Modification of guanosine with cyanopropargylic alcohols

Tetrahedron Letters 55 (2014) 5426–5429

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Modification of guanosine with cyanopropargylic alcohols Valentina V. Nosyreva, Anastasiya G. Mal’kina, Alexander I. Albanov, Boris A. Trofimov ⇑ A.E. Favorsky Irkutsk Institute of Chemistry, Siberian Branch of the Russian Academy of Sciences, 1 Favorsky Str., Irkutsk 664033, Russia

a r t i c l e

i n f o

Article history: Received 16 April 2014 Revised 16 July 2014 Accepted 6 August 2014 Available online 13 August 2014 Keywords: Guanosine Cyanopropargylic alcohols Guanosine cyclic ketals Nucleophilic addition Cyclization

a b s t r a c t Guanosine has been modified with tertiary cyanopropargylic alcohols under mild conditions (1:1.1– 2 molar ratio, K2CO3, DMF, 20–25 °C, 19–50 h) to simultaneously give two modifications. The first product (1:1 adduct) is formed by the stereoselective addition of the amide function of the purine ring to the triple bond (38–43% yields), and the second product is the 1:2 adduct, with a second molecule of cyanopropargylic alcohol having reacted with the two vicinal hydroxy groups of the ribose moiety to give a functionalized 1,3-dioxolane ring (29–50% yields). Ó 2014 Elsevier Ltd. All rights reserved.

Guanosine is an important nucleoside and metabolite1 possessing anti-oxidant and radioprotective properties, and protects DNA in vitro from damage by reactive oxygen species.2 It is incorporated in nucleic acids and biologically active guanyl nucleotides.3 Guanosine derivatives are effective against viruses such as vesicular stomatitis, vaccinia, and herpes viridae.4 Compounds with guanosine modifications are also specific irreversible inhibitors of RNA-polymerase5 and suppress the replication of HIV reverse transcriptase.6 The guanosine derivative, cadeguomycin, inhibits tumor growth and metastasis in association with modification of the immune system.7 Many known drugs (antiviral medicines: aciclovir, ganciclovir, penciclovir, and valaciclovir) are examples of modified guanosines.8 However, these guanosine derivatives are modified at the nitrogen atom at position 9, probably due to the lower reactivity of other positions, for example, the amide nitrogen (N-1). Indeed, the purine moiety has been modified with halo- and unsaturated compounds (C@O, C@C, C„N, and N@CAO) to deliver derivatives in low yields and with incomplete conversion of the guanosine.9 The reaction of guanosine with chloroacetaldehyde gave N2-ethenoguanosine (3-[3,4-dihydroxy-5-(hydroxymethyl) tetrahydro-2-furanyl]-3,5-dihydro-9H-imidazo[1,2-a]purin-9-one) in only 7.5% yield.9b The treatment of guanosine with methyl-Ncyanomethanimidate led to an annelated product involving N-1 (in 39% yield).9c,d When dioxyguanosine was reacted with methyl vinyl ketone, annelation with piperidine ring took place (yield 5– 10%).9e 8-Bromoguanosine was cross-coupled with phenylacetylene in the presence of Pd(PPh3)2Cl2, the yield of the target ⇑ Corresponding author. Tel.: +7 3952 242 1411; fax: +7 395 241 9346. E-mail address: boris_trofi[email protected] (B.A. Trofimov). http://dx.doi.org/10.1016/j.tetlet.2014.08.012 0040-4039/Ó 2014 Elsevier Ltd. All rights reserved.

8-(phenylethynyl)guanosine being 20%. The same reaction, but in the presence of Amberlite IRA-67 (other conditions being similar), gave the cross-coupled product in 83% yield.9h Furthermore, it might be expected that elaboration of easier modifications of other guanosine positions would open a route to additional opportunities for guanosine drug design. Some other modifications of guanosine relate to its ribose moiety. Refluxing guanosine in acetone for five hours in the presence of ZnCl2 gave a 1,3-dioxolane derivative involving the two vicinal groups at positions 20 and 30 .10a,b In addition, the synthesis of a cyclic diguanosine monophosphate, a bacterial signaling molecule, with thiourea, urea, carbodiimide, and guanidinium linkages between the ribose moieties was recently realized.10d The above examples show that guanosine-based medicines originating from guanosine modification remain challenging targets in current drug design. Therefore, the search for novel and straightforward approaches to guanosine modification represents an important task in modern organic synthesis. An uncommon route to new functionalized guanosine derivatives is the modification of guanosine with reactive acetylenic compounds. Indeed, data on the successful modification of guanosine with acetylenes are lacking in the literature. It has been mentioned that guanosine, in contrast to adenosine, does not react with alkyl 4chloro-2-butynoate esters or ethyl propiolate.11 Recently, we succeeded in modifying adenosine and cytidine with cyanopropargylic alcohols, the former reacting with the two vicinal hydroxyls,12a while the latter reacted with all three hydroxyls of the ribose moiety.12b Thus, readily accessible cyanopropargylic alcohols13 prove to be novel and prospective modifying agents for the nucleoside family, allowing simultaneous introduction of hydroxy and cyano

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CN

functions as well as ethylenic and acetal moieties. To extend this chemistry, we decided to investigate whether cyanopropargylic alcohols were also applicable for the modification of guanosine, which has so far remained poorly reactive toward common reactive acetylenes. Here, we report the first examples of guanosine modification with acetylenic compounds, specifically cyanopropargylic alcohols. After optimization, we found that guanosine reacts stereoselectively with cyanopropargylic alcohols 1a–d under mild conditions (30– 50 mol % K2CO3, DMF, 20–25 °C, 19–50 h) to afford simultaneously two modifications (Table 1). The first is 1:1 adducts 2a–d in 38– 43% yields with Z-configuration on the amide moiety of the purine, and with the ribose moiety remaining intact. The second is the 1:2 adducts 3a–d with another molecule of the acetylene 1a–d attached to the ribose moiety via its two vicinal hydroxy groups at positions 20 and 30 to form a 1,3-dioxolane, in 29–50% yield.14 Completion of the reactions was monitored by TLC up to the disappearance of the starting acetylene spot. Owing to different reactivities of the reactants, the reaction times shown in Table 1 vary. The molar ratio of the reactants influences the yields of the modifications and conversion of guanosine (Table 1). At a guanosine:1a molar ratio of 1:1.1 (50 mol % K2CO3), the yields of products 2a and 3a were 43% and 32%, respectively. In the presence of 30 mol % of K2CO3 the yields of products 2a and 3a were 22% and 13%, respectively. The reaction with a two-fold molar excess of acetylene 1a gave 40% and 50% yields of 2a and 3a, respectively, while for acetylenes 1c,d, at the same molar ratios, the yields of 2c,d and 3c,d were 44% and 38% and 35% and 29%, respectively.

Me Me

CN

20-25 o C, 24 h

OH

CN

O

N

N O

NH2

+ R1

CN

K2CO3, DMF

OH 1a-d

OH OH

20-25 oC, 19-50 h

N N O

N R1 NH2 N OH

2a-d

a

c d

OH

N

+

N O

R2

N R1 NH2 N OH

O O R2 CN

OH OH

b

O

R2

O

OH

Me

4

An attempt to accomplish this reaction under solvent-free conditions using a 10-fold excess of solid K2CO315 (guanosine/1/ K2CO3 = 1:1:10 by weight, 20–25 °C, 24 h) led to cyclic dimerization of the starting cyanopropargylic alcohol 1a to form 2,5-di(cyanomethylidene)-1,4-dioxane 4 (Scheme 1, yield 28%). Amazingly, no traces of the expected adducts were discernible in the reaction mixture in this case. Trialkylamines, for example, Et3N, showed no catalytic activity in this reaction. In the case of acetylene 1a, with 50 mol % of Et3N (no solvent, 20–25 °C, 24 h), 1,4-dioxane 4 was isolated in 45% yield. Under neat conditions (reaction temperature and time being the same), no reactions occurred. Under the best conditions found, the reaction time depends on the starting acetylenes 1a–d. In the case of acetylenes 1a,b, the reaction required up to 24 h, whereas the complete consumption of acetylenes 1c,d required 44 and 50 h. This is obviously a consequence of the steric screening of the reaction centers by the bulky cycloalkyl substituents. It is noteworthy that in no case was the step-wise addition of the hydroxy group to the triple bond observed (TLC). It follows that as soon as the first hydroxyl group is added to the triple bond, the neighboring vicinal hydroxyl closes

NC

R2

Me O

Scheme 1. Dimerization of 4-hydroxy-4-methyl-2-pentynenitrile (2a).

NC NH

O

Me

1a

Table 1 Modification of guanosine with cyanopropargylic alcohols 1a–d

N

Me

K2CO3

R1 OH

OH

3a-d

Acetylene

R1

R2

Ratio of guanosine:1 (mol)

Time (h)

Conversion of guanosine (%)

Product

Yield (%)

1a

Me

Me

1:1.1

20

—

1a

Me

Me

1:1.1

19

78

1a

Me

Me

1:2

24

82

1b

Me

Et

1:1.1

20

—

1b

Me

Et

1:2

24

78

1c

(CH2)4

1:1.1

30

—

1c

(CH2)4

1:2

44

72

1d

(CH2)5

1:1.1

30

—

1d

(CH2)5

1:2

50

75

2aa 3aa 2a 3a 2a 3ad 2b 3b 2b 3b 2c 3c 2c 3c 2d 3d 2d 3dd

22b 13b 43c 32c 40c 50c 19b 8b 36c 45c 12b 6b 44c 35c 10b 7b 38c 29c

In the presence of K2CO3 (30 mol %). Yield based on 1H NMR spectroscopy. Isolated yield after preparative column chromatography (based on consumed guanosine). Ratio of diastereomers = 3:1.

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NC

H

Supplementary data CH 3

O

N H3 C NH2 N OH

N N

OH

O OH OH Figure 1. Cross-peak in the 2D NOESY (1H–1H) spectrum of compound 2a.

the dioxolane ring at a much higher rate as compared to the first addition. It is important that both the modifications of guanosine (products 2 and 3) can be easily and cleanly separated by column chromatography. This is an advantage of the reaction that allows two different modifications of a poorly reactive11 nucleoside such as guanosine to be accomplished in one synthetic operation. The structures of the products were confirmed by NMR (1H, 13C, 15 N) spectroscopy using 2D techniques (COSY, NOESY, HMBC, and HSQC) and IR spectroscopy. The products 3a,d exist as mixtures of two diastereomers. The diastereomeric ratio is 3:1 and does not depend on the structure of the starting acetylenes 1a,d. In the 1H NMR spectra of adducts 2a–d and 3a–d, signals for the olefinic protons (@CHCN) were observed at 6.54–6.60 ppm. The protons of the CH2CN group of compounds 3a,c,d appeared as singlets at 3.20–3.23 ppm (major product). For the minor diastereomers (3a,c,d), the protons of this group occurred as singlets in the region 3.05–3.07 ppm. The hydroxyl group protons in compounds 2a,d and 3a,d were identified from 1H NMR spectra recorded at 60 °C and COSY. The signals of the hydroxyl group proton of the R1R2C(OH)C = moiety of compounds 2a and 3a occurred at 5.52–5.53 ppm and those of 2d and 3d were observed at 5.25– 5.27 ppm, respectively. In the 1H NMR spectra of adducts 3a,d, there was a proton signal for the hydroxyl group of the R1R2C(OH)CO2 fragment of the ribose moiety at 4.68 and 4.88 ppm (major product) and at 4.86 and 5.06 ppm (minor product), respectively. The configurational assignment of compound 2 was made using 2D NOESY spectra, where a cross-peak between the olefinic proton of the alkene nitrile fragment and the methyl group protons was observed, thus indicating the formation of one isomer of Z-configuration (Fig. 1). The 13C NMR spectra further confirmed the structures of compounds 2a–d and 3a–d. The IR spectra of synthesized compounds 2a–d and 3a–d were in agreement with their structures: the absorption bands were in the regions 3220–3429 (NH2, OH), 2229–2231 (@CCN), 1701–1707 (C@O), 1578–1660 (C@C, C@N, NH2), and 1040–1119 (COC) cm1. In the IR spectra of compounds 3a–d the bands belonging to the CN group of the (CH2CN) moiety were observed at 2256–2260 cm1. In conclusion, guanosine, a pharmaceutically important though resistant to modifications nucleoside, has been functionalized under mild conditions for the first time by reactions with readily available tertiary cyanopropargylic alcohols to afford two types of derivatives: one being the Z-adduct with the purine ring exclusively, and the other involving both the purine ring and the sugar moiety. It should be emphasized that the functions introduced are physiologically important hydroxyl, cyano, ethylenic, and cyclic acetal groups. The reaction allows these two different modifications of guanosine to be accomplished in one synthetic operation. The new guanosine modifications may be of interest for clinical trials as prospective antiviral, antitumor, and anti-HIV agents. Acknowledgment This work was supported by the Integration Project No. 5.9.

Supplementary data (experimental procedures for the preparation of compounds 2a–d, 3a–d and their spectroscopic characterization) associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.tetlet.2014.08.012. References and notes 1. Becerra, A.; Lazcano, A. Orig. Life Evol. Biosph. 1998, 28, 539. 2. (a) Gudkov, S. V.; Shtarkman, I. N.; Smirnova, V. S.; Chernikov, A. V.; Bruskov, V. I. Dokl. Biochem. Biophys. 2006, 407, 47; (b) Gudkov, S. V.; Shtarkman, I. N.; Smirnova, V. S.; Chernikov, A. V.; Bruskov, V. I. Radiat. Res. 2006, 165, 538; (c) Gudkov, S. V.; Shtarkman, I. N.; Chernikov, A. V.; Usacheva, A. M.; Bruskov, V. I. Dokl. Biochem. Biophys. 2007, 413, 50; (d) Gu, J.; Leszczynski, J.; Schaefer, H. F. Chem. Rev. 2012, 112, 5603. 3. (a) Hamel, E.; Cashel, H. Proc. Nat. Acad. Sci. U.S.A. 1973, 70, 3250; (b) Cass, C. E.; Lowe, J. K.; Manchak, J. M.; Henderson, J. F. Cancer Res. 1977, 37, 3314; (c) Géci, I.; Filichev, V. V.; Pedersen, E. B. Chem. Eur. J. 2007, 13, 6379; (d) Iglesias-Gato, D.; Martín-Marcos, P.; Santos, M. A.; Hinnebusch, A. G.; Tamame, M. Genetics 2011, 187, 105; (e) Zhang, H.; Li, F.; Dever, B.; Li, X.-F.; Le, X. C. Chem. Rev. 2013, 113, 2812. 4. (a) De Clercq, E.; Holy, A. J. Med. Chem. 1979, 22, 510; (b) Collins, P.; Baner, D. J. J. Antimicrob. Chemother. 1979, 5, 431; (c) Fomina, A. N.; Nikolaeva, I. S.; Pushkina, T. V.; Mazan’ko, T. K.; Filatov, F. P. Pharm. Chem. J. 1986, 20, 140; (d) Studentsov, E. P.; Gemkin, D. D.; Burlakov, S.D.; Tets, V. V.; Surkov, K. G. RU Patent 2111967, 1996; Chem. Abstr. 1998, 132, 322076. 5. Khropov, Y. V.; Pinchuk, N. Ya.; Baranova, L. A.; Tunitskaya, V. L.; Kochetkov, S. N. RU Patent 2035468, 1990; Chem. Abstr. 1995, 124, 139766. 6. Korovina, A. N.; Yas’ko, M. V.; Ivanov, A. V.; Handazhinskaya, A. L.; Karamov, E. V.; Kornilova, G. V.; Kukhanova, M. K. Vestn. Mosk. Univ., Ser. 2 Khim. 2008, 49, 108. Chem. Abstr. 2008, 150, 352422. 7. (a) Tanaka, N.; Wu, R. T.; Okabe, T.; Yamashita, H.; Shimazu, A.; Nishimura, T. J. Antibiot. 1982, 35, 272; (b) Yuan, B. D.; Wu, R. T.; Sato, I.; Okabe, T.; Suzuki, H.; Nishimura, T.; Tanaka, N. J. Antibiot. 1985, 38, 642; (c) Kondo, T.; Okamoto, K.; Yamamoto, M.; Goto, T.; Tanaka, N. Tetrahedron 1986, 42, 199; (d) Tumkevicius, S.; Dodonova, J. Chem. Heterocycl. Compd. 2012, 258 (Russian Original, Khim. Geterotsikl. Soed. 2012, 275). 8. (a) Kleemann, A.; Engel, J.; Kutscher, B.; Reichert, D. In Pharmaceutical Substances: Syntheses, Patents, Applications; Thieme: Stuttgart, New York, 2001. pp. 31–32, 954–955, 1575–1576, 2154.; (b) Mashkovskii, M. D. Lekarstvennye Sredstva [Medicines] In ; Novaya Volna: Moscow, 2003; 2,; pp 331–332; (c) Aleksandrova, E V.; Kochergin, P. M. Chem. Heterocycl. Compd. 2009, 1 (Russian Original, Khim. Geterotsikl. Soed. 2009, 3). 9. (a) Loewwengart, G.; Van Duuren, B. L. Tetrahedron Lett. 1976, 3473; (b) Sattsangi, P. D.; Leonard, N. J.; Frihart, C. R. J. Org. Chem. 1977, 42, 3292; (c) Leonard, N. J.; Hosmane, R. S.; Agasimundin, Y. S.; Kostuba, L. J.; Oakes, F. T. J. Am. Chem. Soc. 1984, 106, 6847; (d) Agasimundin, Y. S.; Oakes, F. T.; Kostuba, L. J.; Leonard, N. J. J. Org. Chem. 1985, 50, 2468; (e) Chung, F.-L.; Roy, K. R.; Hecht, S. S. J. Org. Chem. 1988, 53, 14; (f) Nowak, I.; Robins, M. J. Org. Lett. 2003, 5, 3345; (g) Ustinov, A. M.; Stepanova, I. A.; Dubnyakova, V. V.; Zatsepin, T. S.; Nozhevnikova, E. V.; Korshun, V. A. Bioorg. Chem. 2010, 36, 437; (h) Firth, A. G.; Wilson, K.; Baumann, C. G.; Fairlamb, I. J. Nucleosides, Nucleotides, Nucleic Acids 2011, 30, 168; (i) Gillingham, D.; Tishinov, K. Synlett 2013, 24, 893. 10. (a) Hampton, A. J. Am. Chem. Soc. 1961, 83, 3640; (b) Amarnath, V.; Broom, A. D. Chem. Rev. 1977, 77, 183; (c) Youssif, S.; Mohamed, E. K.; Sayed Ahmed, A. F.; Ghoneim, A. A. Bull. Korean Chem. Soc. 2005, 26, 2021; (d) Gaffney, B. L.; Jones, R. A. Org. Lett. 2014, 16, 158. 11. (a) Olomucki, M.; Le Gall, J. Y.; Colinart, S. Tetrahedron Lett. 1984, 25, 3471; (b) Roques, P.; Le Gall, J. Y.; Lacombe, L.; Olomucki, M. J. Org. Chem. 1992, 57, 1579. 12. (a) Trofimov, B. A.; Nosyreva, V. V.; Shemyakina, O. A.; Mal’kina, A. G.; Albanov, A. I. Tetrahedron Lett. 2012, 53, 5769; (b) Nosyreva, V. V.; Mal’kina, A. G.; Volostnykh, O. G.; Albanov, A. I.; Trofimov, B. A. Synthesis 2013, 45, 3263. 13. (a) Landor, S. R.; Demetriou, B.; Grzeskowiak, R.; Pavey, D. J. Organomet. Chem. 1975, 93, 129; (b) Trofimov, B. A.; Andriyankova, L. V.; Shaikhudinova, S. I.; Kazantseva, T. I.; Mal’kina, A. G.; Zhivet’ev, S. A.; Afonin, A. V. Synthesis 2002, 853. 14. General procedure for the synthesis of compounds 2a–d and 3a–d: To a mixture of guanosine (283 mg, 1 mmol) and K2CO3 (69 mg, 0.5 mmol) in DMF (3 mL) was added a tertiary cyanopropargylic alcohol 1a–d (2 mmol) in DMF (2 mL) dropwise over 15 min. The mixture was stirred at 20–25 °C for 19–50 h. The mixture was passed through silica gel (1 cm, 70–230 mesh) using a Schott funnel, eluents: DMF (5 mL) and Et2O (5 mL). The solvents were removed in vacuo. The residue was washed with small portions of Et2O/EtOH (1:1, 5 mL) mixture and dried. The residue was chromatographed on a silica gel column sequentially changing the eluents (CHCl3/EtOH/benzene, 10:1:1, 4:1:1, 2:1:1, CHCl3/EtOH, 1:1, EtOH) to give guanosine (51 mg, 82% conversion in the case of acetylene 1a, 62 mg, 78% conversion in the case of acetylene 1b, 79 mg, 72% conversion in the case of acetylene 1c and 71 mg, 75% conversion in the case of acetylene 1d) and products 2a–d and 3a–d. Pure compounds 2a (Rf = 0.48– 0.50, CHCl3/EtOH, 1:1, silufol) and 3a (Rf = 0.65–0.68, CHCl3/EtOH, 1:1, silufol) were isolated by additional purification using column chromatography on silica gel by consecutive changing of the eluents (CHCl3/EtOH/benzene, 10:1:1,

V. V. Nosyreva et al. / Tetrahedron Letters 55 (2014) 5426–5429 4:1:1, 2:1:1, CHCl3/EtOH, 1:1); compound 2d (Rf = 0.22, CHCl3/EtOH/benzene, 2:1:1, silufol), and 3d (Rf = 0.22–0.25, CHCl3/EtOH/benzene, 10:1:1, silufol). (Z)-3-{2-Amino-9-[3,4-dihydroxy-5-(hydroxymethyl)tetrahydro-2-furanyl]-6oxo-6,9-dihydro-1H-purin-1-yl}-4-hydroxy-4-methyl-2-pentenenitrile (2a). Yield 129 mg (40%); light-yellow powder, mp 132–136 °C. IR (KBr): 3358, 3221, 2979, 2935, 2880, 2231, 1704, 1658, 1628, 1578, 1539, 1461, 1414, 1386, 1360, 1318, 1226, 1194, 1171, 1103, 1072, 1048, 953, 896, 865, 823, 775, 723, 699, 641, 591, 552 cm1. 1H NMR (400.13 MHz, DMSO-d6): d = 8.01 (s, 1H, H-8), 6.91 (br s, 2H, NH2), 6.59 (s, 1H, @CHCN), 5.72 (d, J = 5.6 Hz, 1H, H-10 ), 5.49 (s, 1H, HOC(CH3)2), 5.44 (d, J = 5.8 Hz, 1H, HOC-20 ), 5.12 (d, J = 5.3 Hz, 1H, HOC-30 ), 5.00 (t, J = 3.4 Hz, 1H, HOCH2), 4.44 (ddd, J = 5.6 Hz, J = 5.9 Hz, J = 5.8 Hz, 1H, H20 ), 4.10 (ddd, J = 3.9 Hz, J = 5.9 Hz, J = 5.3 Hz, 1H, H-30 ), 3.88 (m, 1H, H-40 ), 3.59, 3.54 (m, 2H, CH2OH), 1.41, 1.29 (s, 6H, (CH3)2COH). 13C NMR (100.61 MHz, DMSO-d6): d = 163.4 ((CH3)2C(OH)C@), 155.7 (C-6), 152.6 (C-2), 150.5 (C-4), 136.3 (C-8), 115.6 (@CHCN), 115.2 (C-5), 104.8 (@CHCN), 86.3 (C-10 ), 85.4 (C40 ), 73.8 ((CH3)2C(OH)C@), 72.9 (C-20 ), 70.5 (C-30 ), 61.5 (CH2OH), 30.2, 30.1 (CH3). 15N NMR (40.56 MHz, DMSO-d6): d = 235.6 (N-1), 232.2 (N-3), 226.8 (N-9), 146.7 (N-7), 129.3 (CN). Anal. Calcd for C16H20N6O6: C, 48.98; H, 5.14; N, 21.42. Found: C, 49.26; H, 5.24; N, 21.51. (Z)-3-{2-Amino-9-[2-(cyanomethyl)-6-(hydroxymethyl)-2-(1-hydroxy-1methylethyl)tetrahydrofuro[3,4-d][1,3]dioxol-4-yl]-6-oxo-6,9-dihydro-1H-purin1-yl}-4-hydroxy-4-methyl-2-pentenenitrile (3a). Yield 205 mg (50%); lightyellow powder, mp 152–156 °C. IR (KBr): 3417, 3222, 2982, 2938, 2880, 2260, 2230, 1707, 1660, 1627, 1579, 1539, 1465, 1424, 1386, 1363, 1320, 1256, 1225, 1171, 1106, 1057, 964, 904, 855, 826, 775, 663, 641, 590 cm1. 1H NMR (400.13 MHz, DMSO-d6) major diastereomer: d = 8.04 (s, 1H, H-8), 6.98 (br s, 2H, NH2), 6.59 (s, 1H, @CHCN), 6.11 (d, J = 2.3 Hz, 1H, H-10 ), 5.53 (s, 1H, HOC(CH3)2C@), 5.39 (dd, J = 2.3 Hz, J = 7.1 Hz, 1H, H-20 ), 5.23 (dd, J = 4.0 Hz, J = 7.1 Hz, 1H, H-30 ), 5.12 (t, J = 5.4 Hz, 1H, HOCH2), 4.92 (s, 1H,

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(CH3)2C(OH)CO2), 4.32 (dt, J = 5.1 Hz, J = 4.00 Hz, 1H, H-40 ), 3.60, 3.58 (ddd, J = 11.5 Hz, J = 5.4 Hz, J = 5.1 Hz, 2H, CH2OH), 3.20 (s, 2H, CH2CN), 1.41 (s, 6H, (CH3)2C(OH)C@), 1.11 (s, 6H, (CH3)2C(OH)CO2). 13C NMR (100.61 MHz, DMSOd6): d = 163.3 ((CH3)2C(OH)AC@), 155.7 (C-6), 152.7 (C-2), 149.7 (C-4), 136.9 (C-8), 118.2 (OCO), 118.1 (CH2CN), 115.6 (@CHCN), 115.2 (C-5), 104.9 (@CHCN), 88.4 (C-10 ), 87.8 (C-20 ), 87.4 (C-40 ), 84.3 (C-30 ), 74.8 ((CH3)2C(OH)CO2), 72.9 ((CH3)2C(OH)C@), 61.7 (CH2OH), 30.2, 28.8 ((CH3)2C(OH)C@), 25.0 (CH2CN), 24.8 ((CH3)2C(OH)CO2). 1H NMR (400.13 MHz, DMSO-d6) minor diastereomer: d = 8.01 (s, 1H, H-8), 6.99 (br s, 2H, NH2), 6.60 (s, 1H, @CHCN), 6.12 (d, J = 2.3 Hz, 1H, H-10 ), 5.53 (m, 1H, H-20 ), 5.52 (s, 1H, HOC(CH3)2C@), 5.28 (m, 1H, H-30 ), 5.10 (t, J = 4.3 Hz, 1H, HOCH2), 5.06 (s, 1H, (CH3)2C(OH)CO2), 4.42 (m, 1H, H-40 ), 3.59 (m, 2H, CH2OH), 3.06 (s, 2H, CH2CN), 1.42 (s, 6H, (CH3)2C(OH)C@), 1.24 (s, 6H, (CH3)2C(OH)CO2). 13C NMR (100.61 MHz, DMSO-d6): d = 162.5 (CH3)2C(OH)C@), 155.7 (C-6), 152.7 (C-2), 149.7 (C-4), 136.9 (C-8), 118.3 (OCO), 116.6 (CH2CN), 115.7 (@CHCN), 115.2 (C-5), 104.9 (@CHCN), 88.3 (C-10 ), 87.8 (C-20 ), 87.2 (C-40 ), 83.2 (C-30 ), 74.8 ((CH3)2C(OH)CO2), 72.7 ((CH3)2C(OH)C@), 61.6 (CH2OH), 31.0, 28.8 ((CH3)2C(OH)C@), 25.0 (CH2CN), 24.7 ((CH3)2C(OH)CO2). 15N NMR (40.56 MHz, DMSO-d6): d = 235.2 (N-1), 232.2 (N-3), 224.0 (N-9), 146.4 (N-7), 144.5 (CH2CN), 129.4 (@CHCN). Anal. Calcd for C22H27N7O7: C, 52.69; H, 5.43; N, 19.55. Found: C, 52.86; H, 5.24; N, 19.91. 15. (a) Trofimov, B. A.; Sobenina, L. N.; Stepanova, Z. V.; Vakul’skaya, T. I.; Kazheva, O. N.; Aleksandrov, G. G.; Dyachenko, O. A.; Mikhaleva, A. I. Tetrahedron 2008, 64, 5541; (b) Trofimov, B. A.; Sobenina, L. N. Ethynylation of pyrrole nucleus with haloacetylenes on active surfaces. In Targets in Heterocyclic SystemsChemistry and Properties; Attanasi, O. A., Spinelli, D., Eds.; Societa Chemica Italiana: Rome, 2009; Vol. 13, pp 92–119; (c) Sobenina, L. N.; Tomilin, D. N.; Trofimov, B. A. Russ. Chem. Rev. 2014, 83, 475.