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15β-HYDROXYVINCADIFFORMINE, AN ALKALOID FROM THE LEAVES OF RHAZYA STRICTA
3721
Short Reports 13 Kulantharvel, P. and Pelletrer, S W. (1987) Tetrahedron Letters 28, 3883.
Phytochemrstry, Vol. 27, No 11, pp 3721-3723,
14. Pelletrer, S W, Chokshr, H P and Desar, H K (1986) J. Nat Prod. 49, 892
1988
0031-9422/88
Pnnted m GreatBntam
$300+000
0 1988PergamonPressplc
15j-HYDROXYVINCADIFFORMINE, AN ALKALOID OF RHAZYA STRICTA
FROM THE LEAVES
ATTA-UR-RAHMAN*, TALAT FATIMA and SAJIDA KHANUM H. E. J Research Institute of Chemistry, Umverslty of Karachi, Karachi 32, Pakwtan (Recezoed tn revzsedform 24 February 1988) Key Word Index-Rhazya r3C NMR, DEPT
Abstract-A
new alkaloid,
strzcta, Apocynaeeae,
alkaloid;
15fl-hydroxyvincadifformine
15fi-hydroxyvmcadrfformme,
has been isolated
INTRODUCTION Rhazya strzcta (Decaisne) is a small glabrous erect shrub, abundantly distributed in Pakistan [l-3]. The plant is well known m the mdrgenous system of medicine for the treatment of various diseases [4-71. Extracts of R. stricta showed anti-cancer and antineoplastic activity [8-l 1] We have previously reported a number of new alkaloids from the plant [4, 12-213. In contmuatron of our studies on the rsolatron and structure elucidation of new chemical constituents from the leaves of Rhazya stricta, we have isolated a new alkaloid, 15p-hydroxyvmcadrfformme (l), the structure of which has been established by spectroscopic studies.
RESULTSAND DISCUSSION The crude alkaloidal material obtained from the ethanolic extract of the fresh leaves of Rhazya stricta by conventional procedures [4,5] was subjected to column chromatography for prehminary fractionation The fraction obtained on elution with petrolchloroform (1:3) afforded a mixture of alkalords. This mixture was subjected to repeated chromatographrc purification on srhca gel (Merck, GF-254) m petrolchloroform-methanol (14:5.1). The faster moving alkaloid was obtained as a pale yellow amorphous material and gave a UV spectrum characteristic of the amlmoacrylate system. The IR spectrum showed peaks at 3420 (-NH), 3400 (OH) and 1690 cm-r (conjugated ester -N-C=C-CO,Me). The HRMS afforded M at 354 1943 (C,,H,,N,O,), indrcatmg the presence of 10 double bond equivalents in the molecule and a fragmentatton charactenstic of the Aspldosperma skeleton. The ‘HNMR spectrum (CDCI,,
homodecouplmg;
from the leaves of R. stricta.
300 MHz) showed the presence of 26 protons. A threeproton smglet at 63.75 was assigned to the ester methyl protons. Another three proton singlet at 60.67 (J1s,r9 = 7.5 Hz) was assigned to the methyl protons of the ethyl group. Two quartets resonated at 60.96 (Jr,,, is = 7.5 Hz) and 61.11 (J19,r18= 7.5 Hz) for the C-19 methylene protons of the ethyl group, their separate chemical shifts being on account of their prochrral nature. Such a differentiatron between the two methylene protons of the ethyl group has previously been observed in vindolme [22] and bannucme [23]. A multrplet for C-15~~ H appeared at 63.74 Other chemical shift assignments for 15/I-hydroxyvincadrfformine are shown m Table 1. Two dimensional NMR measurements (COSY-45,2D J-resolved) were carrred out to verify the assignments. The coupling mteractrons were established through COSY-45 spectrum while the multiplicities of the overlapping proton srgnals were determined from the 2D Jresolved spectrum. The assignments for the C-18 methyl protons at 6066 could thus be confirmed from the COSY-45 spectrum, which showed strong cross peaks wrth the srgnals at 60.99 for C-19Hcr and at 6 1.11 for C19H /I protons. The srgnal at 63.74 (C-15H a) showed strong cross peaks with the signal at 62.01 (C-14a), while the signal at 62.23 (C-5) showed cross peaks with the srgnal at 6 1.74 C-6 protons The COSY-45 spectrum of 15fi-hydroxyvmcadifformme (1) 1s presented m Fig. 1 with the important interactions indicated. The r3CNMR spectrum (CDCI,, 75 MHz, DEPT) of the compound showed a downfield signal at 6 169.30 for the ester carbonyl group. Another downfield quaternary signal at 6 167.23 was assrgned to the C-2 carbon atom. A downfield signal for the hydroxyl-bearing C-15 methine carbon resonated at 670.51, its downfield chemical shift
Short Reports
3122
Table 1 ‘HNMR spectral data of 15/I-hydroxyvlncadlfforrn~ne (1) H
pw 2.56 dd
3Ha 3HP
3.14ddd
5Ha 5HB 6Ha 6HB 9H 10H 11H
2.14 223 211 1.73 115 685 711
m m dd dd d t t
12H 14HB 14Ha 15Ha 17Ha 17Ha 18H 19Ha 19HB
6 80 d 180 m 200m 3 74 m 264 d 292d 067 t 096q 111 q
21Ha OCH, N-H
396s 375 P 893 s
J 32,38 = 1.5 1 Hz, J,,. 142= 8 0 Hz J38,,5.=S5Hz,J38,,48=98Hz, J 38,3.= 16 3 Hz
Ju
0
J 6.,S.=64Hz,Js,.68=11 5Hz J 6p,6m= 11 55 Hz, JsB, sa =4 3 Hz J 9,10=735 Hz J Io,,1=73Hz, 5,,,,=725Hz 5,,,,,=73Hz J I,.**- -72Hz, .
20
10
J 12 II---72Hz . SPH
/-. /SalI
\ lSoH,‘14atI
/
Pz
1 -t50
.’
J, ,#. , ,* = 15.0 Hz J ,,,,,,~=1467Hz J ,s,19=75Hz J 1sn.,s=75Hz J ,w.,a=735H7.
,*
.
l
,
:
30
9H /'IOH
B
.I 00
100
80
_..
60
40
0
10
PPm
Fig. 1
indicating the presence of 1SP-hydroxyl configuratlon, (in 1%hydroxy compound, the signal occurs at 667.88 [24]). The C-18 methyl carbon atom appeared at 68.45. Other
13C-NMR chemical shift assignments are presented in Table 2. This data led to structure (l), corresponding to
15p-hydroxyvincadlfformme, for the substance. lS/SHydroxyvmcadifformine is known synthetically as an mtermediate in the synthesis of tabersonine [24] EXPERIMENTAL
Table
2. 13C-NMR spectral data of 15/Ghydroxyvmcadifformme (1)
C
ppm
multlphclty
2 3 5 6 7 8 9 10 11 12 13
167.23 5129 47 43” 45 43” 55 57 137 50 121.20 120 70 127 70 109 47 143 35
15 14 16 17 18 19 20 21
7051 31 38 92 97 22 58 845 26 57 43 71 7471
: F
50 98 169 30
4 s
COOK COOMe a Assignments
s
t t t s s d d d d s
General. UV IR The ‘HNMR spectra were recorded at 300 MHz and the ‘“CNMR spectra at 75 MHz TLC expenments were performed on slhca gel (GF-254.0 2 mm) precoated plates Merck) The EtOH extracts of the fresh leaves (95 kg) of Rhazya smcta were concentrated to a gum which was dissolved m 10% HOAc (11) the non-alkaloldal part was removed by extractlon with EtOAc (25 l), the aq acldlc soln baslfied with q NH, (500 ml) to pH 11 and extracted with EtOAc (36 I) to afford the crude alkaloids (350 g) This dlkaloldal material was subjected to CC (alkafold silica gel, 1 40) Elutlon wtth mcreasmg polarltles of C,H,, petrol, CHCl,, EtOAc and MeOH afforded several fractlons. A fraction obtamed on elutlon with petrol-CHCI, (1 3) was found to be a mixture of alkaloids, (5g) Thrs dlkaloldal mixture was further purified on repeated column chromato-
t 4
t :
may be Interchanged.
15 p-
Hydrox~vlncadlftormlne
(1)
Short Reports
graphy over silica gel. Elutton with CHCI,-MeOH (9.1) alTorded a partly purtfied mtxture of alkaloids (30mg) Further purtficatton ytelded the new alkalotd, 15p-hydroxyvmcadilformme (1) (12 mg). 15B-hydroxyvmcadtfformine (1) gave a hght orange colouratton wtth Dragendorff’s reagent and deep blue colouratton wtth certc sulphate spray 158-Hydroxyumcad@brmme (1) [ctlo CHCI, = + 240.57” UV (MeOH). A,,, nm, 205, (log E 3.90) 224 (log E 3.85), 296 (log E 3.99), 327 (log E 4.17), I,,, nm, 218 (log E 4 58), 260 (log E 3.52), 305 (log E 3.20) IR (CHCI,) Y,,, cm _ t, 3420 (-NH), 3400 (-OH) 1690 (ester C=O) HRMS Observed MS rel, 354 (20%, C,,H,,N,O,), 336 1837 (6%, C,,H 24N 20 ZI) 323 1759 (5%, C,,Hs,N,O,), 141 1153 (25% C,H,,NO), 1401075 (lOO%, C,H,,NO). ‘H NMR (CDCI,, 3OOMHz) Table 1 r3CNMR (CDCI,, 75 MHz, DEPT) Table 2
REFERENCES 1 Hooker, J D and Jackson, B D. (1865) Index Kewenszs 4, 705 2. Basset, N G (1958) Ann Bogor 3, 105. 3 Atta-ur-Rahman and Fattma, K. (1982) J. Chem Sot Pak. 4, 121 4 Ahmad, Y., Fatima, K., Quesne, P W. Le and Atta-urRahman (1983) Phytochemlstry 22, 1017. 5. Chopra, R N, Nayar, S. L and Chopra, I. C. (1956) A Glossary of Indtan Medlcmal Plants, p. 212. C S I R , New Delht. 6. Dymock, W., Warden, C J. H. and Hooper, D. H. (1983) Pharmacographla Indlca Vol 3, p. 3911. Kegan, Paul, Trench, Trubner and Company, London
3723
7. Watt, G. (1892) A Dictronary of the Economrc Products of India. W. H. Allen, London. 8. Hooper, D (1906) Pharm. J. 77,258. 9. Siddiqm, S and Bukhan, A Q. S (1972) Nature 235, 393. IO. Mukhopadhyay, S., Handy, G. A., Funayama, S.,and Cordell, G A (1981) J. Nat. Prod. 44, 696 11. Mukhopadhyay, S., Sayed, A. El, Handy, G. A. and Cordell, G. A. (1983) J. Nat Prod. 46, 409. 12. Atta-ur-Rahman and Khanum, S (1984) Phytochemtstry 23, 709. 13 Atta-ur-Rahman and Khanum, S (1984) Heterocycles 22, 2183 14. Atta-ur-Rahman, Habib-ur-Rehman and Mahk, S. (1986) Heterocycles 24, 703 15 Atta-ur-Rahman, Mahk, S and Habm-ur-Rehman (1986) Phytochemtstry 25, 1731 16. Atta-ur-Rahman and Khanum, S (1984) Tetrahedron Letters 25, 3913 17 Atta-ur-Rahman and Khanum, S (1984) Phytochemzstry 24, 2023 18. Atta-ur-Rahman and Zaman, K. (1984) Heterocycles 22, 2023 19. Atta-ur-Rahman and Zaman, K. (1986) Phytochemzstry 25, 1779. 20 Atta-ur-Rahman and Khanum, S. (1985) Heterocycles 26, 405. 21 Atta-ur-Rahman, Khanum, S., Fattma, K ,Ahmad, Y. and Badar, Y (1987) Z. Naturforsch. 42 91. 22. Hunter, B. K., Hall, L. D. and Sanders, J. K. M (1983), J. Chem. Sot., Perkrn Trans. I. 657. 23. Atta-ur-Rahman, Ah, A. and Choudhary, M. I. (1986), J. Chem. Sot., Perkm Trans. I. 923 24. Kuehne, M E., Bornmann, W. G., Earley, W. G. and Marko, I. (1986) J Org Chem 51, 2913.
Short Reports 13 Kulantharvel, P. and Pelletrer, S W. (1987) Tetrahedron Letters 28, 3883.
Phytochemrstry, Vol. 27, No 11, pp 3721-3723,
14. Pelletrer, S W, Chokshr, H P and Desar, H K (1986) J. Nat Prod. 49, 892
1988
0031-9422/88
Pnnted m GreatBntam
$300+000
0 1988PergamonPressplc
15j-HYDROXYVINCADIFFORMINE, AN ALKALOID OF RHAZYA STRICTA
FROM THE LEAVES
ATTA-UR-RAHMAN*, TALAT FATIMA and SAJIDA KHANUM H. E. J Research Institute of Chemistry, Umverslty of Karachi, Karachi 32, Pakwtan (Recezoed tn revzsedform 24 February 1988) Key Word Index-Rhazya r3C NMR, DEPT
Abstract-A
new alkaloid,
strzcta, Apocynaeeae,
alkaloid;
15fl-hydroxyvincadifformine
15fi-hydroxyvmcadrfformme,
has been isolated
INTRODUCTION Rhazya strzcta (Decaisne) is a small glabrous erect shrub, abundantly distributed in Pakistan [l-3]. The plant is well known m the mdrgenous system of medicine for the treatment of various diseases [4-71. Extracts of R. stricta showed anti-cancer and antineoplastic activity [8-l 1] We have previously reported a number of new alkaloids from the plant [4, 12-213. In contmuatron of our studies on the rsolatron and structure elucidation of new chemical constituents from the leaves of Rhazya stricta, we have isolated a new alkaloid, 15p-hydroxyvmcadrfformme (l), the structure of which has been established by spectroscopic studies.
RESULTSAND DISCUSSION The crude alkaloidal material obtained from the ethanolic extract of the fresh leaves of Rhazya stricta by conventional procedures [4,5] was subjected to column chromatography for prehminary fractionation The fraction obtained on elution with petrolchloroform (1:3) afforded a mixture of alkalords. This mixture was subjected to repeated chromatographrc purification on srhca gel (Merck, GF-254) m petrolchloroform-methanol (14:5.1). The faster moving alkaloid was obtained as a pale yellow amorphous material and gave a UV spectrum characteristic of the amlmoacrylate system. The IR spectrum showed peaks at 3420 (-NH), 3400 (OH) and 1690 cm-r (conjugated ester -N-C=C-CO,Me). The HRMS afforded M at 354 1943 (C,,H,,N,O,), indrcatmg the presence of 10 double bond equivalents in the molecule and a fragmentatton charactenstic of the Aspldosperma skeleton. The ‘HNMR spectrum (CDCI,,
homodecouplmg;
from the leaves of R. stricta.
300 MHz) showed the presence of 26 protons. A threeproton smglet at 63.75 was assigned to the ester methyl protons. Another three proton singlet at 60.67 (J1s,r9 = 7.5 Hz) was assigned to the methyl protons of the ethyl group. Two quartets resonated at 60.96 (Jr,,, is = 7.5 Hz) and 61.11 (J19,r18= 7.5 Hz) for the C-19 methylene protons of the ethyl group, their separate chemical shifts being on account of their prochrral nature. Such a differentiatron between the two methylene protons of the ethyl group has previously been observed in vindolme [22] and bannucme [23]. A multrplet for C-15~~ H appeared at 63.74 Other chemical shift assignments for 15/I-hydroxyvincadrfformine are shown m Table 1. Two dimensional NMR measurements (COSY-45,2D J-resolved) were carrred out to verify the assignments. The coupling mteractrons were established through COSY-45 spectrum while the multiplicities of the overlapping proton srgnals were determined from the 2D Jresolved spectrum. The assignments for the C-18 methyl protons at 6066 could thus be confirmed from the COSY-45 spectrum, which showed strong cross peaks wrth the srgnals at 60.99 for C-19Hcr and at 6 1.11 for C19H /I protons. The srgnal at 63.74 (C-15H a) showed strong cross peaks with the signal at 62.01 (C-14a), while the signal at 62.23 (C-5) showed cross peaks with the srgnal at 6 1.74 C-6 protons The COSY-45 spectrum of 15fi-hydroxyvmcadifformme (1) 1s presented m Fig. 1 with the important interactions indicated. The r3CNMR spectrum (CDCI,, 75 MHz, DEPT) of the compound showed a downfield signal at 6 169.30 for the ester carbonyl group. Another downfield quaternary signal at 6 167.23 was assrgned to the C-2 carbon atom. A downfield signal for the hydroxyl-bearing C-15 methine carbon resonated at 670.51, its downfield chemical shift
Short Reports
3122
Table 1 ‘HNMR spectral data of 15/I-hydroxyvlncadlfforrn~ne (1) H
pw 2.56 dd
3Ha 3HP
3.14ddd
5Ha 5HB 6Ha 6HB 9H 10H 11H
2.14 223 211 1.73 115 685 711
m m dd dd d t t
12H 14HB 14Ha 15Ha 17Ha 17Ha 18H 19Ha 19HB
6 80 d 180 m 200m 3 74 m 264 d 292d 067 t 096q 111 q
21Ha OCH, N-H
396s 375 P 893 s
J 32,38 = 1.5 1 Hz, J,,. 142= 8 0 Hz J38,,5.=S5Hz,J38,,48=98Hz, J 38,3.= 16 3 Hz
Ju
0
J 6.,S.=64Hz,Js,.68=11 5Hz J 6p,6m= 11 55 Hz, JsB, sa =4 3 Hz J 9,10=735 Hz J Io,,1=73Hz, 5,,,,=725Hz 5,,,,,=73Hz J I,.**- -72Hz, .
20
10
J 12 II---72Hz . SPH
/-. /SalI
\ lSoH,‘14atI
/
Pz
1 -t50
.’
J, ,#. , ,* = 15.0 Hz J ,,,,,,~=1467Hz J ,s,19=75Hz J 1sn.,s=75Hz J ,w.,a=735H7.
,*
.
l
,
:
30
9H /'IOH
B
.I 00
100
80
_..
60
40
0
10
PPm
Fig. 1
indicating the presence of 1SP-hydroxyl configuratlon, (in 1%hydroxy compound, the signal occurs at 667.88 [24]). The C-18 methyl carbon atom appeared at 68.45. Other
13C-NMR chemical shift assignments are presented in Table 2. This data led to structure (l), corresponding to
15p-hydroxyvincadlfformme, for the substance. lS/SHydroxyvmcadifformine is known synthetically as an mtermediate in the synthesis of tabersonine [24] EXPERIMENTAL
Table
2. 13C-NMR spectral data of 15/Ghydroxyvmcadifformme (1)
C
ppm
multlphclty
2 3 5 6 7 8 9 10 11 12 13
167.23 5129 47 43” 45 43” 55 57 137 50 121.20 120 70 127 70 109 47 143 35
15 14 16 17 18 19 20 21
7051 31 38 92 97 22 58 845 26 57 43 71 7471
: F
50 98 169 30
4 s
COOK COOMe a Assignments
s
t t t s s d d d d s
General. UV IR The ‘HNMR spectra were recorded at 300 MHz and the ‘“CNMR spectra at 75 MHz TLC expenments were performed on slhca gel (GF-254.0 2 mm) precoated plates Merck) The EtOH extracts of the fresh leaves (95 kg) of Rhazya smcta were concentrated to a gum which was dissolved m 10% HOAc (11) the non-alkaloldal part was removed by extractlon with EtOAc (25 l), the aq acldlc soln baslfied with q NH, (500 ml) to pH 11 and extracted with EtOAc (36 I) to afford the crude alkaloids (350 g) This dlkaloldal material was subjected to CC (alkafold silica gel, 1 40) Elutlon wtth mcreasmg polarltles of C,H,, petrol, CHCl,, EtOAc and MeOH afforded several fractlons. A fraction obtamed on elutlon with petrol-CHCI, (1 3) was found to be a mixture of alkaloids, (5g) Thrs dlkaloldal mixture was further purified on repeated column chromato-
t 4
t :
may be Interchanged.
15 p-
Hydrox~vlncadlftormlne
(1)
Short Reports
graphy over silica gel. Elutton with CHCI,-MeOH (9.1) alTorded a partly purtfied mtxture of alkaloids (30mg) Further purtficatton ytelded the new alkalotd, 15p-hydroxyvmcadilformme (1) (12 mg). 15B-hydroxyvmcadtfformine (1) gave a hght orange colouratton wtth Dragendorff’s reagent and deep blue colouratton wtth certc sulphate spray 158-Hydroxyumcad@brmme (1) [ctlo CHCI, = + 240.57” UV (MeOH). A,,, nm, 205, (log E 3.90) 224 (log E 3.85), 296 (log E 3.99), 327 (log E 4.17), I,,, nm, 218 (log E 4 58), 260 (log E 3.52), 305 (log E 3.20) IR (CHCI,) Y,,, cm _ t, 3420 (-NH), 3400 (-OH) 1690 (ester C=O) HRMS Observed MS rel, 354 (20%, C,,H,,N,O,), 336 1837 (6%, C,,H 24N 20 ZI) 323 1759 (5%, C,,Hs,N,O,), 141 1153 (25% C,H,,NO), 1401075 (lOO%, C,H,,NO). ‘H NMR (CDCI,, 3OOMHz) Table 1 r3CNMR (CDCI,, 75 MHz, DEPT) Table 2
REFERENCES 1 Hooker, J D and Jackson, B D. (1865) Index Kewenszs 4, 705 2. Basset, N G (1958) Ann Bogor 3, 105. 3 Atta-ur-Rahman and Fattma, K. (1982) J. Chem Sot Pak. 4, 121 4 Ahmad, Y., Fatima, K., Quesne, P W. Le and Atta-urRahman (1983) Phytochemlstry 22, 1017. 5. Chopra, R N, Nayar, S. L and Chopra, I. C. (1956) A Glossary of Indtan Medlcmal Plants, p. 212. C S I R , New Delht. 6. Dymock, W., Warden, C J. H. and Hooper, D. H. (1983) Pharmacographla Indlca Vol 3, p. 3911. Kegan, Paul, Trench, Trubner and Company, London
3723
7. Watt, G. (1892) A Dictronary of the Economrc Products of India. W. H. Allen, London. 8. Hooper, D (1906) Pharm. J. 77,258. 9. Siddiqm, S and Bukhan, A Q. S (1972) Nature 235, 393. IO. Mukhopadhyay, S., Handy, G. A., Funayama, S.,and Cordell, G A (1981) J. Nat. Prod. 44, 696 11. Mukhopadhyay, S., Sayed, A. El, Handy, G. A. and Cordell, G. A. (1983) J. Nat Prod. 46, 409. 12. Atta-ur-Rahman and Khanum, S (1984) Phytochemtstry 23, 709. 13 Atta-ur-Rahman and Khanum, S (1984) Heterocycles 22, 2183 14. Atta-ur-Rahman, Habib-ur-Rehman and Mahk, S. (1986) Heterocycles 24, 703 15 Atta-ur-Rahman, Mahk, S and Habm-ur-Rehman (1986) Phytochemtstry 25, 1731 16. Atta-ur-Rahman and Khanum, S (1984) Tetrahedron Letters 25, 3913 17 Atta-ur-Rahman and Khanum, S (1984) Phytochemzstry 24, 2023 18. Atta-ur-Rahman and Zaman, K. (1984) Heterocycles 22, 2023 19. Atta-ur-Rahman and Zaman, K. (1986) Phytochemzstry 25, 1779. 20 Atta-ur-Rahman and Khanum, S. (1985) Heterocycles 26, 405. 21 Atta-ur-Rahman, Khanum, S., Fattma, K ,Ahmad, Y. and Badar, Y (1987) Z. Naturforsch. 42 91. 22. Hunter, B. K., Hall, L. D. and Sanders, J. K. M (1983), J. Chem. Sot., Perkrn Trans. I. 657. 23. Atta-ur-Rahman, Ah, A. and Choudhary, M. I. (1986), J. Chem. Sot., Perkm Trans. I. 923 24. Kuehne, M E., Bornmann, W. G., Earley, W. G. and Marko, I. (1986) J Org Chem 51, 2913.