Novel tetrahydro-β-carboline-1-carboxylic acids as inhibitors of mitogen activated protein kinase-activated protein kinase 2 (MK-2)

Article abstract of DOI:10.1016/j.bmcl.2007.05.070

A structure-activity relationship study was conducted on a series of tetrahydro-β-carboline-1-carboxylic acid analogs in order to identify the key functionality responsible for activity against the mitogen-activated protein kinase-activated protein kinase 2 enzyme (MK-2). The compounds were further evaluated for their ability to inhibit TNFα production in U937 cells and in vivo. These compounds represent a novel structural class of compounds capable of inhibiting MK-2 with remarkable selectivity.

Full text of DOI:10.1016/j.bmcl.2007.05.070

Bioorganic & Medicinal Chemistry Letters 17 (2007) 4657–4663
Novel tetrahydro-b-carboline-1-carboxylic acids as inhibitors
of mitogen activated protein kinase-activated
protein kinase 2 (MK-2)
John I. Trujillo,* Marvin J. Meyers,* David R. Anderson, Shridhar Hegde,
Matthew W. Mahoney, William F. Vernier, Ingrid P. Buchler, Kun K. Wu,
Syaluan Yang, Susan J. Hartmann and David B. Reitz
Department of Medicinal Chemistry, Pfizer Global Research and Development, Chesterfield, MO 63017, USA
Received 16 April 2007; revised 21 May 2007; accepted 22 May 2007
Available online 25 May 2007
Abstract—A structure–activity relationship study was conducted on a series of tetrahydro-b-carboline-1-carboxylic acid analogs in
order to identify the key functionality responsible for activity against the mitogen-activated protein kinase-activated protein kinase 2
enzyme (MK-2). The compounds were further evaluated for their ability to inhibit TNFa production in U937 cells and in vivo.
These compounds represent a novel structural class of compounds capable of inhibiting MK-2 with remarkable selectivity.
Ó 2007 Elsevier Ltd. All rights reserved.
The proinflammatory cytokine TNF-a was originally
described as a product of monocytes that induced tumor
lysis1 and since then has been implicated in several
inflammatory diseases in humans.2 The development
of anti-TNFa biological therapies has been described
and been shown to be effective in diseases such as rheu-
matoid arthritis and psoriasis.3 More recently, many
other biological targets have been identified for the inhi-
bition of TNF-a production. The most notable of the
targets is the kinase p38 MAPK.4,5 Several publications
have demonstrated the effect of p38 inhibitors in in vitro
and in vivo models, as well as demonstrating efficacy in
rheumatoid arthritis (RA) patients in a clinical study.6
MK-2 knockout mice with LPS resulted in only a small in-
crease of serum TNFa (10–20%) of the wild type con-
trol.8a Additionally, the MK-2 knockout mice were
observed to be resistant to collagen induced arthritis
(CIA),7b a model well established for the study of (RA).
Furthermore, while MK-2 knockout mice are healthy
and have a normal phenotype, genetic knockout of the
p38 gene is embryonic lethal, suggesting an improved
safety profile for MK-2 inhibition relative to p38. These
observations taken together lend strong support for
MK-2 as an attractive target for treatment of TNF-a med-
iated diseases and rheumatoid arthritis (RA).
Efforts from our laboratories have recently been
reported for a series of aminocyanopyridines and a ser-
ies of pyrrolopyridines as inhibitors of MK-2.9 Through
our compound screening effort, we also identified com-
mercially available b-carboline derivative 1 as a modest
inhibitor of MK-2 (Fig. 1).10 The compound exhibited
The mitogen-activated protein kinase-activated protein
kinase 2 (MK-2) has only recently received a significant
degree of interest for its potential as a target for treatment
of RA.7 MK-2 is a direct substrate of the p38a/b kinases
and has been linked to TNF-a, IL-6, and IFN-c expres-
sion levels.8 A genetic knockout of the MK-2 enzyme in
mice has been described8a and shown to be resistant to dis-
ease in models of arthritis.7b For example, treatment of
Keywords: MAPKAP kinase 2; MK-2; TNF-a; Inflammation; b-Car-
boline-1; Rheumatoid arthritis.
*
Corresponding authors. Tel.: +1 636 244 1937; e-mail:
john.i.trujillo@pfizer.com
Figure 1. Structure of MK-2 inhibitor and in vitro biological activity.
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.05.070

4658
J. I. Trujillo et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4657–4663
an MK-2 IC50 value11 of 2.5 lM and was identified to be
an ATP competitive inhibitor. This structure was of par-
ticular interest to us given its low molecular weight and
unusually good selectivity versus a number of related
kinases12 (see Fig. 1). In order to elucidate the essential
features required for the activity observed and to further
improve the potency of compound 1, an investigation of
the structure–activity relationship between 1 and the
MK-2 enzyme was initiated. It is important to note that
no X-ray crystal structure of the MK-2 enzyme existed
at the time of this work.13 In this report, we describe
the synthesis and key structure–activity relationships
identified during the investigation of this novel series
of tetrahydro-b-caroline-1-carboxylic acids as selective
inhibitors of MK-2. It is also important to note that
SAR for this class of b-carboline carboxylic acids was
conducted on the racemates as the a-carbon was ob-
served to be readily epimerizable.
Scheme 1. Synthesis of compounds 2–11, and 15, 17. Reagents and
conditions: (a) Glyoxylic acid, pH 3–4, H2O/KOH, rt, 12 h; (b)
Me2NCH@CHNO2, TFA, rt; (c) LAH, THF, rt then reflux.
Starting with the A-ring, an investigation of the role of
the methoxy group was conducted (see Table 1). The
syntheses of these analogs are presented in Schemes 1
and 2.14 The position of the methoxy group was found
to be critical as the 6-position was optimal by more than
ten-fold (1, 7, 15, 17). Addition of a second group adja-
cent to the 6-methoxy group was not tolerated (32, 35),
presumably due to twisting of the methoxy group out of
the plane. Substitution of the 6-methoxy for other func-
tionalities was subsequently investigated (8–10, 20, 23,
29). Hydroxy analog 20 and methyl ester 29 proved to
be well tolerated, while other variations, including dele-
tion, resulted in a 10- to 100-fold loss in potency against
MK-2 (8–11, 23, 29). Extension of the methoxy group to
benzyloxy was tolerated to some extent (19), but ace-
tophenone derivative 26 was essentially equipotent to
methoxy, demonstrating the tolerability for larger
groups extending from the A-ring.
Having established the importance of the 6-methoxy
group in the A-ring for potency, we turned our attention
to the C-ring. Oxidation of the tetrahydropyridine to
pyridine was investigated but proved to be incompatible
with MK-2 activity (Scheme 2; 37 and 38). The lack of
potency of compound 38 in particular suggests a
requirement of the acid functionality to exist out of
plane with respect to the ring system. The lack of po-
tency may also be a result of a reduction in basicity of
the nitrogen in the C-ring (secondary amine to pyridine)
(Scheme 3).
Examination of the role of the indole and amino acid
functionalities was also conducted (Table 2). The syn-
thesis of compounds 36–63 is shown in Schemes 4
and 5. The presence of the nitrogen was found to be
critical for potency as demonstrated by carbon analog
65 and oxygen analog 61. While the nitrogen itself was
required for activity, the hydrogen was not, as demon-
strated by N-alkylation with small alkyl groups such as
methyl or ethyl (40 and 49). Larger groups such as iso-
propyl and benzyl, however, resulted in significant
losses in potency (50 and 51), suggesting a sensitive ste-
ric environment.
Table 1. IC50 values for b-carboline derivatives
Compound
Substitution
MK-2 IC50 (lM)
Modification of the carboxylic acid moiety to other
functional groups was also investigated. Conversion of
the carboxylic acid to a methyl ester (36), primary amide
(43), primary alcohol (42) or amine (56) all resulted in a
dramatic loss in potency. Functional groups capable of
mimicking the acid, such as a hydroxamic acid 44 or tet-
razole 58, did not prove to be suitable replacements for
the acid. Addition of a methyl group in the a-position
(54) or incorporation of a methylene spacer between
the C-ring and the carboxylic acid (55) was not toler-
ated. The intolerance for any changes to the carboxylic
acid suggests a key ionic interaction between the inhibi-
tor and MK-2 is critical for binding affinity in this series.
Methylation of the indole NH also proved to be detri-
mental to activity (52), suggesting a potential interaction
1
15
7
6-OMe
5-OMe
2.5
42
7-OMe
8-OMe
>200
>200
53
17
32
35
20
29
23
11
8
6-OMe, 7-OH
6-OMe, 5-Me
6-OH
>200
4.5
6.6
170
87
6-CO2Me
6-OAc
6-H
6-F
6-Cl
26
19
9
10
19
26
6-CONH2
6-OCH2Ph
6-OCH2C(O)Ph
28
12
1.1

J. I. Trujillo et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4657–4663
4659
Scheme 2. Synthesis of compounds 20, 23, 26, 29, 32, and 35. Reagents and conditions: (a) Glyoxylic acid, pH 3–4, H2O/KOH, rt, 12 h; rt; (b) 40 psi
H2, 10% Pd/C, MeOH, rt; (c) K2CO3 then Boc2O, THF, DMAP, rt, 2 h; (d) 40 psi H2, 10% Pd/C, MeOH, rt; (e) Ac2O, pyridine, CH2Cl2, 0 °C to rt,
12 h; (f) 2.0 N HCl in Et2O, rt, 3 h; (g) Boc2O, K2CO3, H2O, rt, 14 h; (h) PhC(O)CH2Br, K2CO3, MEK, 80 °C, 14 h; (i) 2.0 N HCl in Et2O, CH2Cl2,
rt, 14 h; (j) Me2NCH@CHNO2, TFA, rt; (k) NaBH4, THF:MeOH, rt; (l) 10% Pd/C, NH4CO2H, MeOH, reflux, 14, h; (m) 85% H2NNHÃ2H2O, 75 °C,
14 h; (n) glyoxylic acid, H2O, pH 4, 80 °C, 30 min.
Scheme 3. Synthesis of compounds 37 and 38. Reagents and conditions: (a) S, m-xylene, reflux, 4 h; (b) LiOH*H2O, THF:H2O, rt, 4 h.
of the indole N–H with the enzyme, a limited steric envi-
ronment, or distortion of the orientation of the carbox-
ylic acid.
ethyl, in the 3-position (88, 89). Further exploration of
the 3-position demonstrated tolerance for only a select
group of small functional groups capable of H-bonding
(76, 82, 83) (Table 4).
In order to further probe the tolerance of MK-2 to sub-
stitution of the b-carboline C-ring, analogs were pre-
pared to probe the 3- and 4-positions (Table 3 and
Scheme 6). The enzyme exhibited tolerance of only small
substituents such as methyl and ethyl in the 4-position
(70–72), but less tolerant of alkyl substituents, even
Having established the tolerance of 2- and 4-positions in
the C-ring for alkylation with small groups such as
methyl or ethyl, a series of C-ring bridged analogs were
prepared (see Table 5 and Scheme 7). While the three-
carbon bridge between N-2 and C-4 was tolerated

4660
J. I. Trujillo et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4657–4663
Scheme 4. Synthesis of compounds 36, 39–44, 49–52, 54–56, and 58. Reagents and conditions: (a) MeOH/HCl, rt; (b) Na2CO3, Mel, THF:H2O, rt;
(c) KOH(aq), THF:MeOH; (d) for 46: AcCl, Et3N, CH2Cl2, rt; for 47: acetone, MeOH, rt; for 48: PhCHO, MeOH, reflux4, MeOH; (e) for 446: LAH,
THF, rt; for 47 and 48: NaBH4, MeOH, 0 °C; (f) for 49: glyoxylic acid, TFA, CF3CN, rt; for 51 and 52: glyoxylic acid, H2O, rt; (g) acetone,
Na(OAc)3BH, CH2Cl2, rt; (h) NaOH, EtOH; (i) Boc2O, THF, rt; (j) NaH, Mel, DMF, rt; (k) TFA, CH2Cl2, anisole, rt; (l) glyoxylic acid, H2O, rt; (m)
LAH, Et2O, 0 °C to rt; (n) conc. NH4OH(aq); (o) LAH, THF, rt; (p) for 53: (MeO)2CH2CO2Me, CH3CN, TFA, 80 °C; for 54: Me(C@O)CO2H,
MeOH:H2O, 50 °C; (q) LiOH, THF:H2O, rt; (r) 50% NH2OH(aq), THF:H2O, K2CO3, rt; (s) TFAA, Et3CN, CH2Cl2, 0 °C; (t) Me3SnN3, dioxane,
reflux, 5 h; (u) K2CO3, MeOH/H2O, rt. See above mentioned references for further information.
(92), the two-carbon bridge resulted in a 6-fold enhance-
ment in potency, representing the first b-carboline we
identified to achieve submicromolar potency against
MK-2. Furthermore, compound 92 exhibited remark-
able selectivity against a number of structurally related
kinases (IC50, lM: MSK1 > 200, MNK1 > 200,
Scheme 5. Synthesis of compounds 61 and 65. Reagents and conditions: (a) HCOCO2Et, pTsOH, rt to 80 °C, 20 h; (b) MeOH:H2O, KOH, rt, 2 h; (c)
63, EtOH, reflux, 24 h; (d) LiOH, THF:H2O:MeOH, 4 h.

J. I. Trujillo et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4657–4663
4661
Scheme 6. Synthesis of compounds 70–72, 76, 77, 82, 83, 88, and 89. Reagents and conditions: (a) for 67: MeMgBr (3.0 M in Et2O), THF, À50 °C,
2 h then 0 °C, 1 h; for 68: EtMgBr, THF, À78 °C, 3 h; for 69: PhCH2MgCl (2.0 M in Et2O), THF, À50 °C, 2 h then 0 °C, 1 h; (b) 10% Pd/C,
HCO2NH4, MeOH, 80 °C; (c) glyoxylic acid, H2O, pH 4–5, rt, 3 h; (d) for 74: BrCH2C(@NOH)CO2Et, Na2CO3, CH2Cl2, rt, 24 h; for 75: BrCH2C(–
NOH)CF3, Na2CO3, tBuOMe, rt, 24 h; (e) LAH, THF, À50 °C to rt; (f) glyoxylic acid, H2O, pH 4–5, rt, 24 h; (g) NaOMe, Me2SO4, MeNO2, MeOH,
rt, 5 h; (h) for 79: Bn2NH, H2CO, H2O:MeOH, rt then reflux, 3 h; for 80: Me2NH, H2CO, H2O, MeOH, rt; (i) for 81: LAH, THF, 0 °C then reflux;
for 83: HCO2NH4, 10% Pd/C, MeOH, reflux; (j) glyoxylic acid, MeOH/H2O, pH 3–4, 55 °C; (k) HCO2NH4, 10% Pd/C, MeOH, reflux; (l) for 84:
nPrNO2, NaOMe, Me2SO4, MeOH, rt; for 86: EtNO2, NaOMe, Me2SO4, MeOH, rt.
Scheme 7. Synthesis of compounds 92 and 93 and 94–98. Reagents and conditions: (a) For 92: glyoxylic acid, MeOH:H2O, 60 °C, 10 h; for 93: same
as 92 except 45 h; (b) ROH, EDAC, DMAP, rt, 5 h.
JNK2 > 200, p38a > 100, IKK2 > 200), even close
homologs MK-3 and PRAK (IC50, lM: 90 and 24,
respectively).
The potency and selectivity of compound 92 prompted
further investigation in cellular models, specifically
the inhibition of TNFa production in U937 cells.

4662
J. I. Trujillo et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4657–4663
Table 5. IC50 values for b-carboline derivatives 92–97
Table 2. IC50 values for b-carboline derivatives
Compound R1
CO2H
CO2H
R2
X
R3 MK-2 IC50 (lM)
Compound
R
MK-2 IC50 (lM) U937 IC50 (lM)
1
65
61
40
49
50
51
36
43
42
56
44
58
54
55
52
H
H
H
H
H
H
H
H
H
H
H
H
H
NH
CH2
O
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
2.5
92
94
95
96
97
98
H
Et
0.29
29.3
7.09
10.1
3.27
13.0
80.8
5.6
>200
>200
1.0
CO2H
CO2H
Bn
n-Pr
1.7
0.83
NMe
NEt
NiPr
NBn
NH
NH
NH
NH
NH
H
CO2H
CO2H
CO2H
5.8
170
H2C=CHCH2
PhCH(Me)
14.4
10.3
130
47
CO2Me
CONH2
CH2OH
CH2NH2
CONHOH
Tetrazole
CO2H
>200
>200
>200
88
Indeed, when ester prodrugs of 92 were synthesized and
evaluated in the U937 cell assay, a dramatic enhance-
ment in cell potency was observed, suggesting an intra-
cellular conversion of the ester to the active acid. The
most effective compound in the U937 cellular assay
was identified to be compound 96, an n-propyl ester
(see Table 5). The compound was further evaluated in
the rat LPS model (40 mpk, IP) and was shown to inhi-
bit TNF-a production (84%, compound administered
1 h prior to LPS stimulation).
130
>200
>200
Me NH
CH2CO2H
CO2H
H
H
NH
NH
Me >200
Table 3. IC50 values for b-carboline derivatives
In conclusion, a group of b-carbolines capable of inhib-
iting MK-2 has been discovered, and the SAR of the ser-
ies has been described. Key SAR observations include
(1) the importance of an ether functionality at the 6-po-
sition of the A-ring, (2) the critical sensitivity of the car-
boxylic acid, and (3) the enhancement in binding affinity
observed by bridging the C-ring nitrogen with the 4-po-
sition. Compound 92 represents remarkable selectivity
for MK-2 over a number of closely related kinases,
including MK-3 and PRAK. While the carboxylic acids
were inactive in U937 cells, an ester prodrug of 92, com-
pound 96, was found to be effective in modulation of
TNFa production in both U937 cellular assays and in
the rat LPS model.
Compound
R1
R2
MK-2 IC50 (lM)
70
71
72
88
89
77
76
82
83
Me
Et
Bn
H
H
2.2
2.2
H
H
Me
16
7.1
H
Et
CF3
64
H
>200
3.0
5.9
H
CH2OH
CH2NH2
CH2NMe2
H
H
>200
Acknowledgments
Unfortunately, compound 92 did not possess the desired
cellular activity. It was thought that the zwitterionic
character of the amino and carboxylic acid moieties
was limiting the cellular permeability of this compound.
We thank Robert P. Compton, Jeffrey L. Hirsch, Heidi
M. Morgan, Matt J. Saabye, and John F. Schindler for
enzyme support; Heidi R. Hope, Shelia C. Short, and
Jian Zhang for cellular assays; and Gary D. Anderson,
Stephen J. Mnich, Mark A. Thiede, and Elizabeth G.
Webb for in vivo model support.
Table 4. IC50 values for b-carboline derivatives 92 and 93
References and notes
1. Huizinga, T. W. J.; Breedveld, F. C. Rheum. Arthritis
2000, 501.
2. Camussi, G.; Lupia, E. Drugs 1998, 55, 613.
3. (a) Richard-Miceli, C.; Dougados, M. BioDrugs 2001, 15,
251; (b) Braun, J.; Sieper, J. BioDrugs 2003, 17, 187; (c)
Olsen, N. J.; Stein, C. M. N. Eng. J. Med. 2004, 350, 2167.
4. Chen, Z.; Gibson, T. B.; Robinson, F.; Silvestro, L.;
Pearson, G.; Xu, B.; Wright, A.; Vanderbilt, C.; Cobb, M.
Chem. Rev. 2001, 101, 2449.
Compound
n
MK-2 IC50
(lM)
MK-3 IC50
(lM)
PRAK IC50
(lM)
92
93
1
2
0.29
5.1
90
160
24
25

J. I. Trujillo et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4657–4663
4663
5. Pargellis,C.;Regan,J.Curr.Opin.Invest.Drugs2003,4,566.
6. (a) Lee, M. R.; Dominguez, C. Curr. Med. Chem. 2005, 12,
2979; (b) Haddad, J. Curr. Opin. Invest. Drugs 2001, 2,
1070, and references cited therein.
7. (a) Gaestel, M. Nat. Rev. Mol. Cell Biol. 2006, 7, 120; (b)
Hegen, M.; Gaestel, M.; Nickerson-Nutter, C. L.; Lin,
L.-L.; Telliez, J.-B. J. Immunol. 2006, 177, 1913.
8. (a) Kotlyarov, A.; Neininger, A.; Schubert, C.; Eckert,
R.; Birchmeier, C.; Volk, H.; Gaestel, M. Nat. Cell Biol.
1999, 1, 94; (b) Neininger, A.; Kontoyiannis, D.;
Kotylarov, A.; Winzen, R.; Eckert, R.; Volk, H. D.;
Holtmann, H.; Kollias, G.; Gaestel, M. J. Biol. Chem.
2002, 277, 3065.
9. (a) Anderson, D. R.; Hegde, S.; Reinhard, E.; Gomez, L.;
Vernier, W. F.; Lee, L. F.; Liu, S.; Sambandam, A.;
Snider, P.; Masih, L. Bioorg. Med. Chem. Lett. 2005, 15,
1587; (b) Anderson, D. R.; Meyers, M. J.; Vernier, W. F.;
Mahoney, M. W.; Kurumbail, R. G.; Caspers, N.; Poda,
G. I.; Schindler, J. F.; Reitz, D. B.; Mourey, R. J. J. Med.
Chem. 2007, 50, 2647.
10. Meyers, M. J.; Trujillo, J. I.; Vernier, W. F.; Anderson, D.
R.; Reitz, D. B.; Buchler, I. P.; Hegde, S. G.; Mahoney,
M. W.; Wu, K. K. WO 2005009370 A2, 2005.
13. For a report on a MAPKAP kinase 2 structure, see:
Kurumbail, R. G.; Pawlitz, J. P.; Stegeman, R. A.;
Stallings, W. C.; Shieh, H. S.; Mourey, R. M.; Bolten, S.
L.; Broadus, R. M., WO2003076333 A2, 2003 and Ref. 9b.
14. For a review of the Pictet–Spengler reaction, see: Cox, Eric
D.; Cook, James M. Chem. Rev. 1995, 95, 1797.
15. Rinehart, K. L.; Kobayashi, J.; Harbour, G. C.; Gilmore,
J.; Mascal, M.; Holt, T. G.; Shield, L. S.; Lafargue, F.
J. Am. Chem. Soc. 1987, 109, 3378.
16. T, Kurihara; Y, Sakamoto; T, Kimura; H, Ohishi; S,
Harusawa; R, Yoneda; T, Suzutani; M, Azuma Chem.
Pharm. Bull. 1996, 44, 900.
17. Wilkening, D.; Mundy, B. P. Synthetic Commun. 1984, 14,
227.
18. Julia, M.; Lenzi, J. Bull. Soc. Chim. Fr. 1962, 2262.
19. Gilchrist, T. L.; Roberts, T. G. J. Chem. Soc., Perkin
Trans. 1 1983, 1283.
20. (a) Bergman, J. Tetrahedron 1971, 27, 1167; (b) Hermkens,
P. H. H.; van Maarseveen, J. H.; Cobben, P. L. H. M.;
Ottenheijm, H. C. J.; Kruse, C. G.; Scheeren, H. W.
Tetrahedron 1990, 46, 833.
21. Macor, J. E.; Blank, D. H.; Ryan, K.; Post, R. J. Synthesis
1997, 443.
11. For description of MK-2 IC50 assay determinations and
modulation of TNFa production in both U937 cellular
assays and in the rat LPS model, see Ref. 9a.
22. (a) Gremmen, C.; Burm, B. E. A.; Wanner, M. J.;
Koomen, G.-J. Tetrahedron Lett. 1998, 39, 1441; (b)
Gharagozloo, P.; Miyauchi, M.; Birdsall, N. J. M.
Tetrahedron 1996, 52, 10185.
12. For description of kinase selectivity assays, see Ref. 9b.