Discovery of (S)-N-{2-[1-(3-ethoxy-4-methoxy-phenyl)-2- methanesulfonylethyl]-1,3-dioxo-2,3-dihydro-1 H-isoindol-4-yl}acetamide (Apremilast), a potent and orally active phosphodiesterase 4 and tumor necrosis factor-a inhibitor

Article abstract of DOI:10.1021/jm900210d

In this communication, we report the discovery of 1S (apremilast), a novel potent and orally active phosphodiesterase 4 (PDE4) and tumor necrosis factor-α inhibitor. The optimization of previously reported 3-(1,3-dioxo-1,3-dihydroisoindol-2-yl)-3-(3,4-dimethoxyphenyl)propionic acid PDE4 inhibitors led to this series of sulfone analogues. Evaluation of the structure-activity relationship of substitutions on the phthalimide group led to the discovery of an acetylamino analogue 1S, which is currently in clinical trials.

Full text of DOI:10.1021/jm900210d

1522
J. Med. Chem. 2009, 52, 1522–1524
Discovery of (S)-N-{2-[1-(3-Ethoxy-4-methoxy-
phenyl)-2-methanesulfonylethyl]-1,3-dioxo-
2,3-dihydro-1H-isoindol-4-yl}acetamide
(Apremilast), a Potent and Orally Active
Phosphodiesterase 4 and Tumor Necrosis
Factor-r Inhibitor
Figure 1. Structure of 1S.
Hon-Wah Man,*,† Peter Schafer,† Lu Min Wong,†
Rebecca T. Patterson,† Laura G. Corral,‡ Heather Raymon,‡
Kate Blease,‡ Jim Leisten,‡ Michael A. Shirley,‡
Yang Tang,‡ Darius M. Babusis,‡ Roger Chen,†
Dave Stirling,† and George W. Muller†
human peripheral blood mononuclear cells (hPBMC) and in
human whole blood (WB).10 This activity provides us with an
important tool to examine the anti-inflammatory activity of our
PDE4 inhibitors in human cells. We have previously reported
on the development of a novel series of PDE4 and TNF-R
inhibitors derived from 3-(1,3-dioxo-1,3-dihydroisoindol-2-yl)-
3-(3,4-dimethoxyphenyl)propionic acid derivatives.11 Herein, we
report the discovery of 1S (apremilast), a novel orally active
PDE4 and TNF-R inhibitor (Figure 1). 1S is currently in clinical
trials for the treatment of psoriasis.12
Drug DiscoVery Department, Celgene Corporation, 86 Morris
AVenue, Summit, New Jersey 07901, and Departments of
Pharmacology and DMPK, Celgene Signal Research, 4550 Towne
Centre Court, San Diego, California 92121
ReceiVed February 18, 2009
The racemates and enantiomers of phthalimide ꢀ-amino-ꢀ-
aryl acid 4, ester 5, amide 6, and nitrile 7 analogues were
prepared as previously described.11 The ꢀ-aminosulfone 3 was
prepared by treatment of in situ generated imine of 3-ethoxy-
4-methoxybenzaldehyde by lithium hexamethyldisilazide, with
lithium dimethylsulfone and boron trifluoride etherate in a 41%
yield. Resolution of the ꢀ-aminosulfone 3 was achieved by
treatment of 3 with N-acetyl-L-leucine to give the S-isomer 3S
with an ee of 98.4%. The R-isomer 3R could be obtained in a
similar manner using N-acetyl-D-leucine with a 97.6% ee.
Treatment of the corresponding phthalic anhydrides with 3
provided 9-11. Coupling of 3-nitrophthalic anhydride with 3,
followed by hydrogen reduction gave 12. When hydrogenation
was performed in the presence of formaldehyde, the dimethy-
lamino analogue 13 was obtained. Catalytic hydrogenation of
3-nitrophthalic acid followed by acetylation with acetic anhy-
dride afforded 3-N-acetylaminophthalic anhydride. Compound
1S was prepared by condensation of 3-N-acetylaminophthalic
anhydride with ꢀ-aminosulfone 3S in acetic acid in a 75% yield
(Scheme 1). The enantiomeric purity of 1S could be further
enhanced by recrystallization from ethanol to give a sample with
a 99.5% ee.13
Three assays were used to assess the in vitro potency of our
PDE4 inhibitors. PDE4 inhibitory activity was measured with
PDE4 enzyme isolated from U937 cells as previously reported.14
TNF-R inhibitory activity was measured in lipopolysaccharide
(LPS) stimulated hPBMC or WB.11 The hPBMC and WB cells
are human cells, and thus, these cellular assays may have greater
clinical relevance than inhibition of purified PDE4. Furthermore,
the difference between hPBMC and WB TNF-R inhibitory
activity may serve as an indirect indicator of the degree of
protein binding, and the WB data are a more realistic view of
the clinical situation.
Abstract: In this communication, we report the discovery of 1S
(apremilast), a novel potent and orally active phosphodiesterase 4
(PDE4) and tumor necrosis factor-R inhibitor. The optimization of
previously reported 3-(1,3-dioxo-1,3-dihydroisoindol-2-yl)-3-(3,4-
dimethoxyphenyl)propionic acid PDE4 inhibitors led to this series of
sulfone analogues. Evaluation of the structure-activity relationship of
substitutions on the phthalimide group led to the discovery of an
acetylamino analogue 1S, which is currently in clinical trials.
PDE4a is one of 11 known members of the PDE enzyme
family.1 It is a cyclic adenosine monophosphate (cAMP) specific
enzyme, which hydrolyzes cAMP to AMP. Thus, inhibition of
PDE4 leads to an increase of cAMP concentration, which
influences multiple intracellular signaling pathways.2 Since
PDE4 is localized to specific subcellular sites in different parts
of body and cells,3 regulating the localized cAMP gradients in
cells provides opportunities to affect various therapeutic areas,
such as inflammation,4 respiratory,5,6 and central nervous system
diseases.7 Therefore, PDE4 has long been considered to be an
important therapeutic target. Among various activities of PDE4
inhibitors, anti-inflammatory activity probably is the most widely
studied and reported area. One of the driving forces for the anti-
inflammatory activity of PDE4 inhibitors is the inhibition of
production of TNF-R, a key cytokine in the inflammatory
cascade. Elevated levels of TNF-R have been associated with a
number of inflammatory diseases such as psoriasis, rheumatoid
arthritis, and inflammatory bowel disease. In the past decade,
the success of anti-TNF-R biologics has valuated the importance
of the blockade of TNF-R production in the treatment of patients
with various inflammatory diseases.8,9 Monocytes and mac-
rophages are key producers of TNF-R, and PDE4 is a major
PDE enzyme present in these cells. Therefore, production of
TNF-R can be suppressed by inhibition of PDE4 in stimulated
In a previous report, we described a novel series of 3-(1,3-
dioxo-1,3-dihydroisoindol-2-yl)-3-(3,4-dimethoxyphenyl)propi-
onic acid and derivatives with TNF-R inhibitory activity.11 Later,
we reported that inhibition of PDE4 was the mechanism of
action for TNF-R inhibition by this class of compounds.14 In
the effort to optimize the potency, isosteric replacement of the
acid moiety was explored. Replacement of the acid moiety of
4a with a methyl ester 5a resulted in a 6-fold increase in PDE4
* To whom correspondence should be addressed. Phone: (908) 673-9124.
Fax: (908) 673-9124. E-mail: hwman@celgene.com.
†
Celgene Corporation.
Celgene Signal Research.
‡
a Abbreviations: PDE4, phosphodiesterase 4; TNF-R, tumor necrosis
factor-R; SAR, structure-activity relationship; cAMP, cyclic adenosine
monophosphate; hPBMC, human peripheral blood mononuclear cells; WB,
human whole blood; LPS, lipopolysaccharide; CYP450, cytochrome P450.
10.1021/jm900210d CCC: $40.75
2009 American Chemical Society
Published on Web 03/03/2009

Letters
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6 1523
Scheme 1a
Table 1. TNF-R and PDE4 Inhibition (IC50 in µM) by
3-(1,3-Dioxo-1,3-dihydroisoindol-2-yl)-3-(3-alkoxy-4-methoxyphenyl)-
propionic Acid and Derivatives
compd
R
Y
TNF-Ra
WBb
PDE4
4a
4b
5a
5b
6a
6b
7a
7b
8a
8b
Et
COOH
COOH
COOMe
COOMe
CONH2
CONH2
CN
CN
SO2Me
SO2Me
35
36
NDc
NDc
4.0
1.3
cyclopentyl
Et
cyclopentyl
Et
cyclopentyl
Et
cyclopentyl
Et
0.34
0.22
1.6
1.9
1.1
0.04
0.34
0.22
0.26
0.7
1.7
2.7
2.5
0.24
1.6
1.3
1.5
NDc
10.5
NDc
0.49
NDc
2.5
cyclopentyl
NDc
a TNF-R inhibition in LPS-stimulated hPBMC. b TNF-R inhibition in
LPS-stimulated WB. c ND: not determined.
a Reagents and conditions: (a) LiN(SiMe3)2, then Me2SO2/n-BuLi/
BF3Et2O, -78 °C; (b) N-Ac-L-leucine, MeOH; (c) HOAc, reflux.
Table 2. TNF-R and PDE4 Inhibition (IC50 in µM) by Substituted
3-(1,3-Dioxo-1,3-dihydroisoindol-2-yl)-3-(3-ethoxy-4-methoxyphenyl)-
propionic Acid and Derivatives
compd
stereo
Y
Z
TNF-Ra
WBb
PDE4
1
rac
S
R
rac
rac
rac
rac
rac
R
SO2Me
SO2Me
SO2Me
SO2Me
SO2Me
SO2Me
SO2Me
SO2Me
CONH2
CN
NHAc
NHAc
NHAc
OH
OMe
Me
0.19
0.077
0.37
1.47
0.34
0.27
0.37
0.22
3.0
0.44
0.11
NDc
NDc
NDc
2.4
0.082
0.074
0.61
0.82
0.69
0.068
0.12
0.12
0.71
0.13
1S
1R
9
10
11
12
13
14
15
Figure 2. Structures of rolipram and roflumilast.
inhibitory activity and a 50-fold increase in TNF-R inhibitory
activity. The PDE4 inhibitory activity of the amide analogue
6a showed no improvement, but 6a is 13-fold more potent in
the inhibition of TNF-R in hPBMC compared to 4a. Nitrile
analogue 7a was a potent compound, which resulted in a near
32-fold increase in PDE4 inhibitory activity and 145-fold
increase in TNF-R inhibitory activity compared to 4a. The
sulfone moiety is also a good isosteric replacement of the acid
moiety. Sulfone 8a is 6-fold more potent in the inhibition of
PDE4 and 27-fold more potent in the inhibition of TNF-R than
4a. Interestingly, the ethoxy analogues 7a and 8a have similar
potency against TNF-R in hPBMC and WB assays.
The 3,4-dialkoxyphenyl moiety is one of many known
pharmacophores in PDE4 inhibitors.15 Generally, a methoxy
group at the 4-position is the optimal group, and a bulky group,
such as cyclopentoxy, at the 3-position leads to more potent
analogues in a majority of reported series of PDE4 inhibitors,
which contain the 3,4-dialkoxyphenyl moiety. This feature can
be exemplified by rolipram, a prototypical PDE4 inhibitor as
shown in Figure 2. Therefore, to improve the potency, we also
investigated the effect of the 3-alkoxy group. Surprisingly, the
inhibitory activities of TNF-R and PDE4 of ethoxy analogues
appeared to be better or no worse than those of cyclopentoxy
analogues (4-8a vs 4-8b) (Table 1), since literature precedence
suggests that larger alkoxy moieties are preferred. Nevertheless,
the 3-ethoxy-4-methoxyphenyl group was chosen as the standard
group to further optimize the potency.
NH2
2.2
NMe2
NHAc
NHAc
1.4
NDc
1.6
rac
0.12
a TNF-R inhibition in LPS-stimulated hPBMC. b TNF-R inhibition in
LPS-stimulated WB. c ND: not determined.
reported in future publications. Hydroxyl and methoxy substitu-
tions of the 4-hydrogen in 8a resulted in less potent PDE4
inhibitors 9 and 10, respectively. Substitutions of 4-hydrogen
with methyl 11, amino 12, or dimethylamino 13 groups led to
slightly improved PDE4 inhibitory activity and TNF-R inhibitory
activity in hPBMC, compared to those of the parent compound
8a. However, their TNF-R inhibitory activities decreased 6- to
9-fold in WB versus the results in hPBMC. Introduction of a
4-N-acetylamino group led to the sulfone analogue 1, which
was 3-fold more potent than 8a in the PDE4 assay and, more
importantly, 6-fold more potent in the TNF-R assays. Interest-
ingly, the 4-N-acetylamino group in analogues of amide 14 and
nitrile 15 did not provide significant improvement on TNF-R
inhibitory activity compared to their parent compounds 6a and
7a, respectively.
A single enantiomer is preferred in drug development.
Resolution of the racemate of aminosulfone 3 followed by
phthalimide formation yielded the S-isomer 1S that was found
to be the more active enantiomer, which was >5-fold more
potent than the R-isomer 1R. Some of the activity of the 1R
isomer may be due to residual 1S, since 1R contained 1.2% of
1S by chiral HPLC.13b The PDE4 IC50 of 1S from the PDE4
The effect of substitutions on the phthalimide group was
explored to further improve the potency. Below we describe
the effect of substitutions at the 4-position of the phthalimide
ring in the sulfone series (Table 2). The details of SAR will be

1524 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6
Table 3. Pharmacokinetics of 1S in Female Rat
Letters
female rat
dose (mg/kg)
Cmax (ng/mL)
AUC (ng ·h/mL)
t1/2 (h)
F (%)
Cl ((L/h)/kg)
0.5
Vss (L/kg)
IV
oral
5
10
6600
1100
11 000
14 000
3
5
2
64
(3) Houslay, M. D.; Schafer, P.; Zhang, K. Y. J. Phosphodiesterase-4 as
a therapeutic target. Drug DiscoVery Today 2005, 10, 1503–1519.
(4) Banner, K. H.; Trevethick, M. A. PDE4 inhibition: a novel approach
for the treatment of inflammatory bowel disease. Trends Pharmacol.
Sci. 2004, 25, 430–436.
(5) Barnes, P. J. Novel signal transduction modulators for the treatment
of airway diseases. Pharmacol. Ther. 2006, 109, 238–245.
(6) Lipworth, B. J. Phosphodiesterase-4 inhibitors for asthma and chronic
obstructive pulmonary disease. Lancet 2005, 365, 167–175.
(7) O’Donnell, J. M.; Zhang, H.-T. Antidepressant effects of inhibitors
of cAMP phosphodiesterase (PDE4). Trends Pharmacol. Sci. 2004,
25, 158–163.
(8) Boker, A.; Kimball, A. B.; Rolz-Cruz, G. Biologicals in the treatment
of psoriasis. Curr. Opin. InVest. Drugs (Thomson Sci.) 2007, 8, 939–
946.
(9) Tutrone, W. D.; Weinberg, J. M. Biological therapy for psoriasis:
infliximab, etanercept, adalimumab, efalizumab and alefacept. Expert
ReV. Dermatol. 2006, 1, 207–216.
(10) Torphy, T. J. Phosphodiesterase Inhibitors. In Asthma; Lippincott-
Raven: Philadelphia, PA, 1997; pp 1755-1773.
enzymatic assay, and its TNF-R IC50 from hPBMC and WB
cell based assays are very similar. Even though the TNF-R (in
hPBMC) and PDE4 inhibitory activity of 1S is significantly
weaker than that of roflumilast (IC50 of 0.02 and 0.3 nM,
respectively), the TNF-R IC50 in WB is only 2-fold different
between 1S and roflumilast (IC50 ) 50 nM).16 1S was further
profiled for DMPK properties and tested in two in vivo models.
Compound profiling of 1S revealed that it was stable in the
presence of human microsomes (t1/2 > 60 min). It demonstrated
IC50 values of >10 µM against cytochrome P450 (CYP450)
enzymes, including 1A2, 2C9, 2C19, 2D6, and 3A4. 1S was
90% protein bound in human plasma. Oral and intravenous
administration of 1S in female rats showed that it had good
pharmacokinetics with low clearance, a moderate volume of
distribution, and a 64% oral bioavailability (Table 3).
Two models of inflammation were used to characterize the
in vivo efficacy of 1S. The first model, a LPS-induced TNF-R
inhibition model in rats, examined the TNF-R inhibitory ability
of 1S in vivo. A significant suppression of TNF-R production
was observed at oral dose levels as low as 0.01 mg/kg, and the
ED50 was determined to be 0.03 mg/kg. The second model, a
LPS-induced neutrophilia model in rats,17 measured the inhibi-
tion of neutrophils in bronchoalveolar lavage fluid, a common
symptom observed in chronic obstructive pulmonary disease
patients.18 In this model, 1S exhibited an ED50 of 0.3 mg/kg po
in the inhibition of neutrophilia. In a similar model, roflumilast
was reported to have an ED50 of 0.9 mg/kg.19 The in vitro
potency, in vivo oral efficacy, and excellent bioavailability
supported advancement of 1S to clinical development.
In summary, we optimized the structures of a series of 3-(1,3-
dioxo-1,3-dihydroisoindol-2-yl)-3-(3,4-dialkoxyphenyl)propi-
onic acid analogues to enhance PDE4 and TNF-R inhibitory
activity. We discovered that the sulfone moiety is a good isostere
for the carboxylic acid moiety in this series. Substitutions at
the 4-position of the phthalimide group did not have significant
impact on PDE4 inhibitory activity in the enzymatic assay.
However, addition of the N-acetylamino group led to the
discovery of 1S, which had enhanced potency in cell based
assays. 1S demonstrated good rat pharmacokinetics and oral
efficacy in two rat inflammation models. 1S was advanced into
development, and two phase II clinical trials in psoriasis were
reported with significant improvement in treated patients.20
Further details of inhibition of PDE4 subtypes, other proin-
flammatory mediators, and adverse effects will be reported in
future publications.
(11) Muller, G. W.; Corral, L. G.; Shire, M. G.; Wang, H.; Moreira, A.;
Kaplan, G.; Stirling, D. I. Structural modifications of thalidomide
produce analogs with enhanced tumor necrosis factor inhibitory
activity. J. Med. Chem. 1996, 39, 3238–3240.
(12) Gottlieb, A. B.; Strober, B.; Krueger, J. G.; Rohane, P.; Zeldis, J.;
Hu, C. C.; Kipnis, C. An open-label, single-arm pilot study inpatients
with severe plaque-type psoriasis treated with an oral anti-inflammatory
agent, apremilast. Curr. Med. Res. Opin. 2008, 24, 1529–1538.
(13) (a) Schafer, P. H.; Muller, G. W.; Man, H.-W.; Ge, C. Use of (+)-
2-[1-(3-Ethoxy-4-methoxyphenyl)-2-methylsulfonylethyl]-4-acetylami-
noisoindoline-1,3-dione and Compositions Thereof for Inhibiting
TNF-R Production and PDE4 Activity. U.S. Patent 7,427,638,
September 23, 2008. (b) The % ee was determined by chiral HPLC:
Ultron Chiral ES-OVM, 150 mm × 4.6 mm, 0.5 µm, 15:85 EtOH/20
mM KH2PO4 in water, 1 mL/min, 240 nm, 1S tR ) 9 min, 1R tR) 12
min.
(14) Muller, G. W.; Shire, M. G.; Wong, L. M.; Corral, L. G.; Patterson,
R. T.; Chen, Y.; Stirling, D. I. Thalidomide analogs and PDE4
inhibition. Bioorg. Med. Chem. Lett. 1998, 8, 2669–2674.
(15) (a) McKenna, J. M.; Muller, G. W. Medicinal Chemistry of PDE4
Inhibitors. In Cyclic Nucleotide Phosphodiesterases in Health and
Disease; CRC Press/Taylor & Francis: Boca Raton, FL, 2007; pp 667-
699. (b) Kodimuthali, A.; Jabaris, S. S. L.; Pal, M. Recent advances
on phosphodiesterase 4 inhibitors for the treatment of asthma and
chronic obstructive pulmonary disease. J. Med. Chem. 2008, 51, 5471–
5489.
(16) Hatzelmann, A.; Schudt, C. Anti-inflammatory and immunomodulatory
potential of the novel PDE4 inhibitor roflumilast in vitro. J. Pharmacol.
Exp. 2001, 297, 267–279.
(17) (a) Spond, J.; Billah, M. M.; Chapman, R. W.; Egan, R. W.; Hey,
J. A.; House, A.; Kreutner, W.; Minnicozzi, M. The role of neutrophils
in LPS-induced changes in pulmonary function in conscious rats. Pulm.
Pharmacol. Ther. 2004, 17, 133–140. (b) Spond, J.; Chapman, R.;
Fine, J.; Jones, H.; Kreutner, W.; Kung, T. T.; Minnicozzi, M.
Comparison of PDE 4 inhibitors, rolipram and SB 207499 (ariflo), in
a rat model of pulmonary neutrophilia. Pulm. Pharmacol. Ther. 2001,
14, 157–164.
(18) Barbaro, M. P. F.; Carpagnano, G. E.; Spanevello, A.; Cagnazzo,
M. G.; Barnes, P. J. Inflammation, oxidative stress and systemic effects
in mild chronic obstructive pulmonary disease. Int. J. Immunopathol.
Pharmacol 2007, 20, 753–763.
(19) Bundschuh, D. S.; Eltze, M.; Barsig, J.; Wollin, L.; Hatzelmann, A.;
Beume, R. In vivo efficacy in airway disease models of roflumilast, a
novel orally active PDE4 inhibitor. J. Pharmacol. Exp. Ther. 2001,
297, 280–290.
(20) Papp, K.; Bolduc, C.; Rohane, P.; Sutherland, D.; Jones, M.; Hu, A.;
Zeldis, J.; Thaci, D.; Kaufmann, R. A Phase 2 Study Demonstrating
the Efficacy and Safety of the Oral Therapy CC-10004 in Subjects
with Moderate-to-Severe Psoriasis. Presented at the 66th Annual AAD
Meeting, San Antonio, TX, Feb 1-5, 2008.
Supporting Information Available: Elemental analytical data
of 1, 1S, 1R, and 9-13. This material is available free of charge
via the Internet at http://pubs.acs.org.
References
(1) Conti, M.; Richter, W.; Mehats, C.; Livera, G.; Park, J.-Y.; Jin, C.
Cyclic AMP-specific PDE4 phosphodiesterases as critical components
of cyclic AMP signaling. J. Biol. Chem. 2003, 278, 5493–5496.
(2) Odingo, J. O. Inhibitors of PDE4: a review of recent patent literature.
Exp. Opin. Ther. Pat. 2005, 15, 773–787.
JM900210D