Synthesis and CHK1 inhibitory potency of Hymenialdisine analogues

Article abstract of DOI:10.1016/j.bmcl.2008.12.001

A series of thieno[3,2-b]pyrroloazepinones derivatives related to Hymenialdisine were prepared and tested for CHK1 inhibitory activity. Nanomolar inhibitions were achieved when electron-withdrawing substituents were introduced at position 3 of the thiophene ring.

Full text of DOI:10.1016/j.bmcl.2008.12.001

Bioorganic & Medicinal Chemistry Letters 19 (2009) 841–844
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and CHK1 inhibitory potency of Hymenialdisine analogues
Jean-Gilles Parmentier a, Bernard Portevin a, Roy M. Golsteyn b, , Alain Pierré b, John Hickman b,
Philippe Gloanec a, Guillaume De Nanteuil a,
*
a Medicinal Chemistry Division D, Institut de Recherche Servier, 11 rue des Moulineaux, 92150 Suresnes, France
b Division of Cancerology, Institut de Recherche Servier, 125 Chemin de Ronde, 78290 Croissy sur Seine, France
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of thieno[3,2-b]pyrroloazepinones derivatives related to Hymenialdisine were prepared and
tested for CHK1 inhibitory activity. Nanomolar inhibitions were achieved when electron-withdrawing
substituents were introduced at position 3 of the thiophene ring.
Received 22 October 2008
Revised 28 November 2008
Accepted 2 December 2008
Available online 7 December 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Hymenialdisine
CHK1 inhibitor
Over the last decades, DNA damaging agents have been used as
first line agents in cancer chemotherapy. As a consequence of DNA
damage by UV light, radiation, or cytotoxic drugs, tumor cells ar-
rest their cycle in S or G2/M phases to attempt DNA repair or un-
dergo apoptosis.1 Studies have demonstrated that the Serine/
Threonine checkpoint kinase CHK 1 regulates both the G2/M and
intra-S checkpoints and plays an important role in cell-cycle pro-
gression, mainly for the P53-defective cancer cells (50–70% of all
cancers). Since cell-cycle arrest was found to be necessary for
DNA repair following damages induced by genotoxic agents, abro-
gation of the G2/M checkpoint by specific inhibitors should in-
crease the sensitivity of P53 deficient tumours to DNA damaging
agents without enhancing toxicity toward normal proliferating
cells, which are non-mutated P53 proficient cells.2,3
(Z)-Hymenialdisine 1, (Z)-2-debromohymenialdisine 2 and (Z)-
3-bromohymenialdisine 3 (Fig. 1), are three naturally occurring
alkaloids isolated from the marine sponges Hymenialcidon, Acan-
thella, Axinella and Stylissa.4 Structurally these compounds bear
a pyrrole ring, often substituted with one or two bromine atoms,
as well as an imidazolinone. These compounds have been reported
to inhibit a large panel of kinases, among which CHK1.5,6
that improvement was needed in order to obtain a candidate for
development. X-ray crystallography of Debromohymenialdisine
in the ATP binding site of apo human CHK1 suggested that struc-
tural opportunities may still exist to enhance affinity against the
enzyme by filling a pocket in the pyrroloazepinone region.9 Thus,
we decided to explore the replacement of the phenyl ring of the in-
dole system in 4 with other heterocycles. We report here our ef-
forts in preparing and evaluating the potency against CHK1 of
thieno-[3,2-b]-pyrroloazepinones analogues of Z-hymenialdisine 1.
The synthesis of this family of compounds is depicted in Scheme
1. Conveniently substituted 2-formylthiophenes 5 were engaged in
an alkylidenation reaction with ethyl 2-azido acetate in the pres-
ence of sodium ethoxide as a base according to the strategy of
Hemetsberger and Knittel.10 Cyclisation of the ethyl azido thio-
phene carboxylates 6 was realized in refluxing xylene and gave
the ethyl thieno[3,2-b]pyrrole-5-carboxylates 7. The corresponding
acids 8 were obtained by saponification in a mixture of aqueous so-
dium hydroxide and ethanol in quantitative yields. Coupling these
acids with b-alanine ethyl ester gave esters 9 in good yields. A sec-
ond saponification using aqueous potassium hydroxide in refluxing
ethanol allowed us to obtain the acids 10 in very good yields. Cyc-
lisation of these compounds to give the thieno[3,2-b]pyrroloazepi-
nones 11 proved to be more tedious: the expected compounds
were only obtained using a mixture of MeSO3H and P2O5 warmed
at 80–100 °C for 1–4 h with yields ranging from 15% to 93%. Protec-
tion of the nitrogen atom of the pyrrole ring was realized with p-
methoxybenzyl bromide to give compounds 12. At this stage, this
nitrogen atom was also methylated (K2CO3, CH3I, DMF, 63%). To
incorporate the glycocyamidine ring, we capitalized on the obser-
vation made by Tepe et al.,11 and succeeded in condensing thiohy-
dantoine and rhodanine with ketones 12 using the previously
In 2001, Smith-Kline–Beecham patented a series of Myt1 inhib-
itors exemplified here by the indole derivative 4.7 This compound
was later described by Tepe as a potent CHK1 inhibitor and its
preparation was given.8 In our hands, in vitro potency and selectiv-
ity as well as in vivo activity of this family of inhibitors showed
* Corresponding author. Tel.: +33 1 55 72 22 34; fax: +33 1 55 72 24 30.
E-mail address: guillaume.denanteuil@fr.netgrs.com (G. De Nanteuil).
Present address: Department of Biological Sciences, University of Lethbridge,
Lethbridge, Alta., Canada T1K 3M4.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.12.001

842
J.-G. Parmentier et al. / Bioorg. Med. Chem. Lett. 19 (2009) 841–844
N
N
N
N
H2N
Br
H2N
H2N
H2N
O
O
O
O
O
O
N
N
N
N
H
H
H
H
Br
Br
NH
NH
NH
NH
N
N
N
N
H
H
H
H
O
O
1
2
3
4
Figure 1.
reported Ti(IV)-pyridine reagent. Yields of 13 were predominantly
low to moderate. These intermediates were further transformed
into Hymenialdisine analogues by reacting 13 with a variety of
amines in the presence of terbutyl-hydroperoxide as an oxidant,12
to produce 14. 14p and 14q were obtained by condensation with
ethanolamine and 14r with morpholin-1-yl-ethylamine.
Otherwise, ammonia was used. Final deprotection of the thie-
no[3,2-b]pyrroloazepine moiety with trifluoroacetic acid afforded
the final inhibitors 15 after reverse phase HPLC purification. The
preparation of 15o necessitated two more steps, first coupling with
Boc-glycine (HATU, DIEA, DMF, 48 h, rt, 62%) followed by deprotec-
tion with TFA (CH2Cl2, 2 h, rt, 42%).
(Scheme 2). Substitution of the bromide was realized by standard
cyanation conditions resulting in the obtention of 9g, which was
subsequently hydrolysed in acidic media to give 9h. On the other
hand, introduction of a phenyl group at position 3 of 9e was suc-
cessfully realized by a Suzuki–Miyaura reaction involving phenyl
boronic acid according to the conditions described by Buchwald
to produce 9i in excellent yield.13 These intermediates were en-
gaged in the following steps as described above.
IC50 for Chk1 inhibition are given in Table 1. First, it is worth
noting that the potencies of the debromo and dibromo derivatives
2 and 3, as well as the indole analog 4 were found in the same
range of activity at IC50 between 0.10 and 0.47 lM, indicating that
Compound 9e was found to be the starting material for struc-
tural modifications at position 3 on the thieno[3,2-b] pyrrole ring
substitutions on the pyrrole ring as well as its coupling with a phe-
nyl to give the indole ring hardly influenced activity. Hymenialdi-
R1
S
5
S
S
R1
CHO
R1
COOEt
a
b
c
COOEt
N
H
N3
R2
R2
R2
6
7
R1
S
R1
S
R1
S
d
e
H
N
H
COOEt
COOH
N
COOH
N
N
N
H
R2
H
R2
R2
H
O
O
10
8
9
H
N
S
O
X
O
O
R1
R1
R1
S
S
S
g
h
f
NH
NH
NH
N
N
N
R2
R2
R2
R
H
O
O
O
PMB
12
PMB
13
11
N
4HN
R4HN
N
O
O
X
X
R1
S
S
R1
i
j
NH
NH
N
N
R2
R2
H
O
O
PMB
14
15
Scheme 1. Typical synthesis of inhibitors. Reagents and conditions: (a) ethyl 2-azido acetate, EtONa, 2 h, 20–55%; (b) xylene, reflux, 0.5 h, 70–75%; (c) 1 N NaOH, EtOH–H2O,
20 h, 52–95%; (d) b-alanine ethyl ester, DCC-HOBT-DIEA, DMF, 2 h, 80–100%; (e) 1 N KOH, EtOH–H2O, 3–6 h, 85–97%; (f) CH3SO3H–P2O5, 1–4 h, 15–93%; (g) p-methoxybenzyl
bromide (PMB-Br), K2CO3, CH3CN, 4–10 h, 75–85%; (h) thiohydantoine (X = NH) or rhodanine (X = S), TiCl4-pyridine-THF, 3 h at ꢀ5 °C, 11–62%; (i) R4-NH2, tBuOOH–EtOH, 8 h,
12–55%; (j) refluxing TFA, 12 h, 25–52%.

J.-G. Parmentier et al. / Bioorg. Med. Chem. Lett. 19 (2009) 841–844
843
S
S
H
H
N
b
COOEt
COOEt
N
N
H2NOC
N
H
O
NC
H
a
O
9h
S
9g
H
COOEt
N
N
Br
c
H
O
S
9e
H
N
COOEt
N
H
O
9i
Scheme 2. Modifications of 9e at position 3. Reagents and conditions : (a) Cu(CN)2, DMF, 6 h, 41%; (b) 4 N HCl, dioxane, 100 °C, 3 h, 64%; (c) phenyl boronic acid, Pd2(dba)3, 20-
dicyclohexylphosphino-2,6-dimethoxy-1,10-biphenyl (S-Phos), K3PO4, toluene, 10 h, 64%.
Table 1
ChK1 IC50 values for compounds 1–4, 14a–b, 15a–s
R4HN
N
O
X
R1
S
NH
N
R2
O
R3
Compound
R1
R2
R3
R4
X
CHK1 IC50 (lM)
1
2
3
4
—
—
—
—
H
H
H
Cl
H
Cl
Br
H
H
H
H
—
—
—
—
H
Br
H
H
Cl
Cl
H
Br
CN
CONH2
Ph
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
S
S
S
S
NH
NH
NH
NH
1.9*
0.33
0.10
0.47
>10
14a
14b
15a
15b
15c
15d
15e
15f
15g
15h
15i
15j
15k
15l
15m
15n
15o
15q
15r
15s
CH3
4-MeOBn
>10
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
0.15
0.30
0.14
0.89
0.46
0.014
0.029
0.15
1.3
0.23
0.18
0.19
>10
Phenyl ring
Phenyl ring
H
H
H
H
Br
Ph
H
H
H
H
>10
0.26
1.7
0.28
>5
H
H
H
H
Br
H
Br
H
HOOC-CH2
HO(CH2)2
HO(CH2)2
Morpholin-1-ethyl
*
Literature value Ref. 8.
sine 1 was not tested in house, but its IC50 value reported by Tepe
at 1.9 M can be considered if one compares IC50’s for DBH 2 at
0.72 M in Tepe’s paper and 0.33 M in our hands. As far as the
thieno-[3,2-b]-pyrroloazepinone ring system was concerned, we
studied first the alkylation of the nitrogen atom of the central pyr-
role ring. A methyl group in 14a or a p-methoxy benzyl group in
this substitution in 15a restored an inhibitory activity similar to
that of the reference molecules, with an IC50 of 0.15 M. Equivalent
activities were found when a chlorine or a bromine atom were
introduced at position 2 (15b: 0.3 M and 15e: 0.46 M) or when
a chlorine atom was introduced at position 3 (15c: 0.14 M). The
2,3-dichloro substitution present in compound 15d slightly de-
creased the activity with an IC50 of 0.89 M. The breakthrough in
l
l
l
l
l
l
l
14b suppressed the activity, with IC50 greater than 10
lM. This
l
set us to thinking that this nitrogen atom could be engaged in a do-
these structural studies was obtained when bromine or nitrile
nor–acceptor bond with the active site of the enzyme. Suppressing
were introduced at position 3, giving rise to a substantial gain in

844
J.-G. Parmentier et al. / Bioorg. Med. Chem. Lett. 19 (2009) 841–844
activity for inhibitors 15f and 15g with IC50 of 0.014 and 0.029
respectively. Conversion of the nitrile moiety to an amide in 15h
resulted in loss of potency at 0.15 M. Introduction of a phenyl ring
at position 3 in 15i was rather deleterious for activity with an IC50
of 1.3 M, but fusing this phenyl with the thiophene ring in com-
pound 15j restored the activity, giving an IC50 of 0.23 M.
We then turned our attention to the imidazolinone ring. The
protonated nitrogen atom was replaced by a sulphur atom to give
the more lipophilic rhodanine derivative 15k which maintained
l
M,
as in vivo against a panel of tumoral cell lines. These results will
be reported shortly.
l
Acknowledgment
l
l
Sandrine Calis is warmly thanked for her skillful technical
assistance.
References and notes
the inhibitory potency at 0.18
of magnitude in the potency was lost with the 3-bromo derivative
15l (0.19 M vs 0.014 M for 15f). Furthermore, 15m and 15n, the
analogs of compounds 15i and 15j, were found to be barely active,
with activities greater than 10 M.
lM. Curiously enough, one order
1. (a) Zhou, B. B. S.; Bartek, J. J. Nat. Rev. Cancer 2004, 4, 1; (b) Bartek, J.; Lukas, J.
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l
l
l
Finally, substitution of the exocyclic nitrogen atom of the imi-
dazolinone ring gave compounds 15o–s. Activity was decreased
of one order of magnitude compared to the non-substituted parent
compounds 15a or 15f.
In summary, we explored a new series of thieno-[3,2-b]-pyr-
roloazepinones analogues of the natural Hymenialdisine family.
Substitution of the thiophene ring with electron-withdrawing
groups such as bromo or cyano afforded novel inhibitors 15f and
15g which produced a 10-fold improvement in the enzymatic
inhibitory potency at IC50 of 14 and 29 nM against Chk1, respec-
tively. In contrast, small structural variations located on the imi-
dazolinone ring resulted in extensive erosion of inhibitory
activity. 15f was submitted to a selectivity profiling against a panel
of 220 kinases. Like Hymenialdisine and several other CHK1 inhib-
itors, 15f proved to be a multi-targeted kinase inhibitor, giving 52
11. (a) Sharma, V.; Lansdell, T. A.; Jin, G.; Tepe, J. J. J. Med. Chem. 2004, 47, 3700; (b)
Prager, R. H.; Tsopelas, C. Aust. J. Chem. 1992, 45, 1771.
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B.; Golsteyn, R. M.; Pierré, A.; De Nanteuil, G. Tetrahedron Lett. 2003, 44,
9263.
hits (>80% inhibition at 1 lM). Nanomolar IC50 were found for the
inhibition of kinases such as FLT3, GSK3b, ARK5 or SIK. The lead
compounds have also been evaluated at the cellular level as well
13. Walker, S. D.; Barder, T. E.; Martinelli, J. R.; Buchwald, S. L. Angew. Chem. Int.
2004, 43, 1871.