JPH024798A - Cord factor relating compound, production thereof and immunologically enhancing agent containing said compound - Google Patents

Abstract

NEW MATERIAL:A compound expressed by the formula (R<1> represents benzyl and R<2> represents trityl, H, tosyl or acyl; or R<1> represents H and R<2> represents acyl). USE:An immunologically enhancing agent. PREPARATION:A compound expressed by the formula wherein groups R<1> and R<2> both represent H is dissolved in pyridine and tritylated by adding trityl chloride. The reaction liquid is concentrated under reduced pressure, and the resultant residue is passed through a silica gel column and then eluted with a chloroform/methanol mixture, etc. Thus obtained ditritylated compound is dissolved in DMF, etc., and benzylated by adding benzyl bromide.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) The present invention relates to a code factor-related compound, a method for producing the same, and an immune enhancer containing the compound.

(Conventional technology) The compound of the present invention is a novel compound that has not been described in any literature.

Mycolic acid ester of trehalose (α, α-trehalose-6, +6′-cimicolate), called code factor, has immunoenhancing activity, antitumor activity, and host resistance to infection, in addition to lethal toxicity to mice. It is known to have enhancing activity.

The chemical synthesis of α,α-trehalose-6,6'-cimicolate (hereinafter simply referred to as TDM) has already been reported [■, Azuma et al.
and Pharmaceutical ° Bulletin (Che
Mical & PharmaceuticalBul
letin), 33. (1985) 4544-45
55).

(Problem to be solved by the invention) However! When considering TDM as an immune enhancer used in immunotherapy for II tumors, its strong toxicity poses a problem, and it has been desired to eliminate or reduce the toxicity by structural modification of TDM.

(Means for Solving the Problems) Therefore, as a result of extensive research in order to solve the above problems, the present inventors have developed a product in which the sugar skeleton of TDM is replaced with β,β-trehalose or α,β-trehalose. We succeeded in chemically synthesizing it, and when we measured its macrophage activation ability and toxicity, which are indicators of immune-enhancing activity, we obtained favorable results and completed the present invention.

That is, the novel code factor-related compound of the present invention is
It is a compound represented by the following general 2N) or (II).

Formula (1) [In the formula, R1 represents a benzyl group, and R2 represents a trityl group, a hydrogen atom, a tosyl group, or an acyl group (including a mycoloyl group. In the specification of this application, it may be simply referred to as an acyl group) or R' represents a hydrogen atom and R2 represents an acyl group. ] Formula ([) (wherein, R1 represents a benzyl group, R2 represents a benzylidene group or a hydrogen atom, and R″ represents a benzylidene group, a hydrogen atom, a tosyl group, or an acyl group, or R
1 and R2 represent hydrogen atoms, and R3 represents an acyl group. ) The contents of the present invention will be explained in detail below.

(a) -Synthesis of compound of formula (1)-S Compound (11) represented by formula (I) is a known compound β,β-trehalose (10) (E
, Fischer et al., Berichte.

42 (1909) 2776.

] can be synthesized by tritylation and then benzylation. Tritylation of the above compound (10) is
This tritylation reaction can be carried out by treatment with trityl chloride/pyridine, for example at 10-100°
This can be carried out by stirring at a temperature of C for 1 minute to 100 hours.

Furthermore, in the benzylation after tritylation, the tritylated product is mixed with sodium hydride (N
ail) and benzyl bromide.

Furthermore, compound (12) can be synthesized by detritylation of compound (11). This detritylation can be carried out, for example, by stirring in a solvent such as an 80% aqueous acetic acid solution at a temperature of 10 to 100'C for 1 minute to 24 hours.

Compound (13) can be synthesized by tosylating compound (12). This tosylation can be carried out, for example, by treating compound (12) with tosyl chloride/pyridine.

Furthermore, compound (14) can be synthesized by acylating compound (13), and this acylation is carried out by combining mycolic acid in the presence of 18-crown-6 in a solvent such as toluene. This can be done by treatment with potassium.

Compound (15) can be synthesized by debenzylating compound (14), and this debenzylation can be carried out, for example, by adding palladium-
It can be carried out by hydrogen reduction in the presence of a catalyst such as carbon (Pd-C).

(b) - Synthesis of compounds of form (II) - form (n)
Compound (17) represented by is a known compound α, as shown in Scheme 2 below.

β-trehalose (16) CW, N, Hawo
rth et al., Journal of Chemical Society 5o
(1931) 2847. ] can be synthesized by benzylideneation and then benzylation. This compound (16) can be synthesized by benzylideneation, for example, in the presence of P-toluenesulfonic acid in a solvent such as DMF.
- by treatment with dimethoxytoluene. Also, benzylation after benzylideneation can be carried out, for example, by treatment with sodium hydride and benzyl bromide in a solvent such as DMF.

Furthermore, compound (18) can be synthesized by debenzylideneation of compound (17), and this debenzylideneation is carried out at 10 to 100°C in a solvent such as an aqueous acetic acid solution with a concentration of 80%. 1 minute to 24 minutes at a temperature of
This can be carried out by stirring for a period of time.

Compound (19) can be synthesized by tosylating compound (18), and this tosylation can be carried out, for example, by treating compound (1 day) with tosyl chloride/pyridine.

Furthermore, compound (20) can be synthesized by acylating compound (19), and this acylation can be carried out, for example, by 18-crown-
This can be carried out by treatment with potassium mycolate in the presence of 6.

Compound (21) can be synthesized by debenzylating compound (2o), and this debenzylation can be carried out using a catalyst such as palladium black in a CH(f!, -THF-based solvent). This can be carried out by hydrogen reduction in the presence of.

In addition, the compound (11) synthesized in the above step.

(12), (13), (14), (15), (
17), (1B), (19)(20), (21)
Both are new compounds.

Scheme 1 (10) R'=R"=H (11) R'=Bn, R2=Tr (12) R'=Bn, R"=f((13)
R'=Bn, R''=Ts (14) R'=Bn
+ R”=mycoloyl group (15) R’=H, R”=
Mycoloyl group [H: hydrogen atom, Bn: benzyl group, Tr
Nitrityl group, Ts: l-syl group; same applies below]
(Space below) Scheme 2 (16) R'=R2=R3=H (17) R'=Bn, R"=R'=benzylidene group (18) R'=Bn, R"=R"=H (1
9) R'=Bn, R"=H, R'=Ts (20
) R'=Bn, R''=!(, R'= mycoloyl group (21) R'=R''=H, R3= mycoloyl group (Example) The contents of the present invention will be explained in more detail by giving examples below. However, the scope of the present invention is not limited to the following examples.

Example-1 [Compound (10) → Compound (11)] 304.0 mg (0.89 mmol) of compound (10) was dissolved in 6 ml of pyridine, and 594.0 μl of trityl chloride was dissolved.
(2.13 mmol) and stirred at 50°C for 39 hours, further added trityl chloride 148.5 mg (0.5
3 mmol) and stirred at 50°C for 23 hours. This reaction solution was concentrated under reduced pressure, the residue was poured into a column packed with 80 g of silica gel, and the eluate (CHCj!s:
CH30H=10:1, (CH3CHJzN
The ditritylated product obtained (Rf=0.53.CHCffi3:CH30H
=5: Dissolve 1) in DMF20II11, 60%
320 mmol of NaH (8.0 mmol) was added and stirred for 30 minutes while cooling with water. Next, benzyl bromide 0.95 m
j2 (8.0 mmol) was added dropwise, and while cooling with water, 17
Stir for hours. After the reaction, 2mf of CH30H was added, stirred for 30 minutes while cooling with water, and concentrated under reduced pressure to reduce the residue to 80%
The eluate was poured into a column packed with 1 g of silica gel, and the eluent was [toluene.

(CHzCHz) containing 1% N] to obtain 998.6■ of compound (11) (yield: 8
2.2%).

[Properties of compound (11)] TLC (thin layer chromatography) Rf = 0.32 (eluent toluene) Elemental analysis CqJe,,0+ Calculated value as I・H,0 C,79,74; H,6,40 Measured value C, 79
,74; H,6,36 specific optical rotation [α]. = −12
,4' (C= 1.17.CHCf -a'') Example-2 (Compound (11) → Compound (12)) Compound (11
) 939.0■ was dissolved in 25 m2 of acetic acid aqueous solution with a concentration of 80%, and after stirring at 80°C for 2 hours, THF10+nj!
was added and further stirred at 80°C for 1 hour. The reaction solution was concentrated under reduced pressure, and the residue was poured into a column packed with 80 g of silica gel, and the eluate (toluene: ethyl acetate = 2:
Elute and purify in 1) to obtain compound (12) 314.2■
was obtained (yield 51.8%).

[Properties of compound (12)] TLC, Rf = 0.49 (eluent toluene: ethyl acetate = 1: 1) Elemental analysis Calculated value as C54H5l1011 C,
73,45; H,6,62 measurement value C,73,28
1l(,6,63 specific optical rotation [α) o = +10.1°
(C=0.55.CHCf-s)13C-NMR
(CDCj! ri 100.1 (C-1, 'JCH162, 4H2), 84
．． 4 (C-3), 82.1 (C-2), 77.3 (
C-4), 75.2 (C-5), 61.7 (C
-6) Example-3 [Compound (12) → Compound (13)]
Compound (12) 223.6 (0.25 mmol) was dissolved in 2 ml of pyridine, and after cooling on ice, tosyl chloride 144
To the mixture was added 0.8 mmol (0.8 mmol), and the mixture was stirred at 0° C. for 1 hour, and further stirred at room temperature for 19 hours. After the reaction is completed, ethyl acetate 50m/! , 2011 Il of ice water was added for extraction, and the organic layer was treated with 5% hydrochloric acid, saturated brine, saturated aqueous sodium bicarbonate, and saturated brine in this order, and dried over Mg5Oa.

After filtration, it was concentrated under reduced pressure, and the residue was poured into a column packed with 25 g of silica gel, and eluted and purified with an eluent (toluene: ethyl acetate = 10:1) to obtain compound (13) 26.
0.1■ was obtained (yield 86.2%).

[Properties of compound (13)] TLCRf = 0.57 (eluent toluene: ethyl acetate = 6:1) Elemental analysis Calculated value as C611 + 17°0ISS2
C, 68, 55; H, 5, 92 measurement value C, 68,
47; H, 5,96 specific optical rotation [α]. =-4,1°
(C = 0.20. CHCl3) Example-4C Compound (13) → Compound (14)] Compound (13) 116.7 (0.10 mmol) was dissolved in 10 mj2 of toluene, and potassium mycolate (average composition C,,H,5,O,,,Aoyama B
) 240.9 mg (0.20 mmol) and 18-crown-631.7 mg (0.12 mmol) were added and stirred at 100'C for 16 hours. The reaction solution was concentrated under reduced pressure, the residue was dissolved in 5 ml of hexane, filtered, concentrated under reduced pressure, the residue was poured into a column packed with 30 g of silica gel, and the eluate (hexane:ether = 7:4)
The mixture was eluted and purified to obtain 193.0 ml of compound (14) (yield 61.6%).

[Properties of compound (14)] TLCRf = 0.41 (eluent toluene: ethyl acetate lo: 1) Elemental analysis Calculated value as CZ14H37°016 C,
80,34; H, 11,66 measurement value C, 80,1
0; H, 11,64 specific optical rotation [α]. = + 2.
2° (C・0.36. CHCl3) Example-5 [Compound (14) → Compound (15)) Compound (14) 15
4.6 mg THF10n/! Dissolved in 0.5 m of water
150 ■ 10% palladium-carbon catalyst suspended in ff.
and 10mj of ethanol! is added to encapsulate H2, and heated at 50°
After stirring at C for 21 hours, the catalyst was removed by filtration and concentrated under reduced pressure.
10% palladium-carbon catalyst suspended in 5nl 140
mg and 15 mρ of ethanol were added to encapsulate H2, and 50
Stirred at °C for 19 hours. After filtering and removing the catalyst,
It was concentrated under reduced pressure, and the residue was poured into a column packed with 10 g of silica gel, and the eluate (CHCl3:CHaOH=
25: The waxy compound (15) obtained by elution and purification in 1) was dissolved in 1 ran of ether and heated to 0 °C.
C dropwise added to 20 ml of CIhOH to precipitate, centrifuged and dried under reduced pressure to obtain 48.8 ml of compound (15) in the form of a white powder (yield 38.0%).

[Properties of compound (15)] TLCR f 0.46 (eluent CHCl2z:
CH:10H=15:1) Elemental analysis CI? Calculated value as 2H3340+6 C,
? ? , 71; H, 12, 66 measurement value C, 77, 4
0; H, 12,39 specific optical rotation [α]. =+32.0°
(C・0.36.CHC/! 3) IH-NMR (CD
Cffi: CD20D = 30:1) 4.54
6 (d, J 9.21 (z, H-1) Example-6 [Compound (16) → Compound (17)) Compound (16) 262
．． 7 rag (0.77 mmol) in DMF5 mj
l! Add 5 ml of toluene and heat at 50° under reduced pressure.
After azeotropic treatment with C, α,α-dimethoxytoluene 255μ
f (1,7 mmol) and p-)luenesulfonic acid 10
was added and stirred at 60°C under reduced pressure for 1 hour. After cooling,
Add 11,148 mg (3.7 mmol) of 60% Na, stir for 15 minutes while cooling with water, and add 0.44 mg of benzyl bromide.
nu (3.7 mmol) was added dropwise to the solution while cooling with water.
Stir for hours. Furthermore, 60% NaH74[ng and benzyl bromide 0.2211If! , and stirred for 1 hour while cooling with water. After the reaction was completed, 2 ml of CHzol was added, stirred for 30 minutes while cooling with water, concentrated under reduced pressure, and the residue was poured into a column packed with 50 g of silica gel.
IhCHz) J containing 1%] to obtain 602.0 mg of compound (17) (yield 89.2%)
.

[Properties of compound (17)] TLCRf = 0.32 (eluent toluene: ethyl acetate = 10:1) Elemental analysis Calculated value as C3JS4011 C, 73,78; H, 6, 19 Measured value C
,73,75; H,6,21 specific optical rotation (α),
=+10.0° (C= 0.63.CHCj2+)
'H-NMR (CDCl:l) 7.52-7.16 (30H, aromatic p
rotons), 5.553(s, 28. PhCl
lX2) Example-7 (Compound (17) → Compound (1B)) Compound (17) 562.4μ was added to 15m2 of 80% acetic acid aqueous solution
and stirred at 80°C for 1 hour. The reaction solution was concentrated under reduced pressure at 30°C for 910 minutes, and the residue was poured into a column packed with 50 g of silica gel.
: CH30H=10:1) to obtain compound (1B) 294.0■ (yield: 6
5°4%).

[Properties of compound (I8)] TLCRf=0.32 (Eluate Cf1Cl3
: C) bOH=10:1) Elemental analysis Calculated value as C4°H460□ C,68,36,H,6,60 Measured value C,6
7,93,H,6,59 Specific optical rotation [α) n = +1
3.2° (C=0.34.CHCff3) 1 CNM
R(COCl3) 104.6(C-1','Jcl(158,5Hz)
, 99.3 (C-1,'Jc) 1168.2tlz) Example-8 [Compound (18) → Compound (19)] Compound (1B) 124.4■ (0.18 mmol) was dissolved in pyridine (dry ), and after cooling with water, 79.7 μm (0.42 mmol) of tosyl chloride was added, and the mixture was stirred for 21 hours while raising the temperature from 0°C to room temperature. After the reaction was completed, extraction was performed by adding 50 m2 of ethyl acetate and 20 m1 of ice water, and the organic layer was treated with 5% hydrochloric acid, saturated brine, saturated aqueous sodium bicarbonate, and saturated brine in this order, and dried over MgSO4.

After filtration, it was concentrated under reduced pressure, and the residue was poured into a column packed with 12 g of silica gel, and eluted and purified with an eluent (toluene: ethyl acetate = 471) to obtain compound (19) 121.
．． 1 mg was obtained (yield 67.7%).

[Properties of compound (19)] 'rLCRf=o, 5o (eluent toluene:ethyl acetate=11) Elemental analysis Cs41ISeO+sSz・1/3CJ(s
Calculated value as cHa C, 64, 94; II, 5.
87 Measured value C, 65, 11; H, 5, 82 Specific optical rotation [α) o -44,0° (c = 0.20. CH
ix)'HNMR (CDCE 3) 7.80-7.71 (4H, aromatic pro
tons), 7.35-7.16 (2411, aroma
atic protons)” C-NMR (CDCl
3) 103.5(C-1', 'JeH161,4H
z), 99.3 (C-1゜'JcH170, 2Hz) Example-9 [Compound (19) → Compound (20)] Compound (19) 109. ! 11 mg (0.11 mmol)
was dissolved in toluene LOmf, potassium mycolate 26
Add 7.1 mg (0.22 mmol) and 18-crown-634.4 (0.13 mmol) to make 100
Stirred at °C for 15 hours. The reaction solution was concentrated under reduced pressure, the residue was dissolved in 5 ml of CHCj'+, filtered, concentrated under reduced pressure, the residue was poured into a column packed with 35 g of silica gel, and the eluate (toluene: ethyl acetate = 6: 1
) and purified, compound (20) 197.2 m
g (yield 60.1%).

[Properties of compound (20)] TLCRf=0.54 (eluent toluene:ethyl acetate=4:1) Elemental analysis C2°. Calculated value C as 8358016,
79,57; H, 11,95 measurement value C, 79,63
; II, 11.76 specific optical rotation [α]. = +18
．． 6@(C=0.43.CHCf,.

42 hours later) Example-10 (Compound (20) → Compound (21) E Compound (20) 174.8 mg was transferred to CHCft 5 m
35 mg of palladium black catalyst and THF
Add 5 ml and seal 11□, stir at 50°C for 4 hours, remove the catalyst by filtration, concentrate under reduced pressure, pour the residue into a column packed with 12 g of silica gel, and eluate (COCl23: C130H=10:1
) was eluted and purified, resulting in a waxy compound (
2I) to ete)Ltlml? Dissolve and 0 °C CH
, 20 ml of lOH to cause precipitation, centrifugation, and drying under reduced pressure to obtain 123.2 mm of compound (21) in the form of a white powder (yield: 80.1%).

[Properties of compound (21)] TLCRf=0.38 (Eluate ClICl: l
: CH:+0H-10:1) Elemental analysis C+tJ: Calculated value as 1ff40+a C
，? 7.71; H, 12,66 measurement value C, 17,4
9: ■, 12.51 Specific optical rotation [α) o = 19.8°
(C=0.33.CHCn ff) (Experimental Example) As expected, the toxicity of the compound of the present invention was significantly reduced, and examples of experiments conducted regarding its pharmacological activity will be described below.

Experimental Example-1 (Toxicity Test) Yarkoni's method (E, Yarkoni et al., Infe
ct, Immun.

(1977) 552), TDM-related compound 2 and 45 mg of Drakeol 6 VR (Drakeol, mineral oil) were homogenized, and then 455 μl of physiological saline containing 1.1% Tween 80 was added.
The oil-in-water emulsion obtained by adding
．． A dose of 1 mj2 was injected intravenously, and changes in body weight and survival were observed. The results are shown in FIG. 1 and Table 1. A known TDM (α, α) was used as a comparison, and a system to which 70M was not added was used as a control.

(Margin below) Table 1 (Note) Changes in body weight indicate comparisons with before administration.

From FIG. 1 and Table 1, it can be seen that in the known TDM (α, α) system, the number of survivors after 3 weeks was O, whereas the number of survivors for the TDM (α, β) and TDM (β) compounds of the present invention was O. , β) showed a temporary weight loss, but quickly recovered, indicating a significant improvement in toxicity.

Experimental Example 2 (peritoneal macrophage activation test) Kago's method (Arch, Biochem, Biophys, etc.)
140 (1970) 379) in PBS at ioo μg/m
! ! , diluted with 0.5 ml of C57B aged 7 to 9 weeks.
It was administered intraperitoneally to L/6 (female) mice (two mice in each group). After 5 days, the intraperitoneal exudate cells from the mice were collected and plated in a 96-well plate so that 5 lO cells/well were attached to the glass. Next, 104 I25I-UdR-labeled B16-BL6 cells (mouse melanoma) were added as target cells from above the well, and incubated at 37°C for 3 days.
c Cultured in a COz incubator. As a control, P
Intraperitoneal exudate cells from mice to which only BS was administered were used.

On the third day, each person was washed twice with PBS warmed to 37°C.
The remaining viable target cells were collected in 70 μl of 0.00 μl. It was destroyed with IN-NaOH, wiped with a cotton swab, and the radiation dose on the swab was measured with a gamma counter. The results are shown in Table 2.

Table 2 β) and TDM (β, β) are known TDM (α.

It can be seen that it has almost the same ability to activate peritoneal macrophages as α).

(Effects of the Invention) As is clear from the above experimental examples, the compounds of the present invention are useful as immune enhancers for various immunotherapies.

[Brief explanation of the drawing]

FIG. 1 shows the oral changes in body weight of mice intravenously injected with TDM-related compounds. Patent applicant: Towa Kasei Kogyo Co., Ltd.

Claims (1)

[Claims] 1. A compound represented by the following general formula (I). Formula (I) ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (In the formula, R^1 represents a benzyl group, and R^2 represents a trityl group, hydrogen atom, tosyl group, or acyl group, or R^ 1 represents a hydrogen atom, and R^2 represents an acyl group.)2 The compound according to claim 1, wherein R^1 in the general formula (I) is a hydrogen atom and R^2 is a mycolol group. 3 A compound represented by the following general formula (II). Formula (II) ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (In the formula, R^1 represents a benzyl group, R^2 represents a benzylidene group or a hydrogen atom, and R^3 represents a benzylidene group, a hydrogen atom, represents a tosyl group or an acyl group, or R^1 and R^2 represent a hydrogen atom, and R^3 represents an acyl group.) 4 R^1 and R^2 of general formula (II) are 4. The compound according to claim 3, which is a hydrogen atom and R^3 is a mycolol group. 5 (a) β,β-trehalose [compound (10)] is tritylated and then benzylated to produce compound (11) represented by general formula (I) [in general formula (I), R^1 is a benzyl group and R^2 represents a trityl group. ], (b) Detritylation of compound (11) to produce general formula (I
) [In the general formula (I), R^
1 represents a benzyl group, and R^2 represents a hydrogen atom. (c) step of producing a compound (13) represented by the general formula (I) by tosylating the compound (12) [in the general formula (I), R^1 represents a benzyl group, R^2 represents a tosyl group. (d) Step of producing compound (14) represented by general formula (I) by acylating compound (13) [In general formula (I), R^1 represents a benzyl group, R^2 indicates mycolol group. ], (e) debenzylating compound (14) to produce the general formula (I
) [In the general formula (I), R^
1 represents a hydrogen atom, and R^2 represents a mycolol group. ]
1. A method for producing a code factor-related compound, comprising the following steps: 6 (a) α,β-trehalose [compound (16)] is benzylidened and then benzylated to produce compound (17) represented by general formula (II) [in general formula (II), R^1 is a benzyl group and R^2 and R^3 represent benzylidene groups. ], (b) debenzylidene compound (17) to produce the general formula (
Compound (18) represented by II) [in general formula (II), R^
1 represents a benzyl group, and R^2 and R^3 represent a hydrogen atom. ], (c) tosylating compound (18) to produce compound (19) represented by general formula (II) [in general formula (II), R^1 represents a benzyl group, R^2 represents a hydrogen atom, and R^3 represents a tosyl group. ], (d) acylating compound (19) to obtain general formula (II)
Compound (20) represented by formula (II), R^1 represents a benzyl group, R^2 represents a hydrogen atom, and R^3 represents a mycolol group. ], (e) debenzylating compound (20) to produce compound (21) represented by general formula (II) [in general formula (II), R^1 and R^2 are hydrogen atoms; and R^3 represents a mycolol group. ] A method for producing a code factor-related compound, comprising the steps of: 7. An immune enhancer characterized by containing a compound represented by the following general formula (I) as an active ingredient.・Formula (I) ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (In the formula, R^1 represents a benzyl group, and R^2 represents a trityl group, hydrogen atom, tosyl group, or acyl group, or R ^1 represents a hydrogen atom, and R^2 represents an acyl group.)8 An immune enhancer characterized by containing a compound represented by the following general formula (II) as an active ingredient. Formula (II) ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (In the formula, R^1 represents a benzyl group, R^2 represents a benzylidene group or a hydrogen atom, and R^3 represents a benzylidene group, a hydrogen atom, represents a tosyl group or an acyl group, or R^1 and R^2 represent a hydrogen atom, and R^3 represents an acyl group.)