Chem Biol Drug Des 2015; 85: 633–637 Research Letter
Functional Non-Nucleoside Adenylyl Cyclase Inhibitors Marco Lelle1, Abdul Hameed1, Lisa-Maria Ackermann1, Stefka Kaloyanova1, Manfred Wagner1, Filip Berisha2, Viacheslav O. Nikolaev2 and Kalina Peneva1,* 1
Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany 2 Emmy Noether Group of the DFG, Department of Cardiology and Pneumology, European Heart Research €ttingen, Georg August University Medical Institute Go €ttingen, D-37075 Go €ttingen, Center, University of Go Germany *Corresponding author: Kalina Peneva, [email protected] In this study, we describe the synthesis of novel functional non-nucleoside adenylyl cyclase inhibitors, which can be easily modified with thiol containing biomolecules such as tumour targeting structures. The linkage between inhibitor and biomolecule contains cleavable bonds to enable efficient intracellular delivery in the reductive milieu of the cytosol as well as in the acidic environment within endosomes and lysosomes. The suitability of this synthetic approach was shown by the successful bioconjugation of a poor cellpermeable inhibitor with a cell-penetrating peptide. Additionally, we have demonstrated the excellent inhibitory effect of the compounds presented here in a € rster resonance energy transfer-based live-cell Fo assay in human embryonic kidney cells. Key words: adenylyl cyclase inhibitor, cancer treatment, pyridyl disulphide, tumour targeting Received 29 August 2014, revised 3 October 2014 and accepted for publication 7 October 2014
The membrane-bound enzyme adenylyl cyclase (AC) consists of nine different isoforms that catalyse the conversion of adenosine triphosphate into cyclic adenosine monophosphate (cAMP) (1,2). This intracellular process is stimulated by the binding of ligands such as hormones, nutrients, growth factors or neurotransmitters to their specific cell surface receptors (3). The cyclic nucleotide cAMP plays an undisputed role as ubiquitous second messenger and is also involved in the immune response (4). Elevated levels of cAMP are ordinarily harboured by naturally occurring T regulatory cells (Treg) and transferred to T cells via gap junctions (5,6). Thereby, immune response mediated by T cells is efficiently suppressed in patients suffering from ª 2014 John Wiley & Sons A/S. doi: 10.1111/cbdd.12452
malignant melanoma (7). Accumulation of Tregs in tumours is considered to prevent effective antitumor immune responses, consequently leading to tumour growth and progression (8,9). Thus, the manipulation of Tregs represents a promising strategy for the treatment of cancer and has become an emerging target in cancer immunotherapy (7,8). As cAMP is a crucial component in Treg-dependent suppression, the downregulation of this messenger would present a powerful opportunity to combat cancer through T-cells-mediated activation of the immune system. To efficiently decrease intracellular cAMP levels, numerous AC inhibitors have been widely applied. Several adenosine derivatives such as adenosine monophosphate, 2-deoxyadenosine or 9-(tetrahydrofuryl)-adenine were found to potently inhibit different types of ACs (10). However, their systemic application is typically accompanied by severe side-effects and can interfere with DNA synthesis (10,11). These limitations can be successfully circumvented with non-nucleoside-based AC inhibitors. 2-Amino-7-(2-furanyl)7,8-dihydro-5(6H)-quinazolinone and cis-N-((1R,2R)-2-phenylcyclopentyl)-azacyclotridec-1-en-2-amine, also referred to as NKY80 (1) and MDL 12330A (2), are well-studied inhibitors that lack an intact adenine ring (12,13). Nevertheless, both substances, like their adenosine counterparts, exhibit limited cell membrane permeability as well as low solubility in aqueous media (10,11,14). Moreover, 1 and 2 are associated with a poor tumour tissue specificity, which significantly decreases the pharmaceutical relevance for cancer therapy. We have designed and prepared functional derivatives of non-nucleoside AC inhibitors, bearing a 2-pyridyl disulphide functionality for the attachment of cell-permeable (cell-penetrating peptide) or tumour targeting (folic acid, octreotide, antibodies) moieties, that address the aforementioned limitations (Figure 1).
O N
N N O
NH
NH2
1 2
Figure 1: Chemical structure adenylyl cyclase inhibitors.
of
common
non-nucleoside
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and they can serve as prototypic tumour vectors in vivo (17,18). The inhibitor peptide conjugate 7 was isolated by preparative HPLC and displayed an excellent water solubility. This modification approach enables the inhibitor release in the acidic environment within endosomes and lysosomes upon cellular uptake and subsequent cleavage of the hydrazine bond. Additionally, the conjugate can be cleaved by intracellular glutathione, a naturally occurring peptide that is capable of reducing disulphide bonds and its increased concentration in the cytosol has been reported in many tumour cells, thus providing additional selectivity of the conjugates towards cancerous cells. However, AC inhibitor 2 cannot be directly modified with the cross-linking reagent due to the lack of an intrinsic aldehyde or ketone function (Scheme 2).
To introduce the 2-pyridyl disulphide in the inhibitor structure, a small cross-linker with an additional hydrazide group was chosen. The required cross-linking reagent was prepared in two straightforward steps from methyl 3-mercaptopropionate (3). Initially, the methyl ester was transformed into the corresponding hydrazide 4 according to a literature procedure (15). Subsequently, the free thiol group was converted to 2-pyridyl disulphide with an excess of 2,20 -dithiopyridine. Unreacted 2,20 -dithiopyridine was removed by column chromatography, and the desired product 5 was obtained as a colourless solid in good yield. Due to the hydrazide functionality present in 5, aldehydes and ketones can be easily modified with this reagent. Thereby, an acid labile hydrazone bond can be created that is cleavable at slightly acidic pH (pH 5) and has already found extensive use in antibody–drug conjugates applied in vivo (16) (Scheme 1).
Thus, further modification of the original inhibitor molecule was required. A synthetic approach was chosen, which created a 4-bromophenyl derivative of 2. This was achieved by condensation of 2-azacyclotridecanone and a para-substituted cis-2-phenyl-1-amino-cyclopentane. The synthesis of the required cyclopentane derivative 11 was accomplished from 1,4-dibromobenzene as described before (19). Utilization of this synthetic route enabled the preparation of the requested cis amine by a ring opening of cyclopentene oxide with the Grignard reagent of 8 and subsequent inversion via Mitsunobu reaction. However, further modifications were carried out with both cis enantiomers. The following condensation of 11 and 2-azacyclotridecanone mediated by phosphoryl chloride to yield the bromo-substituted inhibitor was accomplished with a procedure described by Grisar et al. (20) for the synthesis of lactamimides. The obtained bromophenyl derivative 12
The AC inhibitor 1 bears a keto group suitable for the condensation with 5. Moreover, the crucial pharmacophore sequence (=C-N=C-NH2) of the inhibitor remains intact (10,13). The modification of the carbonyl was carried out in chloroform with an excess of the cross-linker and catalysed by trifluoroacetic acid. Under these conditions, the functional AC inhibitor precipitated and the product was filtered off. Analysis of the isolated substance by NMR spectroscopy revealed that 6 consists of two isomers, E and Z, respectively. Afterwards, the 2-pyridyl disulphide carrying inhibitor was coupled to the N-terminal cysteine of a cellpenetrating peptide, namely octaarginine. Oligoarginine peptides have been used to transport in vitro a large variety of cargo ranging from small drugs, proteins, polymer conjugates to nanoparticles across the cell membrane,
O
a
SH O
H2N
H N O
3
b
SH
H2N
4
H N
S
S
N
5
O
Scheme 1: Synthesis of cross-linking reagent 5. Reagents and conditions: (a) N2H4xH2O, MeOH, rt, overnight, 83%; (b) 2,20 dithiopyridine, DMF, rt, 4 h, 77%.
NH O O
O N N
O
1
a
N
S
S
NH
NH2
H2N
N
b
S O
N N O
N H
NH2
N
H N O
O N H
H N
O NH2
8
O
S NH
6 (E/Z)
O
NH H2N
O
NH2
N N O
NH2
7 (E/Z)
Scheme 2: Synthesis of functional AC inhibitor 6 and the corresponding cell-penetrating peptide derivative 7. Reagents and conditions: (a) 5, chloroform, TFA, rt, 14 h, 80%; (b) NH2-CGGWRRRRRRRR-NH2, DMF, phosphate buffer, rt, 2 h, 66%.
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Chem Biol Drug Des 2015; 85: 633–637
Functional Non-Nucleoside Adenylyl Cyclase Inhibitors Br Br Br
c
a,b
8
N
Br
OH Br
9()
e
d
O
O
N
NH2
Br
12 ( )
11 ( )
10 ( )
HCl NH
Scheme 3: Synthesis of intermediate 12. Reagents and conditions: (a) Mg, THF, rt, 90 min; (b) cyclopentene oxide, CuI, THF, rt, 14 h, 37%; (c) PPh3, DIAD, phthalimide, THF, rt, overnight, 39%; (d) ethanolamine, 90 °C, 1 h, 78%; (e) 2-azacyclotridecanone, phosphoryl chloride, benzene, reflux, 24 h, 58%.
O Br N
12 ( )
Scheme 4: Synthesis of functional AC inhibitor 15. Reagents and conditions: (a) (Boc)2O, THF, NaHCO3 solution, rt, 14 h; (b) NaN3, L-proline, CuI, DMSO, 100 °C, 24 h, 34%; (c) 4-carboxybenzaldehyde, chloromethylenedimethylammonium chloride, DCM, rt, 2 h; (d) DIPEA, DCM, rt, 14 h, 74%; (e) dioxane, 4 M hydrochloric acid, rt, 2 h; (f) 5, chloroform/methanol (99:1), rt, 12 h, 53%.
O
HCl NH
O
HN
N O N O
13 ( )
N N
14 ( )
O N
S
O
HN N
S
e,f
enables versatile substitutions to create an AC inhibitor accessible for further bioconjugation chemistry (Scheme 3). To modify the inhibitor with cross-linker 5, the amidine group of 12 was protected at first and an aryl amine was prepared through a copper-assisted aromatic substitution reaction with sodium azide (21). Subsequent amide bond formation between 13 and 4-carboxybenzaldehyde, which was transferred into the corresponding acid chloride in the presence of the Vilsmeier reagent, led to the required aldehyde-bearing AC inhibitor derivative. In an initial step, the protective group of this molecule was removed under acidic conditions followed by direct condensation with the cross-linking reagent. The functional non-nucleoside inhibitor 15 was purified by HPLC, and its water solubility is comparable with the commercially available MDL 12330A (2), which enabled further experiments in aqueous buffer or cell culture media without aggregation or precipitation. Characterization by NMR spectroscopy showed two isomers of the obtained hydrazone with an E/Z ratio of 2:1. The 2-pyridyl disulphide group provides the opportunity to further react 15 with thiol containing biomolecules that can be further employed for selective targeting of cancer or immune cells in vivo (Scheme 4). To test the AC inhibitory activity of the new compounds, a €rster resonance energy live-cell assay that relies on a Fo transfer (FRET)-based cAMP biosensor Epac1-camps Chem Biol Drug Des 2015; 85: 633–637
O
c,d
H2N
a,b
HN
15 (E/Z) ( )
N NH
FRET change in response to ISO( %)
25 20 15 10 5 0
control
2
1
6
15
13
Figure 2: AC inhibitory activity of the compounds analysed in live 293A cells. Cells were stimulated with 10 nM of isoproterenol (ISO) after 5- to 7-min preincubation with DMSO (negative control) or various compounds (each at 100 lM concentration). MDL 12330A (1) and NKY-80 (2) were used as positive controls of the gold standard AC inhibitors. Bar graphs show changes of FRET in response to ISO, means SE, n = 10–22 cells per compound.
expressed in 293A cells was used (see Appendix S1) (22). This is the most standard cell line with sufficient expression of endogenous AC routinely used to monitor real-time effects of agonists and antagonists of the cAMP signalling pathway (23). Stimulation of cells with the b-adrenergic receptor agonist isoproterenol (ISO) leads to an AC-dependent increase in cAMP visualized by a change of FRET ratio. Preincubation of cells with the inhibitors for 5–7 min prior to (ISO) administration markedly decreased the cAMP production. The inhibitory effect of the compounds was 635
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comparable to that of the parental inhibitors MDL 12330A and NKY-80 (Figure 2). In addition, we performed luminescent luciferase-based adenosine triphosphate quantification assays (CellTiterGlo; Promega Corporation, Madison, WI, USA) to determine the cytotoxicity of the compounds. A293 cells were incubated with 1, 2, 6 and 15 at a concentration of 50 lM for 72 h in a serum-containing medium, and the obtained IC50 values for all compounds with exception of 2 (12.55 0.69 lM) were higher than 50 lM. These data clearly indicate that the functional non-nucleoside inhibitors described in this work are not cytotoxic. In summary, we have successfully synthesized two different 2-pyridyl disulphide-containing non-nucleoside adenylyl cyclase inhibitors as well as a tumour targeting and cellpermeable bioconjugate and tested their AC inhibitory activity in living cells. In a live-cell FRET-based assay performed in A293 cells, we have shown that the functionalization of the parent molecules does not impede their inhibitory function. This new class of functional inhibitor derivatives can address numerous limitations in the therapeutic application of AC inhibitors such as low cell permeability, poor solubility or tumour tissue selectivity by the facile modification with appropriate biomolecules. The herein utilized bioconjugation chemistry enables efficient inhibitor release within cancer cells due to the applied hydrolysable and reductively cleavable bonds. We believe that these novel functional AC inhibitors can thus establish a unique way in cancer therapy by triggering an antitumor immune response mediated through suppression of the production of intracellular cyclic adenosine monophosphate.
Acknowledgments This work was funded by the Max Planck Society.
Author Contributions M. Lelle and A. Hameed equally contributed to this work.
Conflict of Interest The authors declare no conflict of interest.
References 1. Tang W.J., Hurley J.H. (1998) Catalytic mechanism and regulation of mammalian adenylyl cyclases. Mol Pharmacol;54:231–240.
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2. Hanoune J., Defer N. (2001) Regulation and role of adenylyl cyclase isoforms. Annu Rev Pharmacol Toxicol;41:145–174. 3. Daniel P.B., Walker W.H., Habener J.F. (1998) Cyclic AMP signaling and gene regulation. Annu Rev Nutr;18:353–383. 4. Sprenger J.U., Nikolaev V.O. (2013) Biophysical techniques for detection of cAMP and cGMP in living cells. Int J Mol Sci;14:8025–8046. 5. Bodor J., Bopp T., Vaeth M., Klein M., Serfling E., Hunig T., Becker C., Schild H., Schmitt E. (2012) Cyclic AMP underpins suppression by regulatory T cells. Eur J Immunol;42:1375–1384. 6. Bopp T., Becker C., Klein M., Klein-Hessling S., Palmetshofer A., Serfling E., Heib V. et al. (2007) Cyclic adenosine monophosphate is a key component of regulatory T cell mediated suppression. J Exp Med;204: 1303–1310. 7. Bopp T., Radsak M., Schmitt E., Schild H. (2010) New strategies for the manipulation of adaptive immune responses. Cancer Immunol Immun;59:1443–1448. 8. Zou W.P. (2006) Regulatory T cells, tumour immunity and immunotherapy. Nat Rev Immunol;6:295–307. 9. Klages K., Mayer C.T., Lahl K., Loddenkemper C., Teng M.W.L., Ngiow S.F., Smyth M.J., Hamann A., Huehn J., Sparwasser T. (2010) Selective depletion of Foxp3(+) regulatory T cells improves effective therapeutic vaccination against established melanoma. Cancer Res;70:7788–7799. 10. Pavan B., Biondi C., Dalpiaz A. (2009) Adenylyl cyclases as innovative therapeutic goals. Drug Discov Today;14:982–991. 11. Laux W.H.G., Pande P., Shoshani I., Gao J.Y., Boudou-Vivet V., Gosselin G., Johnson R.A. (2004) Pronucleotide inhibitors of adenylyl cyclases in intact cells. J Biol Chem;279:13317–13332. 12. Seifert R., Lushington G.H., Mou T.C., Gille A., Sprang S.R. (2012) Inhibitors of membranous adenylyl cyclases. Trends Pharmacol Sci;33:64–78. 13. Onda T., Hashimoto Y., Nagai M., Kuramochi H., Saito S., Yamazaki H., Toya Y., Sakai I., Homcy C.J., Nishikawa K., Ishikawa Y. (2001) Type-specific regulation of adenylyl cyclase. Selective pharmacological stimulation and inhibition of adenylyl cyclase isoforms. J Biol Chem;276:47785–47793. 14. Iwatsubo K., Minamisawa S., Tsunematsu T., Nakagome M., Toya Y., Tomlinson J.E., Umemura S., Scarborough R.M., Levy D.E., Ishikawa Y. (2004) Direct inhibition of type 5 adenylyl cyclase prevents myocardial apoptosis without functional deterioration. J Biol Chem;279:40938–40945. 15. Costioli M.D., Berdat D., Freitag R., Andre X., Muller A.H.E. (2005) Investigation of the telomerization kinetics of N-isopropylacrylamide using 3-mercaptopropionic hydrazide as chain transfer agent. Macromolecules;38:3630–3637.
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16. Kratz F., Warnecke A., Schmid B., Chung D.E., Gitzel M. (2006) Prodrugs of anthracyclines in cancer chemotherapy. Curr Med Chem;13:477–523. 17. Duchardt F., Fotin-Mleczek M., Schwarz H., Fischer R., Brock R. (2007) A comprehensive model for the cellular uptake of cationic cell-penetrating peptides. Traffic;8:848–866. 18. Nakase I., Konishi Y., Ueda M., Saji H., Futaki S. (2012) Accumulation of arginine-rich cell-penetrating peptides in tumors and the potential for anticancer drug delivery in vivo. J Controlled Release;159:181– 188. 19. Shepherd T.A., Aikins J.A., Bleakman D., Cantrell B.E., Rearick J.P., Simon R.L., Smith E.C.R., Stephenson G.A., Zimmerman D.M. (2002) Design and synthesis of a novel series of 1,2-disubstituted cyclopentanes as small, potent potentiators of 2-amino-3-(3-hydroxy-5methyl-isoxazol-4-yl)propanoic acid (AMPA) receptors. J Med Chem;45:2101–2111. 20. Grisar J.M., Claxton G.P., Carr A.A., Wiech N.L. (1973) Hypoglycemic cycloalkyl lactamimides. J Med Chem;16:679–683.
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21. Markiewicz J.T., Wiest O., Helquist P. (2010) Synthesis of primary Aryl amines through a copper-assisted aromatic substitution reaction with sodium azide. J Org Chem;75:4887–4890. 22. Nikolaev V.O., Bunemann M., Hein L., Hannawacker A., Lohse M.J. (2004) Novel single chain cAMP sensors for receptor-induced signal propagation. J Biol Chem;279:37215–37218. 23. Borner S., Schwede F., Schlipp A., Berisha F., Calebiro D., Lohse M.J., Nikolaev V.O. (2011) FRET measurements of intracellular cAMP concentrations and cAMP analog permeability in intact cells. Nat Protoc;6:427–438.
Supporting Information Additional Supporting Information may be found in the online version of this article: Appendix S1. Materials and methods.
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Functional Non-Nucleoside Adenylyl Cyclase Inhibitors Marco Lelle1, Abdul Hameed1, Lisa-Maria Ackermann1, Stefka Kaloyanova1, Manfred Wagner1, Filip Berisha2, Viacheslav O. Nikolaev2 and Kalina Peneva1,* 1
Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany 2 Emmy Noether Group of the DFG, Department of Cardiology and Pneumology, European Heart Research €ttingen, Georg August University Medical Institute Go €ttingen, D-37075 Go €ttingen, Center, University of Go Germany *Corresponding author: Kalina Peneva, [email protected] In this study, we describe the synthesis of novel functional non-nucleoside adenylyl cyclase inhibitors, which can be easily modified with thiol containing biomolecules such as tumour targeting structures. The linkage between inhibitor and biomolecule contains cleavable bonds to enable efficient intracellular delivery in the reductive milieu of the cytosol as well as in the acidic environment within endosomes and lysosomes. The suitability of this synthetic approach was shown by the successful bioconjugation of a poor cellpermeable inhibitor with a cell-penetrating peptide. Additionally, we have demonstrated the excellent inhibitory effect of the compounds presented here in a € rster resonance energy transfer-based live-cell Fo assay in human embryonic kidney cells. Key words: adenylyl cyclase inhibitor, cancer treatment, pyridyl disulphide, tumour targeting Received 29 August 2014, revised 3 October 2014 and accepted for publication 7 October 2014
The membrane-bound enzyme adenylyl cyclase (AC) consists of nine different isoforms that catalyse the conversion of adenosine triphosphate into cyclic adenosine monophosphate (cAMP) (1,2). This intracellular process is stimulated by the binding of ligands such as hormones, nutrients, growth factors or neurotransmitters to their specific cell surface receptors (3). The cyclic nucleotide cAMP plays an undisputed role as ubiquitous second messenger and is also involved in the immune response (4). Elevated levels of cAMP are ordinarily harboured by naturally occurring T regulatory cells (Treg) and transferred to T cells via gap junctions (5,6). Thereby, immune response mediated by T cells is efficiently suppressed in patients suffering from ª 2014 John Wiley & Sons A/S. doi: 10.1111/cbdd.12452
malignant melanoma (7). Accumulation of Tregs in tumours is considered to prevent effective antitumor immune responses, consequently leading to tumour growth and progression (8,9). Thus, the manipulation of Tregs represents a promising strategy for the treatment of cancer and has become an emerging target in cancer immunotherapy (7,8). As cAMP is a crucial component in Treg-dependent suppression, the downregulation of this messenger would present a powerful opportunity to combat cancer through T-cells-mediated activation of the immune system. To efficiently decrease intracellular cAMP levels, numerous AC inhibitors have been widely applied. Several adenosine derivatives such as adenosine monophosphate, 2-deoxyadenosine or 9-(tetrahydrofuryl)-adenine were found to potently inhibit different types of ACs (10). However, their systemic application is typically accompanied by severe side-effects and can interfere with DNA synthesis (10,11). These limitations can be successfully circumvented with non-nucleoside-based AC inhibitors. 2-Amino-7-(2-furanyl)7,8-dihydro-5(6H)-quinazolinone and cis-N-((1R,2R)-2-phenylcyclopentyl)-azacyclotridec-1-en-2-amine, also referred to as NKY80 (1) and MDL 12330A (2), are well-studied inhibitors that lack an intact adenine ring (12,13). Nevertheless, both substances, like their adenosine counterparts, exhibit limited cell membrane permeability as well as low solubility in aqueous media (10,11,14). Moreover, 1 and 2 are associated with a poor tumour tissue specificity, which significantly decreases the pharmaceutical relevance for cancer therapy. We have designed and prepared functional derivatives of non-nucleoside AC inhibitors, bearing a 2-pyridyl disulphide functionality for the attachment of cell-permeable (cell-penetrating peptide) or tumour targeting (folic acid, octreotide, antibodies) moieties, that address the aforementioned limitations (Figure 1).
O N
N N O
NH
NH2
1 2
Figure 1: Chemical structure adenylyl cyclase inhibitors.
of
common
non-nucleoside
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and they can serve as prototypic tumour vectors in vivo (17,18). The inhibitor peptide conjugate 7 was isolated by preparative HPLC and displayed an excellent water solubility. This modification approach enables the inhibitor release in the acidic environment within endosomes and lysosomes upon cellular uptake and subsequent cleavage of the hydrazine bond. Additionally, the conjugate can be cleaved by intracellular glutathione, a naturally occurring peptide that is capable of reducing disulphide bonds and its increased concentration in the cytosol has been reported in many tumour cells, thus providing additional selectivity of the conjugates towards cancerous cells. However, AC inhibitor 2 cannot be directly modified with the cross-linking reagent due to the lack of an intrinsic aldehyde or ketone function (Scheme 2).
To introduce the 2-pyridyl disulphide in the inhibitor structure, a small cross-linker with an additional hydrazide group was chosen. The required cross-linking reagent was prepared in two straightforward steps from methyl 3-mercaptopropionate (3). Initially, the methyl ester was transformed into the corresponding hydrazide 4 according to a literature procedure (15). Subsequently, the free thiol group was converted to 2-pyridyl disulphide with an excess of 2,20 -dithiopyridine. Unreacted 2,20 -dithiopyridine was removed by column chromatography, and the desired product 5 was obtained as a colourless solid in good yield. Due to the hydrazide functionality present in 5, aldehydes and ketones can be easily modified with this reagent. Thereby, an acid labile hydrazone bond can be created that is cleavable at slightly acidic pH (pH 5) and has already found extensive use in antibody–drug conjugates applied in vivo (16) (Scheme 1).
Thus, further modification of the original inhibitor molecule was required. A synthetic approach was chosen, which created a 4-bromophenyl derivative of 2. This was achieved by condensation of 2-azacyclotridecanone and a para-substituted cis-2-phenyl-1-amino-cyclopentane. The synthesis of the required cyclopentane derivative 11 was accomplished from 1,4-dibromobenzene as described before (19). Utilization of this synthetic route enabled the preparation of the requested cis amine by a ring opening of cyclopentene oxide with the Grignard reagent of 8 and subsequent inversion via Mitsunobu reaction. However, further modifications were carried out with both cis enantiomers. The following condensation of 11 and 2-azacyclotridecanone mediated by phosphoryl chloride to yield the bromo-substituted inhibitor was accomplished with a procedure described by Grisar et al. (20) for the synthesis of lactamimides. The obtained bromophenyl derivative 12
The AC inhibitor 1 bears a keto group suitable for the condensation with 5. Moreover, the crucial pharmacophore sequence (=C-N=C-NH2) of the inhibitor remains intact (10,13). The modification of the carbonyl was carried out in chloroform with an excess of the cross-linker and catalysed by trifluoroacetic acid. Under these conditions, the functional AC inhibitor precipitated and the product was filtered off. Analysis of the isolated substance by NMR spectroscopy revealed that 6 consists of two isomers, E and Z, respectively. Afterwards, the 2-pyridyl disulphide carrying inhibitor was coupled to the N-terminal cysteine of a cellpenetrating peptide, namely octaarginine. Oligoarginine peptides have been used to transport in vitro a large variety of cargo ranging from small drugs, proteins, polymer conjugates to nanoparticles across the cell membrane,
O
a
SH O
H2N
H N O
3
b
SH
H2N
4
H N
S
S
N
5
O
Scheme 1: Synthesis of cross-linking reagent 5. Reagents and conditions: (a) N2H4xH2O, MeOH, rt, overnight, 83%; (b) 2,20 dithiopyridine, DMF, rt, 4 h, 77%.
NH O O
O N N
O
1
a
N
S
S
NH
NH2
H2N
N
b
S O
N N O
N H
NH2
N
H N O
O N H
H N
O NH2
8
O
S NH
6 (E/Z)
O
NH H2N
O
NH2
N N O
NH2
7 (E/Z)
Scheme 2: Synthesis of functional AC inhibitor 6 and the corresponding cell-penetrating peptide derivative 7. Reagents and conditions: (a) 5, chloroform, TFA, rt, 14 h, 80%; (b) NH2-CGGWRRRRRRRR-NH2, DMF, phosphate buffer, rt, 2 h, 66%.
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Functional Non-Nucleoside Adenylyl Cyclase Inhibitors Br Br Br
c
a,b
8
N
Br
OH Br
9()
e
d
O
O
N
NH2
Br
12 ( )
11 ( )
10 ( )
HCl NH
Scheme 3: Synthesis of intermediate 12. Reagents and conditions: (a) Mg, THF, rt, 90 min; (b) cyclopentene oxide, CuI, THF, rt, 14 h, 37%; (c) PPh3, DIAD, phthalimide, THF, rt, overnight, 39%; (d) ethanolamine, 90 °C, 1 h, 78%; (e) 2-azacyclotridecanone, phosphoryl chloride, benzene, reflux, 24 h, 58%.
O Br N
12 ( )
Scheme 4: Synthesis of functional AC inhibitor 15. Reagents and conditions: (a) (Boc)2O, THF, NaHCO3 solution, rt, 14 h; (b) NaN3, L-proline, CuI, DMSO, 100 °C, 24 h, 34%; (c) 4-carboxybenzaldehyde, chloromethylenedimethylammonium chloride, DCM, rt, 2 h; (d) DIPEA, DCM, rt, 14 h, 74%; (e) dioxane, 4 M hydrochloric acid, rt, 2 h; (f) 5, chloroform/methanol (99:1), rt, 12 h, 53%.
O
HCl NH
O
HN
N O N O
13 ( )
N N
14 ( )
O N
S
O
HN N
S
e,f
enables versatile substitutions to create an AC inhibitor accessible for further bioconjugation chemistry (Scheme 3). To modify the inhibitor with cross-linker 5, the amidine group of 12 was protected at first and an aryl amine was prepared through a copper-assisted aromatic substitution reaction with sodium azide (21). Subsequent amide bond formation between 13 and 4-carboxybenzaldehyde, which was transferred into the corresponding acid chloride in the presence of the Vilsmeier reagent, led to the required aldehyde-bearing AC inhibitor derivative. In an initial step, the protective group of this molecule was removed under acidic conditions followed by direct condensation with the cross-linking reagent. The functional non-nucleoside inhibitor 15 was purified by HPLC, and its water solubility is comparable with the commercially available MDL 12330A (2), which enabled further experiments in aqueous buffer or cell culture media without aggregation or precipitation. Characterization by NMR spectroscopy showed two isomers of the obtained hydrazone with an E/Z ratio of 2:1. The 2-pyridyl disulphide group provides the opportunity to further react 15 with thiol containing biomolecules that can be further employed for selective targeting of cancer or immune cells in vivo (Scheme 4). To test the AC inhibitory activity of the new compounds, a €rster resonance energy live-cell assay that relies on a Fo transfer (FRET)-based cAMP biosensor Epac1-camps Chem Biol Drug Des 2015; 85: 633–637
O
c,d
H2N
a,b
HN
15 (E/Z) ( )
N NH
FRET change in response to ISO( %)
25 20 15 10 5 0
control
2
1
6
15
13
Figure 2: AC inhibitory activity of the compounds analysed in live 293A cells. Cells were stimulated with 10 nM of isoproterenol (ISO) after 5- to 7-min preincubation with DMSO (negative control) or various compounds (each at 100 lM concentration). MDL 12330A (1) and NKY-80 (2) were used as positive controls of the gold standard AC inhibitors. Bar graphs show changes of FRET in response to ISO, means SE, n = 10–22 cells per compound.
expressed in 293A cells was used (see Appendix S1) (22). This is the most standard cell line with sufficient expression of endogenous AC routinely used to monitor real-time effects of agonists and antagonists of the cAMP signalling pathway (23). Stimulation of cells with the b-adrenergic receptor agonist isoproterenol (ISO) leads to an AC-dependent increase in cAMP visualized by a change of FRET ratio. Preincubation of cells with the inhibitors for 5–7 min prior to (ISO) administration markedly decreased the cAMP production. The inhibitory effect of the compounds was 635
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comparable to that of the parental inhibitors MDL 12330A and NKY-80 (Figure 2). In addition, we performed luminescent luciferase-based adenosine triphosphate quantification assays (CellTiterGlo; Promega Corporation, Madison, WI, USA) to determine the cytotoxicity of the compounds. A293 cells were incubated with 1, 2, 6 and 15 at a concentration of 50 lM for 72 h in a serum-containing medium, and the obtained IC50 values for all compounds with exception of 2 (12.55 0.69 lM) were higher than 50 lM. These data clearly indicate that the functional non-nucleoside inhibitors described in this work are not cytotoxic. In summary, we have successfully synthesized two different 2-pyridyl disulphide-containing non-nucleoside adenylyl cyclase inhibitors as well as a tumour targeting and cellpermeable bioconjugate and tested their AC inhibitory activity in living cells. In a live-cell FRET-based assay performed in A293 cells, we have shown that the functionalization of the parent molecules does not impede their inhibitory function. This new class of functional inhibitor derivatives can address numerous limitations in the therapeutic application of AC inhibitors such as low cell permeability, poor solubility or tumour tissue selectivity by the facile modification with appropriate biomolecules. The herein utilized bioconjugation chemistry enables efficient inhibitor release within cancer cells due to the applied hydrolysable and reductively cleavable bonds. We believe that these novel functional AC inhibitors can thus establish a unique way in cancer therapy by triggering an antitumor immune response mediated through suppression of the production of intracellular cyclic adenosine monophosphate.
Acknowledgments This work was funded by the Max Planck Society.
Author Contributions M. Lelle and A. Hameed equally contributed to this work.
Conflict of Interest The authors declare no conflict of interest.
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Supporting Information Additional Supporting Information may be found in the online version of this article: Appendix S1. Materials and methods.
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