,15 - decahydro -
22H-5, 25:16,21-dimetheno-22H-dipyrido[2,3-m:30,20 -
s]pyrrolo[3,4 - p][1,6,12]triazacycloeicosine - 22,24(23H) -
dione (21). To a solution of 20 (6 mg, 0.009 mmol) in
CH2Cl2 (1.0 mL) was added methanesulfonic acid (0.2
mL). After the mixture was stirred at 20 ꢁC for 2 h,
ammonium hydroxide was added carefully to basify the
mixture. It was extracted with EtOAc (2 ꢄ 10 mL), and
the organic layers were combined, washed with water (5
mL), brine (5 mL), dried (Na2SO4) and concentrated.
The product was purified by column chromatography
on silica gel (eluting with MeOH/CH2Cl2/NH4OH) to
give 21 (4 mg, 90%) as an orange solid: 1H NMR
(300 MHz, CDCl3) d 8.35 (m, 2H), 7.90 (m, 1H), 7.71
(m, 1H), 7.54 (s, 1H), 7.35 (s, 1H), 7.07 (dd, J=7.8, 4.9
Hz, 1H), 7.00 (dd, J=7.3, 4.7 Hz, 1H), 4.24 (m, 4H),
2.37 (m, 2H), 2.30 (m, 2H), 2.04 (m, 2H), 1.73 (t, J=6.2
Hz, 4H), 1.24 (m, 4H), 0.95–1.02 (m, 5H); MS (ES) m/z:
497 (M+H+); FAB-HRMS (M+H+). calcd 497.2665,
found 497.2682.
8.1.15. 23-[(2,4-Dimethoxyphenyl)methyl]-6,7,8,9,10,11,
12,13,14,15 - decahydro - 5,25:16,21 - dimetheno - 22H - di-
pyrido[2,3 - m:30,20 - s]pyrrolo[3,4 - p][1,6,12]triazacyclo-
eicosine-22,24(23H)-dione (18). To a solution of 17 (27
mg, 0.037 mmol) in CH2Cl2 (2 mL) was added TFA
(1.0 mL). The mixture was stirred at 20 ꢁC for 30 min.
Ammonium hydroxide was carefully added to adjust the
pH of the mixture to 10. It was extracted with EtOAc (3
ꢄ 10 mL). The organic layers were combined, washed
with water (10 mL), brine (5 mL), dried (Na2SO4) and
concentrated to give 18 (22 mg, 100%) as an orange
1
solid: H NMR (300 MHz, CD3OD) d 8.27 (m, 2H),
7.77 (d, J=8.0 Hz, 1H), 7.68 (d, J=8.0 Hz, 1H), 7.61 (s,
1H), 7.52 (s, 1H), 7.13 (d, J=8.3 Hz, 1H), 7.07 (m, 2H),
6.53 (s, 1H), 6.45 (d, J=8.5 Hz, 1H), 4.77 (s, 2H), 4.26
(m, 4H), 3.84 (s, 3H), 3.83 (s, 3H), 2.44 (t, J=7.1 Hz,
2H), 2.15 (t, J=6.8 Hz, 2H), 1.78 (m, 4H), 1.31 (m, 2H),
1.20 (m, 2H), 1.01 (m, 2H); MS (ES) m/z: 619 (M+H+).
8.1.19. 5-Oxo-undecanedioic acid diethyl ester (22). A
mixture of 4-oxo-1,9-nonanedicarboxylic acid (240 mg,
1.04 mmol), absolute ethanol (3.0 mL) and concentrated
HCl(1.0 mL) was heated under reflux for 20 h. After
8.1.16. 6,7,8,9,10,11,12,13,14,15 - decahydro - 22H -
5,25:16,21 - dimetheno - 22H - dipyrido[2,3 - m:30,20 - s]pyr-
rolo[3,4 - p][1,6,12]triazacycloeicosine-22,24(23H)-dione

1250
L. Shen et al. / Bioorg. Med. Chem. 12 (2004) 1239–1255
the mixture was cooled to 20 ꢁC, it was diluted with
EtOAc (25 mL), and neutralized with saturated aqueous
NaHCO3. The organic layer was separated, washed with
water (5 mL), brine (5 mL), dried (Na2SO4) and con-
trione (27). A mixture of 3 (55 mg, 0.13 mmol), Cs2CO3
(370 mg, 1.13 mmol) and DMF (25 mL) was heated to
100 ꢁC. A DMF (5 mL) solution of 26 (84 mg, 0.23
mmol) was added via syringe pump over 1.5 h. After the
addition was complete, the mixture was stirred at 20 ꢁC
for 2 h. It was quenched with saturated ammonium
chloride (30 mL), and extracted with methylene chloride
(2 ꢄ 30 mL). The organic phases were combined,
washed with water (3 ꢄ 20 mL), brine (30 mL), dried
(Na2SO4) and concentrated. The product was purified
by column chromatography (eluting with EtOAc/hex-
1
centrated to give 22 (270 mg, 91%) as colorless oil: H
NMR (300 MHz, CDCl3) d 4.09–4.16 (m, 4H), 2.10–2.50
(m, 10H), 1.89 (q, J=7.0 Hz, 2H), 1.54–1.68 (m, 4H),
1.25 (t, J=7.1 Hz, 6H); MS (ES) m/z: 309 (M+Na).
8.1.20. 6-[2-(3-Ethoxycarbonyl-propyl)-[1,3]dioxolan-2-
yl]-hexanoic acid ethyl ester (23). A mixture of 22 (270
mg, 0.94 mmol), ethylene glycol (0.24 mL, 4.30 mmol),
triethylorthoformate (0.48 mL, 2.89 mmo)l and TsOH
monohydrate (14 mg, 0.074 mmol) was refluxed for 45
min, and cooled to 20 ꢁC. It was diluted with saturated
aqueous NaHCO3, and extracted with diethylether (2 ꢄ
20 mL). The organic layers were combined, washed
again with NaHCO3, dried (Na2SO4) and concentrated
1
ane) to give 27 (11 mg, 16%) as an orange solid: H
NMR (300 MHz, CD3OD) d 8.24–8.30 (ddd, J=6.0,
4.7, 1.2 Hz, 2H), 7.82–7.85 (dd, J=8.0, 1.3 Hz, 1H),
7.80 (s, 1H), 7.58 (s, 1H), 7.40 (dd, J=8.1, 1.3 Hz, 1H),
7.09 (dd, J=8.0, 4.7 Hz, 1H), 6.96 (dd, J=8.1, 4.8 Hz,
1H), 4.34 (t, J=5.8 Hz, 2H), 4.20 (t, J=6.2 Hz, 2H),
3.14 (s, 3H), 2.32 (t, J=7.1 Hz, 2H), 2.11 (t, J=6.8 Hz,
2H), 1.69–1.84 (m, 4H), 1.37–1.41 (m, 2H), 1.18–1.31
(m, 2H), 1.07–1.16 (m, 2H), 0.90–1.04 (m, 2H); MS (ES)
m/z: 510 (M+H+).
1
to give 23 (310 mg, 100%) as colorless oil: H NMR
(300 MHz, CDCl3) d 4.08–4.16 (m, 4H), 3.92 (s, 4H),
2.26–2.33 (m, 4H), 1.58–1.72 (m, 8H), 1.30–1.35 (m, 4H),
1.25 (t, J=7.2 Hz, 6H); MS (ES) m/z: 353 (M+Na).
8.1.25. 8,9,11,12,13,14,15,16 - Octahydro - 6H,23H -
5,26:17,22 - dimethenodipyrido[2,3 - b:30,20 - h]pyrrolo[3,4 -
e][1,10]diazacycloheneicosine-10,23,25(7H,24H)-trione
(28). Compound 27 (12 mg, 0.023 mmol) was converted
into 28 (2 mg, 10%) using the same procedure described
for converting 9 to 10. Compound 28 was isolated as an
8.1.21. 6-[2-(4-Hydroxy-butyl)-[1,3]dioxolan-2-yl]-hexan-1-
ol (24). To a THF solution of LiAlH4 (1.0 M, 1.50
mmol) was added 23 (330 mg, 0.94 mmol) in THF (5.0
mL) solution. After the mixture was stirred at 20 ꢁC for
2 h, it was quenched with water, extracted with diethyl
ether (3 ꢄ 20 mL). The organic layers were combined,
dried (Na2SO4) and concentrated to give 24 (210 mg,
91%) as colorless liquid: 1H NMR (300 MHz, CDCl3) d
3.93 (s, 4H), 3.62–3.67 (m, 4H), 1.34–1.67 (m, 16H); MS
(ES) m/z: 269 (M+Na).
1
orange solid: H NMR (300 MHz, CDCl3) d 8.37 (d,
J=5.0 Hz, 1H), 8.32 (d, J=4.6 Hz, 1H), 7.84 (d, J=8.0
Hz, 1H), 7.70 (s, 1H), 7.49 (s, 1H), 7.42 (m, 1H), 7.08
(dd, J=8.0, 4.6 Hz, 1H), 6.95 (dd, J=8.0, 4.5 Hz, 1H),
4.34 (t, J=6.1 Hz, 2H), 4.20 (t, J=6.2 Hz, 2H), 2.30 (t,
J=7.1 Hz, 2H), 2.12 (t, J=6.7 Hz, 2H), 1.73–1.84 (m,
4H), 1.34–1.41 (m, 2H), 1.10–1.22 (m, 4H), 0.85–1.08
(m, 2H); MS (ES) m/z: 496 (M+H+); FAB-HRMS
(M+H+). calcd 495.2805, found 495.2810.
8.1.22. 1,11-Dihydroxy-undecan-5-one (25). A mixture of
24 (210 mg, 0.85 mmol), water (3.4 mL), H2SO4 (6 M,
0.5 mL) and acetone (0.3 mL) was refluxed for 1.5 h.
After the mixture was concentrated, the residue was
extracted with CH2Cl2 (3 ꢄ 15 mL). The organic
extracts were combined, dried (Na2SO4) and con-
8.1.26. 4-[2-(4-Hydroxy-butyl)-[1,3]dioxolan-2-yl]-butan-1-
ol (30). A mixture of diethyl5-oxoazeal te (318 mg, 1.23
mmol), TsOH monohydrate (19 mg, 0.10 mmol), ethyl-
ene glycol (0.35 mL, 6.20 mmol) and triethyl ortho-
formate (0.62 mL, 3.72 mmol) was heated to reflux for 1
h, and cooled to 20 ꢁC. It was diluted with saturated
aqueous NaHCO3 (5 mL), extracted with diethylether
(3 ꢄ 15 mL). The organic layers were combined, washed
with saturated NaHCO3, dried (Na2SO4) and con-
centrated to give crude 29 (370 mg, 100%): MS (ES) m/
z: 325 (M+Na). To a THF solution of LiAlH4 (2.9 mL,
1 M in THF, 2.90 mmol) was added a solution of crude
29 (370 mg, 1.23 mmol) in THF (6 mL). The mixture
was stirred at 20 ꢁC for 2 h, and water was added to
quenched excess LiAlH4. It was extracted with diethyl
ether (3 ꢄ 20 mL) and the combined diethylether
extracts were dried over Na2SO4 and concentrated. The
crude product was purified by column chromatography
eluting with EtOAc to give 30 (168 mg, 63%) as color-
less oil: 1H NMR (300 MHz, CDCl3) d 3.94 (s, 4H), 3.65
(t, J=6.3 Hz, 4H), 1.43–1.67 (m, 12H); MS (ES) m/z:
241 (M+Na).
1
centrated to give 25 (121 mg, 71%) as a white solid: H
NMR (400 MHz, CDCl3) d 3.64 (s, 2H), 3.63 (t, J=6.5
Hz, 4H), 2.39–2.46 (m, 4H), 1.25–1.78 (m, 12H); MS
(ES) m/z: 225 (M+Na).
8.1.23. Methanesulfonic acid 11-methanesulfonyloxy-7-
oxo-undecyl ester (26). To a methylene chloride (2.5
mL) solution of 25 (120 mg, 0.59 mmol) was added
triethylamine (0.41 mL, 2.97 mmol) and MsCl (0.23 mL,
2.97 mmol) at 0 ꢁC. The mixture was stirred at 20 ꢁC for
2 h, and quenched with water. The layers were sepa-
rated, and the aqueous phase was extracted with
CH2Cl2 (2 ꢄ 20 mL). The organic phases were com-
bined, washed sequentially with 5 mL of 5% HCl, water
and 5% NaHCO3. It was dried (Na2SO4) and con-
centrated to give 26 (169 mg, 80%): 1H NMR
(300 MHz, CDCl3) d 4.20–4.25 (m, 4H), 3.64 (m, 2H),
3.01 (s, 3H), 3.00 (s, 3H), 2.34–2.49 (m, 4H), 1.32–1.78
(m, 10H); MS (ES) m/z: 381 (M+Na).
8.1.24. 8,9,11,12,13,14,15,16 - Octahydro - 24 - methyl -
6H,23H-5,26:17,22-dimethenodipyrido[2,3-b:30,20 -h]pyr-
rolo[3,4-e][1,10]diazacycloheneicosine-10,23,25(7H,24H)-
8.1.27. Methanesulfonic acid 9-methanesulfonyloxy-5-
oxo-nonyl ester (31). To a solution of 30 (151 mg, 0.69

L. Shen et al. / Bioorg. Med. Chem. 12 (2004) 1239–1255
1251
mmol) in methylene chloride (2 mL) was added triethy-
lamine (0.48 mL, 3.45 mmol) and MsCl (0.27 mL, 3.45
mmol) at 0 ꢁC. The mixture was stirred at 20 ꢁC for 3 h,
and quenched with water. The layers were separated,
and the organic phase was washed with 5% HCl, water,
5% NaHCO3 and brine sequentially. It was then dried
over Na2SO4 and concentrated. The product was pur-
ified by column chromatography eluting with EtOAc/
hexane to give 31 (192 mg, 84%) as a light brown oily
and DMF (12 mL) was added a DMF (4 mL) solution
of 34 (33 mg, 0.10 mmol) at 60 ꢁC over 30 min. After the
addition was completed, the mixture was stirred at 80 ꢁC
for 3 h, and then 20 ꢁC for 17 h. It was quenched with
aqueous NH4Cl(10 mL), and extracted with EtOAc (2
ꢄ 45 mL). The organic layers were combined, washed
with water (3 ꢄ 20 mL), brine (20 mL), dried (Na2SO4)
and concentrated. Purification with column chromato-
graphy eluting with acetone/CH2Cl2 gave 35 (27 mg,
64%) as an orange solid: 1H NMR (400 MHz, CDCl3) d
8.33 (dd, J=4.6, 1.3 Hz, 2H), 7.69 (d, J=7.9 Hz, 2H),
7.39 (s, 2H), 7.25 (s, 1H), 6.99 (dd, J=8.0, 4.7 Hz, 2H),
6.46 (s, 1H), 6.44 (d, J=2.3 Hz, 1H), 4.85 (s, 2H), 4.47–
4.51 (m, 2H), 4.08–4.14 (m, 2H), 3.86 (s, 3H), 3.79 (s,
3H), 3.49 (d, J=4.8 Hz, 1H), 3.26 (m, 1H), 1.73–1.83
(m, 4H), 0.93–1.04 (m, 8H); MS (ES) m/z: 620
(M+H+).
1
solid: H NMR (300 MHz, CDCl3) d 4.21 (m, 4H), 3.01
(s, 6H), 2.48 (m, 2H), 1.43–1.77 (m, 10H); MS (ES) m/z:
353 (M+Na).
8.1.2.8. 7,8,9,11,12,13,14-heptahydro-6H,21H-5,24:15,20
-dimethenodipyrido[2,3-b:30,20-h]pyrrolo[3,4-e][1,10]diaza-
cyclononadecine-10,21,23(22H)-trione (33). To a mixture
of 5 (19 mg, 0.040 mmol), Cs2CO3 (160 mg, 0.50 mmol)
and DMF (6 mL) was added dropwise a solution of 31
(24 mg, 0.072 mmol) in DMF (3 mL) at 70 ꢁC. After
stirring at 70 ꢁC for 4 h, it was cooled in an ice bath, and
quenched with aqueous NH4Cl. It was extracted with
EtOAc (2 ꢄ 30 mL), and the organic extracts were
combined, washed with water (3 ꢄ 15 mL), brine (15
mL), dried (Na2SO4) and concentrated to give the crude
32. The crude 32 was mixed with methylene chloride (1
mL), and methanesulfonic acid (0.3 mL) was added.
The mixture was stirred at 20 ꢁC for severalhours until
no 32 was detected by MS. It was cooled in an ice bath,
and carefully quenched with ammonium hydroxide. The
mixture was extracted with EtOAc (3 ꢄ 15 mL), and the
extracts were washed with water (10 mL), brine (10
mL), dried (Na2SO4) and concentrated. The product
was purified by column chromatography on silica gel
(eluting with MeOH/CH2Cl2) to give 33 (12 mg, 67%
from 31) as an orange solid: 1H NMR (300 MHz,
CDCl3) d 8.34 (d, J=3.9 Hz, 2H), 7.80 (d, J=7.9 Hz,
2H), 7.63 (s, 2H), 7.05 (dd, J=8.0, 4.7 Hz, 2H), 4.26 (t,
J=6.0 Hz, 4H), 2.10 (t, J=7.0 Hz, 4H), 1.71–1.80 (m,
4H), 1.32–1.39 (m, 4H); MS (ES) m/z: 468 (M+H+).
8.1.31. 7,8,9,10,11,12,13,14-Octahydro-10-hydroxy-6H,
21H - 5,24:15,20 - dimethenodipyrido[2,3 - b:30,20 - h]pyr-
rolo[3,4-e].[1,10]diazacyclononadecine-21,23(22H)-dione
(36) To a solution of 35 (18 mg, 0.029 mmol) in CH2Cl2
(2 mL) was added methanesulfonic acid (0.2 mL). The
mixture was stirred at 20 ꢁC for 15 h, and carefully
quenched with ammonium hydroxide. It was then
extracted with EtOAc (2 ꢄ 20 mL), and the combined
organic extracts were washed with water (10 mL), brine
(10 mL), dried (Na2SO4) and concentrated. Purification
with column chromatography on silica gel (eluting with
acetone/CH2Cl2) gave 36 (5 mg, 38%) as an orange
1
solid: H NMR (300 MHz, CDCl3) d 8.35 (dd, J=4.7,
1.5 Hz, 2H), 7.69 (dd, J=7.9, 1.1 Hz, 2H), 7.49 (s, 1H),
7.40 (s, 2H), 7.03 (dd, J=8.0, 4.7 Hz, 2H), 4.48–4.56 (m,
2H), 4.10–4.18 (m, 2H), 3.28 (m, 1H), 1.40–1.87 (m,
4H), 0.79–1.11 (m, 8H); MS (ES) m/z: 470 (M+H+).
.
.
Anal. calcd for C27H27N5O3 2HCl 1.7H2O: C, 56.59; H,
5.70; N, 12.22. Found: C, 56.63; H, 5.47; N, 12.13.
8.1.32. 2,6-Bis-[4-(tert-butyl-dimethyl-silanyloxy)-butyl]-
pyridine (37). To a solution of 2,6-lutidine (0.5 mL,
4.30 mmol) in THF (15 mL) was added n-BuLi (6.4 mL,
1.6 M in hexane, 10.3 mmol) at ꢀ78 ꢁC. The deep red
solution was kept stirring at ꢀ78 ꢁC for 30 min, and 3-
bromo-propoxy-tert-butyldimethylsilane (2.4 mL, 10.3
mmol) was added. The mixture was warmed to 20 ꢁC for
18 h, quenched with water (2 mL), and concentrated
under reduced pressure. The residue was diluted with
water (15 mL), extracted with hexane (3ꢄ20 mL). The
organic extracts were combined, dried (Na2SO4) and
concentrated. Purification by column chromatography
(eluting with hexane/EtOAc) gave 37 (0.55 g, 30%) as
.
.
Anal. calcd for C27H25N5O3 2HCl 2H2O: C, 56.45; H,
5.09; N, 12.19. Found: C, 56.20; H, 5.23; N, 11.81.
8.1.29. Methanesulfonic acid 5-hydroxy-9-methanesulfo-
nyloxy-nonyl ester (34). To a mixture of NaBH4 (33
mg, 0.88 mmol) in ethanol (0.8 mL) was added a solu-
tion of 31 (48 mg, 0.14 mmol) in THF (1 mL) and eth-
anol(1 mL). The mixture was stirred at 20 ꢁC for 40
min, and quenched with water (1 mL) and acetic acid (1
mL). The mixture was then extracted with CH2Cl2 (3 ꢄ
10 mL), and the combined organic layers were washed
with water (10 mL), saturated aqueous NaHCO3 (10
mL), dried (Na2SO4) and concentrated. The product
was purified by column chromatography (eluting with
EtOAc/hexane) to give 34 (38 mg, 79%) as colorless oil:
1H NMR (300 MHz, CDCl3) d 4.25 (t, J=6.4 Hz, 4H),
3.62 (m, 1H), 3.01 (s, 6H), 1.74–1.82 (m, 4H), 1.47–1.62
(m, 8H); MS (ES) m/z: 355 (M+Na).
1
colorless oil: H NMR (300 MHz, CDCl3) d 7.45 (m,
1H), 6.91 (m, 2H), 3.59 (t, J=6.4 Hz, 4H), 2.73 (m, 4H),
1.70 (m, 4H), 1.57 (m, 4H), 0.85 (s, 18H), 0.00 (s, 12H);
MS (ES) m/z: 452 (M+H+).
8.1.33. 4-[6-(4-Hydroxy-butyl)-pyridin-2-yl]-butan-1-ol
(38). To a mixture of 37 (0.55 g, 1.20 mmol) in THF
(3.0 mL) was added TBAF (2.6 mL, 1 M in THF, 2.60
mmol). It was stirred at 20 ꢁC for 3 h, and concentrated
under reduced pressure. Purification by column chro-
matography on silica gel (eluting with ethyl acetate
containing 5% Et3N) gave 38 (254 mg, 95%) as color-
8.1.30. 22-[(2,4-Dimethoxyphenyl)methyl]-7,8,9,10,11,12,
13,14-octahydro-10-hydroxy-6H,21H-5,24:15,20-dime-
thenodipyrido[2,3 - b:30,20 - h]pyrrolo[3,4 - e].[1,10]diaza-
cyclononadecine-21,23(22H)-dione (35) To a mixture of
5 (33 mg, 0.069 mmol), Cs2CO3 (450 mg, 1.38 mmol)

1252
L. Shen et al. / Bioorg. Med. Chem. 12 (2004) 1239–1255
1
less oil: H NMR (300 MHz, CDCl3) d 7.53 (t, J=7.6
Hz, 1H), 6.98 (d, J=7.6 Hz, 2H), 3.70 (t, J=6.0 Hz,
4H), 2.83 (t, J=7.4 Hz, 4H), 1.85 (m, 4H), 1.64 (m, 4H);
MS (ES) m/z: 224 (M+H+).
(m, 2H), 7.43–7.54 (m, 3H), 6.99 (dd, J=7.9, 4.7 Hz,
2H), 6.87 (d, J=7.4 Hz, 2H), 4.28 (t, J=6.2 Hz,
4H), 2.65 (m, 4H), 1.81 (m, 4H), 1.46 (m, 4H); MS
(ES) m/z: 517 (M+H+); FAB-HRMS (M+H+).
calcd 517.2352, found 517.2343.
8.1.34. Methanesulfonic acid 4-[6-(4-methanesulfony-
loxy-butyl)-pyridin-2-yl]-butyl ester (39). To a solution
of diol 38 (254 mg, 1.14 mmol) in CH2Cl2 (4 mL) was
added triethylamine (0.95 mL, 6.84 mmol) at 0 ꢁC, fol-
lowed by MsCl (0.35 mL, 4.56 mmol). After the mixture
was stirred at 20 ꢁC for 1.5 h, it was diluted with diethyl
ether (20 mL) and washed with 5% HCl(5 mL). The
layers were separated, and the organic phase was dis-
carded. The aqueous phase was diluted with CH2Cl2 (10
mL), and treated with 5% NaOH (5 mL) till it turned
basic. The mixture was extracted with CH2Cl2 (3ꢄ20
mL), and the organic extracts were combined, washed
with brine (10 mL), dried (Na2SO4) and concentrated.
Purification with column chromatography eluting with
hexane/EtOAc gave 39 (162 mg, 38%) as light brown
liquid: MS (ES) m/z: 380 (M+H+).
8.1.37. 3-[5-(3-Hydroxy-propyl)-thiophen-2-yl]-propan-1-
ol (43). To a mixture of thiophene (0.50 mL, 6.25
mmol), anhydrous hexane (2.0 mL), TMEDA (2.2 mL,
14.4 mmol) was added n-BuLi (9 mL, 1.6 M in hexane,
14.4 mmol) at 20 ꢁC. After it was refluxed for 30 min,
the mixture was cooled to ꢀ40 ꢁC and diluted with
anhydrous THF (5 mL). A THF (3 mL) solution of (3-
bromopropoxy)-tert-butyldimethylsilane (3.3 mL, 14.4
mmol) was added. The mixture was warmed slowly to
20 ꢁC overnight, quenched with water (3 mL) and con-
centrated. The residue was diluted with hexane (50 mL),
washed with water (15 mL), brine (15 mL), dried
(Na2SO4) and concentrated under reduced pressure to
give crude 42 (2.60 g): MS (ES) e/z: 451 (M+Na). The
crude 42 was dissolved in THF (6 mL), and TBAF (18
mL, 1 M in THF, 18.0 mmol) was added. After the
mixture was stirred at 20 ꢁC for 2 h, it was concentrated
under reduced pressure. Purification with column chro-
matography on silica gel eluting with ethyl acetate/hex-
ane gave 43 (0.44g, 30%) as colorless oil: 1HNMR
(300 MHz, CDCl3) d 6.60 (s, 2H), 3.71 (t, J=6.3 Hz,
4H), 2.87 (t, J=7.4 Hz, 4H), 1.87–1.96 (m, 4H); MS
(ES) e/z: 223 (M+Na).
8.1.35. 6,7,8,9,15,16,17,18 - Octahydro - 26 - methyl - 25H -
5,28:19,24 - dimetheno - 10,14 - nitrilodipyrido[2,3 - b:30,20 -
h]pyrrolo[3,4 - e][1,10]diazacyclotricosine-25,27(26H)-dione
(40). A mixture of 3 (74 mg, 0.21 mmol), Cs2CO3 (290
mg, 0.89 mmol) and DMF (30 mL) was heated to
100 ꢁC. A solution of 39 (100 mg, 0.26 mmol) in DMF
(7 mL) was added via syringe pump over 2 h. After the
addition was completed, the mixture was stirred at 20 ꢁC
for 18 h. It was quenched with saturated ammonium
chloride, and extracted with ethyl acetate (3ꢄ50 mL).
The organic extracts were combined, washed with water
(3ꢄ30 mL), brine (30 mL), dried (Na2SO4) and con-
centrated. The residue was purified by column chroma-
tography (eluting with acetone/methylene chloride) to
give 40 (14 mg, 22%) as an orange solid and recover 39
(21 mg): 1H NMR (300 MHz, CD3OD) d 8.14 (d, J=4.6
Hz, 1H), 7.54–7.73 (m, 8H), 6.97 (m, 2H), 4.30 (t, J=5.6
Hz, 4H), 3.14 (s, 3H), 2.65 (m, 4H), 1.73 (m, 4H), 1.31
(m, 4H); MS (ES) m/z: 531 (M+H+).
8.1.38. Methanesulfonic acid 3-[5-(3-methanesulfony-
loxy-propyl)-thiophen-2-yl]-propyl ester (44). A solution
of 43 (0.44 g, 2.20 mmol) in CH2Cl2 (5 mL) was cooled
in an ice bath, and triethylamine (3.0 mL, 22.0 mmol)
and MsCl(1.7 mL, 22.0 mmo)l were added. The mix-
ture was stirred at 20 ꢁC for 30 min, and then quenched
with water ( 2 mL). It was extracted with CH2Cl2 (15
mL), and the combined organic extracts were combined,
dried (Na2SO4) and concentrated. Purification with col-
umn chromatography on silica gel eluting with ethyl
acetate/hexane gave 44 (245 mg, 31%) as light brown
1
oil: HNMR (300 MHz, CDCl3) d 6.63 (s, 2H), 4.26 (t,
J=6.2 Hz, 4H), 3.00 (s, 6H), 2.91 (t, J=7.3 Hz, 4H),
2.07-2.14 (m, 4H); MS (ES) e/z: 379 (M+Na).
8.1.36. 6,7,8,9,15,16,17,18-Octahydro-25H-5,28:19,24-di-
metheno - 10,14 - nitrilodipyrido[2,3 - b:30,20 - h]pyrrolo[3,4 -
e][1,10]diazacyclotricosine-25,27(26H)-dione (41). A mix-
ture of 40 (14 mg, 0.026 mmol), KOH (360 mg, 6.43
mmol) and ethanol (3 mL) was refluxed for 2 days.
After it was cooled to 20 ꢁC, the solvent was removed
under reduced pressure. The residue was dissolved in
water (5 mL), acidified with 10% citric acid and stirred
at 20 ꢁC for 10 min. It was extracted with methylene
chloride (3ꢄ20 mL). The organic extracts were com-
bined, dried (Na2SO4) and concentrated. The residue
was mixed with ammonium acetate (2.5 g), and heated
to 140 ꢁC for 3 h. After it was cooled to 20 ꢁC, water (10
mL) was added, and it was then basified with 20%
NaOH. The mixture was extracted with EtOAc (3ꢄ20
mL), and the organic extracts were combined, washed
with water (20 mL), brine (10 mL), dried (Na2SO4)
and concentrated. The product was purified by col-
umn chromatography (eluting with acetone/CH2Cl2)
8.1.39. 23-[(2,4-Dimethoxyphenyl)methyl]-6,7,8,13,14,
15-hexahydro-9,12-epithio-5,25:16,21-dimethenodipyrido
[2,3-b:30,20-h]pyrrolo[3,4-e][1,10]diazacyclotricosine-22,24
(23H)-dione (45). To a mixture of 5 (42 mg, 0.088
mmol), Cs2CO3 (550 mg, 1.68 mmol) and DMF (15 mL)
at 70 ꢁC was added a DMF (3 mL) solution of 44 (56
mg, 0.15 mmol). The mixture was stirred at 90 ꢁC for 3.5
h, and then 20 ꢁC for 18 h. It was quenched with aqu-
eous NH4Cl(5 mL), diulted with water (10 mL) and
extracted with EtOAc (3ꢄ20 mL). The organic layers
were combined, washed with water (3ꢄ10 mL), brine
(10 mL), dried (Na2SO4) and concentrated. Purification
with column chromatography eluting with acetone/
1
CH2Cl2 gave 45 (11 mg, 39%) as an orange solid: H
NMR (300 MHz, CDCl3) d 8.30 (dd, J=4.7, 1.4 Hz,
2H), 7.79 (d, J=7.9 Hz, 2H), 7.33 (s, 2H), 6.97 (dd,
J=8.0, 4.7 Hz, 2H), 6.59 (s, 1H), 6.47 (m, 2H), 6.33 (s,
2H), 4.85 (s, 2H), 4.33 (t, J=5.4 Hz, 4H), 3.87 (s, 3H),
1
to give 41 (4 mg, 30%) as a yellow solid: H NMR
(300 MHz, CDCl3) d 8.28 (d, J=4.0 Hz, 2H), 7.68

L. Shen et al. / Bioorg. Med. Chem. 12 (2004) 1239–1255
1253
3.78 (s, 3H), 2.82 (m, 4H), 2.27 (m, 4H); MS (ES) m/z:
644 (M+H+).
mM EDTA, 0.1 mg/mL histone-H1, 10 mM MgAce-
tate, and 0.2 mCuries per well 33P-g-ATP (2000–3000 Ci/
mmol). After incubation for 40 min at room tempera-
ture, the reaction was terminated by the addition of 5
mL of a 3% phosphoric acid solution. 10 mL of the
reaction mixture was then spotted onto a P30 filtermat
and washed three times for 5 min in 75 mM phosphoric
acid and once in methanolprior to drying and counting
on a scintillation counter.
8.1.40. 6,7,8,13,14,15-Hexahydro-9,12-epithio-5,25:16,21
-dimethenodipyrido[2,3-b:30,20-h]pyrrolo[3,4-e][1,10]diaza-
cyclotricosine-22,24(23H)-dione (46). To a solution of 45
(11 mg, 0.017 mmol) in CH2Cl2 (2 mL) was added
methanesulfonic acid (0.2 mL). The mixture was stirred
at 20 ꢁC for 15 h, and carefully quenched with ammo-
nium hydroxide. It was then extracted with EtOAc
(2ꢄ15 mL), and the combined organic extracts were
washed with water (3ꢄ10 mL), brine (10 mL), dried
(Na2SO4) and concentrated. Purification with column
chromatography on silica gel (eluting with acetone/
8.2.4. Rsk3 kinase assay. A kinase reaction mixture was
prepared containing 8 mM MOPS (pH 7.0), 0.2 mM
EDTA, 30 mM KKKNRTLSVA, 10 mM MgAcetate,
and 0.2 mCuries per well 33P-g-ATP (2000–3000 Ci/
mmol). After incubation for 40 min at room tempera-
ture, the reaction was terminated by the addition of 5
mL of a 3% phosphoric acid solution. 10 mL of the
reaction mixture was then spotted onto a P30 filtermat
and washed three times for 5 min in 75 mM phosphoric
acid and once in methanolprior to drying and counting
on a scintillation counter.
1
CH2Cl2) gave 46 (3.5 mg, 50%) as an orange solid: H
NMR (300 MHz, CDCl3) d 8.32 (dd, J=4.7, 1.5 Hz,
2H), 7.76 (m, 2H), 7.33 (s, 2H), 7.00 (dd, J=8.0, 4.7 Hz,
2H), 6.34 (s, 2H), 4.35 (t, J=5.6 Hz, 4H), 2.83 (t, J=5.7
Hz, 4H), 2.28 (m, 4H); MS (ES) m/z: 494 (M+H+).
.
Anal. calcd for C28H23N5O2S 0.39H2O: C, 66.91; H,
4.81; N, 13.93. Found: C, 67.25; H, 4.57; N, 13.54.
8.2.5. Protein kinase selectivity panel (Upstate Biotech
Inc.). Protein kinase selectivity assays were performed
as previously described.31,40 Briefly, protein kinases
were assayed for their ability to phosphorylate the
appropriate peptide/protein substrates in the presence
of 10 mM compound. Assays were done using 100 mM
ATP and were linear with respect to time. IC50 values
were determined from at least three separate experi-
ments and the Ki valuesꢂSEM were calculated using
the Cheng–Prussof equstion.
8.2. Biology
8.2.1. GSK-3ꢀ kinase assay. Compounds were tested
for the ability to inhibit recombinant rabbit GSK-3b
(New England Biolabs) using the following protocol.
Protein phosphatase inhibitor-2 (PPI-2, Calbiochem)
phosphorylation was measured using a standard fil-
tration assay (MultiScreen-DV/Millipore). Briefly, the
test compounds were added to a reaction mixture con-
taining PPI-2 (45 ng), GSK-3b (0.75 units) and 33P-ATP
(1 mCi) in 50 mM Tris–HCl(pH 8.0), 10 mM MgCl ,
2
0.1% BSA, 1 mM DTT, and 100 mM activated Sodium
Orthovanadate. The totalATP concentration was 25
mM. After 90 min incubation at 30 ꢁC, the phosphoryl-
ated PPI-2 was precipitated using one volume of 20%
trichloroacetic acid (TCA). Filter plates were subse-
quently washed with 10% TCA and radioactivity was
quantified using a TopCount Scintillation Counter
(Packard). IC50 values were determined from at least
three separate experiments and the Ki valuesꢂSEM
were calculated using the Cheng–Prussof equation.
8.2.6. Glycogen synthase assay. Compounds were tested
for the ability to increase glycogen synthase (GS) activ-
ity in living cells (HEK293 cells). To do this, cell
extracts were prepared from cells treated with com-
pounds or vehicle and GS activity measured using a
modified protocol.17 Briefly, cells were serum (and glu-
cose) starved for 3 h and treated with the appropriate
compounds for 90 min at 37 ꢁC. Cells were then washed,
scraped and collected by centrifugation prior to lysis
using three freeze/thaw cycles. Lysates were then clar-
ified by centrifugation and the supernatants assayed for
GS activity. To do this, 14C-UDP glucose incorporation
into glycogen was measured in the absence or presence
of glucose 6-phosphate. The EC50 for GS activation was
then determined and compared with lithium.
8.2.2. PKCßII assays. Compounds were tested for the
ability to inhibit recombinant human PKCßII using the
following protocol. Histone H1 phosphorylation was
measured using a standard filtration assay. Briefly, the
test compounds were added to a reaction mixture con-
taining histone H1 (0.1 mg/mL), the PKCßII (5-10 mU)
and 33P-ATP (1 mCi) in 20 mM Hepes (pH 7.4), 0.03%
Triton X-100, 0.1 mM CaCl2, 10 mM MgAcetate, 0.1
mg/mL phosphatidylserine, and 10 mg/mL diacylgly-
cerol. The total ATP concentration was 100 mM. After
40 min incubation at room temperature, the reaction
was stopped by the addition of 5 mL of a 3% phosphoric
acid solution. Reaction mix was then spotted onto a P30
filtermat and the filters were washed three times for 5 min
with 75 mM phosphoric acid. The filtermat was then
washed one time with MeOH and dried prior to counting.
8.2.7. Molecular docking procedures. The X-ray struc-
ture of unbound GSK-3b (1 h8f.pdb)35 was used as the
target structure for docking. Its ATP-binding site was
determined by superimposition to the X-ray structure of
CDK2 and staurosporine complex (1aq1.pdb)36 and
defined as the spatialregion occupied by staurosporine.
Conformationalsearching was first conducted for com-
pound 46, using the Monte Carlo Multiple Minimum
(MCMM)41 method implemented in MacroModel,42
leading to 260 low-energy and unique conformers. The
cutoff of energy window was set as 50 kj/mol, and the
Monte Carlo steps were set to 10,000 times. Conforma-
tionalstructures were not considered unique unelss the
least squares superimposition of heavy atoms found one
8.2.3. CDK2 kinase assay. A kinase reaction mixture
was prepared containing 8 mM MOPS (pH 7.0), 0.2

1254
L. Shen et al. / Bioorg. Med. Chem. 12 (2004) 1239–1255
or more pairs of equivalent atoms separated by more
than 0.25 A. All of the 260 conformational structures
were then individually docked into the ATP binding-site
of GSK-3b, using the program Glide.43 Both the com-
pound 46 conformationalstructure and the GSK-3 b
protein structure were held rigid during the docking.
The docking poses were evaluated by the empirical
scoring function, GlideScore.44 The GSK-3b structure
complexed with the best-scoring pose of compound 46
was further optimized by full energy minimization, and
then treated as the binding structure of GSK-3b and com-
pound 46 (Supporting Information). All of the molecular
mechanism calculations were done with the program
MacroModel,42 using OPLS_AA force field.45 The effect
of aqueous solution was treated by GB/SA model.46
References and notes
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GSK-3b, glycogen synthase kinase-3b; AMPK, AMP-
activated protein kinase; Blk, B lymphocyte kinase;
CAMKII, calmodulin-dependent protein kinase II;
CAMKIV, calmodulin-dependent protein kinase IV;
CDK1/cyclinB, cycline-dependent protein kinase 1;
CDK2/cycA, cyclin-dependent protein kinase 2; CDK5/
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Src kinase; Fes, cellular product of the fes proto-onco-
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JNK3, c-Jun N-terminalkinase 3; Lck, ylmphocyte
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mitogen-activated protein kinase 1; MAPK2, mitogen-
activated protein kinase 2; MAPKAP-K2, MAPK-acti-
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regulated/activated kinase; c-Raf, cellular product of raf
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