Synthesis of highly substituted dibenzo[b,f]azocines and their evaluation as protein kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2006.07.078

Synthetic routes towards highly substituted eight membered ring heterocycles fused to aryl rings such as the dibenzo[b,f]azocine system are still lacking. Herein, we present a convenient convergent synthetic route towards this heterocyclic class of compou

Full text of DOI:10.1016/j.bmcl.2006.07.078

Bioorganic & Medicinal Chemistry Letters 16 (2006) 5360–5363
Synthesis of highly substituted dibenzo[b,f]azocines and their
evaluation as protein kinase inhibitors
Leggy A. Arnold and R. Kiplin Guy*
St. Jude Children’s Research Hospital, Department of Chemical Biology and Therapeutics, 332 N. Lauderdale St.,
Memphis, TN 38105-2794, USA
Received 13 June 2006; revised 21 July 2006; accepted 24 July 2006
Available online 4 August 2006
Abstract—Synthetic routes towards highly substituted eight membered ring heterocycles fused to aryl rings such as the dib-
enzo[b,f]azocine system are still lacking. Herein, we present a convenient convergent synthetic route towards this heterocyclic class
of compounds with possible variations at positions 4, 7, and 11. One member of a library of dibenzo[b,f]azocines with different sub-
stituents at position 11 was identified to inhibit protein kinase A activity (IC50 = 122 lM) but not protein kinase C.
Ó 2006 Elsevier Ltd. All rights reserved.
Medium-size heterocycles fused to aryl rings are found
in many drugs and preclinical leads. An important scaf-
fold is the dibenzo[b,f]azepine present in psychotropic
drugs like Imipramine, Desipramine and Bonnecore.1
While the mechanism of action of this class of com-
pounds has been associated with protein kinase activity,
especially cAMP-dependent kinase (PKA) and
phospholipid-dependent protein kinase C (PKC),2 little
Figure 1. Protein kinase inhibitors H-89 and H-7.
is known about their mechanism of influence on the sig-
nal transduction mediated by these kinases.3,4 Recently,
we described a novel strategy to synthesize these hetero-
cycles along with the synthesis of dibenzo[b,f]azocines.5
In order to explore their potential activity on PKA
and PKC, we produced and tested a focused library uti-
lizing this route. We used isoquinolinesulfonyl based
protein kinase inhibitors H-896 and H-77 as controls
Scheme 1. Synthesis of 4. Reagents and conditions: (i) a—Mg (2
for the activity studies as they have been exhaustively
studied and are known to inhibit the ATP binding site
of the PKs (Fig. 1).8 Closely related heterocycles have
also been applied as inhibitors of neuronal Na+ chan-
nels,9 ligands for human EP1 prostanoid receptor10
and arginine vasopressin antagonists.11
equiv), 1,2-dibromoethane (cat.), THF, 60 °C, 12 h; b—ethyl pyruvate
(1 equiv), THF, 0–25 °C, 3 h; (ii) P2O5 (2 equiv), benzene, 80 °C, 8 h;
(iii) N-bromosuccinimide (1.5 equiv), benzoyl peroxide (cat.), CCl4,
80 °C, 18 h.
Commercially available 2,6-dichlorotoluene was con-
verted into the corresponding mono Grignard reagent
in the presence of magnesium metal and 1,2-dibromo-
ethane as an activating agent. Addition of ethyl pyru-
vate at 0 °C gave the tertiary alcohol 2 in 60% yield.
The dehydration of 2 was carried out using P2O5 in ben-
zene giving analytically pure 3 in good yield. Subsequent
benzylic bromination of 3 with NBS gave 4 in 80% yield.
The dibenzo[b,f]azocine scaffold was synthesized using
precursor 4 and 8 (Schemes 1 and 2).
Keywords: Dibenzo[b,f]azocine; Kinase inhibitor.
*
The synthesis of 8 was realized in three steps with an
overall yield of 41% (Scheme 2). This synthesis includes
Corresponding author. Tel.: +1 901 495 5714; fax: +1 901 495
5715; e-mail: kip.guy@stjude.org
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.07.078

L. A. Arnold, R. Kiplin Guy / Bioorg. Med. Chem. Lett. 16 (2006) 5360–5363
5361
amine proceeded smoothly at 90 °C.13 The ring closure
product 10 could be isolated in 80% yield after 6 h.
The introduction of a vinyl substituent at position 7
was realized using a Stille coupling employing a new cat-
alytic system reported by Fu et al.14 Under these condi-
tions compound 10 reacted with tributyl(vinyl)tin giving
compound 11 in 73% yield after 16 h at 100 °C. Reduc-
tion with diisobutylaluminium hydride followed by an
acid work-up resulted in the formation of allylic alcohol
12. Selective hydrogenation using Pt/C at 35 psi (H2)
gave compound 13 in 95% yield.
Scheme 2. Synthesis of 8. Reagents and conditions: (i) Na2CO3 (5
equiv), Bu4NBr (1 equiv), Pd(PPh3)4 (0.015 equiv), water/toluene (1:2),
100 °C, 18 h; (ii) 10% Pt/C (0.02 equiv of Pt), H2 (36 psi), ethyl acetate,
rt, 14 h; (iii) a—sodium hexamethyldisilazide (2.2 equiv), THF, rt,
15 min; b—Boc-anhydride (1.1 equiv), rt, 3 h.
Compound 13 was used as starting material to synthe-
size a library of dibenzo[b,f]azocines. The allylic alcohol
underwent a Mitsunobu reaction with different phenols
in the presence of di-tert-butylazodicarboxylate (Scheme
4).15 The results are summarized in Table 1.
the introduction of a 2-phenylvinyl substituent under
Suzuki coupling reaction conditions. Because of the
great variety of commercially available boronic acid
derivatives this reaction is well suited for the introduc-
tion of various substituents. Unprotected 2,6-dibro-
moaniline and trans-2-phenylvinylboronic acid reacted
in a two-phase system (water/toluene) using the phase-
transfer catalyst Bu4NBr. In the presence of 1.5 mol%
The phenols used were mono-substituted bearing func-
tional groups like nitro, ester, chloro, fluoro, methoxy,
tetrakis-(triphenylphosphine)palladium compound
6
could be isolated in 62% yield after 18 h at 100 °C.
The major side products were the dialkylated aniline
(15%) and unreacted starting material (15%). Subse-
quent hydrogenation of 6 in the presence of a Pt/C cat-
alyst gave 7 in very good yield. Protection of the aniline
with di-tert-butyldicarbonate was realized by using two
equivalents of NaHMDS, a sterically hindered strong
base.12 Compound 8 was isolated in 71% yield.
Scheme 4. Synthesis of dibenzo[b,f]azocine library. Reagents and
conditions: (i) a—di-tert-butylazodicarboxylate (1.5 equiv), PPh3 (1.5
equiv), THF, rt, 3 h; b—trifluoroacetic acid (5 equiv), DCM, rt, 12 h.
The synthesis of dibenzo[b,f]azocine 13 was carried out
in five steps using compounds 4 and 8 as starting mate-
rials (Scheme 3). Deprotonation of the aniline 8 in the
presence of sodium hydride followed by the slow addi-
tion of 4 at À50 °C gave 9 in 50% yield. One side reac-
tion is the debromination of 4 which became more
dominant at higher temperature. The intramolecular
Heck reaction of 9 in the presence of palladium acetate,
tetraethylammonium chloride and dicyclohexylmethyl-
Table 1. Summary of dibenzo[b,f]azocine library
Entry Phenol (ArOH)
Product Overall yielda (%)
1
2-Nitrophenol
3-Nitrophenol
4-Nitrophenol
14a
14b
14c
59 (90)
41 (76)
51 (68)
46 (74)
25 (67)
35 (80)
43 (91)
63 (70)
48 (78)
66 (88)
51 (81)
56 (85)
38 (70)
39 (70)
55 (87)
76 (91)
49 (83)
79 (84)
39 (64)
2
3
4
2-Hydroxymethylbenzoate 14d
3-Hydroxymethylbenzoate 14e
4-Hydroxymethylbenzoate 14f
5
6
7
2-Chlorophenol
3-Chlorophenol
4-Chlorophenol
2-Fluorophenol
3-Fluorophenol
4-Fluorophenol
2-Methoxyphenol
3-Methoxyphenol
4-Methoxyphenol
Trifluoro o-cresol
Trifluoro m-cresol
Phenol
14g
14h
14i
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
14j
14k
14l
14m
14n
14o
14p
14q
14r
14s
14t
14u
14v
14w
14x
2-Acetamidophenol
3-Acetamidophenol
4-Acetamidophenol
2-Hydroxypyridine
3-Hydroxypyridine
4-Hydroxypyridine
b
—
b
—
Scheme 3. Synthesis of 13. Reagents and conditions: (i) a—8 (1 equiv),
NaH (1.1 equiv), dimethylformamide, rt, 1 h; b—4 (1.1 equiv), À50 °C
to rt, 18 h; (ii) Pd(OAc)2 (0.03 equiv), Et4NCl (1 equiv), Cy2NMe (1.5
equiv), N,N-dimethylacetamide, 90 °C, 6 h; (iii) tributyl(vinyl)tin (1
equiv), Pd(t-Bu3)2 (0.045 equiv), CsF (2 equiv), dioxane, 100 °C, 16 h;
(iv) DIBAL-H (2 equiv), toluene, À78 °C to rt, 3 h; (v) 10% Pt/C (0.07
equiv of Pt), H2 (36 psi), ethyl acetate, rt, 14 h.
–(<10)c
–(<10)c
–(<10)c
a Isolated yield and yield of Mitsunobu reaction in parentheses.
b Complex reaction mixture.
c Less than 10% conversion.

5362
L. A. Arnold, R. Kiplin Guy / Bioorg. Med. Chem. Lett. 16 (2006) 5360–5363
trifluoromethyl and acetamido substituents in ortho,
meta and para positions to the hydroxy functionality.
Most of the phenols could be coupled successfully ex-
cept hydroxypyridines and m-and p-acetamidophenols
(Table 1). The hydroxypyridines gave less than 10% con-
version after 12 h and the 3- and 4-acetamidophenols
gave complex reaction mixtures (Table 1, entries 20–
24). The products 14a–s were separated from di-tert-
butyl hydrazinedicarboxylate, the reduction product of
di-tert-butylazodicarboxylate, and triphenylphosphine
oxide by column chromatography and isolated in 64–
91% yield. Subsequent deprotection in the presence of
TFA gave the corresponding compounds 14a–s in 25–
79% yield over two steps.
Figure 3. Dose–response analysis of 14s, H-89 and H-7 using a
luminescence based PKA activity assay. The conditions are identical to
Figure 2. Compounds were serial diluted in DMSO. IC50 values were
obtained by fitting data to the following equation (y = min + (max À
min)/1 + (x/IC50) Hill slope). j (14s), . (no kinase), Ç (H-89), d (H-
7), m (DMSO).
We investigated the ability of these compounds to inhib-
it the phosphorylation of kemptide in the presence of
PKA. The screen was carried out using a commercially
available luminescence kinase assay (Promega). This as-
say quantifies the remains of adenosine triphosphate
(ATP) by converting luciferin to oxyluciferin in the pres-
ence of luciferase. The results are depicted in Figure 2.
bition constant of H-7 could not be calculated because
of the lack of saturation at the measured concentrations.
Thus, 14s is a fairly good inhibitor of PKA activity in
comparison with the control compounds tested.
Among compounds 14a–s of the dibenzo[b,f]azocine li-
brary only compound 14s inhibited partially the phos-
phorylation of kemptide in the presence of PKA at
100 lM. The other derivatives showed no inhibition.
Control experiments were carried out using protein ki-
nase inhibitors H-7 and H-89. H-89 fully inhibited
PKA activity, whereas H-7 was only partially active.
Furthermore, we included DMSO as negative control
and the experiment in the absence of PKA as a positive
control.
To define the biological mechanism of action of the
small molecules, we examined the activity of PKA in
the presence of 14s and H-89 at increasing concentra-
tions of ATP (Fig. 4).
Three concentrations of ATP (25, 5, and 1 lM) were
examined in the presence of 41 lg/ml PKA and 10 lM
kemptide. The analysis showed that the IC50 values of
14s and H-89 increased with increasing concentration
ATP. In case of APT-competitive inhibitor H-89, this
behaviour has been reported.6
To quantify the inhibition of PKA by small molecules
we performed dose response studies of 14s, H-89 and
H-7 (Fig. 3). The small molecules were diluted and incu-
bated with kemptide and PKA in the presence of ATP.
The unconsumed ATP was quantified. Under these as-
say conditions we calculated the following IC50 values:
1.2 lM (H-89) and 122 lM (14s), respectively. The inhi-
Finally, we investigated the influence of the compounds
14a–s on the protein kinase C. In this case, we added
phosphatidylserine, diacylglycol and CaCl2 to the buffer
solution to activate PKC. The results are depicted in
Figure 5.
Figure 2. Luminescence based PKA kinase screen. A solution of PKA
(41 lg/ml), kemptide (10 lM) and compound (100 lM) in 9 ll reaction
buffer (40 mM Tris, pH 7.5, 20 mM MgCl2 and 0.1 mg/ml BSA) was
equilibrated for 1 h. Ten minutes after the addition of 1 ll ATP
(1 lM), 10 ll Kinase-GloÒ plus was added and luminescence was
measured. Values represents means of two replicates. . (no kinase),
Ç (H-89), d (H-7), m (DMSO), j compound.
Figure 4. Kinetic analysis of 14s and H-89. The conditions are
identical to Figure 2. H-89 and 14s were serial diluted in DMSO.
Kinase activity was determined by normalizing data to luminescence
signal measured for full activity (DMSO) and no activity (exclusion of
kinase). j (25 lM), d (5 lM) and m (1 lM).

L. A. Arnold, R. Kiplin Guy / Bioorg. Med. Chem. Lett. 16 (2006) 5360–5363
5363
was detected in the presence of 14s. The activity of the
acetamido functionality regarding the other functional-
ities employed in this library is remarkable. This is the
first example of a protein kinase inhibitor based on a
dibenzo[b,f]azocine scaffold.
We plan to synthesize a second library addressing the
suggested two positions of diversity. Furthermore, we
will explore the influence of these compounds on the
activity of different protein kinases.
Acknowledgments
Figure 5. Luminescence based PKC kinase screen. A solution of PKC
(0.33 lg/ml), neurogranin (20 lM) and compound (100 lM) in 4.5 ll
reactionbuffer (20 mM Tris, pH7.5, 10 mM MgCl2, and 0.1 mg/ml BSA,
250 lM EGTA and 400 lM CaCl2) and 4.5 ll PKC lipid activator
solution (Upstate; 0.5 mg/ml phosphatidylserine, 0.05 mg/ml diacylgly-
col, 20 mM MOPS, pH 7.2, 25 mM b-glycerol phosphate, 1 mM
dithiothreitol and 1 mM CaCl2) was equilibrated for 1 h. Thirty minutes
after the addition of 1 ll ATP (1 lM), 10 ll Kinase-GloÒ Plus was added
and luminescence was measured. Values represents means of two
replicates. . (no kinase), Ç (H-89), d (H-7), m (DMSO), j compound.
This work was supported by the HHMI Research
Resources Program Grant No. 76296-549901, the NIH
(R01 No. DK58080), and the Sandler Research
Foundation.
References and notes
1. Kessel, B. J.; Simpson, M. G. In Comprehensive Textbook
of Psychiatry; Kaplan, H. I., Sadock, B. J., Eds.; Williams
and Wilkins: Baltimore, 1995; Vol. 2, p 2095.
2. Tardito, D.; Perez, J.; Tiraboschi, E.; Musazzi, L.;
Racagni, G.; Popoli, M. Pharmacol. Rev. 2006, 58, 115.
3. Taylor, S. S.; Kim, C.; Vigil, D.; Haste, N. M.; Yang, J.;
Wu, J.; Anand, G. S. Biochim. Biophys. Acta 2005, 1754,
25.
4. Newton, A. C. Biochem. J. 2003, 370, 361.
5. Arnold, L. A.; Luo, W.; Guy, R. K. Org. Lett. 2004, 6,
3005.
6. Chijiwa, T.; Mishima, A.; Hagiwara, M.; Sano, M.;
Hayashi, K.; Inoue, T.; Naito, K.; Toshioka, T.; Hidaka,
H. J. Biol. Chem. 1990, 265, 5267.
The compounds 14a–s could not inhibit the phosphory-
lation of neurogranin in the presence of PKC and ATP.
We used the kinase inhibitors H-7 and H-89 as control
compounds that showed only partial inhibition of
PKC at a concentration of 100 lM. The negative con-
trol (DMSO) exhibits still a substantial amount of
ATP (2,500,000 counts) due to the low concentration
of PKC. However, this concentration was sufficient to
give clear controls (DMSO and no kinase). In contrast
to H-7 and H-89 compound 14s showed no activity
towards PKC although it was identified to inhibit PKA.
7. Hidaka, H.; Inagaki, M.; Kawamoto, S.; Sasaki, Y.
Biochemistry 1984, 23, 5036.
We present herein a convenient synthesis for highly
substituted dibenzo[b,f]azocines. The starting materials
to synthesize dibenzo[b,f]azocine scaffolds are readily
available in three steps. The presented synthetic route
leaves three independent positions available for the
introduction of different substituents at positions 4, 7
and 11 (Scheme 4). Compound 5 can be coupled with
different boronic acid derivatives under the developed
Suzuki reaction conditions. Furthermore, scaffold 10
can be reacted with commercially available organotin
compounds under Stille reaction conditions. The final
position of diversity was successfully employed in the
synthesis of a dibenzo[b,f]azocine library (compounds
14a–s).
8. Engh, R. A.; Girod, A.; Kinzel, V.; Huber, R.; Bosse-
meyer, D. J. Biol. Chem. 1996, 271, 26157.
9. Maillard, M. C.; Perlman, M. E.; Amitay, O.; Baxter, D.;
Berlove, D.; Connaughton, S.; Fischer, J. B.; Guo, J. Q.;
Hu, L. Y.; McBurney, R. N.; Nagy, P. I.; Subbarao, K.;
Yost, E. A.; Zhang, L.; Durant, G. J. J. Med. Chem. 1998,
41, 3048.
10. Ruel, R.; Lacombe, P.; Abramovitz, M.; Godbout, C.;
Lamontagne, S.; Rochette, C.; Sawyer, N.; Stocco, R.;
Tremblay, N. M.; Metters, K. M.; Labelle, M. Bioorg.
Med. Chem. Lett. 1999, 9, 2699.
11. Sum, F. W.; Dusza, J.; Santos, E. D.; Grosu, G.; Reich,
M.; Du, X.; Albright, J. D.; Chan, P.; Coupet, J.; Ru, X.;
Mazandarani, H.; Saunders, T. Bioorg. Med. Chem. Lett.
2003, 13, 2195.
12. Kelly, T. A.; Mcneil, D. W. Tetrahedron Lett. 1994, 35,
9003.
13. Gurtler, C.; Buchwald, S. L. Chem. Eur. J. 1999, 5, 3107.
14. Littke, A. F.; Schwarz, L.; Fu, G. C. J. Am. Chem. Soc.
2002, 124, 6343.
We showed that one member of this library (compound
14s) could successfully inhibit the phosphorylation of
kemptide in the presence of PKA. Additionally, this
compound exhibits selectivity among the serine/threo-
nine kinases PKA and PKC. No inhibition of PKC
15. Mitsunobu, O. Synthesis 1981, 1.