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Iridoid aglycones from Deutzia scabra
0031~9422/84 $3.00+0.00
Phyrochemistry, Vol. 23, No. 4. pp. 803-807, 1984.
PergamonFTeasLtd.
Printedin GreatBritain.
IRIDOID AGLYCONES FROM DEUTZIA SCABRA PAOLA ESPOSITO, CARLO IAVARONE,ALINA SEN and CORRADOTROGOLO* Centro di Studio per la Chimica delle Sostanze Organiche Naturali de1 C.N.R., Roma, Dipartimento di Chimica dell’Universith di Roma, ‘La Sapien&, P. le Aldo Moro, 24185 Roma, Italy (Revised received 4 August 1983)
Key Word Index-Deutzia scabra; Saxifragaceae; scabrogenin; deutziogekn; natural iridoid aglycones.
Ahstrac&--Scabrogenin and deutziogenin, the stable aglycones of known iridoid glucosides scabroside and deutzioside, have been isolated from Deutzia scab-a. Their structures and mutarotational equilibria were investigated by detailed analysis of ‘H NMR and 13C NMR data.
RESULTSAND
INTRODWWON
The present paper deals with the isolation from Deutzia (1) and scabrogenin (2), the stable aglycones of iridoid glucosides deutzioside (3) [l] and scabroside (4) [Z], respectively, both co-occurring in the plant. The probable presence of 1 in Mentzelia decapetala was suggested by Danielson et al. [3]. As far as we know, this is the second Pase of isolation of free iridoid aglycones from natural sources after the well known genipin [4]. In fact the other reports published on this topic refer to the isolation of 3,4_dihydroiridoid aglycones neomatatabiol and isoneomatatabiol [5] and of silvestroside III [6], the aglycone of a bisiridoid diglycoside. All other known iridoid aglycones, including compound 1, have been obtained by enzymatic hydrolysis of parent glucosides [l, 73. scabra (Saxifragaceae) of deutziogenin
The occurrence in the leaves of Deutzia scabra of 1 and 2, the free aglycones of 3 and 4, respectively, was first suggested by paper chromatography of an ethanolic extract of fresh leaves prepared immediately after their collection. Two spots were observed which were identical in colour (vanillin reagent) with those of 3 and 4 but with higher R, values (1,0.75,pink; 2,0.66, grey-brown; 3,0.40, pink; 4, 0.21, grey-brown). The ethanolic extract was carefully concentrated in uacuo, at low temperature and in neutral conditions, to an aqueous suspension which was continuously extracted with petrol to remove chlorophyll and apolar compounds. The residue from the aqueous fraction gave, after repeated chromatographies on cellulose and silica gel, pure compounds 1 and 2.
*To whom correspondence should be addressed. OH.,
!Ie
R R’
la
2a R = R’=H,R’=OH
R=H,R’=OH
lb R=OH,R’=H
2b R’=R~=H,R --OH 5a R=H,p=OAc,R’=Ac
5b R=OAc,R’=H,R2=Ac
0 -B
-D
-
DISCUSSION
choose
4
3 803
804
P. ESPOSITOet al.
From the examination of the ‘H NMR and ’ %ZNMR spectra (Tables 1 and 2) it was evident that 1 and 2 were the aglycones of the parent iridoid glucosides 3 and 4. Both compounds were present as a mixture of the /3- and a-epimers at C-l in a ratio of about 2: 1, as shown by the integral trace of the corresponding signal multiplicities in their ‘H NMR spectra and by the similar intensity ratio observed in the “C NMR spectra (PND) for each pair of signals relative to different carbons. The assignment of the set of more intense ‘H NMR signals to the protons of the B-form was achieved by the correct identification of their Jl,9 values relative to the a and fl H-l protons in the pairs l/3 (1, J1.,9 = 10.0 Hz, J 1B,9 = 3,5Hz; 3, Jla,P = lO.OHz) and 2/4 (2, Jlaa9 = 10.5 Hz, J,,,? = 3.8 Hz; 4, J1.,9 = 9.2 Hz). Analogous differentiation of the a- and j?-epimeric forms by the 13C NMR spectral approach based only upon the analysis of direct J,,.-, coupling constant values of the hemiacetalic C-l carbbns was not so reliable, in agreement with a previous report of Tietze et al. [8] on C-l epimeric iridoid aglycone methyl ethers. In fact the ‘gated decoupled’ spectrum of 2 in D20 showed a J,, H_l value of 165 Hz for the more intense C-l resonance which was attributed to the B-form (Zb). This value was different = 172 Hz) of the parent glucoside (4) to that (J,, H_1a which has the 0-glucosyl unit at C-l in the same /3configuration. The :ess intense C-l resonance in the spectrum of 2, due to a-epimer (2a), had a JC_,,H_Ivalue of 172 Hz. Also, in scabrogenin acetate (5) the lowest J,,,,_, value (165 Hz) was linked to the more intense C-l doublet (/Lepimer, Sb) and the highest value (174 Hz) to the less intense doublet of the a-epimer (Sa). The possibility of an inversion due to mutarotational equilibration occurring during the long period the sample remained in the probe during the accumulation time required for the measurement of the ‘gated decoupled spectrum and thus leading to a preponderance of the aform, may be excluded because of the identical ‘H NMR spectra of 2 registered on the same sample immediately before and after taking the ‘gated decoupled’ spectrum. An analogous comparison of J,_,,,_, values of the corresponding pair 1 and 3 was hindered by the very low solubility of 3 in DzO which did not allow the recording of a satisfactory ‘gated decoupled’ spectrum of the parent glucoside 3. However, there was a perfect agreement of the ‘gated spectrum’ of 1 with that of 2, showing a Jcv,,,_, of 165 Hz for the more intense C-l signal (p-epimer) and a J,, H-l of 172 Hz for the less intense one (a-epimer) (Table 2). In an attempt to overcome the solubility problem of compounds in DzO a second set of ‘gated decoupled spectra were recorded for compounds l-4 in DMSO-de. However, the results (Table 2) were rather confusing since the Jw,H-1 values of the parent glucosides 3 (166 Hz) and 4 (165 Hz) were both intermediate with respect to the values measured for the pairs of epimeric C-l doublets of the corresponding aglycones 1 (162 and 171 Hz) and 2 (162 and 172 Hz), respectively. Thus, the criteria for distinguishing a- and pepimeric forms of iridoid aglycones (e.g. 1 and 2) using their ‘H NMR spectra by comparing their J1,9 values with those of the parent iridoids seems straightforward and can be diagnostic and is not solvent dependent (Table 1). However, the analysis of these compounds using their “C NMR spectra appears to be misleading and valueless showing, either in DzO or in DMSO-d,, rather different
J CIH_l values
between the B-aglycone and its parent iridbid having the same b-configuration at C-l. The appreciable shielding effect (ca 3-4 ppm) observable in both solvents for the C-l of a-aglycones with respect to the B-ones is worthy of note. The deshielding of the aglycone C-l carbon due to the glucosidation of hydroxyl1 (‘the glucosidation shift’) may be calculated as A&, = 6(R glucoside) - (/I-aglycone, RH); in the pairs lb/3 and 2bJ4 its values are 3.32 and 2.34 ppm in D20 and1.87 and 1.61 ppm in DMSO-de, respectively. The possibility that 1 and 2 may be artefacts is very unlikely owing to the mild conditions used for the extraction (ethanol at room temperature) and to their constant presence in extracts prepared and investigated immediately after the collection of plant material. The extractions, repeated several times on different sets of samples and in different conditions (pH and evaporation temperature) always led to the same yields in aglycones. Confirmation of the good stability of these aglycones was provided by the recovery of 1 and 2 chromatographitally unaffected after the measurement of the “C NMR spectra: their DzO solutions were only slightly coloured after 40 hr standing at a probe temperature of 36”. In addition 1 and 2 proved to be very stable at low temperature even after several months. The unexpected and misleading decrease of the J,, H_la observed in the comparison of scabroside (4) with thk /3form of its aglycone (2b) requires an investigation of the behaviour of other selected glucoside/&aglycone pairs for a possible rationalization of this conformational effect. EXPERIMENTAL
General techniques were as described earlier [ 111. Isolation ofthe iridoidfiaction. Paper chromatography (PC) of various EtOH extracts of fresh leavesof Deutzia scabra, obtained under different conditions (time, temp, pH) immediately after their harvest, showed the presence in constant amount of two sparingly polar compounds behaving like the aglycones of iridoid glucosides, namely deutzioside (3) and scabroside (4). In fact a PC, developed with n-BuOH-HOAc-H,O’ (63: 10:27) and visualized with vanillin reagent, showed at least five spots two of which were identical in colour to the iridoid glucosides present in the plant but with higher R, values: 0.75 (1, pink, deutziogenin), 0.66 (2, grey-brown, scabrogenin), 0.40 (3, pink, deutzioside), 0.34 (pink, deutziol) [ 12],0.21 (4, grey-brown, scabroside). Fresh aerial part of Deutzia scabra (6.4 kg) were collected in July 1980 in the Botanical Garden of the University of Rome (a reference specimen A 28-45 has been deposited there) and extracted with 9O”/dEtOH (15 1.)for 1 day at room temp and pH 7. The extract was coned at low temp to an aq. suspension and then continuously extracted with petrol 40-70” (2 1.).The residue was chromatographed on cellulose (350 g) in n-BuOH satd with Hz0 and afforded the following fractions: A 1 and 2 (_ 1:2), 1.5 g; B 1 (small amounts) and 2, 1.8 g; C compounds with lower R, value (deutziol, 3, 4), 10 g. Isolation of a- and @deutziogenin (1). Fraction A (1.5 g) chromatographed over 150g of silica gel in CHCl,-MeOH (95:5) afforded crude 1 (410 mg), still contaminated by 2, which was rechromatographed on silica gel (40g) in Et@-EtOAc (95:5)and gave pure l(60 mg)as anamorphous mixture ofSand a-epimers which crystallized very slowly. For analytical purposes only, a sample was crystallized from EtOAc, mp 145-153” (dec). (Found: C, 58.80; H, 6.63. C9H1204 requires C, 58.69; H, 6.57”/.) Isolation of a- and B-scabrogenin (2). Fraction E (1.8 g) chromatographed over 180 g of silica gel in CHCl,-MeOH (95:5)
sfs = Singlet with fine structure; J in Hz. *Covered by HDO signal present in DMSO-ds. tTaken from ref. [7]. *Taken from ref. [9]. #These signals are mutually overlapped. (/Taken from ref. [2]. TOverlapped by the H-5 signal of the /J-form. **Tentative assignment.
1.56$s
1.56sjs
3H-11
1.57sfs
2.4Odd J,., = 10.0 J,*, = 7.7
2.45 dd Jle9 = 3.6 Js,g = nm
H-8
2.12 dd J,e9 = 3.5 J,,, = 8.3
3.7-3.5
H-9
3.53.2*
3.7-3.5
Js,6 = 7.5 J 6.7 = 1.5
4.05 dd
3.1-3.5
J 3.6 = 7.7 = 8.4
= 7.5 = 7.5
= 7.5 = 1.5
1.57sfs 1.64s
2.55 dd J,,, = 10.0 J5,9 = 1.5
2.19dd
J6,, = 1.5 J,,* = 2.5 3.73d J,.* = 2.5
3.62 dd
J,,, J,,,
4.12dd
J,,, J,,,
2.07 t
6.151~1
4.80d J,,9 = 10.0
DzDS
J,., = 9.5 JSeg = 7.4
3.5-3.2.
3.53.2*
J,,,
3.81d
1.82t J,,, = 7.4 JS.g = 7.4
= 7.5 = 7.7
1.96 bt
6.05 sfs
J,., J,.,
6.106
3.5-3.2*
4.03 d
4.18d
1.827
JSe, = 7.5
2.1-2.7
5.92 $s
5.98 sfs
4.6Od J,,, = 10.0 4.4Od J,,, = 9.5
5.261 J,,, = 3.6
5.41 d J,,9 = 3.5
3.7-3.5
H-7
H-6
H-S
H-3
H-l
DMSO-de
DMSO-d6
DzO
DsO
lb
la
= 10.0
J 3,Me = 0.5
1.6Od
Jle9 = 10.0 J,,, = 7.5
2.31 dd
3.50d J 7.8 = 2.1
3.39 m
J 5,6 = 8.0 J,,, = 1.5
3.89 dd
JSF6 = 8.0 J5,9 = 7.5 J ,.Me = 0.5
1.91 m
J,,, = 0.5 J 3.m = 0.5
6.13m
J,,,
4.68 d
DMSO-d,t
3
1.6Osfs
2.78 d J,,9 = 3.8
3.8-3.6
3.8-3.6
4.304
6.136
5.54d J ,.9 = 3.8
DsO
1.49sfs
2.41 d J,,9 = 4.0
3.g3.1
3.8-3.1
4.00 bs
5.93 s@
5.25d J,., = 4.0
DMSO-de
2r
1.6Osf
2.52d J1,9 = 10.5
3.8-3.6
3.8-3.6
J6,, = 1.5
4.34&d
6.20 sfs
4.82d J 1.9 = 10.5
DsO
Table 1. ‘H NMR spectra (90 MHz) of iridoid aglycones and glucosides of Deutzia scabra
= 9.2
= 1.5
2.626 J ,,9 = 9.2
1.62sfs
1.49sfs
3.9-3.7
3.9-3.7
J,,,
4.33 d
6.25 sfs
J,,,
5.12d
DsO ”
2.241 J 1.9 = 10.0
3.8-3.1
3.8-3.1
4.00 bs
6.02 sfs
4.541 J,,, = 10.0
DMSO-I,
2b
= 10.0
1.5Osj~
J,.,
2.31 d
3.8-3.2
3.8-3.2
4.01**
6.08 sf
4.82 d J,,g = 10.0
DMSO-$
4
89.39 d (172) 133.03d (190) 112.99s 38.296 (132) 17.95 d (142) 61.06d (191) 51.63 d (192) 41.64d (Ml) 16.61 q (129)
(128)
16.7Oq
(186) nm
(188) 54.96 d
(142) 59.10d
76.51 d
(190) 109.78s nm
(171) 132.83d
88.62 d
DMSO-d,
(129)
(140) 16.11q
(191) 44.15d
(192) 56.66d
(142) 59.50 d
(135) 78.53 d
(190) 112.99s 41.13d
(165) 135.95.d
93.41 d
DrO
(128)
t 16.05 q
(189) 43.24d
(189) 54.18d
(141) 51.48 d
t 77.61 d
(188) 110.75s 40.39d
(162) 135.74d
92.95 d
DMSO&
lb
16.01 q
42.68d
56.24 d
59.68 d
78.58 d
113.48s 41.12d
135.75d
96.19 d
DsO
(128)
t 15.91 q
t 41.6Od
(190) 53.81 d
(141) 57.52 d
t 77.06d
(189) 111.42s 40.12d
(166) 135.07d
94.82d
DMSO-I,
3
(130)
(138) 11.84q
(193) 50.00 d
(191) 56.441
(142) 60.93 d
71.34d
(192) 113.88s 72.18s
(172) 136.43d
914Od
DrO
*Chemical shifts are expressed on the TMS scale according to the following equation S,,, the nearest f 1 Hz. IOverlapped by DMSO-d, signals. *Tentative assignment. §Taken from ref. [ 111. mn, Not measurable.
‘-11
c-9
C-8
c-7
c-6
c-5
c-3 C-4
‘-’
Dz0
la
(130)
(136) 11.52q
(193) 52.34d
(191) 55.96d
(142) 5958d
77.65 d
(191) 115.25s 74.13s
(165) 138.18d
94.541
DrO
(130)
(138) 11.52q
(190) 51.53d
(191) 54.44d
(142) 58.8Od
76.96 d
(187) 114.70s 72.11 s
(162) 136.58d
94.18d
DMSO-d,
2b
(129)
(140) 11.53q
(192) 50.19 d
(192) 56.07 d
(142) 59.78 d
77.06 d
(193) 115.65 s 74.52 s
(172) 137.89d
96.88 d
D#X
(130)
(nm) 11.34q
(189) 49.90 d
(190) 54.OOd
(141) 57.91 d
76.55 d
(189) 115.43 s 72.17s
(165) 135.94d
95.79 d
DMSO-I,
4
(128)
(run) 11.79q
47:84d
(nm) ’ 54.57$d
57.301
nm
(190) 113.18s mn
(174) 136.3Od
88.15 d
5a CDCl,
(128)
(142) 11.43q
(193) 49.29 d
(194) 54.5ld
5i.:d
78.22 d
(192) 113.18s 73.33 s
(165) 137.961
90.62 d
Sb CDCI,
= ?idioxane + 67.4 ppm. Values in parentheses are coupling constants, which have been quoted to
(130)
(138) 11.97q
(190) 49.45 d
(190) 55.02d
(142) 59.42 d
16.12d
(187) 112.59s 71.14s
(172) 135.161
90.49d
DMSO-d,
2a
Table 2. 13C NMR spectra (20 MHz) of iridoid aglyeones and glucosides of Dcutzia scabra*
Iridoid aglycones from Deutzia gave pure 2 (0.6 g) as an amorphous mixture (2: 1) of fi- and aepimers. For analytical purposes a sample was rechromatographed in the same conditions. (Found: C, 53.85; H.6.10. C,HisOs requires C, 53.99, H, 6.04 T,.) Di-0-acetylscabrogenin (5). Compound 2 (a- and b-epimers, 80 mg) was acetylated with pyridine and AcsO for 1 hr at room temp. After addition of MeOH the soln was left for 20 min and then evaporated to dryness. The residue, chromatographed on silica gel (7 g) in Et,O-CsH, (1: 1) afforded pure 5 (60 mg) as an inseparable mixture of 01-and /I-epimers.
REFERENCES 1. Bonadies, F., Esposito, P. and Guiso, M. (1974) Gazz. Chim. Ital. 104, 17. 2. Esposito, P. and Guiso, M. (1973) Gazz. Chim. Ital. 103,517. 3. Danielson, T. J., Hawes, E. M. and Bliss, C. A. (1975) J. Chromatogr. 103,216.
scabra
807
4. Djerassi, C., Nakano, T., James, A. N., Zalkov, L. H., Eisenbraun, E. J. and Shoolery, J. N. (1961) J. Org. Chem. 26, 1192. 5. Hyeon, S. B., Isoe, S. and Salran, T. (1968)Tetrahedron Letters 5325. 6. Jensen, S. R., Lyse-Petersen, S. E. and Nielsen, B. J. (1979) Phytocbemistry 18, 273. 7. Danielson, T. J., Hawes, E. M. and Bliss, C. A. (1973) Can. J. Chem. 51, 760. 8. Tie-, L. F., Niemeyer, LJ.,Marx, P. and Ghlsenkamp, K. H. (1980) Tetrahedron 36, 1231. 9. Jensen, S. R., Mikkelsen, C. B. and Nielsen, B. J. (1981) Phytocbemistry 20, 71. 10. Bianco, A., Caciola, P., Guiso, M., Iavarone, C. and Trogolo, C. (1981) Gmz. Chim. Ital. 111, 201. 11. Bonini, C., Davini, E., Iavarone, C. and Trogolo, C. (1981) Phytochemistry 20, 1587. 12. Esposito, P., Guiso, M., Nicoletti, M. and De Luca, C. (1976) Gazz. Chim. Ital. 106, 57.
Phyrochemistry, Vol. 23, No. 4. pp. 803-807, 1984.
PergamonFTeasLtd.
Printedin GreatBritain.
IRIDOID AGLYCONES FROM DEUTZIA SCABRA PAOLA ESPOSITO, CARLO IAVARONE,ALINA SEN and CORRADOTROGOLO* Centro di Studio per la Chimica delle Sostanze Organiche Naturali de1 C.N.R., Roma, Dipartimento di Chimica dell’Universith di Roma, ‘La Sapien&, P. le Aldo Moro, 24185 Roma, Italy (Revised received 4 August 1983)
Key Word Index-Deutzia scabra; Saxifragaceae; scabrogenin; deutziogekn; natural iridoid aglycones.
Ahstrac&--Scabrogenin and deutziogenin, the stable aglycones of known iridoid glucosides scabroside and deutzioside, have been isolated from Deutzia scab-a. Their structures and mutarotational equilibria were investigated by detailed analysis of ‘H NMR and 13C NMR data.
RESULTSAND
INTRODWWON
The present paper deals with the isolation from Deutzia (1) and scabrogenin (2), the stable aglycones of iridoid glucosides deutzioside (3) [l] and scabroside (4) [Z], respectively, both co-occurring in the plant. The probable presence of 1 in Mentzelia decapetala was suggested by Danielson et al. [3]. As far as we know, this is the second Pase of isolation of free iridoid aglycones from natural sources after the well known genipin [4]. In fact the other reports published on this topic refer to the isolation of 3,4_dihydroiridoid aglycones neomatatabiol and isoneomatatabiol [5] and of silvestroside III [6], the aglycone of a bisiridoid diglycoside. All other known iridoid aglycones, including compound 1, have been obtained by enzymatic hydrolysis of parent glucosides [l, 73. scabra (Saxifragaceae) of deutziogenin
The occurrence in the leaves of Deutzia scabra of 1 and 2, the free aglycones of 3 and 4, respectively, was first suggested by paper chromatography of an ethanolic extract of fresh leaves prepared immediately after their collection. Two spots were observed which were identical in colour (vanillin reagent) with those of 3 and 4 but with higher R, values (1,0.75,pink; 2,0.66, grey-brown; 3,0.40, pink; 4, 0.21, grey-brown). The ethanolic extract was carefully concentrated in uacuo, at low temperature and in neutral conditions, to an aqueous suspension which was continuously extracted with petrol to remove chlorophyll and apolar compounds. The residue from the aqueous fraction gave, after repeated chromatographies on cellulose and silica gel, pure compounds 1 and 2.
*To whom correspondence should be addressed. OH.,
!Ie
R R’
la
2a R = R’=H,R’=OH
R=H,R’=OH
lb R=OH,R’=H
2b R’=R~=H,R --OH 5a R=H,p=OAc,R’=Ac
5b R=OAc,R’=H,R2=Ac
0 -B
-D
-
DISCUSSION
choose
4
3 803
804
P. ESPOSITOet al.
From the examination of the ‘H NMR and ’ %ZNMR spectra (Tables 1 and 2) it was evident that 1 and 2 were the aglycones of the parent iridoid glucosides 3 and 4. Both compounds were present as a mixture of the /3- and a-epimers at C-l in a ratio of about 2: 1, as shown by the integral trace of the corresponding signal multiplicities in their ‘H NMR spectra and by the similar intensity ratio observed in the “C NMR spectra (PND) for each pair of signals relative to different carbons. The assignment of the set of more intense ‘H NMR signals to the protons of the B-form was achieved by the correct identification of their Jl,9 values relative to the a and fl H-l protons in the pairs l/3 (1, J1.,9 = 10.0 Hz, J 1B,9 = 3,5Hz; 3, Jla,P = lO.OHz) and 2/4 (2, Jlaa9 = 10.5 Hz, J,,,? = 3.8 Hz; 4, J1.,9 = 9.2 Hz). Analogous differentiation of the a- and j?-epimeric forms by the 13C NMR spectral approach based only upon the analysis of direct J,,.-, coupling constant values of the hemiacetalic C-l carbbns was not so reliable, in agreement with a previous report of Tietze et al. [8] on C-l epimeric iridoid aglycone methyl ethers. In fact the ‘gated decoupled’ spectrum of 2 in D20 showed a J,, H_l value of 165 Hz for the more intense C-l resonance which was attributed to the B-form (Zb). This value was different = 172 Hz) of the parent glucoside (4) to that (J,, H_1a which has the 0-glucosyl unit at C-l in the same /3configuration. The :ess intense C-l resonance in the spectrum of 2, due to a-epimer (2a), had a JC_,,H_Ivalue of 172 Hz. Also, in scabrogenin acetate (5) the lowest J,,,,_, value (165 Hz) was linked to the more intense C-l doublet (/Lepimer, Sb) and the highest value (174 Hz) to the less intense doublet of the a-epimer (Sa). The possibility of an inversion due to mutarotational equilibration occurring during the long period the sample remained in the probe during the accumulation time required for the measurement of the ‘gated decoupled spectrum and thus leading to a preponderance of the aform, may be excluded because of the identical ‘H NMR spectra of 2 registered on the same sample immediately before and after taking the ‘gated decoupled’ spectrum. An analogous comparison of J,_,,,_, values of the corresponding pair 1 and 3 was hindered by the very low solubility of 3 in DzO which did not allow the recording of a satisfactory ‘gated decoupled’ spectrum of the parent glucoside 3. However, there was a perfect agreement of the ‘gated spectrum’ of 1 with that of 2, showing a Jcv,,,_, of 165 Hz for the more intense C-l signal (p-epimer) and a J,, H-l of 172 Hz for the less intense one (a-epimer) (Table 2). In an attempt to overcome the solubility problem of compounds in DzO a second set of ‘gated decoupled spectra were recorded for compounds l-4 in DMSO-de. However, the results (Table 2) were rather confusing since the Jw,H-1 values of the parent glucosides 3 (166 Hz) and 4 (165 Hz) were both intermediate with respect to the values measured for the pairs of epimeric C-l doublets of the corresponding aglycones 1 (162 and 171 Hz) and 2 (162 and 172 Hz), respectively. Thus, the criteria for distinguishing a- and pepimeric forms of iridoid aglycones (e.g. 1 and 2) using their ‘H NMR spectra by comparing their J1,9 values with those of the parent iridoids seems straightforward and can be diagnostic and is not solvent dependent (Table 1). However, the analysis of these compounds using their “C NMR spectra appears to be misleading and valueless showing, either in DzO or in DMSO-d,, rather different
J CIH_l values
between the B-aglycone and its parent iridbid having the same b-configuration at C-l. The appreciable shielding effect (ca 3-4 ppm) observable in both solvents for the C-l of a-aglycones with respect to the B-ones is worthy of note. The deshielding of the aglycone C-l carbon due to the glucosidation of hydroxyl1 (‘the glucosidation shift’) may be calculated as A&, = 6(R glucoside) - (/I-aglycone, RH); in the pairs lb/3 and 2bJ4 its values are 3.32 and 2.34 ppm in D20 and1.87 and 1.61 ppm in DMSO-de, respectively. The possibility that 1 and 2 may be artefacts is very unlikely owing to the mild conditions used for the extraction (ethanol at room temperature) and to their constant presence in extracts prepared and investigated immediately after the collection of plant material. The extractions, repeated several times on different sets of samples and in different conditions (pH and evaporation temperature) always led to the same yields in aglycones. Confirmation of the good stability of these aglycones was provided by the recovery of 1 and 2 chromatographitally unaffected after the measurement of the “C NMR spectra: their DzO solutions were only slightly coloured after 40 hr standing at a probe temperature of 36”. In addition 1 and 2 proved to be very stable at low temperature even after several months. The unexpected and misleading decrease of the J,, H_la observed in the comparison of scabroside (4) with thk /3form of its aglycone (2b) requires an investigation of the behaviour of other selected glucoside/&aglycone pairs for a possible rationalization of this conformational effect. EXPERIMENTAL
General techniques were as described earlier [ 111. Isolation ofthe iridoidfiaction. Paper chromatography (PC) of various EtOH extracts of fresh leavesof Deutzia scabra, obtained under different conditions (time, temp, pH) immediately after their harvest, showed the presence in constant amount of two sparingly polar compounds behaving like the aglycones of iridoid glucosides, namely deutzioside (3) and scabroside (4). In fact a PC, developed with n-BuOH-HOAc-H,O’ (63: 10:27) and visualized with vanillin reagent, showed at least five spots two of which were identical in colour to the iridoid glucosides present in the plant but with higher R, values: 0.75 (1, pink, deutziogenin), 0.66 (2, grey-brown, scabrogenin), 0.40 (3, pink, deutzioside), 0.34 (pink, deutziol) [ 12],0.21 (4, grey-brown, scabroside). Fresh aerial part of Deutzia scabra (6.4 kg) were collected in July 1980 in the Botanical Garden of the University of Rome (a reference specimen A 28-45 has been deposited there) and extracted with 9O”/dEtOH (15 1.)for 1 day at room temp and pH 7. The extract was coned at low temp to an aq. suspension and then continuously extracted with petrol 40-70” (2 1.).The residue was chromatographed on cellulose (350 g) in n-BuOH satd with Hz0 and afforded the following fractions: A 1 and 2 (_ 1:2), 1.5 g; B 1 (small amounts) and 2, 1.8 g; C compounds with lower R, value (deutziol, 3, 4), 10 g. Isolation of a- and @deutziogenin (1). Fraction A (1.5 g) chromatographed over 150g of silica gel in CHCl,-MeOH (95:5) afforded crude 1 (410 mg), still contaminated by 2, which was rechromatographed on silica gel (40g) in Et@-EtOAc (95:5)and gave pure l(60 mg)as anamorphous mixture ofSand a-epimers which crystallized very slowly. For analytical purposes only, a sample was crystallized from EtOAc, mp 145-153” (dec). (Found: C, 58.80; H, 6.63. C9H1204 requires C, 58.69; H, 6.57”/.) Isolation of a- and B-scabrogenin (2). Fraction E (1.8 g) chromatographed over 180 g of silica gel in CHCl,-MeOH (95:5)
sfs = Singlet with fine structure; J in Hz. *Covered by HDO signal present in DMSO-ds. tTaken from ref. [7]. *Taken from ref. [9]. #These signals are mutually overlapped. (/Taken from ref. [2]. TOverlapped by the H-5 signal of the /J-form. **Tentative assignment.
1.56$s
1.56sjs
3H-11
1.57sfs
2.4Odd J,., = 10.0 J,*, = 7.7
2.45 dd Jle9 = 3.6 Js,g = nm
H-8
2.12 dd J,e9 = 3.5 J,,, = 8.3
3.7-3.5
H-9
3.53.2*
3.7-3.5
Js,6 = 7.5 J 6.7 = 1.5
4.05 dd
3.1-3.5
J 3.6 = 7.7 = 8.4
= 7.5 = 7.5
= 7.5 = 1.5
1.57sfs 1.64s
2.55 dd J,,, = 10.0 J5,9 = 1.5
2.19dd
J6,, = 1.5 J,,* = 2.5 3.73d J,.* = 2.5
3.62 dd
J,,, J,,,
4.12dd
J,,, J,,,
2.07 t
6.151~1
4.80d J,,9 = 10.0
DzDS
J,., = 9.5 JSeg = 7.4
3.5-3.2.
3.53.2*
J,,,
3.81d
1.82t J,,, = 7.4 JS.g = 7.4
= 7.5 = 7.7
1.96 bt
6.05 sfs
J,., J,.,
6.106
3.5-3.2*
4.03 d
4.18d
1.827
JSe, = 7.5
2.1-2.7
5.92 $s
5.98 sfs
4.6Od J,,, = 10.0 4.4Od J,,, = 9.5
5.261 J,,, = 3.6
5.41 d J,,9 = 3.5
3.7-3.5
H-7
H-6
H-S
H-3
H-l
DMSO-de
DMSO-d6
DzO
DsO
lb
la
= 10.0
J 3,Me = 0.5
1.6Od
Jle9 = 10.0 J,,, = 7.5
2.31 dd
3.50d J 7.8 = 2.1
3.39 m
J 5,6 = 8.0 J,,, = 1.5
3.89 dd
JSF6 = 8.0 J5,9 = 7.5 J ,.Me = 0.5
1.91 m
J,,, = 0.5 J 3.m = 0.5
6.13m
J,,,
4.68 d
DMSO-d,t
3
1.6Osfs
2.78 d J,,9 = 3.8
3.8-3.6
3.8-3.6
4.304
6.136
5.54d J ,.9 = 3.8
DsO
1.49sfs
2.41 d J,,9 = 4.0
3.g3.1
3.8-3.1
4.00 bs
5.93 s@
5.25d J,., = 4.0
DMSO-de
2r
1.6Osf
2.52d J1,9 = 10.5
3.8-3.6
3.8-3.6
J6,, = 1.5
4.34&d
6.20 sfs
4.82d J 1.9 = 10.5
DsO
Table 1. ‘H NMR spectra (90 MHz) of iridoid aglycones and glucosides of Deutzia scabra
= 9.2
= 1.5
2.626 J ,,9 = 9.2
1.62sfs
1.49sfs
3.9-3.7
3.9-3.7
J,,,
4.33 d
6.25 sfs
J,,,
5.12d
DsO ”
2.241 J 1.9 = 10.0
3.8-3.1
3.8-3.1
4.00 bs
6.02 sfs
4.541 J,,, = 10.0
DMSO-I,
2b
= 10.0
1.5Osj~
J,.,
2.31 d
3.8-3.2
3.8-3.2
4.01**
6.08 sf
4.82 d J,,g = 10.0
DMSO-$
4
89.39 d (172) 133.03d (190) 112.99s 38.296 (132) 17.95 d (142) 61.06d (191) 51.63 d (192) 41.64d (Ml) 16.61 q (129)
(128)
16.7Oq
(186) nm
(188) 54.96 d
(142) 59.10d
76.51 d
(190) 109.78s nm
(171) 132.83d
88.62 d
DMSO-d,
(129)
(140) 16.11q
(191) 44.15d
(192) 56.66d
(142) 59.50 d
(135) 78.53 d
(190) 112.99s 41.13d
(165) 135.95.d
93.41 d
DrO
(128)
t 16.05 q
(189) 43.24d
(189) 54.18d
(141) 51.48 d
t 77.61 d
(188) 110.75s 40.39d
(162) 135.74d
92.95 d
DMSO&
lb
16.01 q
42.68d
56.24 d
59.68 d
78.58 d
113.48s 41.12d
135.75d
96.19 d
DsO
(128)
t 15.91 q
t 41.6Od
(190) 53.81 d
(141) 57.52 d
t 77.06d
(189) 111.42s 40.12d
(166) 135.07d
94.82d
DMSO-I,
3
(130)
(138) 11.84q
(193) 50.00 d
(191) 56.441
(142) 60.93 d
71.34d
(192) 113.88s 72.18s
(172) 136.43d
914Od
DrO
*Chemical shifts are expressed on the TMS scale according to the following equation S,,, the nearest f 1 Hz. IOverlapped by DMSO-d, signals. *Tentative assignment. §Taken from ref. [ 111. mn, Not measurable.
‘-11
c-9
C-8
c-7
c-6
c-5
c-3 C-4
‘-’
Dz0
la
(130)
(136) 11.52q
(193) 52.34d
(191) 55.96d
(142) 5958d
77.65 d
(191) 115.25s 74.13s
(165) 138.18d
94.541
DrO
(130)
(138) 11.52q
(190) 51.53d
(191) 54.44d
(142) 58.8Od
76.96 d
(187) 114.70s 72.11 s
(162) 136.58d
94.18d
DMSO-d,
2b
(129)
(140) 11.53q
(192) 50.19 d
(192) 56.07 d
(142) 59.78 d
77.06 d
(193) 115.65 s 74.52 s
(172) 137.89d
96.88 d
D#X
(130)
(nm) 11.34q
(189) 49.90 d
(190) 54.OOd
(141) 57.91 d
76.55 d
(189) 115.43 s 72.17s
(165) 135.94d
95.79 d
DMSO-I,
4
(128)
(run) 11.79q
47:84d
(nm) ’ 54.57$d
57.301
nm
(190) 113.18s mn
(174) 136.3Od
88.15 d
5a CDCl,
(128)
(142) 11.43q
(193) 49.29 d
(194) 54.5ld
5i.:d
78.22 d
(192) 113.18s 73.33 s
(165) 137.961
90.62 d
Sb CDCI,
= ?idioxane + 67.4 ppm. Values in parentheses are coupling constants, which have been quoted to
(130)
(138) 11.97q
(190) 49.45 d
(190) 55.02d
(142) 59.42 d
16.12d
(187) 112.59s 71.14s
(172) 135.161
90.49d
DMSO-d,
2a
Table 2. 13C NMR spectra (20 MHz) of iridoid aglyeones and glucosides of Dcutzia scabra*
Iridoid aglycones from Deutzia gave pure 2 (0.6 g) as an amorphous mixture (2: 1) of fi- and aepimers. For analytical purposes a sample was rechromatographed in the same conditions. (Found: C, 53.85; H.6.10. C,HisOs requires C, 53.99, H, 6.04 T,.) Di-0-acetylscabrogenin (5). Compound 2 (a- and b-epimers, 80 mg) was acetylated with pyridine and AcsO for 1 hr at room temp. After addition of MeOH the soln was left for 20 min and then evaporated to dryness. The residue, chromatographed on silica gel (7 g) in Et,O-CsH, (1: 1) afforded pure 5 (60 mg) as an inseparable mixture of 01-and /I-epimers.
REFERENCES 1. Bonadies, F., Esposito, P. and Guiso, M. (1974) Gazz. Chim. Ital. 104, 17. 2. Esposito, P. and Guiso, M. (1973) Gazz. Chim. Ital. 103,517. 3. Danielson, T. J., Hawes, E. M. and Bliss, C. A. (1975) J. Chromatogr. 103,216.
scabra
807
4. Djerassi, C., Nakano, T., James, A. N., Zalkov, L. H., Eisenbraun, E. J. and Shoolery, J. N. (1961) J. Org. Chem. 26, 1192. 5. Hyeon, S. B., Isoe, S. and Salran, T. (1968)Tetrahedron Letters 5325. 6. Jensen, S. R., Lyse-Petersen, S. E. and Nielsen, B. J. (1979) Phytocbemistry 18, 273. 7. Danielson, T. J., Hawes, E. M. and Bliss, C. A. (1973) Can. J. Chem. 51, 760. 8. Tie-, L. F., Niemeyer, LJ.,Marx, P. and Ghlsenkamp, K. H. (1980) Tetrahedron 36, 1231. 9. Jensen, S. R., Mikkelsen, C. B. and Nielsen, B. J. (1981) Phytocbemistry 20, 71. 10. Bianco, A., Caciola, P., Guiso, M., Iavarone, C. and Trogolo, C. (1981) Gmz. Chim. Ital. 111, 201. 11. Bonini, C., Davini, E., Iavarone, C. and Trogolo, C. (1981) Phytochemistry 20, 1587. 12. Esposito, P., Guiso, M., Nicoletti, M. and De Luca, C. (1976) Gazz. Chim. Ital. 106, 57.