Hantzsch synthesis of pyrazolo[1′,2′:1,2]pyrazolo[3,4-b] pyridines: Partial agonists of the calcitonin receptor

Article abstract of DOI:10.1021/jo050370b

Small molecule calcitonin receptor agonists are of potential utility in the treatment and prevention of osteoporosis. Bicycloeneamine 1 was a useful intermediate in the synthesis of pyrazolopyridine calcitonin receptor partial agonists 2a-f. Dihydropyridi

Full text of DOI:10.1021/jo050370b

We recently developed a convenient synthesis of the
unusual bicyclic eneamine 15 and herein describe the
application of this compound to the synthesis of pyrazolo-
[1′,2′:1,2]pyrazolo[3,4-b]pyridinecarboxamides 2a-f, a
potent series of calcitonin receptor partial agonists and
achiral isosteres of the corresponding (S)-proline-derived
congeners (e.g., compound 3).
Hantzsch Synthesis of
Pyrazolo[1′,2′:1,2]pyrazolo[3,4-b]pyridines:
Partial Agonists of the Calcitonin Receptor
Eric E. Boros,* David J. Cowan, Richard F. Cox,
Makda M. Mebrahtu, Michael H. Rabinowitz,†
James B. Thompson, and Lawrence A. Wolfe, III
GlaxoSmithKline Research and Development, Five Moore
Drive, Research Triangle Park, North Carolina 27709
eric.e.boros@gsk.com
Received February 28, 2005
Small molecule calcitonin receptor agonists are of potential
utility in the treatment and prevention of osteoporosis.
Bicycloeneamine 1 was a useful intermediate in the syn-
thesis of pyrazolopyridine calcitonin receptor partial agonists
2a-f. Dihydropyridines 10a-c were conveniently prepared
by reaction of 1 with Knoevenagel adducts 9a-c, or in the
case of 10d, by a three component reaction with 1, â-keto-
ester 7b, and aldehyde 8c. Oxidation of 10a-d to pyridines
11a-d and subsequent amide formation afforded the title
compounds.
Osteoclast cells digest bone matrix and release calcium
and phosphorus into the blood through a process known
as resorption. When left unchecked, resorption can lead
to the diseased states of osteopenia and ultimately,
osteoporosis. Human calcitonin, a 32 amino acid peptide
hormone, binds to high affinity G-protein coupled recep-
tors on the osteoclast, and its release from the thyroid
gland inhibits resorption and effects a reduction in blood
calcium levels.1 Moreover, administration of injectible
and nasal spray formulations of salmon calcitonin2 is
known to increase bone mineral density, reduce fracture
risk, and ameliorate musculoskeletal pain in humans.3
For these reasons, small molecule calcitonin receptor
agonists are of interest as biological tools and therapeutic
agents in the treatment and prevention of osteoporosis.4
Partial agonists of the calcitonin receptor may also reduce
or modulate the side effects associated with a full agonist.
The functionally diverse pyrrolopyridinecarboxamide
3 represents a rare class of potent small molecule
calcitonin receptor partial agonists.6 Compound 3 and its
analogues are prepared on solid support or in solution
by Hantzsch dihydropyridine methodology as previously
described.6,7 Oxidation of the intermediate dihydropyri-
dine 4 and subsequent deprotection, cyclization, and
amide formation generates the target 3 (eq 1).6
* Corresponding author. Phone: (919) 483-8116; fax: (919) 315-
0430.
† Current Address: Johnson and Johnson Pharmaceutical Research
and Development, 3210 Merryfield Row, San Diego, CA 92121.
(1) Mehta, N. M.; Malootian, A.; Gilligan, J. P. Curr. Pharm. Design
2003, 9, 2659-2676.
(2) Yamauchi, H.; Suzuki, H.; Orima, H. J. Bone Miner. Metab. 2003,
21, 198-204.
(3) Avioli, L. V. Trends Endocrinol. Metab. 1997, 8, 89-92.
(4) For recent syntheses of calcitonin peptide analogues and natural
product calcitonin agonists, see: (a) Hongbin, Y. Y.; Men, H.; Lee, C.
J. Am. Chem. Soc. 2004, 126, 14720-14721. (b) Kapurniotu, A. Curr.
Med. Chem. 2004, 11, 2845-2865. (c) Taylor, J. W.; Jin, Q. K.; Sbacchi,
M.; Wang, L.; Belfiore, P.; Garnier, M.; Kazantzis, A.; Kapurniotu, A.;
Zaratin, P. F.; Scheideler, M. A. J. Med. Chem. 2002, 45, 1108-1121.
10.1021/jo050370b CCC: $30.25 © 2005 American Chemical Society
Published on Web 05/21/2005
J. Org. Chem. 2005, 70, 5331-5334
5331

SCHEME 1 a
a Key: (i) Meldrum’s acid, DCC, DMAP, CH2Cl2; (ii) EtOH, reflux; (iii) C6H6, piperidine, Dean-Stark; (iv) 1‚HCl (generated in situ),
EtOH, NaOEt, reflux; (v) ceric ammonium nitrate, CH3CN; (vi) RNH2, EDC‚HCl, HOBT, DMF; (vii) 1‚HCl, 7b, 8c, EtOH, NaOEt, reflux.
During a medicinal chemistry effort to evaluate core-
template isosteres of 3, we wished to prepare the novel
aza-analogues 2, in which the stereogenic carbon of 3 is
replaced by nitrogen.8 A Hantzsch synthesis9 of dihydro-
pyridines 10 was proposed, starting from Knoevenagel
adducts 9 and enamine 1 (eq 2). In contrast to the
Bicyclic eneamine 1 was generated from 1-cyanoacetyl-
pyrazolidine as previously described.5 Syntheses of 9a-c
were accomplished in three steps from the appropriate
hydrocinnamic acids 5a-b as shown in Scheme 1.
Coupling of 5a-b with Meldrum’s acid afforded the
tricarbonyl intermediates 6a-b, which were heated in
refluxing ethanol to generate â-ketoesters 7a-b.11
Condensation of 7a with aldehydes 8a-c provided
Knoevenagel12 adducts 9a-c and set the stage for their
Hantzsch cyclization to dihydropyridines 10a-c.
Syntheses of 10a-c were accomplished by treating
9a-c with 1‚HCl (generated in situ from 1-cyanoacetyl-
pyrazolidine hydrochloride5) followed by the addition of
NaOEt (Scheme 1). Under these conditions, dihydro-
pyridines 10a-c were obtained in 41-64% yields. To
streamline the process, a three-component Hantzsch
reaction was investigated with aldehyde 8c, â-keto ester
7b, and 1‚HCl (eq 3). Heating these reactants with
NaOEt in EtOH at reflux afforded 10d in 51% yield.
(7) Bhandari, A.; Li, B.; Gallop, M. A. Synthesis 1999, 1951-1960.
(8) For examples of isosteric stereogenic carbon replacement in
antibacterial agents and HIV-protease inhibitors, see: (a) Snyder, L.
B.; Meng, Z.; Mate, R.; D’Andrea, S. V.; Marinier, A.; Quesnelle, C. A.;
Gill, P.; DenBleyker, K. L.; Fung-Tomc, J. C.; Frosco, M.; Martel, A.;
Barrett, J. F.; Bronson, J. J. Bioorg. Med. Chem. Lett. 2004, 14, 4735-
4739. (b) Getman, D. P.; DeCrescenzo, G. A.; Heintz, R. M.; Reed, K.
L.; Talley, J. J.; Bryant, M. L.; Clare, M.; Houseman, K. A.; Marr, J.
J.; Mueller, R. A.; Vazquez, M. L.; Shieh, H.-S.; Stallings, W. C.;
Stegeman, R. A. J. Med. Chem. 1993, 36, 288-291.
(9) For reviews of the Hantzsch dihydropyridine synthesis and
related multicomponent cyclocondensation reactions, see: (a) Sausins,
A.; Duburs, G. Heterocycles 1988, 27, 269-289. (b) Simon, C.; Con-
stantieux, T.; Rodriguez, J. Eur. J. Org. Chem. 2004, 4957-4980.
(10) Greenwald, R. B. U.S. Patent 4,128,425 (1978); Chem. Abstr.
1979, 90, 95418n.
synthesis of 3 (eq 1), which requires late stage deprotec-
tion and cyclization steps, the synthesis of 2 generates
the fused tricyclic core in one step. Interestingly, applica-
tions of 1 in organic synthesis are limited;5,10 however,
its compact, electron-rich structure appeared well-suited
for the Hantzsch protocol.
(5) Boros, E. E.; Bouvier, F.; Randhawa, S.; Rabinowitz, M. H. J.
Heterocycl. Chem. 2001, 38, 613-616.
(6) Bhandari, A.; Boros, E. E.; Cowan, D. J.; Handlon, A. L.; Hyman,
C. E.; Oplinger, J. A.; Rabinowitz, M. H.; Turnbull, P. S. International
Patent Application WO 03,076,440 (2003); Chem. Abstr. 2003, 139,
261291.
(11) Oikawa, Y.; Sugano, K.; Yonemitsu, O. J. Org. Chem. 1978, 43,
2087-2088.
(12) For a review of the Knoevenagel condensation, see Jones, G.
Org. React. 1967, 15, 204-599.
5332 J. Org. Chem., Vol. 70, No. 13, 2005

TABLE 1. Human Calcitonin Activation Properties of
the corresponding proline-derived analogues (e.g., com-
pound 3), and their exemplification further diversifies the
portfolio of existing small molecule calcitonin mimetics.
Additional details concerning the synthesis and biological
activity of 2a-f, 3, and their congeners will be reported
in due course.
Pyrazolopyridines 2a-f and Pyrrolopyridine 3a
compd
EC50 (nM)
% hCal maxb
nc
2a
2b
2c
2d
2e
2f
3.1 ( 0.7
4.9 ( 0.2
2.5 ( 0.4
11 ( 2
27 ( 2
29 ( 2
28 ( 2
34 ( 1
48 ( 17
22 ( 1
38 ( 3
3
3
3
3
5
3
5
23 ( 8
Experimental Procedures
7 ( 4
8 ( 5
3
General Procedure for Synthesis of Dihydropyridines
10a-c from Knoevenagel Adducts 9a-c. A stirred solution
of 1-cyanoacetylpyrazolidine hydrochloride5 (2.4 mmol) in EtOH
(5 mL) was heated at reflux for 30 min to generate 1‚HCl.
Compound 9 (2 mmol) and solid NaOEt (2 mmol) were added,
and the reflux was maintained an additional 2 h. The stirred
mixture was cooled to room temperature and diluted with water
(75 mL), and the precipitated product (10) was collected by
filtration. This material was used in the next step without
further purification.
a Data are expressed as means ( SE. b Percentage of the
maximal human calcitonin response. c Number of measurements.
Knoevenagel adduct 9d is a likely intermediate in the
reaction, although additional mechanisms involving reac-
tion of 1 with 7b or 8c are plausible.
5-{3-[(Ethoxy)carbonyl]-2-[2-(4-fluorophenyl)ethyl]-5-
oxo-1,5,8,9-tetrahydro-4H,7H-pyrazolo[1′,2′:1,2]pyrazolo-
[3,4-b]pyridin-4-yl}thiophene-2-carboxylic acid (10a). This
compound was prepared from 9a (61% yield): 1H NMR (d6-
DMSO) δ 12.74 (br s, 1H), 9.92 (s, 1H), 7.46 (br s, 1H), 7.24 (m,
2H), 7.08 (t, 2H, J ) 8.5), 6.77 (br s, 1H), 5.01 (s, 1H), 4.01 (q,
2H, J ) 7), 3.45 (m, 2H), 3.38 (m, 1H), 3.29 (m, 1H), 2.90 (m,
2H), 2.80 (m, 2H), 2.29 (m, 2H), 1.09 (t, 3H, J ) 7); ES+ MS:
370 (20), 498 (M + H+, 70).
Hantzsch Synthesis of Dihydropyridine 10d from 1, 7b,
and 8c. A stirred mixture of 1‚HCl5 (1.2 g, 6.83 mmol), 7b (1.91
g, 6.63 mmol), 8c (995 mg, 6.63 mmol), and NaOEt (451 mg,
6.63 mmol) in EtOH (45 mL) was heated at reflux for 8 h. Water
(50 mL) and EtOAc (50 mL) were added, and the mixture was
stirred for 30 min. The suspended solids were collected by
filtration, triturated with EtOAc, and dried under high vacuum
to afford 10d as an off-white solid (1.83 g, 51% yield). This
material was used without further purification.
4-(3-[(Ethoxy)carbonyl]-5-oxo-2-{2-[4-(trifluoromethyl)-
phenyl]ethyl}-1,5,8,9-tetrahydro-4H,7H-pyrazolo[1′,2′:1,2]-
pyrazolo[3,4-b]pyridin-4-yl)benzoic Acid (10d). 1H NMR (d6-
DMSO) δ 12.63 (br s, 1H), 9.77 (s, 1H), 7.77 (d, 2H, J ) 8), 7.65
(d, 2H, J ) 8), 7.46 (d, 2H, J ) 8), 7.21 (d, 2H, J ) 8), 4.76 (s,
1H), 3.87 (q, 2H, J ) 7), 3.41 (m, 2H), 3.22 (m, 2H), 2.96 (m,
4H), 2.24 (m, 2H), 0.95 (t, 3H, J ) 7); ES+ MS: 542 (M + H+,
100).
Dihydropyridines 10a-d were judged to be 86-98%
pure based on HPLC and were used in the next step
without further purification. Oxidation of 10a-d with
ceric ammonium nitrate in CH3CN gave the correspond-
ing pyridines 11a-d in 66-76% yields. Conversion of
11a-d to carboxamides 2a-f was achieved with EDC‚
HCl, HOBT, and the requisite primary amine in DMF.
The desired products 2a-f were purified by flash chro-
matography on silica gel in 50-91% yields and were fully
characterized.
As shown in Table 1, the title compounds are effective
calcitonin receptor partial agonists with potencies com-
parable to the chiral proline-derived analogue 3. All
compounds in Table 1 displayed 1-2 digit nanomolar
potency values (EC50), and 22-48% maximum efficacy,
relative to human calcitonin (i.e., the percentage of
maximal human calcitonin response).13,14 Aromatic and
heteroaromatic rings at the 4-pyridyl position were well-
tolerated as were a variety of 4′-carboxamide groups. The
corresponding 4′-carboxylic acids 11a-d were signifi-
cantly less active under similar assay conditions.
In summary, bicyclic eneamine 1 was of general utility
in both two- and three-component Hantzsch syntheses
of pyrazolopyridine calcitonin receptor partial agonists
2a-f. The title compounds represent achiral isosteres of
General Procedure for Oxidation of Dihydropyridines
10a-d to Pyridines 11a-d. A solution of ceric ammonium
nitrate (2 mmol) in 3:1 actonitrile/water (4 mL) was added
dropwise to a stirred solution of 10 (1 mmol) in acetonitrile (5
mL) at room temperature. The reaction mixture was stirred for
4 h and concentrated at reduced pressure. Trituration of the
crude product with water/ethanol provided 11 as a filterable
solid. This material was used without further purification.
5-{3-[(Ethoxy)carbonyl]-2-[2-(4-fluorophenyl)ethyl]-5-
oxo-8,9-dihydro-5H,7H-pyrazolo[1′,2′:1,2]pyrazolo[3,4-b]-
pyridin-4-yl}thiophene-2-carboxylic Acid (11a). This com-
pound was prepared from 10a (67% yield): 1H NMR (d6-DMSO)
δ 13.30 (br s, 1H), 7.69 (d, 1H, J ) 3.8), 7.22 (m, 3H), 7.06 (t,
2H, J ) 9), 4.04 (q, 2H, J ) 7), 3.87 (t, 2H, J ) 67), 3.79 (t, 2H,
J ) 7), 3.00 (m, 4H), 2.55 (m, 2H, J ) 7), 0.94 (t, 3H, J ) 7).
General Procedure for Synthesis of Calcitonin Agonists
2a-f from 11a-d. Solid EDC‚HCl (0.16 mmol) was added to a
stirred solution of 11 (0.15 mmol), the requisite primary amine
(0.15 mmol), and HOBT (0.25 mmol) in DMF (2.5 mL). The
reaction mixture was stirred at room temperature overnight,
concentrated at reduced pressure, and reconstituted in CH2Cl2.
The organic phase was washed with water and purified by silica
gel chromatography (eluting with 0-5% MeOH/CH2Cl2) to afford
2 as a light yellow solid.
(13) Human calcitonin was cloned according to procedures described
in ref 14.
(14) Chen, W. J.; Armour, S.; Way, J.; Chen, G.; Watson, C.; Irving,
P.; Cobb, J.; Kadwell, S.; Beaumont, K.; Rimele, T.; Kenakin, T. Mol.
Pharmacol. 1997, 52, 1164-1175.
Ethyl
2-[2-(4-Fluorophenyl)ethyl]-4-(5-{[(2-furanyl-
methyl)amino]carbonyl}-2-thienyl)-5-oxo-8,9-dihydro-
5H,7H-pyrazolo[1′,2′:1,2]pyrazolo[3,4-b]pyridine-3-carbox-
ylate (2a). This compound was prepared from 11a and furfu-
J. Org. Chem, Vol. 70, No. 13, 2005 5333

rylamine (84% yield): 1H NMR (d6-DMSO) δ 9.08 (t, 1H, J ) 6),
7.76 (d, 1H, J ) 4), 7.56 (s, 1H), 7.21 (m, 3H), 7.06 (t, 2H, J )
9), 6.37 (t, 1H, J ∼ 3), 6.27 (d, 1H, J ∼ 3), 4.43 (d, 2H, J ) 6),
4.05 (q, 2H, J ) 7), 3.86 (t, 2H, J ) 7), 3.79 (t, 2H, J ) 7), 3.00
(m, 4H), 2.54 (m, 2H, J ) 7), 0.96 (t, 3H, J ) 7); ES+ MS: 575
(M + H+, 100); Anal. Calcd for C30H27FN4O5S: C, 62.71; H, 4.74;
N, 9.75. Found: C, 62.49; H, 4.75; N, 9.81.
3.86 (m, 4H), 2.99 (m, 5H), 2.85 (m, 1H), 2.56 (m, 2H, J ) 7),
2.43 (m, 1H), 1.92 (m, 1H), 1.08 (t, 3H, J ) 7); ES+ MS: 595 (M
+ H+, 100); Anal. Calcd for C34H31FN4O5: C, 68.68; H, 5.25; N,
9.42. Found: C, 68.40; H, 5.33; N, 9.35.
Ethyl 4-(4-{[(2-Furanylmethyl)amino]carbonyl}phenyl)-
5-oxo-2-{2-[4-(trifluoromethyl)phenyl]ethyl}-8,9-dihydro-
5H,7H-pyrazolo[1′,2′:1,2]pyrazolo[3,4-b]pyridine-3-carbox-
ylate (2e). This compound was prepared from 11d and
furfurylamine (50% yield): 1H NMR (d6-DMSO) δ 9.05 (br t, 1H,
J ∼ 6), 7.88 (d, 2H, J ) 8), 7.61 (d, 2H, J ) 8), 7.55 (s, 1H), 7.42
(d, 2H, J ) 8), 7.38 (d, 2H, J ) 8), 6.37 (m, 1H, J ∼ 3), 6.26 (d,
1H, J ∼ 3), 4.46 (d, 2H, J ∼ 6), 3.88 (q, 2H, J ) 7), 3.85 (t, 2H,
J ) 7), 3.76 (t, 2H, J ) 7), 3.11 (s, 4H), 2.53 (m, 2H, J ) 7), 0.76
(t, 3H, J ) 7); ES+ MS: 619 (M + H+, 100); Anal. Calcd for
Ethyl 2-[2-(4-Fluorophenyl)ethyl]-5-oxo-4-(5-{[(3-pyri-
dinylmethyl)amino]-carbonyl}-2-thienyl)-8,9-dihydro-
5H,7H-pyrazolo[1′,2′:1,2]pyrazolo[3,4-b]pyridine-3-carbox-
ylate (2b). This compound was prepared from 11a and 3-(ami-
nomethyl)pyridine (71% yield): 1H NMR (d6-DMSO) δ 9.21 (t,
1H, J ) 6), 8.52 (s, 1H), 8.44 (d, 1H, J ) 5), 7.76 (d, 1H, J ) 4),
7.69 (d, 1H, J ) 8), 7.34 (dd, 1H, J ) 8, 5), 7.23 (d, 1H, J ) 4),
7.21 (m, 2H), 7.06 (t, 2H, J ) 9), 4.46 (d, 2H, J ) 6), 4.05 (q, 2H,
J ) 7), 3.86 (t, 2H, J ) 7), 3.79 (t, 2H, J ) 7), 3.00 (m, 4H), 2.54
(m, 2H, J ) 7), 0.96 (t, 3H, J ) 7); ES+ MS: 586 (M + H+, 100);
Anal. Calcd for C31H28FN5O4S: C, 63.58; H, 4.82; N, 11.96.
Found: C, 63.69; H, 4.89; N, 11.88.
C
33H29F3N4O5‚(0.5 H2O): C, 63.15; H, 4.82; N, 8.93. Found: C,
63.13; H, 4.72; N, 8.99.
Ethyl
2-[2-(4-Fluorophenyl)ethyl]-4-(4-{[(2-furanyl-
methyl)amino]carbonyl}-phenyl)-5-oxo-8,9-dihydro-
5H,7H-pyrazolo[1′,2′:1,2]pyrazolo[3,4-b]pyridine-3-carbox-
ylate (2f). This compound was prepared from 11c and furfury-
lamine (91% yield): 1H NMR (d6-DMSO) δ 9.06 (t, 1H, J ) 6),
7.88 (d, 2H, J ) 8), 7.56 (s, 1H), 7.38 (d, 2H, J ) 8), 7.22 (t, 2H,
J ∼ 9), 7.07 (t, 2H, J ∼ 9), 6.37 (s, 1H), 6.26 (d, 1H, J ) 3), 4.46
(d, 2H, J ) 6), 3.89 (m, 4H), 3.77 (t, 2H, J ) 7), 3.06 (m, 2H),
2.98 (m, 2H), 2.54 (m, 2H, J ) 7), 0.78 (t, 3H, J ) 7); ES+ MS:
569 (M + H+, 100); Anal. Calcd for C32H29FN4O5‚(0.5 H2O): C,
66.54; H, 5.24; N, 9.70. Found: C, 66.52; H, 5.13; N, 9.70.
Ethyl
2-[2-(4-Fluorophenyl)ethyl]-4-[5-({[(3-fluoro-
phenyl)methyl]amino}-carbonyl)-2-thienyl]-5-oxo-8,9-
dihydro-5H,7H-pyrazolo[1′,2′:1,2]pyrazolo[3,4-b]pyridine-
3-carboxylate (2c). This compound was prepared from 11a and
3-fluorobenzylamine (80% yield): 1H NMR (d6-DMSO) δ 9.19 (t,
1H, J ) 6), 7.78 (d, 1H, J ) 4), 7.36 (m, 1H), 7.23-7.20 (m, 3H),
7.14-7.04 (m, 5H), 4.45 (d, 2H, J ) 6), 4.05 (q, 2H, J ) 7), 3.86
(t, 2H, J ) 7), 3.79 (t, 2H, J ) 7), 3.00 (m, 4H), 2.54 (m, 2H, J
) 7), 0.96 (t, 3H, J ) 7); ES+ MS: 603 (M + H+, 100); Anal.
Calcd for C32H28F2N4O4S: C, 63.78; H, 4.68; N, 9.30. Found: C,
63.63; H, 4.62; N, 9.27.
Supporting Information Available: General experimen-
tal methods; procedures for the calcitonin reporter assay;
procedures for synthesis of 7a-b and 9a-c; 1H NMR data for
7a-b, 9a-c, 10b-c, and 11b-d; MS data for 10b-c; 1H NMR
spectra of 10a-d and 11a-d. This material is available free
of charge via the Internet at http://pubs.acs.org.
Ethyl 4-(5-{[(1R)-2,3-Dihydro-1H-inden-1-ylamino]car-
bonyl}-2-furanyl)-2-[2-(4-fluorophenyl)ethyl]-5-oxo-8,9-di-
hydro-5H,7H-pyrazolo[1′,2′:1,2]pyrazolo[3,4-b]pyridine-3-
carboxylate (2d). This compound was prepared from 11b and
(R)-1-aminoindane (81% yield): 1H NMR (d6-DMSO) δ 8.56 (d,
1H, J ) 8), 8.03 (d, 1H, J ) 4), 7.37 (d, 1H, J ) 4), 7.26-7.15
(m, 6H), 7.06 (t, 2H, J ) 9), 5.47 (q, 1H, J ) 8), 4.31 (m, 2H),
JO050370B
5334 J. Org. Chem., Vol. 70, No. 13, 2005