Discovering a new analogue of thalidomide which may be used as a potent modulator of TNF-α production

Article abstract of DOI:10.1016/j.ejmech.2009.03.018

A new series of imide derivatives related to thalidomide were synthesized and evaluated as modulators of TNF-α production. These derivatives enhance TNF-α production using human leukemia HL-60 cells induced with 12-O-tetradecanoylphorbol 13-acetate (TPA), while inhibiting TNF-α production induced with okadaic acid (OA) in the same cell line. The diphenylmaleimide derivative 2f, was found to be the most active product, producing a strong modulation of the cytokine level.

Full text of DOI:10.1016/j.ejmech.2009.03.018

European Journal of Medicinal Chemistry 44 (2009) 3533–3542
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Discovering a new analogue of thalidomide which may be used
as a potent modulator of TNF-
a
production
a
,
b
,
c
c
*
**
´
˜
Miguel Fernandez Brana , Nuria Acero
, Loreto Anorbe , Dolores Munoz Mingarro ,
˜ ˜
d
c
´
Francisco Llinares , Gema Domınguez
a
´
Instituto Canario de Investigacio´n del Cancer, Brasil 25, 28110 Algete, Madrid, Spain
b Departamento de Ciencias Ambientales y Recursos Naturales, Universidad CEU San Pablo, Urbanizacio´n Montepr´ıncipe, 28668 Boadilla del Monte, Madrid, Spain
c Departamento de Qu´ımica, Universidad CEU San Pablo, Urbanizacio´n Montepr´ıncipe, 28668 Boadilla del Monte, Madrid, Spain
d Departamento de Biolog´ıa Celular, Bioqu´ımica y Biolog´ıa Molecular, Universidad CEU San Pablo, Urbanizacio´n Montepr´ıncipe, 28668 Boadilla del Monte, Madrid, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of imide derivatives related to thalidomide were synthesized and evaluated as modulators
Received 11 February 2009
Received in revised form
13 March 2009
Accepted 16 March 2009
Available online 26 March 2009
of TNF-
a
production. These derivatives enhance TNF-
a
production using human leukemia HL-60 cells
production induced
induced with 12-O-tetradecanoylphorbol 13-acetate (TPA), while inhibiting TNF-
a
with okadaic acid (OA) in the same cell line. The diphenylmaleimide derivative 2f, was found to be the
most active product, producing a strong modulation of the cytokine level.
Ó 2009 Elsevier Masson SAS. All rights reserved.
Keywords:
Thalidomide
Diphenylmaleimide derivate
TNF-
a
1. Introduction
identified as an endotoxin-induced serum factor that causes
hemorrhagic necrosis of transplanted solid tumors [13]. TNF-
a
Thalidomide (ThalomidÔ, Celgene Corp) (Fig. 1) is a synthetic
phthalimido glutamic acid derivative developed as a sedative-
hypnotic agent to treat emesis in pregnancy [1]. Despite its early
successful clinical results, use was halted because of its teratogenic
properties [2]. Nevertheless, there has been a resurgence of interest
in the drug in recent years due to its potential usefulness in the
treatment of Erythema Nodosum Leprosum [3,4] multiple myeloma
[5], AIDS, and various cancers [6]. It has been discovered that
thalidomide has various biological activities, including, the inhibi-
plays a critical role in several physiological immune systems, and
can cause severe damage when it is produced in excess. Therefore,
TNF-a can be regarded as possessing both favorable and unfavor-
able effects. The favorable effects include direct tumor killing
action, stimulation of the host’s immune system, and acting as
a growth factor for normal B-cells. The unfavorable effects include
induction of tissue inflammation,
a tumor-promoting action,
stimulation of human immunodeficiency virus (HIV) replication,
and induction of insulin resistance. These pleiotropic effects of TNF-
tion of tumor necrosis factor-
anti-inflammatory, anti-angiogenic [9], and cycloxygenase inhibi-
tory activities [10]. The TNF- production inhibitory activity was
a
(TNF-
a
) [7,8] production, as well as
a demonstrate that cytokine production enhancers and inhibitors
could be of use as biological response modifiers under various
circumstances [14,15].
a
initially considered to be one of the key thalidomide action mech-
anisms [11], though the precise molecular mechanism(s) involved
remain unclear. An interesting review of thalidomide activity and
other less teratogenic homologous has recently been published [12].
On this basis, we have synthesized a new series of imide
thalidomide derivatives with the aim of creating bidirectional
regulators of TNF-a production. As the regulation of this cytokine
production was found to be inducer-specific, compounds were
tested using the TPA-stimulated HL-60 and OA-stimulated HL-60
assay system [16].
Tumor necrosis factor-a (TNF-a), an important cytokine
produced by activated monocytes/macrophagues, has been
In this paper we describe the synthesis and biological activity of
a new series of thalidomide homologues. In order to improve TNF-
* Corresponding author. Tel.: þ34916282961.
** Corresponding author. Tel.: þ34913724711; fax: þ34913510496.
E-mail addresses: mifbrana@gmail.com (M.F. Bran˜a), nacemes@ceu.es
(N. Acero).
a
production modulator activity, we explored structural modifica-
tions of the thalidomide phthaloyl ring (compounds 1a–f). The
choice of changes made to the phthalimido moiety was determined
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.03.018

3534
M.F. Bra˜na et al. / European Journal of Medicinal Chemistry 44 (2009) 3533–3542
In Scheme 3, we describe the synthesis of glutarimides 1a–f.
O
Glutaric anhydrides 9a, d–e were heated in the presence of
ammonium carbonate to obtain high yields of 1a, d–e. Compound
1f was obtained by refluxing 3f with a (1:1) mixture of acetic
anhydride:acetyl chloride. Finally, compound 3b was directly con-
verted into 1b by heating at 195–250 ꢀC in the absence of solvent
and high yields of 1c was obtained by hydrogenation of 1b.
N-Substituted glutarimides 2a–g were synthesized by treating
the corresponding anhydride with N,N-dimethylethylenediamine
followed by cyclization, as depicted in Scheme 4. In this case the
amidoacid intermediate was not isolated, and was directly con-
verted into the N-substituted glutarimide by reaction with a (1:1)
mixture of acetic anhydride:acetyl chloride at reflux temperature.
All compounds were isolated as the water-soluble hydrochloride.
Finally, compound 2c was obtained by hydrogenation of the nitro
derivative 2b (Scheme 4).
O
N
NH
O
O
Fig. 1. Thalidomide.
by our experience in other areas of cancer research [17,18]. To
investigate the contribution of basic residue, a dimethylaminoethyl
group was introduced to the glutarimidic nitrogen. This basic
residue may also increase water solubility (compounds 2a–g). The
open ring intermediate, compounds 3a–g, were also evaluated.
Polyamines and diamines play key roles in several of biological
processes and possess a variety of pharmacological properties.
Thus, we synthesized a new series of bis-glutarimides (compounds
6a, d, f–g) consisting of two imide units joined by N,N-bis(2-ami-
noethyl)-N-methylamine. Moreover, two dimeric type thalidomide
homologues, 4–5, were obtained (Fig. 2).
Compound 11 was obtained by refluxing pyromellitic anhydride
10 with L-glutamic acid in the presence of pyridine. The solid
obtained was transformed into the final compound by heating in
acetic anhydride. However, all attempts to obtain glutarimides 4 by
reaction of 11 with ammonia or ammonium carbonate failed. We
also prepared 4, obtaining good yield (83%) by direct reaction of 10
with of 3-aminopiperidine-2,6-dione hydrochloride 12, following
the procedure of Muller et al. [22], as outlined in Scheme 5.
2. Results and discussion
Scheme
6
describes the synthesis of N,N0 bis-substituted
2.1. Chemistry
phthalimide 5. This compound was obtained by direct reaction of
11 with N,N-dimethylethylenediamine, in a 1:2 ratio, followed by
reaction with a (1:1) mixture of acetic anhydride:acetyl chloride at
reflux temperature.
Finally, compounds 6a, d, f–g were synthesized using a similar
procedure, starting from glutaric anhydrides 9a, d, f–g and N,N-
bis(2-aminoethyl)-N-methylamine in a 2:1 ratio, as outlined in
Scheme 7.
Thalidomide was prepared following the procedure described
by Jo¨nsson [19]. Syntheses of the final compounds are described in
Schemes 1–7. Anhydride 7d was obtained by reaction of 2,3-
naphthalenedicarboxylic acid at reflux in acetic anhydride, and 7e
by the Hershberg and Fieser procedure [20]. The other anhydrides
are commercially available. Intermediates 7a–b, d–f were synthe-
sized according the procedure of King and Kidd [21]. These
compounds reacted with L-glutamic acid in pyridine at reflux,
followed by cyclization in acetic anhydride, to obtain compounds
9a–b, d–f.
2.2. In vitro antiproliferative activity
Scheme 2 describes the synthesis of amidoacids 3a–f. The
appropriate glutaric anhydrides 9a, d–f reacted with gaseous
ammonia in dioxane to give a high yield compounds 3a–b, d–f. The
synthesis of compound 3c was carried out by hydrogenation of 3b
to give 89% yield.
All compounds and thalidomide were tested for cytotoxicity in
a MTT assay [23] against several human cell lines: colon carcinoma
DLD-1 and HT-29, Burkitt lymphoma ST-486, leukemia HL-60 and
endothelial HUVEC. No antiproliferative effect was detected
(IC50 > 50
m
M in all cases).
O
O
O
Z
Z
Z
O
N
O
N
N
O
NH2
O
O
O
OH
O
N
O
N
H
O
2a-g
3a-g
1a-f
N
CH3
H3C
NH2
NO2
Z =
a
c
e
g
b
d
f
O
O
O
O
O
Z
Z
O
CH3
N
O
N
N
O
N
N
N
N
R
N
N
R
O
O
O
O
O
O
O
O
4: R = H
5: R = CH2CH2N(CH3)2
6a, d, f-g
Fig. 2. Compounds 1a–f, 2a–g, 3a–g, 4, 5, 6a–g.

M.F. Bran˜a et al. / European Journal of Medicinal Chemistry 44 (2009) 3533–3542
3535
O
TNF-a production was detected. Similar activity is observed with
both, 2g and 2a (1,8-naphthalimide). However, when phthalimide
is substituted with 2,3-naphthalimide, 2d, the compound inhibi-
tion effect of the TNF-a production is similar to thalidomide, and
better than 2g.
O
O
O
Z
H2N
O
a, b
OH
N
O
Z
+
OH
O
O
O
O
7a-b, d-f
8
9a-b, d-f
Finally, the diphenylmaleimide group produces a compound
with a potent regulatory activity over cytokine production. 2f has
Scheme 1. Reagents: (a) pyridine,
D
; (b) Ac2O, D.
a stimulatory effect over TNF-
(672%) and shows inhibitory activity in OA treated cells (6%) (Fig. 4).
Pharmacologic inhibition of TNF- production or blockade has
a production, in cells treated with TPA
a
2.3. TNF-
a production-regulatory activity
been a highly effective approach in the treatment of several
immunologically mediated diseases, including rheumatoid arthritis
[26], Crohn’s disease [27], and psoriatic arthritis [28]. Nevertheless,
HL-60 cells do not produce detectable amounts of TNF-
a
under
normal cell culture conditions, but begin to produce it in response
to okadaic acid (OA) or 12-O-tetradecanoylphorbol 13-acetate
(TPA). The cytokine concentration produced by HL-60 cell under
our standard experimental conditions (5 ꢁ105 cells/mL, incubated
with 10 nM TPA or 50 nM OA during 16 h) was defined as 100% of
production [24]. The effect of compounds 1–6 were represented as
it has recently become clear that blockade of TNF-
a action is
profoundly immunosuppressive [29,30], and may result in reac-
tivation of tuberculosis [31] and histoplasmosis [32], as well as the
emergence of B-cell lymphomas [33]. Due to its modulating
capacity, 2f should be considered for further studies, as an alter-
native to thalidomide and other regulatory substances (usually
the percentage, of TNF-
TPA or OA) in the presence of each compound. The TNF-
a
produced by stimulated HL-60 cells (with
concen-
blockers) of TNF-a levels.
a
tration in cell culture medium was quantified using an ELISA. Each
assay was performed in triplicate, the means appear in Table 1.
3. Conclusions
None of the tested compounds induced detectable TNF-a produc-
In conclusion, the phthalimide substitution with diphenylma-
leimide moiety appears to be the better option for a TNF- produc-
tion modulation of all synthesized compounds. The most important
aspect of this compound activity is its strong inhibitory effect.
Therefore, the 2f molecular structure may be considered as a new
tion by themselves. Cell number was estimated at the end of the
assay. No differences in cell number between treatments with
thalidomide and their derivatives were observed.
a
All treatments show TPA-induced TNF-
a production-enhancing
activity and OA-induced TNF- production-inhibiting activity.
a
leader in the design of new TNF-a inhibitors, through modification
of the lateral basic chain and the introduction of substituents into
one or both phenyl rings. At this point, further experimental work is
Therefore, all imide derivates show the same bidirectional modu-
lation of the cytokine production as thalidomide. The dose–
response curves for enhancing and inhibiting activities of the most
required to improve TNF-a production modulation.
active components on TNF-a production are shown in Fig. 3a,b. The
effective concentration ranges of these compounds are roughly the
same for the TPA-induced HL-60 and OA-induced HL-60 assay
systems.
4. Experimental protocols
In view of these results and structural modifications realized, we
may summarize that the introduction of the basic chain into the
thalidomide glutarimide ring, 2g, produces a noticeable reduction
in ability to regulate cytokine production. Therefore, imidic NH may
4.1. Chemistry
Melting points (uncorrected) were determined on a Stuart
Scientific SMP3 apparatus. Infrared (IR) spectra were recorded with
a Perkin–Elmer 1330 infrared spectrophotometer. 1H and 13C NMR
values were recorded on a Bruker 300-AC instrument. Chemical
contribute to this activity. Overproduction of TNF-a is associated
with a wide range of pathological conditions, which has led to
recent efforts to discover ways to downregulate its production [25].
The thalidomide structural duplication produces an interesting
result. The bis-thalidomide joined by a bis-amine 6g, exhibiting
better results than 2g, but only for the stimulation of cytokine
production. However, when 2g was duplicated using an aromatic
shifts (d) are expressed in parts per million relative to internal
tetramethylsilane; coupling constants (J) are in hertz. Mass spectra
were run on a HP 5989A spectrometer. Elemental analyses (C, H, N)
were performed on a Perkin–Elmer 2400 CHN apparatus at the
Microanalyses Service of the University Complutense of Madrid; all
the reported values are within ꢂ0.4% of the theoretical
bis-imide, 5,
a
small improvement in the inhibition of
O
O
Z
Z
O
a
N
N
O
NH2
O
O
OH
O
O
O
3a-b, d-f
9a, d-f
O
N
O
O
O
b
N
O
H2N
NH2
O2N
NH2
O
O
O
OH
OH
3b
3c
Scheme 2. Reagents: (a) (i) NH3 (g), dioxane, RT; (ii) H3Oþ; (b) H2, Pd/C 10%, DMF, 50 psi.

3536
M.F. Bra˜na et al. / European Journal of Medicinal Chemistry 44 (2009) 3533–3542
O
O
O
Z
Z
Z
O
b
N
O
a
N
O
N
O
NH2
O
O
O
OH
O
N
H
O
O
9a, d-e
1a, d-f
3f
O
O
N
c
O
d
O
N
N
O2N
O2N
NH2
H2N
O
O
O
O
O
O
OH
N
O
O
N
H
H
3b
1b
1c
Scheme 3. Reagents: (a) (NH4)2CO3,
D; (b) Ac2O, AcCl,
D
; (c) D
, 250 ꢀC; (ii) H3Oþ; (d) H2, Pd/C 10%, DMF, 50 psi.
compositions. Thin-layer chromatography (TLC) was run on Merck
silica gel 60 F-254 plates. Unless stated otherwise, starting mate-
rials used were high-grade commercial products.
4.1.1.2. Synthesis of 2-(2,6-dioxotetrahydro-2H-pyran-3-yl)-5-nitro-
1H-benzo[de]isoquinoline-1,3(2H)-dione 9b. Following the general
procedure for the synthesis of glutaric anhydrides, from 3-nitro-
1,8-naphthalic anhydride (10.00 g, 50.50 mmol), refluxed for 24 h,
14.00 g (78%) of 9b were obtained as a pale solid (mp >270 ꢀC dec).
4.1.1. General procedure for the synthesis of glutaric anhydrides 9
A mixture of the corresponding anhydride 7 (10.0 mmol) and
1H NMR (DMSO-d6)
d 2.11–2.17 (m, 1H, CH2), 2.44–2.59 (m, 1H,
L
-glutamic acid (15.1 mmol) was refluxed in dry pyridine (35 mL).
CH2), 2.91–3.00 (m, 1H, CH2), 3.12–3.24 (m, 1H, CH2), 6.13 (dd, 1H,
CH, J1 ¼11.6 Hz, J2 ¼ 6.1 Hz), 8.11 (t,1H, Ar, J ¼ 7.9 Hz), 8.74 (d,1H, Ar,
J ¼ 7.3 Hz), 8.86 (d, 1H, Ar, J ¼ 7.3 Hz), 9.01 (bs, 1H, Ar), 9.57 (s, 1H,
Then the mixture was cooled to room temperature and the solvent
was evaporated to dryness. Acetic anhydride (12 mL) was added
and the mixture was heated under reflux for an additional 0.5 h.
After cooling, the precipitate was collected by filtration. The solid
was recrystallized from anhydride acetic.
Ar). 13C NMR (DMSO-d6)
d 20.1, 29.0, 49.2, 121.9, 123.3, 123.7, 129.4,
129.5, 130.5, 130.9, 134.7, 137.1, 145.9, 161.8, 162.2, 165.6, 166.8. IR
(KBr) 1810, 1770, 1715, 1680, 1540, 1350, 1340.
4.1.1.1. Synthesis of 2-(2,6-dioxotetrahydro-2H-pyran-3-yl)-1H-ben-
zo[de]isoquinoline-1,3(2H)-dione 9a. Following the general proce-
dure for the synthesis of glutaric anhydrides, from 1,8-naphthalic
anhydride (2.00 g, 10.10 mmol), refluxed for 40 h, 1.43 g (46%) of 9a
were obtained as a yellow solid (mp 276–278 ꢀC). 1H NMR (DMSO-
4.1.1.3. Synthesis of 2-(2,6-dioxotetrahydro-2H-pyran-3-yl)-1H-ben-
zo[f]isoindole-1,3(2H)-dione 9d. Following the general procedure
for the synthesis of glutaric anhydrides, from 2,3-naphthalic
anhydride (4.15 g, 20.92 mmol), refluxed for 2 h, 4.84 g (75%) of 9d
were obtained as a white solid (mp >270 ꢀC dec). 1H NMR (DMSO-
d6)
d
1.90–2.18 (m, 1H, CH2), 2.43–2.57 (m, 1H, CH2), 2.90–2.98 (m,
d6) d 2.19–2.24 (m, 1H, CH2), 2.64–2.78 (m, 1H, CH2), 3.01–3.26 (m,
1H, CH2), 3.12–3.24 (m, 1H, CH2), 6.11 (dd, 1H, CH, J1 ¼11.6 Hz,
2H, CH2), 5.59 (dd, 1H, CH, J1 ¼12.8 Hz, J2 ¼ 5.5 Hz), 7.82 (dd, 2H, Ar,
J2 ¼ 6.7 Hz), 7.93 (t, 2H, Ar, J ¼ 7.9 Hz), 8.55 (d, 4H, Ar, J ¼ 7.9 Hz). 13
C
J1 ¼5.8 Hz, J2 ¼ 2.7 Hz), 8.28 (dd, 2H, Ar, J1 ¼6.1 Hz, J2 ¼ 3.0 Hz),
NMR (DMSO-d6)
d
20.2, 29.0, 48.9, 121.3, 127.3, 127.4, 131.2, 131.5,
8.58 (s, 2H, Ar). 13C NMR (DMSO-d6)
d 20.4, 29.4, 47.8, 125.2, 126.6,
135.1, 165.9, 162.8, 167.0. IR (KBr) 1810, 1765, 1700, 1660.
129.6, 130.4, 135.0, 165.6, 166.3, 166.3. IR (KBr) 1815, 1770, 1710.
O
O
Z
Z
N
O
a
N
O
N
O
O
O
O
O
9a-b, d-g
2a-b, d-g
N
H3C
CH3
O
O
b
N
O
N
O
H2N
O2N
O
O
N
O
O
N
2c
2b
N
N
H3C
H3C
CH3
CH3
Scheme 4. Reagents: (a) (i) H2NCH2CH2N(CH3)2, DMF, RT; (ii) Ac2O, AcCl,
D; (b) H2, Pd/C 10%, DMF, 50 psi.

M.F. Bran˜a et al. / European Journal of Medicinal Chemistry 44 (2009) 3533–3542
3537
O
O
O
O
O
H2N
a
OH
O
O
+
O
N
N
O
O
O
O
OH
O
O
O
O
O
O
10
8
11
b or c
O
O
O
O
H2N
O
d
Cl
O
O
+
O
N
N
O
HN
NH
N
H
O
O
O
O
O
O
O
H
10
12
4
Scheme 5. Reagents: (a) (i) pyridine,
D; (ii) Ac2O, D; (b) (i) NH3; (ii) Ac2O, AcCl, D; (c) (NH4)2CO3, D; (d) NaAcO, AcOH.
4.1.1.4. Synthesis of 2-(2,6-dioxotetrahydro-2H-pyran-3-yl)-1H-ben-
zo[e]isoindole-1,3(2H)-dione 9e. Following the general procedure
for the synthesis of glutaric anhydrides, from 1,2-naphthalic
anhydride (2.50 g, 12.61 mmol), refluxed for 2 h, 3.34 g (83%) of 9e
were obtained as a pale solid (mp 234–236 ꢀC). 1H NMR (DMSO-d6)
127.5, 131.3, 131.4, 134.8, 163.3, 171.0, 173.4. IR (KBr) 3400, 3170,
1700, 1650. MS (ESI), m/z 327 [M þ H]þ Anal. (C17H14N2O5$0.1H2O)
C, H, N.
4.1.2.2. Synthesis of 5-amino-2-(5-nitro-1,3-dioxo-1H-benzo[de]iso-
quinolin-2(3H)-yl)-5-oxopentanoic acid 3b. Following the general
procedure for the synthesis of amidoacids, from 9b (3.00 g,
8.47 mmol), 2.67 g (85%) of 3b were obtained as a yellow solid (mp
d
2.13–2.22 (m, 1H, CH2), 2.59–2.74 (m, 1H, CH2), 2.96–3.20 (m, 2H,
CH2), 5.52 (dd, 1H, CH, J1 ¼13.1 Hz, J2 ¼ 5.8 Hz), 7.79–7.91 (m, 2H,
Ar), 7.98 (d, 1H, Ar, J ¼ 7.9 Hz), 8.23 (d, 1H, Ar, J ¼ 7.9 Hz), 8.49 (d, 1H,
Ar, J ¼ 8.6 Hz), 8.79 (d,1H, Ar, J ¼ 7.9 Hz). 13C NMR (DMSO-d6)
d
20.6,
237–239 ꢀC). 1H NMR (DMSO-d6)
d
2.04–2.46 (m, 4H, 2 ꢁ CH2), 5.53
29.6, 47.7, 118.5, 123.8, 126.3, 127.1, 129.1, 129.2, 130.1, 130.7, 136.0,
136.2, 165.8, 166.3, 167.1, 167.9. IR (KBr) 1815, 1770, 1710.
(dd, 1H, CH, J ¼ 9.8 Hz, J ¼ 4.9 Hz), 6.62 (bs, 1H, NH2), 7.13 (bs, 1H,
NH2), 8.09 (t, 1H, Ar, J ¼ 7.9 Hz), 8.71 (d, 1H, Ar, J ¼ 7.3 Hz), 8.83 (d,
1H, Ar, J ¼ 7.9 Hz), 8.99 (d, 1H, Ar, J ¼ 2.4 Hz), 9.53 (d, 1H, Ar,
4.1.1.5. Synthesis of 1-(2,6-dioxotetrahydro-2H-pyran-3-yl)-3,4-diphenyl-
1H-pyrrole-2,5-dione 9f. Following the general procedure for the
synthesis of glutaric anhydrides, from 2,3-diphenylmaleic anhydride
(2.00 g, 7.99 mmol), refluxed for 24 h, 2.10 g (73%) of 9f were obtained
J ¼ 2.5 Hz), 12.87 (bs, 1H, OH). 13C NMR (DMSO-d6)
d 24.1, 31.8, 53.3,
122.1, 123.5, 123.5, 129.4, 129.6, 130.1, 130.9, 134.5, 136.7, 145.9,
162.2,162.7,170.8,173.6. IR (KBr) 3460, 3200,1710,1675,1530,1340.
MS (EI), m/z 353 (MþꢃH2O, 8), 323 (18), 242 (68), 212 (100). Anal.
(C17H13N3O7$1.1H2O) C. H, N.
as a yellow solid (mp 219–221 ꢀC). 1H NMR (DMSO-d6)
d 2.10–2.19 (m,
1H, CH2), 2.52–2.67 (m, 1H, CH2), 2.94–3.18 (m, 2H, CH2), 5.45 (dd, 1H,
CH, J1 ¼12.8 Hz, J2 ¼ 5.5 Hz), 7.42–7.44 (m, 10H, Ar). 13C NMR (DMSO-
4.1.2.3. Synthesis of 5-amino-2-(5-amino-1,3-dioxo-1H-benzo[de]i-
soquinolin-2(3H)-yl)-5-oxopentanoic acid 3c. A solution of 3b
(0.20 g, 0.54 mmol) in DMF (30 mL) was treated with 10% Pd/C
(0.07 g) and hydrogenated for 7 h at 50 psi. The mixture was filtered
through a celite pad. The solvent was removed under reduced
pressure, the residue was disgregated in AcOEt and filtered to afford
3c (0,16 g, 89%) as a yellow solid (mp >283 ꢀC dec). 1H NMR (DMSO-
d6) d 20.4, 29.4, 47.9, 128.0, 128.5, 129.5, 129.9, 136.2, 165.6, 166.2, 168.9.
IR (KBr) 1820, 1770, 1710.
4.1.2. General procedure for the synthesis of amidoacids 3
Gaseous NH3 was bubbled through a suspension of anhydride 9
(10.00 mmol) in dioxane (30 ml) for 1.5 h. The precipitate formed
was filtered. The solid was dissolved in H2O (10.0 mL) and acidified
to pH 1 with 37% HCl, the precipitate was filtered, washed with H2O
and dried. The solid was recrystallized from glacial acetic acid.
d6)
d
1.98–2.27 (m, 3H, 2 ꢁ CH2), 2.37–2.44 (m, 1H, CH2), 5.50 (dd,
1H, CH, J ¼ 10.1 Hz, J ¼ 4.6 Hz), 6.07 (bs, 2H, NH2Ar), 6.67 (bs, 1H,
NH2), 7.16 (bs, 1H, NH2), 7.32 (d, 1H, Ar, J ¼ 2.5 Hz), 7.64 (t, 1H, Ar,
J ¼ 7.6 Hz), 7.97 (d, 1H, Ar, J ¼ 2.5 Hz), 8.07–8.10 (m, 2H, Ar), 12.73
4.1.2.1. Synthesis of 5-amino-2-(1,3-dioxo-1H-benzo[de]isoquinolin-
2(3H)-yl)-5-oxopentanoic acid 3a. Following the general procedure
for the synthesis of amidoacids, from 9a (1.00 g, 3.24 mmol), 0.91 g
(86%) of 3a were obtained as a white solid (mp >265 ꢀC dec). 1H
(bs, 1H, OH). 13C NMR (DMSO-d6)
d 24.1, 31.6, 52.5, 112.0, 120.7,
121.4, 122.1, 122.2, 125.9, 127.1, 131.9, 133.6, 148.0, 163.5, 163.7, 171.1,
173.4. IR (KBr) 3420, 3380, 3200, 1695, 1650. MS (ESI), m/z 364
[M þ Na]þ Anal. (C17H15N3O5$0.65H2O) C, H, N.
NMR (DMSO-d6)
d 2.05–2.19 (m, 2H, CH2), 2.22–2.32 (m, 1H, CH2),
2.40–2.46 (m, 1H, CH2), 5.55 (dd, 1H, CH, J1 ¼9.5 Hz, J2 ¼ 4.6 Hz),
6.67 (bs, 1H, NH2), 7.16 (bs, 1H, NH2), 7.92 (dd, 2H, Ar, J ¼ 7.9 Hz,
J ¼ 7.3 Hz), 8.52 (d, 2H, Ar, J ¼ 7.9 Hz), 8.54 (d, 2H, Ar, J ¼ 7.3 Hz),
4.1.2.4. Synthesis of 5-amino-2-(1,3-dioxo-1H-benzo[f]isoindol-
2(3H)-yl)-5-oxopentanoic acid 3d. Following the general procedure
for the synthesis of amidoacids, from 9d (0.50 g, 1.62 mmol), 0.48 g
(91%) of 3d were obtained as a white solid (mp >223 ꢀC dec).
12.80 (bs, 1H, OH). 13C NMR (DMSO-d6)
d
24.1, 31.7, 52.6, 121.7, 127.4,
O
O
O
O
a
O
N
O
O
N
O
N
N
N
N
O
O
O
O
O
O
O
O
O
O
11
5
CH3
N
H3C
N
CH3
H3C
Scheme 6. Reagents: (a) (i) 0.5 H2NCH2CH2N(CH3)2, DMF, RT; (ii) H3Oþ; (iii) Ac2O, AcCl,
D.

3538
M.F. Bran˜a et al. / European Journal of Medicinal Chemistry 44 (2009) 3533–3542
O
O
O
Z
Z
Z
O
CH3
N
O
a
N
N
N
O
N
N
O
O
O
O
O
O
O
9a, d, f-g
6a, d, f-g
Scheme 7. Reagents: (a) (i) 0.5 H2NCH2CH2N(CH3)CH2CH2NH2, DMF, RT; (ii) H3Oþ; (iii) Ac2O, AcCl,
D.
1H NMR (DMSO-d6)
d
2.12 (t, 2H, CH2, J ¼ 7.3 Hz), 2.24–2.43 (m, 2H,
d 24.0, 31.5, 51.7, 128.4, 128.6, 129.7, 129.9, 136.0, 169.7, 170.5, 173.2.
CH2), 4.83 (dd, 1H, CH, J ¼ 10.4 Hz, J ¼ 4.9 Hz), 6.74 (bs, 1H, NH2),
7.22 (bs, 1H, NH2), 7.81 (dd, 2H, Ar, J1 ¼6.1 Hz, J2 ¼ 3.7 Hz), 8.30 (dd,
IR (KBr) 3430, 3200, 1750, 1720, 1690. MS (EI), m/z 360 (MþꢃH2O,
26), 249 (63), 178 (100). Anal. (C21H18N2O5$0.8H2O) C, H, N.
2H, Ar, J1 ¼6.1 Hz, J2 ¼ 3.7 Hz), 8.59 (s, 2H, Ar),13.18 (bs,1H, OH). 13
C
NMR (DMSO-d6)
d
24.0, 51.5, 31.4, 124.9, 127.0, 129.5, 130.4, 135.2,
4.1.3. General procedure for the synthesis of glutarimides 1
A mixture of the corresponding anhydride 9 (1.00 mmol) and
(NH4)2CO3 (0.62 mmol) was heated for 15–30 min at 190–250 ꢀC.
Then, the mixture was cooled to room temperature and the crude
was recrystallized from glacial acetic acid.
167.1,170.5,173.2. IR (KBr) 3480, 3340,1780,1710,1660. MS (ESI), m/
z 349 [M þ Na]þ Anal. (C17H14N2O5) C, H, N.
4.1.2.5. Synthesis of 5-amino-2-(1,3-dioxo-1H-benzo[e]isoindol-
2(3H)-yl)-5-oxopentanoic acid 3e. Following the general procedure
for the synthesis of amidoacids, from 9e (0.50 g, 1.62 mmol), 0.37 g
(70%) of 3e were obtained as a yellow solid (mp 239–241 ꢀC). 1H
4.1.3.1. Synthesis of 2-(2,6-dioxopiperidin-3-yl)-1H-benzo[de]iso-
quinoline-1,3(2H)-dione 1a. Following the general procedure for
the synthesis of glutarimides, from 9a (0.26 g, 0.85 mmol), 0.12 g
(45%) of 1a were obtained as a white solid (mp >300 ꢀC dec). 1H
NMR (DMSO-d6)
d
2.15 (t, 2H, CH2, J ¼ 7.3 Hz), 2.26–2.47 (m, 2H,
CH2), 4.80 (dd, 1H, CH, J1 ¼10.4 Hz, J2 ¼ 4.9 Hz), 6.73 (bs, 1H, NH2),
7.21 (bs, 1H, NH2), 7.77–7.89 (m, 2H, Ar), 7.94 (d, 1H, Ar, J ¼ 8.5 Hz),
8.21 (d, 1H, Ar, J ¼ 7.9 Hz), 8.46 (d, 1H, Ar, J ¼ 8.5 Hz), 8.80 (d, 1H, Ar,
NMR (DMSO-d6)
d 2.02–2.09 (m, 1H, CH2), 2.52–2.67 (m, 2H, CH2),
2.89–3.02 (m, 1H, CH2), 5.85 (dd, 1H, CH, J1 ¼11.6 Hz, J2 ¼ 5.5 Hz),
7.88–7.95 (m, 2H, Ar), 8.47 (d, 1H, Ar, J ¼ 7.3 Hz), 8.52 (d, 2H, Ar,
J ¼ 8.5 Hz), 8.58 (d, 1H, Ar, J ¼ 6.7 Hz), 11.10 (bs, 1H, NH). 13C NMR
J ¼ 8.5 Hz), 13.10 (bs, 1H, OH). 13C NMR (DMSO-d6)
d 24.1, 31.4, 51.3,
118.5,123.9,126.4,127.2,129.1,129.3,130.1,130.8,135.8,136.3,167.9,
168.7, 170.7, 173.2. IR (KBr) 3440, 3200, 1775, 1710. 1660, MS (ESI),
m/z 349 [M þ Na]þ Anal. (C17H14N2O5$0.2CH3COOH) C, H, N.
(DMSO-d6)
d 21.4, 30.9, 50.5, 121.5, 121.8, 127.4, 127.4, 127.5, 131.0,
131.3, 131.6, 134.9, 134.9, 162.6, 163.5, 170.3, 172.9. IR (KBr) 3200,
1700, 1660. MS (ESI), m/z 331 [M þ Na]þ Anal. (C17H12N2O4) C, H, N.
4.1.2.6. Synthesis of 5-amino-2-(2,5-dioxo-3,4-diphenyl-2,5-dihydro-
1H-pyrrol-1-yl)-5-oxopentanoic acid 3f. Following the general
procedure for the synthesis of amidoacids, from 9f (0.80 g,
2.21 mmol), 0.60 g (75%) of 3f were obtained as a yellow solid (mp
4.1.3.2. Synthesis of 2-(2,6-dioxopiperidin-3-yl)-5-nitro-1H-benzo-
[de]isoquinoline-1,3(2H)-dione
1b. Compound
3b
(1.49 g,
4.01 mmol) was heated for 15 min at 250 ꢀC, then cooled to room
temperature, and finally disgregated in AcOEt. The suspension was
filtered and the solid was purified by recrystallization from glacial
acetic acid to afford 1b (0.42 g, 30%) as a yellow solid (mp >298 ꢀC).
237–239 ꢀC). 1H NMR (DMSO-d6)
d
2.14–2.36 (m, 4H, 2 ꢁ CH2), 4.71
(dd, 1H, CH, J1 ¼9.8 Hz, J2 ¼ 5.5 Hz), 6.78 (bs, 1H, NH2), 7.28 (bs, 1H,
NH2), 7.40–7.43 (m,10H, Ar),13.24 (bs,1H, OH). 13C NMR (DMSO-d6)
1H NMR (DMSO-d6)
d 2.02–2.10 (m, 1H, CH2), 2.58–2.64 (m, 2H,
CH2), 2.89–3.02 (m, 1H, CH2), 5.87 (dd, 1H, CH, J1 ¼11.9 Hz,
J2 ¼ 5.8 Hz), 8.07–8.14 (m, 1H, Ar), 8.66 and 8.77 (d, d, 1H, Ar,
J ¼ 7.3 Hz), 8.85 (d, 1H, Ar, J ¼ 8.6 Hz), 8.93 and 9.04 (d, d*, 1H, Ar,
J ¼ 2.5 Hz, J* ¼ 1.8 Hz), 9.56 (bs, 1H, Ar), 11.10 (bs, 1H, NH). 1H NMR
Table 1
Effects of thalidomide and imide derivatives (30
human leukemia HL-60 cells induced with 10 nM TPA or 50 nM OA. TNF-
tration in treatments without thalidomide or derivate compounds (only with TPA or
OA) was defined as 100%.
m
M) in TNF-
a
production (%) by
concen-
a
(DMSO-d6, 60 ꢀC)
d 2.06–2.13 (m, 1H, CH2), 2.55–2.68 (m, 2H, CH2),
2.88–3.00 (m, 1H, CH2), 5.84 (dd, 1H, CH, J1 ¼11.6 Hz, J2 ¼ 5.5 Hz),
8.08 (t,1H, Ar, J ¼ 7.9 Hz), 8.72 (bs,1H, Ar), 8.81 (d,1H, Ar, J ¼ 7.9 Hz),
9.01 (bs, 1H, Ar), 9.48 (d, 1H, Ar, J ¼ 2.4 Hz), 10.90 (bs, 1H, NH). 13C
Compound
% TNF-
a
TPA
OA
49
1a
1b
1c
1d
1e
1f
2a
2b
2c
2d
2e
2f
2g
3a
3b
3c
158
248
138
166
161
147
107
124
132
149
105
672
125
116
86
140
258
146
146
294
138
159
164
NMR (DMSO-d6)
d 21.3, 21.4, 30.9, 50.9, 50.9, 122.0, 122.3, 123.4,
47
46
138
78
47
91
48
47
50
59
6
93
55
51
49
60
111
66
34
77
99
53
123.4, 123.6, 123.8, 129.4, 129.5, 129.5, 130.2, 130.3, 131.0, 134.2,
134.8, 136.9, 146.0, 161.6, 162.0, 162.4, 162.8, 170.1, 172.9. IR (KBr)
3180, 1700, 1665, 1530, 1335, 1325. MS (EI), m/z 353 (Mþ, 33), 323
(48), 242 (100), 212 (79). Anal. (C17H11N3O6$0.2CH3COOH) C, H, N.
4.1.3.3. Synthesis of 5-amino-2-(2,6-dioxopiperidin-3-yl)-1H-ben-
zo[de]isoquinoline-1,3(2H)-dione 1c. A solution of 1b (0.20 g,
0.57 mmol) in DMF (30 mL) was treated with 10% Pd/C (0.07 g) and
hydrogenated for 4.5 h at 50 psi. The mixture was filtered through
a celite pad. The solvent was removed under reduced pressure, the
residue was disgregated in AcOEt and filtered to afford 1c (0,16 g,
88%) as a red solid (mp >299 ꢀC). 1H NMR (DMSO-d6)
d 1.99–2.03
3d
3e
3f
4
5
6g
(m, 1H, CH2), 2.50–2.60 (m, 2H, CH2), 2.87–2.98 (m, 1H, CH2), 5.80
(dd,1H, J1 ¼11.9 Hz, J2 ¼ 5.8 Hz), 6.07 (bs, 2H, NH2), 7.33 (bs, 1H, Ar),
7.61–7.68 (m, 1H, Ar), 7.90–8.16 (m, 3H, Ar), 11.00 (s, 1H, NH). 1H
NMR (DMSO-d6, 60 ꢀC)
d 2.00–2.09 (m, 1H, CH2), 2.58–2.63 (m, 2H,
CH2), 2.85–2.98 (m, 1H, CH2), 5.78 (dd, 1H, CH, J1 ¼11.3 Hz,
Thalidomide
J2 ¼ 5.8 Hz), 5.91 (bs, 2H, NH2Ar), 7.35 (s, 1H, Ar), 7.64 (t, 1H, Ar,

M.F. Bran˜a et al. / European Journal of Medicinal Chemistry 44 (2009) 3533–3542
3539
A
B
TPA induced HL60 cells
OA induced HL60 cells
120
100
80
60
40
20
0
800
600
400
200
0
-6,2 -5,9 -5,6 -5,3 -5 -4,7 -4,4 -4,1
-6,2 -5,9 -5,6 -5,3 -5 -4,7 -4,4 -4,1
log M
log M
1b
2f
3d
4
1b
2f
3d
4
Fig. 3. Dose–response curves of most active compounds. Horizontal scale: concentration of added test compound. Vertical scale: amount of TNF-
production-enhancing activity. HL-60 cells were treated with 10 nM TPA in the presence of a test compound. TNF-
with TPA) was defined as 100%. B) OA-induced TNF- production-inhibiting activity. HL-60 cells were treated with 50 nM OA in the presence of a test compound. TNF-
concentration in treatments without test compounds (only with TPA) was defined as 100%.
a
(%). A) TPA-induced TNF-
a
a concentration in treatments without test compounds (only
a
a
J ¼ 7.9 Hz), 7.95–8.15 (m, 2H, Ar), 8.06 (d, 1H, Ar, J ¼ 8.5 Hz), 10.82
(bs, 1H, NH). 13C NMR (DMSO-d6)
21.4, 30.9, 50.4, 112.2, 112.2,
120.5, 120.6, 121.2, 121.6, 121.7, 122.0, 122.3, 122.4, 125.6, 126.2,
127.1, 127.1, 132.0, 132.1, 133.6, 148.0, 148.0, 162.8, 163.0, 163.6, 163.8,
170.3, 172.9. IR (KBr) 3450, 3360, 3200, 1690, 1655. MS (ESI), m/z
324 [M þ H]þ Anal. (C17H13N3O4$0.6H2O) C, H, N.
was refluxed for 24 h. After cooling, the formed solid was collected
by filtration and recrystallized from glacial acetic acid to afford 1f
(0.22 g, 55%) as a yellow solid (mp 288–290 ꢀC). 1H NMR (DMSO-d6)
d
d
2.07–2.12 (m, 1H, CH2), 2.45–2.64 (m, 2H, CH2), 2.84–2.96 (m, 1H,
CH2), 5.14 (dd, 1H, CH, J1 ¼12.8 Hz, J2 ¼ 4.9 Hz), 7.40–7.45 (m, 10H,
Ar), 11.15 (s, 1H, NH). 13C NMR (DMSO-d6)
22.0, 30.8, 49.2, 128.2,
d
128.5, 129.5, 129.9, 136.1, 169.4, 169.9, 172.7. IR (KBr) 3280, 1770,
4.1.3.4. Synthesis of 2-(2,6-dioxopiperidin-3-yl)-1H-benzo[f]isoindole-
1,3(2H)-dione 1d. Following the general procedure for the
synthesis of glutarimides, from 9d (1.00 g, 3.23 mmol), 0.48 g (48%)
of 1d were obtained as a white solid (mp >298 ꢀC). 1H NMR (DMSO-
1710. MS (EI), m/z 360.
4.1.4. General procedure for the synthesis of N-substituted
glutarimides 2
d6)
d
2.08–2.15 (m, 1H, CH2), 2.55–2.66 (m, 2H, CH2), 2.87–2.99 (m,
4.1.4.1. Procedure A. To a mixture of the corresponding anhydride 9
(10.00 mmol) and DMF (60 mL) was added N,N-dimethylethy-
lendiamine (10.00 mmol) at room temperature until a precipitate
appeared. Then, the precipitate was collected by filtration, treated
with 14 mL of a mixture of acetic anhydride:acetyl chloride (1:1),
and refluxed. After cooling, the solid was collected by filtration,
disgregated in AcOEt and filtered. The solid obtained was recrys-
tallized from DMF–AcOEt.
1H, CH2), 5.24 (dd, 1H, CH, J1 ¼12.8 Hz, J2 ¼ 5.5 Hz), 7.81 (dd, 2H, Ar,
J1 ¼6.1 Hz, J2 ¼ 3.1 Hz), 8.30 (dd, 2H, Ar, J1 ¼6.1 Hz, J2 ¼ 3.7 Hz), 8.60
(s, 2H, Ar), 11.18 (s, 1H, NH). 13C NMR (DMSO-d6)
d 21.9, 30.9, 49.1,
125.0, 126.9, 129.5, 130.4, 135.1, 166.8, 169.8, 172.8. IR (KBr) 3200,
1760, 1710. MS (EI), m/z 308 (Mþ, 84), 197 (100), 154 (40), 126 (93).
Anal. (C17H12N2O4) C, H, N.
4.1.3.5. Synthesis of 2-(2,6-dioxopiperidin-3-yl)-1H-benzo[e]isoindole-
1,3(2H)-dione 1e. Following the general procedure for the synthesis
of glutarimides, from 9e (1.00 g, 3.23 mmol), 0.53 g (53%) of 1e were
4.1.4.1.1. Synthesis of 2-(3-(1,3-dioxo-1H-benzo[de]isoquinolin-
2(3H)-yl)-2,6-dioxopiperidin-1-yl)-N,N-imethylethanaminium chlo-
ride 2a. Following the general procedure A for the synthesis of
N-substituted glutarimides, from 9a (2.00 g, 6.47 mmol), refluxed
for 16 h, 1.58 g (59%) of 2a were obtained as a pale solid (mp 273–
obtained as a yellow solid (mp >280 ꢀC). 1H NMR (DMSO-d6)
d 2.10–
2.14 (m, 1H, CH2), 2.54–2.66 (m, 2H, CH2), 2.87–2.98 (m, 1H, CH2),
5.22 (dd, 1H, CH, J1 ¼12.8 Hz, J2 ¼ 5.5 Hz), 7.78–7.88 (m, 2H, Ar), 7.96
(d, 1H, Ar, J ¼ 8.6 Hz), 8.22 (d, 1H, Ar, J ¼ 8.6 Hz), 8.48 (d, 1H, Ar,
J ¼ 7.9 Hz), 8.79 (d, 1H, Ar, J ¼ 8.6 Hz), 11.18 (s, 1H, NH). 13C NMR
275 ꢀC). 1H NMR (DMSO d6)
d 2.08–2.14 (m, 1H, CH2), 2.52–2.67 (m,
1H, CH2), 2.73–2.89 (m,1H, CH2), 2.81 (s, 6H, 2 ꢁ CH3), 3.05–3.17 (m,
1H, CH2), 3.23 (t, 2H, CH2N, J ¼ 6.1 Hz), 4.08 (t, 2H, CH2N, J ¼ 6.1 Hz),
6.03 (dd,1H, CH, J1 ¼12.2 Hz, J2 ¼ 6.1 Hz), 7.88–7.97 (m, 2H, Ar), 8.47
(d, 1H, Ar, J ¼ 7.3 Hz), 8.54 (d, 2H, Ar, J ¼ 8.6 Hz), 8.61 (d, 1H, Ar,
(DMSO-d6)
d 22.2, 31.1, 49.0, 118.5, 123.8, 126.3, 127.1, 129.1, 129.2,
130.1, 130.8, 135.8, 136.2, 167.7, 168.4, 170.2, 172.9. IR (KBr) 3200,
1770, 1710. MS (EI), m/z 308 (Mþ, 85), 197 (100), 154 (40), 126 (66).
Anal. (C17H12N2O4) C, H, N.
J ¼ 7.3 Hz), 10.23 (bs, 1H, NH). 13C NMR (DMSO d6)
d 20.5, 31.2, 34.6,
42.6, 51.0, 54.0, 121.5, 121.7, 127.4, 127.5, 131.1, 131.4, 131.7, 135.1,
162.8, 163.3, 170.3, 172.1. IR (KBr) 2540, 1740, 1690, 1670. MS (EI), m/
z 379 (Mþ, 4),126 (6), 71 (35), 58 (100). Anal. (C21H22ClN3O4) C, H, N.
4.1.4.1.2. Synthesis of N,N-dimethyl-2-(3-(5-nitro-1,3-dioxo-1H-
benzo[de]isoquinolin-2(3H)-yl)-2,6-dioxopiperidin-1-yl)ethanaminium
chloride 2b. Following the general procedure A for the synthesis of
N-substituted glutarimides, from 9b (1.00 g, 2.82 mmol), refluxed for
48 h, 0.44 g (37%) of 2b were obtained as a pale solid (mp >270 ꢀC
4.1.3.6. Synthesis of 3-(2,5-dioxo-3,4-diphenyl-2,5-dihydro-1H-pyr-
rol-1-yl)piperidine-2,6-dione 1f. A mixture of 3f (412 mg,
1.09 mmol), acetic anhydride (1.1 mL), and acetyl chloride (1.1 mL)
O
dec). 1H NMR (DMSO-d6)
d 2.08–2.13 (m, 1H, CH2), 2.51–2.84 (m, 2H,
N
O
CH2), 2.80 (bs, 6H, 2 ꢁ CH3), 3.04–3.16 (m, 1H, CH2), 3.20 (t, 2H, CH2N,
J ¼ 5.8 Hz), 4.06 (t, 2H, CH2N, J ¼ 5.8 Hz), 6.04 (dd,1H, CH, J1 ¼12.2 Hz,
J2 ¼ 5.5 Hz), 8.05–8.13 (m, 1H, Ar), 8.64 and 8.77 (d, d*, 1H, Ar,
J ¼ 6.7 Hz, J* ¼ 7.3 Hz), 8.84 (d, 1H, Ar, J ¼ 7.9 Hz), 8.91 and 9.03 (d, d,
1H, Ar, J ¼ 1.8 Hz), 9.55 (s, 1H, Ar), 10.27 (bs, 1H, NH). 1H NMR (DMSO-
N
O
O
CH3
N
H3C
2f
Fig. 4. Compound 2f.
d6, 60 ꢀC)
d 2.08–2.17

3540
M.F. Bran˜a et al. / European Journal of Medicinal Chemistry 44 (2009) 3533–3542
(m, 1H, CH2), 2.55–2.89 (m, 2H, CH2), 2.80 (s, 6H, 2 ꢁ CH3), 3.06–3.14
(m, 1H, CH2), 3.23 (t, 2H, CH2N, J ¼ 6.1 Hz), 4.09 (t, 2H, CH2N,
J ¼ 6.1 Hz), 6.05 (dd, 1H, CH, J1 ¼12.2 Hz, J2 ¼ 5.5 Hz), 8.10 (t, 1H, Ar,
J ¼ 7.3 Hz), 8.74 (bs, 1H, Ar), 8.84 (d, 1H, Ar, J ¼ 8.5 Hz), 9.00 (bs, 1H,
1H, Ar, J ¼ 8.6 Hz), 10.28 (bs, 1H, NH). 13C NMR (DMSO-d6)
d
21.4, 31.5, 34.8, 42.3, 49.6, 53.7, 118.5, 123.8, 126.3, 127.1,
129.1, 129.2, 130.1, 130.7, 135.9, 136.2, 167.5, 168.3, 170.1, 172.2.
IR (KBr) 2600, 1765, 1730, 1700, 1680. MS (EI), m/z 379 (Mþ, 3),
126 (6), 71 (17), 58 (100). Anal. (C21H22ClN3O4$0.9H2O) C, H, N.
4.1.4.2.3. Synthesis of 2-(3-(2,5-dioxo-3,4-diphenyl-2,5-dihydro-
1H-pyrrol-1-yl)-2,6-dioxopiperidin-1-yl)-N,N-dimethylethanaminium
chloride 2f. Following the general procedure B for the synthesis of
N-substituted glutarimides, from 9f (0.50 g, 1.38 mmol), refluxed
for 2.5 h, 0.57 g (88%) of 2f were obtained as a yellow solid (mp
Ar), 9.51 (s, 1H, Ar), 10.36 (bs, 1H, NH). 13C NMR (D2O)
d 22.8, 22.8,
32.8, 33.4, 37.9, 45.5, 46.5, 54.3, 54.4, 57.9, 123.5, 123.8, 125.0, 125.3,
126.2, 126.8, 131.5, 131.6, 132.0, 132.0, 132.9, 132.9, 133.0, 133.1, 137.4,
138.0, 139.6, 139.8, 147.9, 148.0, 165.3, 165.5, 166.0, 166.3, 174.1, 177.0.
IR (KBr) 2480, 1730, 1705, 1670, 1530, 1330. MS (ESI), m/z 425
[M þ H]þ. Anal. (C21H21ClN4O6$0.25H2O) C, H, N.
4.1.4.1.3. Synthesis of 2-(3-(5-amino-1,3-dioxo-1H-benzo[de]iso-
quinolin-2(3H)-yl)-2,6-dioxopiperidin-1-yl)-N,N-dimethylethanami-
nium chloride 2c. A mixture of 2b (110 mg, 0.24 mmol) in DMF/
MeOH (35 mL, 6/1, v/v) was treated with 10% Pd/C (0.04 g) and
hydrogenated for 4.5 h at 50 psi. The mixture was filtered through
a celite pad. The solvent was removed under reduced pressure, the
residue was disgregated in AcOEt and filtered to afford 2c (0,94 g,
91%) as a orange solid (mp >268 ꢀC dec). 1H NMR (DMSO-d6)
>259 ꢀC dec). 1H NMR (DMSO-d6)
d 2.13–2.17 (m, 1H, CH2), 2.50–
2.66 (m, 1H, CH2), 2.75–2.84 (m, 1H, CH2), 2.79 (s, 6H, 2 ꢁ CH3),
3.00–3.12 (m, 1H, CH2), 3.19 (t, 2H, CH2N, J ¼ 5.8 Hz), 4.03 (t, 2H,
CH2N, J ¼ 5.8 Hz), 5.29 (dd,1H, CH, J1 ¼13.1 Hz, J2 ¼ 5.2 Hz), 7.43 (bs,
10H, Ar), 10.33 (bs, 1H, NH). 13C NMR (DMSO-d6)
d 21.2, 31.4, 34.8,
42.3, 49.9, 53.8, 128.2, 128.7, 129.6, 130.0, 136.3, 169.4, 169.9, 172.0.
IR (KBr) 2600, 1770, 1710, 1680. MS (EI), m/z 431 (Mþ, 3), 178 (8), 71
(17), 58 (100). Anal. (C25H26ClN3O4) C, H, N.
d
2.06–2.09 (m, 1H, CH2), 2.49–2.64 (m, 1H, CH2), 2.75–2.77 (m, 1H,
4.1.4.2.4. Synthesis of 2-(3-(1,3-dioxoisoindolin-2-yl)-2,6-dioxo-
piperidin-1-yl)-N,N-dimethylethanaminium chloride 2g. Following
the general procedure B for the synthesis of N-substituted gluta-
rimides, from 9g (2.00 g, 7.72 mmol), refluxed for 24 h, 1.83 g (65%)
of 2g were obtained as a white solid (mp 238–240 ꢀC). 1H NMR
CH2), 2.80 (s, 6H, 2 ꢁ CH3), 3.03–3.15 (m,1H, CH2), 3.20 (t, 2H, CH2N,
J ¼ 5.8 Hz), 4.06 (t, 2H, CH2N, J ¼ 5.8 Hz), 5.98 (dd, 1H, CH,
J1 ¼11.9 Hz, J2 ¼ 5.8 Hz), 6.10 (bs, 1H, NH2), 6.12 (bs, 1H, NH2), 7.35
(s, 1H, Ar), 7.61–7.70 (m, 1H, Ar), 7.91–8.19 (m, 3H, Ar), 10.20 (bs, 1H,
NH). 1H NMR (DMSO-d6, 60 ꢀC)
d
2.06–2.13 (m, 1H, CH2), 2.50–2.65
(DMSO-d6) d 2.11–2.16 (m, 1H, CH2), 2.52–2.67 (m, 1H, CH2), 2.74–
(m, 1H, CH2), 2.73–2.85 (m, 1H, CH2), 2.81 (s, 6H, 2 ꢁ CH3), 3.00–
3.30 (m, 3H, CH2 þ CH2N), 4.07 (t, 2H, CH2N, J ¼ 6.1 Hz), 5.94 (bs, 2H,
NH2), 5.95 (dd, 1H, CH, J1 ¼12.5 Hz, J2 ¼ 5.8 Hz), 7.36 (s, 1H, Ar),
7.61–7.67 (m, 1H, Ar), 7.92 (bs, 1H, Ar), 8.07 (d, 1H, Ar, J ¼ 7.9 Hz),
2.89 (m, 1H, CH2), 2.78 (s, 6H, 2 ꢁ CH3), 3.01–3.13 (m, 1H, CH2), 3.19
(t, 2H, CH2N, J ¼ 5.8 Hz), 4.03 (t, 2H, CH2N, J ¼ 6.1 Hz), 5.34 (dd, 1H,
CH, J1 ¼13.1 Hz, J2 ¼ 5.2 Hz), 7.89–7.97 (m, 4H, Ar), 10.50 (bs, 1H,
NH). 13C NMR (DMSO-d6)
d 21.1, 31.4, 34.8, 42.4, 49.6, 53.8, 123.5,
8.15 (bs, 1H, Ar). 13C NMR (DMSO-d6)
d
20.6, 31.2, 34.7, 42.6, 50.9,
131.2, 135.0, 167.1, 169.9, 172.0. IR (KBr) 2540, 1780, 1720, 1685. MS
(EI), m/z 329 (Mþ, 3), 76 (6), 71 (13), 58 (100), Anal. (C17H20ClN3O4)
C, H, N.
54.0, 112.3, 120.6, 121.2, 121.5, 121.8, 122.0, 122.2, 122.4, 125.8, 126.3,
127.1,127.2, 133.2,133.6,148.0,148.1,163.0, 163.2, 163.5, 163.7, 170.4,
172.1. IR (KBr) 3380, 3320, 3200, 2550, 1730, 1680, 1650. MS (ESI),
m/z 395 [M þ H]þ. Anal. (C21H23ClN4O4$1.25H2O) C, H, N.
4.1.5. Synthesis of 2,6-bis(2,6-dioxopiperidin-3-yl)pyrrolo[3,4-
f]isoindole-1,3,5,7(2H,6H)-tetraone 4
4.1.4.2. Procedure B. To a mixture of the corresponding anhydride 9
(10.00 mmol) and DMF (60 mL) was added N,N-dimethylethy-
lendiamine (10.00 mmol) and the mixture was stirred for 24 h at
room temperature. Then, the solvent was evaporated under
reduced pressure and the resulting residue was treated with 14 mL
of a mixture of acetic anhydride:acetyl chloride (1:1), and refluxed.
After cooling, the reaction mixture was concentrated in vacuo, and
the crude product was recrystallized from DMF–AcOEt.
A mixture of the benzene-1,2,4,5-tetracarboxylic dianhydride
(0.10 g,
0.46 mmol),
3-aminopiperidine-2,6-dione
(0.15 g,
0.92 mmol) and sodium acetate (0.08 mg,1.01 mmol) was heated at
reflux in an anhydrous atmosphere for 16 h. The solution was
allowed to cool to room temperature and concentrated in vacuo.
Water was then added to the residue with vigorous stirring to give
a precipitate, which was collected by filtration, washed with water
and dried to offer 4 (0.17 mg, 83%) as a grey solid (mp >300 ꢀC). 1H
4.1.4.2.1. Synthesis of 2-(3-(1,3-dioxo-1H-benzo[f]isoindol-2(3H)-
yl)-2,6-dioxopiperidin-1-yl)-N,N-dimethylethanaminium 2d. Following
the general procedure B for the synthesis of N-substituted gluta-
rimides, from 9d (1.00 g, 3.23 mmol), refluxed for 24 h, 0.64 g (47%) of
2d were obtained as a pale solid (mp 283–284 ꢀC). 1H NMR (DMSO-
NMR (DMSO-d6)
d
2.10–2.13 (m, 2H, 2 ꢁ CH2), 2.50–2.66 (m, 4H,
2 ꢁ CH2), 2.86–2.98 (m, 2H, 2 ꢁ CH2), 5.27 (dd, 2H, 2 ꢁ CH,
J1 ¼12.5 Hz, J2 ¼ 5.2 Hz), 8.36 (s, 2H, Ar), 11.21 (s, 2H, 2 ꢁ NH). 13
C
NMR (DMSO-d6) d 21.9, 30.9, 49.5, 118.3, 136.8, 165.5, 169.6, 172.8. IR
(KBr) 3240, 1780, 1720. MS (ESI), m/z 437 [M ꢃ H]þ. Anal.
d6)
d
2.14–2.18 (m, 1H, CH2), 2.62–2.87 (m, 2H, CH2), 2.80 (s, 6H,
(C20H14N4O8) C, H, N.
2 ꢁ CH3), 3.01–3.13 (m, 1H, CH2), 3.21 (t, 2H, CH2N, J ¼ 6.1 Hz), 4.05 (t,
2H, CH2N, J ¼ 6.1 Hz), 5.40 (dd, 1H, CH, J1 ¼12.8 Hz, J2 ¼ 5.5 Hz), 7.81–
7.84 (m, 2H, Ar), 8.29–8.33 (m, 2H, Ar), 8.61 (s, 2H, Ar), 10.23 (bs, 1H,
4.1.6. Synthesis of 2,20-(3,30-(1,3,5,7-tetraoxopyrrolo[3,4-f]isoindole-
2,6(1H,3H,5H,7H)-diyl)bis(2,6-dioxopiperidine-3,1-diyl))bis(N,N-
dimethylethanaminium) chloride 5
NH). 13C NMR (DMSO-d6)
d 21.1, 31.4, 34.8, 42.3, 49.7, 53.7, 125.1, 126.9,
129.6, 130.4, 135.1, 166.7, 169.9, 172.1. IR (KBr) 2500, 1770, 1710, 1680.
MS (ESI), m/z 380 [M þ H]þ. Anal. (C21H22ClN3O4$0.75H2O) C, H, N.
4.1.4.2.2. Synthesis of 2-(3-(1,3-dioxo-1H-benzo[e]isoindol-
2(3H)-yl)-2,6-dioxopiperidin-1-yl)-N,N-dimethylethanaminium
To a mixture of anhydride 11 [34] (1.00 g, 2.27 mmol) and dry
DMF (15 mL) was added N,N-dimethylethylendiamine (0.5 mL,
4.55 mmol) and the mixture was stirred for 24 h at room temper-
ature. Then, the solvent was evaporated under reduced pressure
and the resulting residue was treated with 10 mL of a mixture of
acetic anhydride:acetyl chloride (1:1), and refluxed for 24 h. After
cooling, the solid was collected by filtration and recrystallized from
DMF–AcOEt to afford 5 (0,78 g, 52%) as a white solid (mp >280 ꢀC
chloride 2e. Following the general procedure
B for the
synthesis of N-substituted glutarimides, from 9e (1.66 g,
5.38 mmol), refluxed for 24 h, 1.58 g (71%) of 2e were obtained
as a yellow solid (mp 243–245 ꢀC). 1H NMR (DMSO-d6)
d 2.16–
2.21 (m, 1H, CH2), 2.63–2.89 (m, 2H, CH2), 2.78 (bs, 6H,
2 ꢁ CH3), 3.02–3.14 (m, 1H, CH2), 3.18 (t, 2H, CH2N, J ¼ 5.5 Hz),
4.04 (t, 2H, CH2N, J ¼ 5.8 Hz), 5.37 (dd, 1H, CH, J1 ¼13.1 Hz,
J2 ¼ 5.2 Hz), 7.78–7.90 (m, 2H, Ar), 7.96 (d, 1H, Ar, J ¼ 7.9 Hz),
8.23 (d, 1H, Ar, J ¼ 8.6 Hz), 8.49 (d, 1H, Ar, J ¼ 8.6 Hz), 8.79 (d,
dec). 1H NMR (DMSO-d6)
d
2.15–2.19 (m, 2H, 2 ꢁ CH2), 2.54–2.69
(m, 2H, 2 ꢁ CH2), 2.73–2.89 (m, 2H, 2 ꢁ CH2), 2.78 (s, 12H, 4 ꢁ CH3),
3.03–3.15 (m, 2H, 2 ꢁ CH2), 3.20 (t, 4H, 2 ꢁ CH2N, J ¼ 6.1 Hz), 4.05 (t,
4H, 2 ꢁ CH2N, J ¼ 6.1 Hz), 5.45 (dd, 2H, 2 ꢁ CH, J1 ¼13.1 Hz,
J2 ¼ 5.2 Hz), 8.38 (s, 2H, Ar), 10.51 (bs, 2H, 2 ꢁ NH). 13C NMR

M.F. Bran˜a et al. / European Journal of Medicinal Chemistry 44 (2009) 3533–3542
3541
(DMSO-d6)
d
21.0, 31.4, 34.9, 42.4, 50.1, 53.8, 118.3, 136.8, 165.3,
4.1.7.4. Synthesis of 2-(3-(1,3-dioxoisoindolin-2-yl)-2,6-dioxopiperidin-
1-yl)-N-(2-(3-(1,3-dioxoisoindolin-2-yl)-2,6-dioxopiperidin-1-yl)ethyl)-
N-methylethanaminium chloride 6g. Following the general procedure
for the synthesis of HCl salt N-substituted bis-glutarimides, from 9g
(0.50 g, 1.93 mmol), refluxed 24 h, 0.25 g (40%) of HCl salt-6d were
169.6, 172.0. IR (KBr) 2600, 1775, 1720, 1680. MS (EI), m/z 580 (Mþ,
5), 71 (4), 58 (100). Anal. (C28H34Cl2N6O8$1.5H2O) C, H, N.
4.1.7. General procedure for the synthesis of HCl salt N-substituted
bis-glutarimides 6
obtained as a white solid (mp > 210 ꢀC dec). 1H NMR (D2O)
d 1.85 (bs,
To a solution of the corresponding anhydride 9 (1.62 mmol) and
dry DMF (10 mL) was added N,N-(2-aminoetil)-N-metiletilendi-
amine (0.81 mmol) and the mixture was stirred for 48 h at room
temperature. After evaporation in vacuo water (2 mL) was added,
and the pH value was adjusted to 1 by adding 10% HCl. Then, the
solvent was evaporated under reduced pressure and the resulting
residue was treated with 4 mL of a mixture of acetic anhy-
dride:acetyl chloride (1:1), and refluxed. After cooling, the solid
was collected by filtration, washed thoroughly with Ac2O and
Et2O and filtered to afford HCl salt-6. The solid was recrystallized
from DMF.
2H, 2 ꢁ CH2), 2.26–2.41 (m, 2H, 2 ꢁ CH2), 2.62–2.65 (m, 4H, 2 ꢁ CH2),
2.83 (s, 3H, CH3), 3.27 (bs, 4H, 2 ꢁ CH2N), 3.97 (bs, 4H, 2 ꢁ CH2N), 4.96
(dd, 2H, 2 ꢁ CH, J1 ¼12.4 Hz, J2 ¼ 5.2 Hz), 7.52–7.54 (m, 8H, Ar). 13C
NMR (D2O) d 23.5, 33.5, 38.1, 44.4, 52.4, 57.6, 126.3, 133.3, 137.7, 171.5,
174.4, 177.0. IR (KBr) 2500, 1780, 1715, 1680. MS (ESI), m/z 600
[M þ H]þ. Anal. (C31H30ClN5O8$0.7DMF) C, H, N.
4.2. Biological assays
4.2.1. Cell and measurement of TNF-
a
HL-60 cells were incubated at 37 ꢀC in a 5% CO2 atmosphere
using RPMI 1640 medium (Gibco, Grand Island, NY, U.S.A.) sup-
plemented with 10% v/v BFS (Bovine Foetal Serum), 2 mM
4.1.7.1. Synthesis of 2,20-(1,10-(2,20-(methylazanediyl)bis(ethane-2,1-
diyl))bis(2,6-dioxopiperidine-3,1-diyl))bis(1H-benzo[de]isoquinoline-
1,3(2H)-dione) 6a. Following the general procedure for the
synthesis of N-substituted bis-glutarimides, from 9a (0.50 g,
1.62 mmol), refluxed for 72 h, 0.48 g (81%) of HCl salt-6a were
obtained. This salt was converted into the free base using a satu-
rated 5% NaHCO3 solution which was filtered and washed with hot
EtOH. The solid was purified by recrystallization from CHCl3/Et2O to
afford 6a (0.38 g, 68%) as a pale solid (mp >154 ꢀC dec). 1H NMR
L
-glutamine, 100 U/mL penicillin and 100 mL/mL streptomycin. Cells
in exponential growth (5 ꢁ105 cells/mL) were then treated with OA
(50 nM) or TPA (10 nM) and with or without each compound
(30
morphology were evaluated microscopically. Cells were collected
by centrifugation (2000 rpm during 10 min.). TNF- supernatant
concentration was quantified using a human TNF- ELISA (Amer-
sham Co.) following supplier protocol. The TNF- concentration in
mM), during 16 h in 24 well plates. Then cell number and cell
a
a
a
(DMSO-d6 þ TFA)
d
2.02–2.15 (m, 2H, 2 ꢁ CH2), 2.50–2.72 (m, 2H,
those treatments without thalidomide or derivate compounds
(only with TPA or OA) was defined as the 100%. All experiments
were performed at least three times.
2 ꢁ CH2), 2.76–3.10 (m, 4H, 2 ꢁ CH2), 3.01 (s, 3H, CH3), 3.36–3.46
(m, 4H, 2 ꢁ CH2N), 4.14–4.16 (m, 4H, 2 ꢁ CH2N), 5.94–6.00 (m, 2H,
2 ꢁ CH), 7.80–7.95 (m, 4H, Ar), 8.44–8.62 (m, 8H, Ar). 13C NMR
(DMSO-d6) d 20.6, 31.1, 36.3, 42.3, 51.0, 53.4, 121.4,121.7,121.8,127.3,
127.4, 131.1, 131.3, 131.3, 131.6, 134.9, 162.7, 163.3, 170.0, 171.8. IR
(KBr) 1730, 1680, 1660. MS (ESI), m/z 700 [M þ H]þ. Anal.
(C39H33N5O8$0.6CHCl3) C, H, N.
Acknowledgment
´
We thank Direccion General de Estudios Superiores (Grant No.
PB98-055, CYTED), Laboratorios Knoll (BASF Pharma), Universidad
CEU-San Pablo for financial support and Brian Crilly for linguistic
assistance. L. An˜orbe acknowledges Comunidad de Madrid for
a predoctoral fellowship.
4.1.7.2. Synthesis of 2-(3-(1,3-dioxo-1H-benzo[f]isoindol-2(3H)-yl)-2,6-
dioxopiperidin-1-yl)-N-(2-(3-(1,3-dioxo-1H-benzo[f]isoindol-2(3H)-yl)-2,6-
dioxopiperidin-1-yl)ethyl)-N-methylethanaminium chloride 6d. Following
the general procedure for the synthesis of HCl salt N-substituted bis-
glutarimides, from 9d (0.50 g, 1.62 mmol), refluxed 48 h, 0.48 g (81%) of
HCl salt-6d were obtained as a pale solid (mp > 272 ꢀC dec). 1H NMR
References
(DMSO-d6)
d
2.11–2.20 (m, 2H, 2 ꢁ CH2), 2.60–2.73 (m, 2H, 2 ꢁ CH2),
[1] W. Kunz, H. Keller, H. Muckter, Arzneimittelforschung 6 (1956) 426–430.
[2] W. Lenz, R.A. Pfeiffer, W.D. Kosenow, J. Hayman, Lancet 279 (1962) 45–46.
[3] J. Sheskin, Clin. Pharmacol. Ther. 6 (1965) 303–306.
[4] E.P. Sampaio, G. Kaplan, A. Miranda, J.A. Nery, C.P. Miguel, S.M. Viana, J. Infect.
Dis. 168 (1993) 408–414.
[5] S.V. Rajkumar, E. Blood, D. Vesole, R. Fonseca, P.R. Greipp, J. Clin. Oncol. 24
(2006) 431–436.
[6] V. Eleutherakis-Papaiakovou, A. Bamias, M.A. Dimopoulos, Ann. Oncol. 15
(2004) 1151–1160.
2.81–2.90 (m, 2H, 2 ꢁ CH2), 2.89 (bs, 3H, CH3), 2.99–3.09 (m, 2H, 2 ꢁ CH2),
3.25 (bs, 4H, 2 ꢁ CH2N), 4.08 (bs, 4H, 2 ꢁ CH2N), 5.34–5.43 (m, 2H,
2 ꢁ CH), 7.81 (dd, 4H, Ar, J1 ¼6.1 Hz, J2 ¼ 3.1 Hz), 8.29 (dd, 4H, Ar,
J1 ¼6.1 Hz, J2 ¼ 3.1 Hz), 8.60 (bs, 4H, Ar), 10.33 (bs, 1H, NH). 13C NMR
(DMSO-d6 þ TFA)
d 21.2, 31.4, 34.5, 41.1, 49.7, 52.6, 125.2, 127.0, 129.7,
130.6, 135.3, 166.9, 169.9, 172.2. IR (KBr) 2500, 1770, 1710, 1685. MS (ESI),
m/z 700 [M þ H]þ. Anal. (C39H34ClN5O8$0.6H2O) C, H, N.
[7] E.P. Sampaio, E.N. Sarno, R. Galilly, Z.A. Cohn, G. Kaplan, J. Exp. Med. 173 (1991)
699–703.
4.1.7.3. Synthesis of 2-(3-(2,5-dioxo-3,4-diphenyl-2,5-dihydro-1H-
pyrrol-1-yl)-2,6-dioxopiperidin-1-yl)-N-(2-(3-(2,5-dioxo-3,4-diphenyl
2,5-dihydro-1H-pyrrol-1-yl)-2,6-dioxopiperidin-1-yl)ethyl)-N-methyl-
ethanaminium chloride 6f. Following the general procedure for the
synthesis of HCl salt N-substituted bis-glutarimides, from 9d (0.50 g,
1.39 mmol), refluxed 16 h, 0.43 g (74%) of HCl salt-6f were obtained
[8] M.V. De Almeida, F.M. Teixeira, V.N. De Souza, G.W. Amarante, C.C.S. Alves,
S.H. Cardoso, A.M. Mattos, A.P. Ferreira, H.C. Teixeira, Chem. Pharm. Bull. 55
(2007) 223–226.
[9] R.L. D’Amato, M.S. Loughan, E. Flynn, J. Folkman, Proc. Natl. Acad. Sci. U.S.A. 91
(1994) 4082–4085.
[10] T. Nakamura, T. Noguchi, H. Kobayashi, H. Miyachi, Y. Hashimoto, Chem.
Pharm. Bull. 54 (2006) 1709–1714.
[11] A.L. Moreira, E.P. Sampaio, A. Zmuidzinas, P. Frindt, K.A. Smith, G. Kaplan, J.
Exp. Med. 177 (1993) 1675–1680.
1
as a yellow solid (mp > 261 ꢀC dec). H NMR (DMSO-d6, 60 ꢀC)
[12] M. Melchert, A. List, Int. J. Biochem. Cell Biol. 39 (2007) 1489–1499.
[13] H. Miyachi, A. Azuma, E. Hioki, S. Iwasaki, Y. Kobayashi, Y. Hashimoto, Bio-
chem. Biophys. Res. Commun. 224 (1996) 426–430.
[14] Y. Shibata, K. Sasaki, Y. Hashimoto, S. Iwasaki, Biochem. Biophys. Res. Commun.
205 (1994) 1992–1997.
[15] H. Miyachi, A. Azuma, A. Ogasawara, E. Uchimura, N. Watanabe, Y. Kobayashi,
F. Kato, M. Kato, Y. Hashimoto, J. Med. Chem. 40 (1997) 2858–2865.
[16] H. Miyachy, A. Azuma, E. Hioki, S. Iwasaki, Y. Kobayashi, Y. Hashimoto, Chem.
Pharm. Bull. 44 (1996) 1980–1982.
d
2.12–2.17 (m, 2H, 2 ꢁ CH2), 2.50–2.65 (m, 2H, 2 ꢁ CH2), 2.78–2.88
(m, 2H, 2 ꢁ CH2), 2.85 (bs, 3H, CH3), 2.96–3.07 (m, 2H, 2 ꢁ CH2),
3.26–3.32 (m, 4H, 2 ꢁ CH2N), 4.05 (bs, 4H, 2 ꢁ CH2N), 5.24 (dd, 2H,
2 ꢁ CH, J1 ¼12.8 Hz, J2 ¼ 5.5 Hz), 7.41 (bs, 20H, Ar),10.28 (NH,1H).13
C
NMR (DMSO-d6) d 21.2, 31.3, 34.3, 40.2, 49.9, 52.2, 128.2, 128.7, 129.6,
130.0, 136.3, 169.4, 169.9, 172.0. IR (KBr) 2500, 1770, 1710, 1690. MS
(ESI), m/z 805 [M þ H]þ. Anal. (C47H42ClN5O8) C, H, N.

3542
M.F. Bran˜a et al. / European Journal of Medicinal Chemistry 44 (2009) 3533–3542
[17] M.F. Bran˜a, M. Cacho, M.A. Garcı´a, B. De Pascual, A. Ramos, M.T. Domı´nguez,
J.M. Pozuelo, C. Abradelo, M.F. Rey-Stolle, M. Yuste, M. Ban˜ez, J.C. Lacal, J. Med.
Chem. 47 (2004) 1391–1399.
[26] H. Mitoma, T. Horiuchi, H. Tsukamoto, Y. Tamimoto, Y. Kimoto, A. Uchino, K. To,
S. Harashima, N. Hatta, M. Harada, Arthritis Rheum. 58 (2008) 1248–1257.
[27] J. Pane´s, F. Gomollo´n, C. Taxonera, J. Hinojosa, J. Clofent, P. Nos, Drugs 67
(2007) 2511–2537.
´
´
´
[18] M.F. Bran˜a, A. Fernandez, M. Garrido, M.L. Lopez Rodrıguez, M.J. Morcillo,
A.M. Sanz, Chem. Pharm. Bull. 37 (1989) 2710–2712.
[28] U. Wollina, G. Hansel, A. Koch, J. Scho¨nlebe, E. Ko¨stler, G. Haroske, Am. J. Clin.
Dermatol. 9 (2008) 1–14.
[29] A. Rijkeboer, A. Voskuyl, M. Van Agtmael, Scand. J. Infect. Dis. 39 (2007)
80–83.
[30] J.C. Lagier, D. Andriamanantena, J. Damiano, J.M. Dot, B. Chaudier, J. Margery,
Rev. Mal. Respir. 24 (2007) 1159–1160.
[31] A. Gupta, A.C. Street, F.A. Macrae, Med. J. Aust. 188 (2008) 168–170.
[32] V.V. Jain, T. Evans, M.W. Peterson, Respir. Med. 100 (2006) 1291–1293.
[33] A.E. Adams, J. Zwicker, C. Curiel, M.E. Kadin, K.R. Falchuk, R. Drews, T.S. Kupper,
J. Am. Acad. Dermatol. 51 (2004) 660–662.
[19] N.A. Jo¨nsson, Acta Pharm. Suec. 9 (1972) 425–430.
[20] E.B. Hershberg, L.F. Fieser, Org. Synth. Coll. 2 (1943) 194.
[21] F.E. King, D.A. Kidd, J. Chem. Soc. 4 (1949) 3315–3319.
[22] G.W. Muller, D.I. Stirling, R.S.-C. Chen. Pat. WO 9803502, 1998.
[23] A. Moreira, D. Friedlander, B. Shif, G. Kaplan, D. Zagzag, J. Neuro-Oncol. 43
(1999) 109–114.
[24] H. Miyachi, A. Ogasawara, A. Azuma, Y. Hashimoto, Bioorg. Med. Chem. 5
(1997) 2095–2102.
[25] L.M. Lima, P. Castro, A.L. Machado, C.A.M. Fraga, C. Lugnier, V.L. Gonçalves de
Moraes, E.J. Barreiro, Bioorg. Mol. Chem. 10 (2002) 3067–3073.
[34] Y. Iwakura, C.P. Yang, K. Uno, Makromol. Chem. 175 (1974) 137–159.