2-Amino-9-aryl-3-cyano-4-methyl-7-oxo-6,7,8,9-tetrahydropyrido[2′,3′:4,5]thieno[2,3-b]pyridine derivatives as selective progesterone receptor agonists

Article abstract of DOI:10.1016/j.bmcl.2009.07.100

High throughput screening of the corporate compound collection led to the identification of a novel series of 2-amino-9-aryl-3-cyano-4-methyl-7-oxo-6,7,8,9-tetrahydropyrido[2′,3′:4,5]thieno[2,3-b]pyridine derivatives as selective PR agonists. Initial SAR

Full text of DOI:10.1016/j.bmcl.2009.07.100

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4916–4919
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
2-Amino-9-aryl-3-cyano-4-methyl-7-oxo-6,7,8,9-tetrahydropyrido[20,30:4,5]thie-
no[2,3-b]pyridine derivatives as selective progesterone receptor agonists
a
b
b
b
Yonghui Wang a, , Chaya Duraiswami , Kevin P. Madauss , Thuy B. Tran , Shawn P. Williams ,
Su-Jun Deng b, Todd L. Graybill a, Marlys Hammond c, David G. Jones b, Eugene T. Grygielko c,
Jeffrey D. Bray c, Scott K. Thompson c
*
a R&D, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, PA 19426, USA
b R&D, GlaxoSmithKline, 5 Moore Drive, Research Triangle Park, NC 27709, USA
c R&D, GlaxoSmithKline, 709 Swedeland Road, King of Prussia, PA 19406, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
High throughput screening of the corporate compound collection led to the identification of a novel series of
2-amino-9-aryl-3-cyano-4-methyl-7-oxo-6,7,8,9-tetrahydropyrido[20,30:4,5]thieno[2,3-b]pyridine deriv-
atives as selective PR agonists. Initial SAR exploration leading to potent and selective agonists 9 and 11,
X-ray crystal structure of 9 bound to PR-LBD and preliminary developability data are described.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 5 February 2009
Revised 16 July 2009
Accepted 18 July 2009
Available online 23 July 2009
Keywords:
Progesterone
Progesterone receptor
Agonists
Selective agonists
Nuclear receptor
Endometriosis
The progesterone receptor (PR) is a member of the nuclear
receptor super-family and is a ligand dependent transcription fac-
tor.1 PR and its endogenous ligand, progesterone (P4, 1 in Fig. 1),
play an important role in uterine ovulation and implantation.2 Syn-
thetic PR ligands are commonly used in hormone replacement
therapy, oral contraception, and treatment of endometriosis and
uterine fibroids.3 Endometriosis is a painful disease characterized
by the growth of endometrial tissue outside the uterus. It is asso-
ciated with dysmenorrhea, chronic pelvic pain, fatigue and a host
of other symptoms. Progestins such as P4 and medroxyprogester-
one acetate (MPA, 2) act by opposing estrogen-stimulated endome-
trial tissue proliferation in the treatment of endometriosis. Some
side-effects associated with this therapy such as weight gain, break
through bleeding, and mood disturbances, are associated with the
full agonist activity or poor steroid receptor selectivity of the prog-
estins.3–5 There has been great interest in development of selective
non-steroidal PR agonists, and several small molecule non-steroi-
dal PR agonists have been reported in the literature (e.g., 3–5).6,7
Herein we describe discovery of a novel series of 2-amino-9-aryl-
3-cyano-4-methyl-7-oxo-6,7,8,9-tetrahydropyrido[20,30:4,5]thie-
no[2,3-b]pyridine derivatives as selective PR agonists.
O
O
Me
Me
OAc
Me
Me
H
H
H
H
H
H
O
O
Me
2
1
Me Me
O
NC
NC
N
N
Me
N
Me
S
N
N
H
S
H
4
3
Cl
NH2
O
Cl
N
Me
CN
Me
F
S
O
N
Me
Me
N
H
* Corresponding author at present address: Medicinal Chemistry, R&D China,
GlaxoSmithKline, 898 Halei Road, Shanghai 201203, China. Tel.: +1 86 21
61590761; fax: +1 86 21 61590730.
H
6
5
E-mail address: yonghui.2.wang@gsk.com (Y. Wang).
Figure 1. Steroidal (1, 2) and non-steroidal (3–6) PR agonists.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.07.100

Y. Wang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4916–4919
4917
High throughput screening (HTS) of the corporate compound col-
lection using a PR binding assay8 led to the identification of the tricy-
clic pyridine 6 as a hit with a PR pIC50 of 6.7. The compound was
subsequently found to be active in a PR CV-1 agonism assay9 and
in the more definitive T47D alkaline phosphatase cellular assay10
with a pEC50 of 6.2 and 7.1, respectively. Furthermore, compound
6 showed great selectivity for PR over AR (AR pIC50 <5.0 in the bind-
ing assay, AR pEC50 <5.0 in the CV-1 agonism assay).
In order to assess the tractability of the HTS hit, several close ana-
logs of 6 were synthesized via multi-component condensation of
malononitrile, acetone and N-methylmorpholinium tetrahydropyri-
dine-6-thiolates (7). The required tetrahydropyridine-6-thiolates 7
were generated in situ from arylaldehydes, cyanothioacetamide
and Meldrum’s acid in the presence of N-methylmorpholine
(NMM) (Scheme 1).11 These tricyclic pyridine analogs (8a–8i)12 were
tested in the PR binding and the CV-1 agonism assays to provide an
initial SAR.
As shown in Table 1, the nature and position of substituents on
the 9-aryl moiety of these tricyclic compounds had a significant ef-
fect on PR potency. The analogs with substituents on the ortho-po-
sition of phenyl (6, 8b–8d) increased binding affinities in the order
of 2-OMe < 2-Me < 2-F < 2-Cl. Introduction of a second halogen on
the 3-, 4-, 5-, or 6-position of 2-Cl-phenyl group attenuated its PR
binding affinity (8f–8i).
The synthetic route described in Scheme 1 provided analogs,
such as compound 6, as a 1:1 mixture of enantiomers owing to
the chiral center on 9-position of the tricyclic ring. Computational
docking studies13 were conducted to predict the more active iso-
mer and postulate plausible binding modes for both isomers with-
in PR. Docking studies predicted the S-isomer to be the more active
enantiomer as the lactam NH group of the S-isomer could form a
hydrogen bond with Asn-719 while the R-isomer could not due
to steric constraints. The two enantiomers of the compound 6 were
then separated by chiral HPLC and absolute stereochemistry of
each isomer was subsequently determined by VCD analysis.14 Both
S- and R-isomers, 9 and 10, were tested in the PR binding and the
CV-1 agonism assays. Consistent with the computational docking
predictions, the S-isomer (9) was found to be much more potent
(PR binding pIC50 = 6.8 and CV-1 pEC50 = 6.7) than the R-isomer
(10, PR binding pIC50 = 5.2, CV-1 pEC50 <5.0). Compound 9 also
showed good activity in the T47D alkaline phosphatase cellular as-
say with a pEC50 of 7.4.
NH2
NH2
Cl
Cl
N
N
CN
Me
CN
Me
S
S
O
O
N
N
H
H
10
9
To investigate the binding mode of the tricyclic pyridines, the
crystal structure of 9 bound to the PR-LBD (ligand binding domain)
was solved to a resolution of 2.24 Å.15 Figure 2 displays an overlay
of progesterone 1 and compound 9 bound to PR-LBD and shows
that compound 9 occupies the same binding pocket as PR’s natural
ligand, progesterone. The charged clamp region of Q725 and R766
overlays well with little notable movement. The 2-chlorophenyl
moiety pushes the angle of the ligand towards M909, which is a
residue associated with decreased agonism.16 The ligand slightly
moves M909, but the Ca carbon is not moved from the agonist po-
sition in progesterone. The distance from the amide nitrogen in
compound 9 and the oxygen on N719 is 2.7 Å, which confirmed
the docking predictions for the S-isomer.
Both the docked binding mode and crystal structure of 9 sug-
gested that the binding affinity might be enhanced by replacement
of the 2-amino group with a less polar group. To test this hypoth-
esis, both 2-chloro-analog 11 and 2-hydroxyl-analog 12 were syn-
thesized from 6 via Sandmeyer reaction (Scheme 2).17 Compound
11 demonstrated greater potency than compound 6 in both the
PR binding and the CV-1 assays (pIC50 = 7.3, pEC50 = 9.1, respec-
tively) while compound 12 found to be inactive in both assays. In
addition, compound 11 was found to be more potent than 6 in
T47D alkaline phosphatase cellular assay with a pEC50 of 8.6. Fur-
Ar
Ar
O
NH2
CN
S
S
NH2
+
a
O
7
CN
Ar
N
O
O
N
CN
Me
H
NMMH+
b
O
Me
Me
S
+
O
N
H
O
O
NC
Me
CN
8a-8i
Me
Scheme 1. Reaction conditions: (a) NMM, ethanol, rt then reflux; (b) ethanol,
reflux.
Table 1
PR binding, CV-1 agonism and T47D alkaline phosphatase data for the tricyclic pyridine compounds
X
2
Ar
N
9
CN
Me
S
O
N
H
a
a
a
a
Compds
Ar
X
PR binding pIC50
PR CV-1 pEC50
AR binding pIC50
PR T47D pEC50 (% P4)
8a
8b
6
8c
8d
8e
8f
8g
8h
8i
Ph
2-F–Ph
2-Cl–Ph
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
Cl
5.7
6.2
6.7
5.9
<5.0
<5.0
6.1
6.1
6.0
5.9
6.8
5.2
7.3
<5.0
<5.0
5.4
6.2
<5.0
<5.0
< 5.0
<5.0
<5.0
<5.0
5.4
<5.0
—
<5.0
<5.0
—
—
6.4 (47)
7.1 (80)
—
—
—
—
—
—
6.1 (13)
7.4 (83)
—
8.6 (116)
—
2-Me–Ph
2-OMe–Ph
3-OMe–Ph
2,3-Di-Cl–Ph
2,4-Di-Cl–Ph
2,5-Di-Cl–Ph
2-Cl-6-F–Ph
(S)-2-Cl–Ph
(R)-2-Cl–Ph
2-Cl–Ph
—
<5.0
<5.0
<5.0
<5.0
<5.0
<5.0
5.7
9
6.7
<5.0
9.1
10
11
12
2-Cl–Ph
OH
<5.0
<5.0
a
Values are the mean of P2 determinations.

4918
Y. Wang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4916–4919
Natalie L. Binstead for PK study, Raymond V. Merrihew and our
other colleagues in the Screening and Compound Profiling group
for performing the PR and AR binding and agonism assays.
References and notes
1. Mangelsdorf, D. J.; Thummel, C.; Beato, M.; Herrlich, P.; Schütz, G.; Umesono,
K.; Blumberg, B.; Kastner, P.; Mark, M.; Chambon, P.; Evans, R. M. Cell 1995, 83,
835.
2. Edgren, R. A.; Sturtevant, F. M. Am. J. Obstet. Gynecol. 1976, 125, 1029.
3. (a) Spitz, I. M. Expert Rev. Obstet. Gynecol. 2007, 2, 227; (b) Olive, D. L.; Pritts, E.
A. N. Eng. J. Med. 2001, 345, 266; (c) Sitruk-Ware, R. Maturitas 2004, 47, 277.
4. Chwalisz, K.; Garg, R.; Brenner, R.; Slayden, O.; Winkel, C.; Elger, W. Reprod. Biol.
Endocrinol. 2006, 4, S8.
5. Slayden, O. D.; Zelinski, M. B.; Chwalisz, K.; Hess-Stumpp, H.; Brenner, R. M.
Hum. Reprod. 2006, 21, 3081.
6. (a) Zhi, L.; Tegley, C. M.; Kallel, E. A.; Marschke, K. B.; Mais, D. E.; Gottardis, M.
M.; Jones, T. K. J. Med. Chem. 1998, 41, 291; (b) Edwards, J. P.; West, S. J.;
Marschke, K. B.; Mais, D. E.; Gottardis, M. M.; Jones, T. K. J. Med. Chem. 1998, 41,
303; (c) Zhi, L.; Tegley, C. M.; Marschke, K. B.; Mais, D. E.; Jones, T. K. J. Med.
Chem. 1999, 42, 1466; (d) Fensome, A.; Bender, R.; Chopra, R.; Cohen, J.; Collins,
M. A.; Hudak, V.; Malakian, K.; Lockhead, S.; Olland, A.; Svenson, K.; Terefenko,
E. A.; Unwalla, R. J.; Wilhelm, J. M.; Wolfrom, S.; Zhu, Y.; Zhang, Z.; Zhang, P.;
Winneker, R. C.; Wrobel, J. J. Med. Chem. 2005, 48, 5092; (e) Kurihara, K.; Shinei,
R.; Tanabe, K.; Tabata, Y.; Kurata, Y.; Hoshiko, S.; Okonogi, T. Bioorg. Med. Chem.
2006, 14, 4862; (f) Zhang, P.; Terefenko, E.; Kern, J.; Fensome, A.; Trybulski, E.;
Unwalla, R.; Wrobel, J.; Lockhead, S.; Zhu, Y.; Cohen, J.; LaCava, M.; Winneker, R.
C.; Zhang, Z. Bioorg. Med. Chem. 2007, 15, 6556; (g) Dols, P. P. M. A.; Folmer, B. J.
B.; Hamersma, H.; Kuil, C. W.; Lucas, H.; Ollero, L.; Rewinkel, J. B. M.; Hermkens,
P. H. H. Bioorg. Med. Chem. 2008, 16, 1461.
7. Allan, G.; Macielag, M. Expert Opin. Ther. Patents 1999, 9, 955.
8. ThePR binding assaywas performed accordingto themanufacturers protocol(PR
Competitor Assay Kit, Red—(Invitrogen—Product No. P2962)) with minor
amendments. Briefly, 40 nM PR-Ligand Binding Domain, 2 nM Fluormone PL
Red and 1 mM DTT were dissolved and mixed in Complete PR RED Buffer
supplemented with 2 mM CHAPS. Ten microlitres of the mix were dispensed to
each well of Greiner low volume plates, containing compounds at the required
concentration. The plates were spun for 1 min at 200 g, covered to protect the
reagents from light, and then incubated at room temperature for approximately
2 h. Plates were read on an Acquest using a 530–25 nm excitation and 580–
10 nm emission interference filter and a 561 nm Dichroic mirror.
Figure 2. Superposition of
backbone and key backbone residues are shown in the color of the ligand to which
they correspond.
9 (blue) and 1 (green) crystal structures. Enzyme
Cl
OH
Cl
Cl
N
N
a
CN
CN
Me
6
+
Me
S
S
O
N
O
N
H
H
12
11
Scheme 2. Reagents: (a) NaNO2, HCl, H2O.
ther, compound 11 demonstrated good selectivity for PR over AR
(AR pIC50 = 5.7 in the binding assay, pEC50 <5.0 in the CV-1 agonism
assay). Docking studies showed that the chloro group in 11 can
form good hydrophobic interactions with Leu-763, Val-760 and
Met-801 as well as an electrostatic interaction with the sulfur of
Met-801. The amino group in 6 can also form a weak hydrogen
bond with Met-801, however, this interaction is absent with the
hydroxyl analog 12 or its tautomer.
9. The CV-1 functional assay measures compound-mediated interaction of the PR-
B isoform with the MMTV luciferase reporter to calculate compound potency
and efficacy in BacMam transduced, progesterone-stimulated (4 nM) CV-1
cells. The antagonist response is expressed as a pIC50 (RU-486 pIC50 = 9.8, 100%
efficacy).
10. The T47D alkaline phosphatase PR agonist assay was performed according to
Ref.16. In short, 120
lL of a T47D cell suspension was seeded into a 96-well
plate and cells were allowed to attach to the plate overnight. On the next day,
the cells were treated with compound and incubated overnight. On the
following day, 100 lL of pNPP-SPAP was added and there were allowed to
stand in the dark for 2 h. Optical density was then measured at a wavelength of
405 nm on a plate reader.
Compound 6 was evaluated in general selectivity, CYP450, hERG,
and in vitro and in vivo PK studies. Compound 6 demonstrated high
selectivity in a CEREP screen of 50 receptors, transporters and ion-
channels (<10% of inhibition at 1 lM against all 50 targets in the pa-
11. (a) Nesterov, V. N.; Krivokolysko, S. G.; Dyachenko, V. D.; Dotsenko, V. V.;
Litvinov, V. P. Russ. Chem. Bull. 1997, 46, 990; (b) Dotsenko, V. V.; Krivokolysko,
S. G.; Litvinov, V. P.; Chernega, A. N. Russ. Chem. Bull. 2002, 51, 362; (c)
Dotsenko, V. V.; Krivokolysko, S. G.; Chernega, A. N.; Litvinov, V. P. Russ. Chem.
Bull. 2003, 52, 969.
12. All tricyclic pyridines were prepared and tested as 1:1 mixtures of two
enantiomers unless stated otherwise.
13. The co-crystal structure of progesterone in PR (PDB code 1A28) was used as the
starting point for the initial docking calculations using the program, Flo+,
version 0203. Residues within a 15 Å sphere of progesterone in the binding site
nel). In a CYP450 screen, compound 6 showed no or little inhibition
for 4 major P450 isozymes (pIC50 <5.0 for 1A2, 2C19, 2D6, 3A4) and
moderate inhibition for 2C9 (pIC50 = 5.9). Compound 6 did not show
activity in a hERG binding assay. In vitro human and rat liver micro-
some stability studies revealed moderate intrinsic clearance for
compound 6 (human: 0.019 mL/min/mg liver; rat 0.083 mL/min/
mg liver). Compound 6 also demonstrated moderate clearance and
oral bioavailability (Clb = 24 mL/min/kg, F = 36%, T1/2 = 1.1 h,
Vdss = 2.5 L/kg) in in vivo mouse PK studies (0.5 mg/kg iv, 1.0 mg/
kg po). These PR potency, selectivity and initial developability data
suggest that this series constitutes a reasonable starting point for
further medicinal chemistry investigation.
In summary, we have identified a novel series of tricyclic pyri-
dines as non-steroidal small molecular PR agonists. Initial SAR
exploration led to potent and selective PR agonists such as 9 and
11. The crystal structure of 9 bound to the PR-LBD was solved
and provided direction for subsequent lead optimization.
were retained. The mcdock algorithm, which relies on
a Monte Carlo
perturbation/fast search/energy minimization algorithm was used. Only
those residues that were known to be flexible from other crystal structures
were made flexible during the energy minimization step. Two thousand steps
of perturbation were performed and the 25 top-ranked poses were retained.
Visual inspection of the interactions made by the ligand within the active site
and the relative strain energies of the ligand calculated using Flo+ in each pose
were used to determine the best docked pose. Also see: McMartin, C.; Bohacek,
R. S. J. Comput. Aided Mol. Des. 1997, 11, 333.
14. (a) The two enantiomers of the compound 6 were separated by chiral HPLC via
the following (SFC) conditions: 20
lm, 10 mm AD column, 10 mL/min total
flow, 35% MeOH/CHCl3 (4:1) and 65% CO2, 140 bar, 40 °C, UV @ 280 nm; (b)
Stereochemistry of 9 and 10 was assigned by Vibrational Circular Dichroism
(VCD) analysis of both enantiomers.
15. Thecrystalof PRLBDboundto compound 9hada datasetcollectedat 17-IDof the
Acknowledgements
APS, and were solved using the programs HKL2000, CCP4 (MOLREP), COOT, and REFMAC
.
16. Madauss, K. P.; Grygielko, E. T.; Deng, S.-J.; Sulpizio, A. C.; Stanley, T. B.; Wu, C.;
Short, S. A.; Thompson, S. K.; Stewart, E. L.; Laping, N. J.; Williams, S. P.; Bray, J.
D. Mol. Endocrinol. 2007, 21, 1066.
The authors would like to thank Minghui Wang for NMR, Eric S.
Wentz for chiral HPLC separation, Doug J. Minick for VCD analysis,

Y. Wang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4916–4919
4919
17. Synthesis of 11 and 12: A solution of 375 mg (5.43 mmol) of NaNO2 in water
(5 mL) was slowly added to a suspension of 200 mg (0.54 mmol) of 6 in concd
HCl (10 mL) with stirring over a period of 1 h. The mixture was stirred at rt for
another 12 h and then filtered. The filtered solid was washed with water and
dried under vacuum. The HPLC purification of the crude products yielded
52 mg of 11 and 36 mg of 12. Compound 11: 1H NMR (400 MHz, DMSO-d6) d
ppm 2.71 (s, 3H) 3.39 (dd, J = 16.67, 8.34 Hz, 2H) 4.94 (dd, J = 8.34, 1.77 Hz, 1H)
6.70 (dd, J = 7.71, 1.64 Hz, 1H) 7.11–7.24 (m, 1H) 7.29 (td, J = 7.64, 1.64 Hz, 1H)
7.55 (dd, J = 7.83, 1.26 Hz, 1H) 11.58 (s, 1H); MS m/e 388.1 (MH+).