Carbohydrate Research 376 (2013) 15–23
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Synthesis of 60 -acylamido-60 -deoxy-a-D-galactoglycerolipids Chunxia Li ⇑, Yihua Sun, Jun Zhang, Zhimin Zhao, Guangli Yu, Huashi Guan Key Laboratory of Marine Drugs, Ministry of Education, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, PR China
a r t i c l e
i n f o
Article history: Received 15 December 2012 Received in revised form 28 January 2013 Accepted 17 February 2013 Available online 26 February 2013 Keywords: Glycolipid Myt1-kinase Remote neighboring participation a-Selectivity
a b s t r a c t Aminoglycoglycerolipid 1a isolated from an algal extract showed activity against the enzyme Myt1 kinase with an IC50 value of 0.12 lg/mL. Its analogues, 60 -acylamido-60 -deoxy-a-D-galactoglycerolipids (2a–g) were synthesized in an efficient way with high stereoselectivity. The key step was to employ a 4-OAc protecting group of the galactosyl donor 14 as a remote neighboring participation group to give the glycoside with high a-anomeric selectivity (a:b = 32:1) in the glycosylation. The preliminary bioactivity screening showed that compound 2g exhibited good inhibition against Myt1 kinase. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction The human Myt1 kinase is a Thr-14 and Tyr-15 specific regulator of the cdc2/cyclin B kinase activity in the cell cycle. Inhibition of Myt1 kinase is predicted to cause premature activation of cdc2.1,2 It has been reported that premature activation of cdc2 leads to mitotic catastrophe and cell death.1,3,4 So inhibitors of this enzyme would thus be expected to kill rapidly proliferating cells and abrogate normal cell cycle checkpoints of cancer cells. Such inhibitors would be attractive for the treatment of cancer in the future. In 2005, aminoglycoglycerolipid 1a (Fig. 1), which was isolated from an algal species showed high activity against the enzyme Myt1-kinase with an IC50 value of 0.12 lg/mL.5 Due to the unique structure and inhibition of Myt1-kinase, 1a had been synthesized by our group and Schmidt et al.6–8 Glycoglycerolipids demonstrated various biological activities, such as tumor growth and DNA polymerase inhibition,9,10 fatty acid synthase inhibiton,11 glucose-lowing effect,12 and anti-inflammatory action.13 Colombo et al. had evaluated a series of glycoglycerolipids for their anti-tumor promoting activity in vitro and vivo.14–16 Reports showed that the minor structural changes of the lipophilic acyl chain and the type of glycosyl in glycolipids affected the antitumor activity of glycolipids. In order to acquire detailed information of the structure–activity relationship (SAR) of glycoglycerolipid 1a, we synthesized its analogues 1b–h (Fig. 1) with different acyl chains.7 Recently, through changes in the configuration of glycosidic bond (a, b), saccharide unit (glu, gal, Man) and acyl chains (palmitoyl, capryloyl), 6 derivatives of glyco-
⇑ Corresponding author. Tel.: +86 532 82032030; fax: +86 532 82033054. E-mail address:
[email protected] (C. Li). 0008-6215/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.carres.2013.02.008
glycerolipid 1a were synthesized and tested in a binding assay together with a set of common kinase inhibitors against a full-length Myt1 expressed in a human cell line.17 But these compounds showed no significant effects in the binding assay. For most of the bioactive natural glycoglycerolipids, the sugar moiety was the galactosyl residue. Colombo et al. had found that the axial configuration of the hydroxyl in the 4-position of the sugar of glycoglycerolipids was important for a remarkable anti-tumor-promoting activity.14 These prompted us to synthesize a series of 60 -acylamido-60 -deoxy-a-D-galactoglycerolipids analogues 2a–g (Fig. 1) by substituting the glucosyl with galactosyl and altering the acyl chains, and ascertain the structural features responsible for the activity. In this paper, we introduced an intelligent method for the synthesis of these compounds and evaluated their Myt1 kinase inhibition activity. 2. Results and discussion The main challenge in the synthesis of 2a–g was to efficiently construct the a-configured glycosidic bond in the glycosylation. The aminoglycoglycerolipids 1a–h have been synthesized with high stereoselectivity by employing thioglycoside or trichloroacetimidate as donor.7 Now we tried to verify whether the same synthetic strategy could be applied to the synthesis of 2a–g. Thiogalactoside 8a and trichloroacetimidate 8b were initially investigated as the donors. Synthesis of 8a and 8b starting from the known 1-thio-b-D-galactopyranoside 318 was shown in Scheme 1. Tritylation of the primary hydroxyl of the thiogalactoside 3 yielded the triol 4, which was perbenzylated to yield 5. Acid-catalyzed removal of the trityl group provided the desired alcohol 6 in an overall yield of 60% over the three steps.19 Treat-
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HO HO
a: b: c: d: e: OR f: g OR : h:
NHR O
1
OH O
R = palmitoyl R = hexanoyl R = octanoyl R = lauroyl R = myristoyl R = stearoyl R = isovaleryl R = hydrocinnamoyl
OH NHR O HO OH O 2
a: b: c: d: e: f: g:
OR OR
R = hexanoyl R = octanoyl R = lauroyl R = myristoyl R = palmitoyl R = stearoyl R = isovaleryl
Figure 1. Structures of natural aminoglycoglycerolipid 1a and its analogues 1b–h and 2a–g.
ment of thiogalactoside 6 with p-toluenesulfonyl chloride produced tosylate 7. Then 7 reacted with sodium azide in DMF to afford 8a in only 26% yield. We supposed that steric hindrance of 4OBn may influence the substitution by azide. Then 8a was converted to trichloroacetimidate 8b following a standard procedure to undertake the initial investigation. Glycosylation of trichloroacetimidate 8b and (S)-isopropylideneglycerol catalyzed by TMSOTf in absolute ether furnished a 2:5 inseparable mixture of 9a (H-1: d 4.93, J 3.8 Hz) and 9b (H-1: d 4.39, J 7.7 Hz), whereas the glycosylation of thioglycoside 8a under NIS–TMSOTf in the presence of DTBMP afforded a better ratio (a/b = 9:5) without S-chiral center racemization (Scheme 2). Although the ratio was improved, the donor thioglycoside 8a was obtained in a low yield (Scheme 1), and the anomeric stereoselectivity of the glycosylation was far from our expectation. So the strategy used in the synthesis of glucosyl type aminoglycoglycerolipids 1a–h failed in the practical synthesis of 2a–g. But it was encouraging that these results gave us a clue that a thioglycoside may become a hopeful glycosyl donor for the stereoselective glycosylation. Then a major effort was made to find a suitable galactosyl donor, which should be designed to obtain a high a/b ratio as well as a satisfactory yield for the preparation of a-galactosyl glycerol. Cox and Besra prepared 6-N-derivatized a-galactosyl ceramides using a glycosyl iodide donor, and found that small changes in the sugar substitution pattern affected the donors’ activity greatly.20 During a one-pot a-galactosylation method using Appel agents in DMF, Nishida and Kobayashi showed that the donors with the 6-O-acyl group can promote a-selectivity.21 Boons reported that iodonium-ion promoted glycosylations in 1,4-dioxane/toluene with galactosyl donors having a neighboring group participating functionality at C-4 gave exceptionally high a-anomeric selectivity.22 Based on these facts, a new galactosyl donor 14 which was equipped with an acetyl group on the C-4 position was rationally designed. The synthesis of 14 began from 3 in six steps as shown in Scheme 3. Treatment of galactopyranoside 3 with benzaldehyde dimethyl acetal yielded the 4,6-O-benzylidene derivative. The remaining free hydroxyl groups were benzylated to afford compound 10 in 80% yield for the two steps. Then the benzylidene group was removed to furnish 11 with the 4-OH and 6-OH exposed. Compound 11 was tosylated with p-toluenesulfonyl chloride in pyridine to give 12 selectively in 70% yield.23 Treatment of 12 with sodium azide in DMF in the presence of 15-crown-5 gave 13 in 76% yield. Here our supposition of the steric hindrance of 4OH OH
OH OTr
O
HO
a
STol 3
OH
STol
b
STol
BnO 7
OBn
e
3
BnO
STol 8a
OBn
STol 6
3
f
O
BnO OBn O 8b
O
BnO
OBn OBn N
O
c
STol 5
OBn N
O
OBn OH O
BnO
4 OH
OBnOTs d
OBn OTr
O
HO
OBn in the production of 8 (Scheme 1) was confirmed. Then 13 was O-acetylated to afford the 6-azido-6-deoxy thiogalactoside donor 14. Subsequently, the coupling reaction between 14 and the acceptor (S)-isopropylideneglycerol in various solvents was carefully examined using NIS–TMSOTf as promoters at room temperature, meanwhile, DTBMP was added to prevent (S)-isopropylideneglycerol from racemization.24–27 The results were summarized in Table 1. Racemization of the (S)-isopropylideneglycerol residue was not observed in all the reactions, which were identified by 1H NMR. First, the glycosylation of (S)-isopropylideneglycerol with donor 14 was carried out in dioxane/toluene (3:1) (entry 1). There was no change in the reaction mixture during 48 h. When CH2Cl2 served as the solvent, products 15a and 15b were obtained in 92% yield with a modest a/b ratio of 5:1 (entry 2). This stereoselectivity was further raised to a/b 25:1 when a mixed solvent CH2Cl2/ Et2O (1:3) was used (entry 3). The best result was achieved using Et2O as solvent which gave almost exclusively the 15a isomer (15a:15b = 32:1) (entry 4). These results supported the notion that the 4-OAc protecting group of the galactosyl donor can perform remote neighboring group participation to give galactosides with high a-anomeric selectivity, and that using Et2O can enhance this effect. Having completed the synthesis of 15a, we continued to synthesize the final compounds 2a–g (Scheme 4). The acetyl group of 15a was converted to the benzyl group to produce 16. Hydrolysis of the isopropylidene group with TsOH and then reduction of the azido group by the Staudinger reaction yielded the appropriate amino derivative 17. Introduction of acyl chains on C1, C2 and C60 – NH2 of 17 by condensation with the corresponding acyl chloride in the presence of 4-N,N-dimethylaminopyridine (DMAP) gave 18a–g in 67–90% yield. Then removal of the benzyl groups by hydrogenolysis with H2/Pd(OH)2 generated the final compounds 2a–g. The structures of the synthetic samples were identified by 1H, 13C NMR spectroscopy, and HRESIMS. Glycoglycerolipids 1a–h and 2a–g were assayed for inhibitory activity against Myt1 kinase based on the chemiluminescence method.28 The profiling data for various compounds against Myt1 protein kinase were shown in Table 2. Compared with the reference compound staurosporine and 1a, compound 2g showed good inhibition activity. This result indicated that the galactosyl and isovaleryl groups exerted positive effect on the inhibition of Myt1 kinase. From this table, we found that the activities of the galacto
OBn
NH CCl3
Scheme 1. Reagents and conditions: (a) TrCl, DMAP, Py, 80 °C; (b) BnBr, NaH, DMF, 66% for two steps; (c) AcOH–EtOH, 90 °C, 91%; (d) CH2Cl2, Et3N, DMAP, TsCl, 95%; (e) NaN3, DMF, 65 °C, 26%; (f) NBS, acetone/water (9:1), then DBU, CNCCl3, CH2Cl2 72%.
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OBn N 3
O
BnO 8a
OBn
STol
OBn N 3 a for 8a
or OBn
N3
8b
BnO
O
O O
BnO
O
9β
O
OBn
9α : 9β = 9 : 5 for 8a
NH
OBn O
+
O
OBn O 9α
b for 8b O
BnO
OBn N 3
O
9α : 9β = 2 : 5 for 8b CCl3
Scheme 2. Reagents and conditions: (a) (S)-isopropylideneglycerol, DTBMP, NIS–TMSOTf, Et2O, 0 °C to rt, 5 h, 83%; (b) (S)-isopropyleneglycerol, TMSOTf, Et2O, 0 °C, 30 min, 86%.
Ph OH
OO
OH O
HO
a STol
OH
BnO
3
OH
OH O
OH O
b STol
OBn
BnO
OH
O BnO
d
OBn
N3
OAc N3 O
BnO
STol
OBn
STol
O
e BnO
OBn
13
12
STol
11
10
OTs
c
OBn
STol
14
Scheme 3. Reagents and conditions: (a) PhCH(OCH3)2, DMF, TsOHH2O, 50 °C, then BnBr, NaH, DMF, 80% for the two steps; (b) TsOHH2O, MeOH, 50 °C, 88%; (c) TsCl, Py 45 °C to rt, 70%; (d) NaN3, DMF, 15-crown-5, 65 °C, 76%; (e) Ac2O, CH2Cl2, Et3N, DMAP, 92%.
Table 1 Glycosylation of (S)-isopropylideneglycerol with 14 in various solventsa
OAc N 3 BnO
OAc STol OBn
14
a b c
O
O
+
HO
O
N3
OAc O
BnO
(S)-isopropylideneglycerol
OBn O 15α α
+
O
N3
O
O O
BnO
O
OBn
O
15β
Entry
Solvents
Time
Yieldb
Ratio (15a:15b)c
1 2 3 4
Dioxane/toluene = 3:1 CH2Cl2 CH2Cl2/Et2O = 1:3 Et2O
— 3h 5h 16 h
— 92% 90% 90%
— 5:1 25:1 32:1
All reactions were carried out with 1.2 equiv (S)-isopropylideneglycerol in the presence of N-iodosuccinimide (1.5 equiv), TMSOTf (0.6 equiv) and DTBMP (1.0 equiv). Isolated yield. Determined by 600 MHz 1H NMR.
series were better than their gluco counterparts generally. We inferred that the galactosyl residue performed positive effect on the activity and a suitable acyl group would enforce this effect. Further structural optimization and more detailed bioassay are underway.
Et2O. The preliminary bioactivity screening showed that compound 2g had good inhibition against Myt1 kinase. A detailed structure antitumor activity relationship study of these analogues will be reported later. 4. Experimental
3. Conclusions 4.1. General methods In conclusion, a series of 60 -acylamido-60 -deoxy-a-D-galactoglycerolipids were synthesized in an efficient way with high stereoselectivity (a:b = 32:1) by employing 14 as a glycosyl donor in
Solvents were purified in a conventional manner. The boiling range of petroleum ether was 60–90 °C. Thin layer chromatogra-
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OAc N 3
OBn N3 O
a
BnO 15α
c
OBn O
O
O OBn O 16
O
OBn NHR O BnO OBn O 18
b
BnO
d OR OR
O O
OH NHR O HO OH O 2
OR3 OR3
OBn NH2 O BnO OBn O 17
a: b: c: d: e: f: g:
OH OH
R = hexanoyl R = octanoyl R = lauroyl R = myristoyl R = palmitoyl R = stearoyl R = isovaleryl
Scheme 4. Reagents and conditions: (a) MeONa, MeOH, then BnBr, NaH, DMF, 82% for two steps; (b) TsOH, MeOH, then PPh3, THF–H2O, 88% for the two steps; (c) acyl chloride, Py, DMAP, 67–90%; (d) Pd(OH)2/C, H2, THF/i-PrOH (9:1), 82–90%.
Table 2 Activity change (%) of Myt1 in the presence of various compounds Compound
Activity change %
Compound
Activity change %
1b 1c 1d 1e 1a 1f 1g 1h
2 4 5 32 34 32 35 38
2a 2b 2c 2d 2e 2f 2g Staurosporine
9 15 24 23 42 40 60 65
phy (TLC) was performed on precoated HSGF254 plates (Yantai, China). Flash column chromatography was performed on silica gel (200–300 mesh, Qingdao, China). Optical rotation was determined with a Perkin–Elmer Model 241 MC polarimeter. IR spectroscopy was performed on a Nicolet Nexus 470 FT-IR spectrometer using the KBr pellet technique. 1H NMR and 13C NMR spectra were taken on a JEOL JNM-ECP 600 spectrometer with tetramethylsilane (Me4Si) as the internal standard, and chemical shifts were recorded as d values. Mass spectra were recorded on a Global Q-TOF mass spectrometer and IonSpec 4.7 Tesla FTMS (MALDI/DHB). 4.2. Synthetic chemistry 4.2.1. p-Tolyl-2,3,4-tri-O-benzyl-6-O-trityl-1-thio-b-Dgalactopyranoside (5) To a mixture of tetrol 3 (5.3 g, 18.5 mmol) and trityl chloride (7.7 g, 27.8 mmol) in pyridine (50 mL) was added catalytic DMAP (0.23 g, 1.85 mmol). The reaction mixture was stirred at 80 °C for 5 h, and then concentrated in vacuo. The residue was dissolved in CH2Cl2 (100 mL), washed sequentially with aq HCl (1 M, 2 100 mL), satd aq NaHCO3 (2 100 mL) and brine (100 mL). The organic phase was collected, dried with Na2SO4 and concentrated in vacuo to afford the crude triol 4. The crude residue 4 was dissolved in DMF (50 mL) and BnBr (11 mL, 92.5 mmol) was added. The solution was cooled to 0 °C and sodium hydride (4.5 g, 60%, 111 mmol) was added portionwise to the stirred solution over 5 min. The reaction mixture was allowed to warm to room temperature. After 6 h, MeOH (40 mL) was added and the solution was stirred for 15 min. The reaction mixture was then concentrated in vacuo (co-evaporation with toluene, 3 50 mL) and the residue was dissolved in CH2Cl2 (100 mL). The resulting solution was washed with brine (3 50 mL), dried (Na2SO4) and concentrated in vacuo. The residue was purified by flash column chromatography (1:12 EtOAc–petroleum ether) to afford fully protected thioglycoside 5 (9.8 g, 66%) as a white crystalline solid; 1H NMR (600 MHz, CDCl3): d 6.95–7.46 (m, 34H, –Ar),
4.50–4.88 (m, 6H, 3 PhCH2), 4.71 (d, 1H, J 9.9 Hz, H-1), 3.90 (d, 1H, J 2.7 Hz, H-4), 3.87 (t, 1H, J 9.4 Hz, H-2), 3.57 (dd, 1H, J 9.5, 6.0 Hz, H-6), 3.54 (dd, 1H, J 9.3, 2.7 Hz, H-3), 3.34 (app. t, J 6.4 Hz, 1H, H-5), 3.24 (dd, 1H, J 9.5, 6.7 Hz, H-60 ), 2.28 (s, 3H, ArCH3); LR-ESI-MS m/z calcd for [M+Na]+ 821.3; found 821.3. 4.2.2. p-Tolyl-2,3,4-tri-O-benzyl-1-thio-b-D-galactopyranoside (6)29 Thioglycoside 5 (2.0 g, 2.51 mmol) was suspended in a mixture of AcOH:EtOH (20 mL:10 mL) and refluxed at 90 °C for 16 h. Then the reaction mixture was concentrated in vacuo (co-evaporation with toluene) and the residue was dissolved in CH2Cl2 (200 mL), washed sequentially with satd aq NaHCO3 (200 mL), brine (200 mL), dried (Na2SO4) and concentrated in vacuo to afford alcohol 6 (1.26 g, 91%) as a white crystalline solid; 1H NMR (600 MHz, CDCl3): d 7.01–7.52 (m, 19H, –Ar), 4.62–4.99 (m, 6H, 3 PhCH2), 4.56 (d, 1H, J 9.7 Hz, H-1), 4.25 (dd, 1H, J 11.2, 7.0 Hz, H-6), 4.10 (dd, 1H, J 11.3, 5.6 Hz, H-60 ), 3.91 (t, 1H, J 9.4 Hz, H-2), 3.83 (d, 1H, J 2.1 Hz, H-4), 3.59 (dd, 1H, J 9.3, 2.7 Hz, H-3), 3.56 (app.t, J 6.3 Hz, 1H, H-5), 2.30 (s, 3H, ArCH3); LR-ESI-MS m/z calcd for [M+Na]+ 579.2; found 579.2. 4.2.3. p-Tolyl-2,3,4-tri-O-benzyl-6-O-p-tosyl-1-thio-b-Dgalactopyranoside (7) To a mixture of 6 (1.1 g, 2.0 mmol) and TsCl (1.5 g, 8.0 mmol) in CH2Cl2 (20 mL) were added Et3N (0.55 mL, 4.0 mmol) and catalytic DMAP (24 mg, 0.2 mmol). The reaction mixture was stirred at room temperature for 5 h, and then washed sequentially with aq HCl (1 M) and brine. The organic phase was collected, dried with Na2SO4 and concentrated. The residue was purified by column chromatography (8:1 petroleum ether–EtOAc) to provide 7 (1.32 g, 95%) as a white solid; 1H NMR (600 MHz, CDCl3): d 6.99– 7.74 (m, 23H, –Ar), 4.47–4.95 (m, 6H, 3 PhCH2), 4.52 (d, 1H, J 9.6 Hz, H-1), 4.11 (dd, 1H, J 9.8, 6.2 Hz, H-6), 4.05 (dd, 1H, J 9.8, 6.7 Hz, H-60 ), 3.90 (d, 1H, J 2.2 Hz, H-4), 3.83 (t, 1H, J 9.4 Hz, H-2), 3.63 (app.t, J 6.4 Hz, 1H, H-5), 3.57 (dd, 1H, J 9.2, 2.6 Hz, H-3), 2.40 (s, 3H, ArCH3), 2.30 (s, 3H, ArCH3); LR-ESI-MS m/z calcd for [M+Na]+ 733.2; found 733.3. 4.2.4. p-Tolyl-6-azido-2,3,4-tri-O-benzyl-6-deoxy-1-thio-b-Dgalactopyranoside (8a)30 A mixture of 7 (0.8 g, 1.1 mmol) and sodium azide (0.37 g, 5.5 mmol) in dry DMF (15 mL) was stirred at 65 °C overnight. Then the solvent was removed in vacuo and the residue was dissolved in EtOAc. The excess of sodium azide and sodium tosylate was removed by filtration. The filtrate was evaporated and purified by silica gel column (12:1 petroleum ether–EtOAc) to afford 8a (0.17 g, 26%) as a white solid; 1H NMR (CDCl3, 600 MHz): d 7.04–7.48 (m, 19H, Ar), 4.60–5.03 (m, 6H, 3 PhCH2), 4.56 (d, 1H, J 9.9 Hz, H-
C. Li et al. / Carbohydrate Research 376 (2013) 15–23
1), 3.89 (t, 1H, J 9.4 Hz, H-2), 3.79 (d, 1H, J 2.1 Hz, H-4), 3.58–3.62 (m, 2H, H-3, H-6), 3.43–3.45 (m, 1H, H-5), 3.15 (dd, 1H, J 12.1, 5.5 Hz, H-60 ), 2.32 (s, 3H, ArCH3); LR-ESI-MS m/z calcd for [M+Na]+ 604.2; found 604.3. 4.2.5. 3-O-(60 -azido-20 ,30 ,40 -tri-O-benzyl-60 -deoxy-a-Dgalactosyl)-1,2-isopropylidene-sn-glycerol (9a) 4.2.5.1. For donor 8a. A mixture of the glycosyl donor 8a (137 mg, 0.23 mmol) and freshly dried 4 Å molecular sieves was stirred in dry Et2O (10 mL) at 0 °C under a N2 atmosphere, then NIS (77 mg, 0.35 mmol) was added. After stirring for 10 min, DTBMP (48 mg, 0.23 mmol) was added, followed by the addition of acceptor (S)-1,2-isopropylideneglycerol (34 lL, 0.28 mmol) and TMSOTf (50 lL, 0.14 mmol). The reaction mixture was stirred at room temperature under a N2 atmosphere for 5 h, when TLC showed that the reaction was complete. The mixture was quenched by the addition of Et3N, filtered through a pad of Celite. The filtrate was concentrated under reduced pressure and the residue was dissolved in CH2Cl2. The solution was washed with 10% Na2S2O3 solution, followed by satd aq NaHCO3. The organic layer was dried over Na2SO4, then the solvent was removed in vacuo, and the residue was purified by column chromatography (8:1 petroleum ether–EtOAc) to afford a mixture of 9a and 9b (115 mg, 83%, a/b = 9:5) as a syrup; 1H NMR (CDCl3, 600 MHz): d H-1 for 9a: 4.93 (d, J 3.8 Hz), d H-1 for 9b: 4.40 (d, J 7.7 Hz). HRESI-MS m/z calcd for C33H39O7N3 Na 612.2686 [M+Na]+, found 612.2680. 4.2.5.2. For donor 8b. To a stirred solution of 8a (120 mg, 0.20 mmol) in acetone (4.5 mL) and H2O (0.5 mL) at ambient temperature was added NBS (110 mg, 0.60 mmol). After 10 min, the reaction was quenched with satd aq NaHCO3 and the mixture concentrated. The crude intermediate was diluted with CHCl3 (15 mL) and washed sequentially with satd aq NaHCO3 (10 mL), brine (2 10 mL), dried over Na2SO4 and concentrated. The residue was purified by silica gel column chromatography to afford a white solid (82.0 mg). The white solid was added CCl3CN (0.11 mL, 1.1 mmol) and DBU (13.7 lL, 0.092 mmol) in CH2Cl2 (4 mL) at 0 °C. After 4 h, the solution was concentrated and the residue was purified by silica gel column chromatography (10:1 petroleum ether–EtOAc) to afford 8b (92 mg, 72%) as a syrup. To a solution of trichloroacetimidate 8b (50.0 mg, 0.081 mmol) and (S)-1,2-isopropylideneglycerol (13 lL, 0.097 mmol) in dry Et2O (4 mL) was added freshly dried powdered 4 Å MS and the mixture was cooled to 0 °C. TMSOTf (3.3 lL, 0.016 mmol) was added to the solution, and the mixture was stirred at 0 °C for 0.5 h. The mixture was diluted with EtOAc (40 mL) and filtered through Celite. The organic layer was washed sequentially with satd aq NaHCO3 and brine, dried (Na2SO4), and concentrated. The residue was purified by column chromatography on silica gel (8:1 petroleum ether– EtOAc) to furnish 9a and 9b (43.6 mg, 86%, a/b = 2:5) as a syrup; 1 H NMR (CDCl3, 600 MHz): d H-1 for 9a: 4.93 (d, J 3.8 Hz), d H-1 for 9b: 4.40 (d, J 7.7 Hz). 4.2.6. p-Tolyl-4,6-O-Benzylidene-2,3-di-O-benzyl-1-thio-b-Dgalactopyranoside (10)31 Compound 3 (0.5 g, 1.7 mmol) was dissolved in dry DMF (5 mL), then benzaldehyde dimethyl acetal (0.3 mL, 2.0 mmol) and TsOH (65 mg, 0.34 mmol) were added. After stirring at 50 °C for 6 h, the mixture was neutralized by the addition of Et3N, and then evaporated to dryness to provide a foamy solid. The crude foamy solid (0.60 g, about 1.3 mmol) was dissolved in DMF (5 mL) and then cooled to 0 °C. Sodium hydride (156 mg, 60%, 3.9 mmol) was added, followed by BnBr (0.4 mL, 3.9 mmol). The reaction was monitored by TLC and was complete after 2 h at 0 °C. The mixture was diluted with water (15 mL) and extracted with CH2Cl2
19
(15 mL 3). All the organic extracts were combined, dried over Na2SO4 and concentrated. The residue was recrystallized in ethanol to afford 10 (0.77 g, 80%) as a white solid; 1H NMR (600 MHz, CDCl3): d 7.25–7.62 (m, 19H, –Ar), 5.48 (s, 1H, PhCH), 4.68–4.73 (m, 4H, 2 PhCH2), 4.56 (d, 1H, J 9.3 Hz, H-1), 4.37 (dd, 1H, J 12.1, 1.6 Hz, H-6), 4.14 (d, 1H, J 2.8 Hz, H-4), 3.97 (dd, 1H, J 12.1, 1.7 Hz, H-60 ), 3.83 (t, 1H, J 9.3 Hz, H-2), 3.62 (dd, 1H, J 9.3, 3.3 Hz, H-3), 3.40–3.42 (m, 1H, H-5), 2.30 (s, 3H, ArCH3); LR-ESI-MS m/z calcd for [M+Na]+ 577.2; found 577.1. 4.2.7. p-Tolyl-2,3-di-O-benzyl-1-thio-b-D-galactopyranoside (11) A mixture of compound 10 (1.2 g, 2.1 mmol) and TsOHH2O (0.41 g, 2.1 mmol) in MeOH (30 mL) was stirred at room temperature for 2 h. The reaction solution was diluted with EtOAc (100 mL), and the mixture was consecutively washed by satd aq NaHCO3 and brine. The organic layer was dried over NaSO4, filtered, and concentrated in vacuo to get a residue, which was recrystallized in EtOH to afford 11 (0.86 g, 88%) as a foamy solid; 1 H NMR (600 MHz, CDCl3): d 7.02–7.39 (m, 14H, –Ar), 4.62–4.77 (m, 4 H, 2 PhCH2), 4.51 (d, 1H, J 9.4 Hz, H-1), 3.97 (d, 1H, J 2.8 Hz, H-4), 3.88 (dd, 1H, J 11.5, 6.6 Hz, H-6), 3.72 (dd, 1H, J 11.5, 3.8 Hz, H-60 ), 3.64 (t, 1H, J 9.4 Hz, H-2), 3.50 (dd, 1H, J 8.8, 2.8 Hz, H-3), 3.39 (m, 1H, H-5), 2.24 (s, 3H, ArCH3), 2.08 (br s, 2H, 2 –OH); LR-ESI-MS m/z calcd for [M+Na]+ 489.2; found 489.1. 4.2.8. p-Tolyl-2,3-di-O-benzyl-6-O-p-tosyl-1-thio-b-Dgalactopyranoside (12) To a solution of 11 (750 mg, 1.6 mmol) in dry pyridine (15 mL) under N2 at 45 °C, was added TsCl (340 mg, 1.76 mmol). Then the temperature was raised to ambient temperature. CH2Cl2 (30 mL) was added after 6 h and the mixture was washed with water and aq HCl (0.1 M). The organic phase was dried and concentrated and the residue was purified by chromatography on silica gel (1:2 EtOAc–petroleum ether) to give 12 (697 mg, 70%) as a white 1 solid; ½a22 H NMR (600 MHz, CDCl3): d D +10.2 (c 1.5, CHCl3); 7.07–7.79 (m, 18H, Ar), 4.66–4.81 (m, 4H, 2 PhCH2), 4.50 (d, 1H, J 9.9 Hz, H-1), 4.25 (dd, 1H, J 10.4, 6.1 Hz, H-6), 4.19 (dd, 1H, J 10.4, 7.1 Hz, H-60 ), 3.98 (d, 1H, J 3.3 Hz, H-4), 3.71 (t, 1H, J 9.3 Hz, H-2), 3.62–3.65 (m, 1H, H-5), 3.54 (dd, 1H, J 8.8, 3.3 Hz, H-3), 2.41 (s, 3H, ArCH3), 2.31 (s, 3H, ArCH3); 13C NMR (150 MHz, CDCl3): d 21.3, 21.9, 66.3, 68.5, 72.6, 75.4, 75.9, 76.9, 82.2, 88.1, 128.1–128.8, 129.8–130.1 (resonance overlap), 132.7, 132.8, 137.6, 138.0, 138.3, 145.2; LR-ESI-MS m/z calcd for [M+Na]+ 643.2; found 643.1. 4.2.9. p-Tolyl-6-azido-2,3-di-O-benzyl-6-deoxy-1-thio-b-Dgalactopyranoside (13) To a solution of 12 (500 mg, 0.80 mmol) in dry DMF (12 mL), were added 15-crown-5 (0.16 mL, 0.80 mmol) and NaN3 (200 mg, 3.2 mmol). The reaction mixture was stirred at 65 °C for 8 h, then cooled to rt, and water (20 mL) and CH2Cl2 (80 mL) were added. The organic layer was dried over Na2SO4, filtered and concentrated. The residue was purified on silica gel (1:4 EtOAc–petroleum ether) to give 13 (300 mg, 76%) as a thick oil; 1 ½a22 ): 3383.5 (mO–H), 3030.0 D +26.0 (c 4.0, CHCl3); IR (film, cm (mAr-H), 2106.6 (mN„N), 1092.9 cm1(mC–O); 1H NMR (600 MHz, CDCl3): d 7.10–7.49 (m, 14H, Ar), 4.86 (d, 1H, J 9.9 Hz, PhCH2), 4.74 (d, 1H, J 9.9 Hz, PhCH2), 4.69 (br s, 2H, PhCH2, H-1), 4.55 (d, 1H, J 9.9 Hz, PhCH2), 3.94 (br s, 1H, H-4, J 4.4 Hz), 3.65–3.70 (m, 2H, H-2, H-6), 3.57(dd, 1H, J 8.8, 3.3 Hz, H-3), 3.47–3.49 (m, 1H, H-5), 3.39 (dd, 1H, J 13.2, 5.5 Hz, H-60 ), 2.33 (s, 3H, ArCH3); 13 C NMR (150 MHz, CDCl3): d 21.4, 51.3, 67.1, 72.7, 76.0, 76.9, 77.0, 82.5, 88.6, 128.1–128.8, 129.7–129.9 (resonance overlap), 133.1, 137.6, 138.2, 138.3; LR-ESI-MS m/z calcd for [M+Na]+ 514.2; found 514.2.
20
C. Li et al. / Carbohydrate Research 376 (2013) 15–23
4.2.10. p-Tolyl-4-O-acetyl-6-azido-2,3-di-O-benzyl-6-deoxy-1thio-b-D-galactopyranoside (14) To a solution of 13 (1.0 g, 2.0 mmol) in CH2Cl2 (15 mL), were added Ac2O (0.38 mL, 4.0 mmol) and Et3N (0.55 mL, 4.0 mmol) and catalytic DMAP (24 mg, 0.2 mmol). The reaction mixture was stirred at room temperature for 0.5 h, then washed sequentially with aq HCl (1 M) and brine. The organic phase was dried and concentrated and the residue was purified by column chromatography on silica gel to provide 14 (0.99 g, 92%) as a white solid; ½a22 D +3.7 (c 1.4, CHCl3); IR (film, cm1): 3027.1 (mAr-H), 2103.7 (mN„N), 1743.9 (mC@O), 1230.3, 1102.3 (mC–O–C); 1H NMR (600 MHz, CDCl3): d 7.10–7.49 (m, 14H, Ar), 5.46 (d, 1H, J 4.4 Hz, H-4), 4.81 (d, 1H, J 9.8 Hz, PhCH2), 4.75 (d, 1H, J 9.8 Hz, PhCH2), 4.72 (d, 1H, J 11.0 Hz, PhCH2), 4.61 (d, 1H, J 7.7 Hz, H-1), 4.60 (d, 1H, J 11.0 Hz, PhCH2), 3.61–3.64 (m, 3H, H-2, H-3, H-5), 3.53 (dd, 1H, J 12.1, 7.7 Hz, H-6), 3.22 (dd, 1H, J 12.1, 4.4 Hz, H-60 ), 2.33 (s, 3H, ArCH3), 2.17 (s, 3H, –COCH3); 13C NMR (150 MHz, CDCl3): d 21.1, 21.3, 51.4, 67.5, 72.3, 76.0, 76.9, 77.1, 81.1, 88.7, 128.1–128.7, 129.6–129.9 (resonance overlap), 133.2, 137.6, 138.2, 138.4, 170.1; LR-ESI-MS m/z calcd for [M+Na]+ 556.2; found 556.1. 4.2.11. 3-O-(40 -O-Acetyl-60 -azido-20 ,30 -di-O-benzyl-60 -deoxy-a-Dgalactosyl)-1,2-isopropylidene-sn-glycerol (15a) A mixture of the glycosyl donor 14 (2.0 g, 3.8 mmol) and freshly dried 4 Å molecular sieves was stirred in dry Et2O (150 mL) at room temperature under a N2 atmosphere. The reaction mixture was cooled in an ice bath and NIS (1.28 g, 5.7 mmol) was added. After stirring for 10 min, DTBMP (780 mg, 3.8 mmol) was added, followed by the addition of acceptor (S)-1,2-isopropylideneglycerol (0.57 mL, 4.6 mmol) and TMSOTf (0.4 mL, 2.3 mmol). The reaction mixture was stirred at 0 °C under a N2 atmosphere for 12 h, when TLC showed that the reaction was complete. The mixture was quenched by the addition of Et3N, filtered through a pad of Celite. The filtrate was removed under reduced pressure and the residue was dissolved in CH2Cl2. The solution was washed with 10% Na2S2O3, followed by satd aq NaHCO3. The organic layer was dried over Na2SO4, then the solvent was removed in vacuo, and the residue was purified by column chromatography on silica gel to afford 15a and 15b (1.82 g, 90%, a/b = 32:1) as a thick oil; for 15a: ½a22 D +89.0 (c 2.5, CHCl3); 1H NMR (600 MHz, CDCl3): d 7.27–7.36 (m, 10H, Ar), 5.46 (d, 1H, J 2.8 Hz, H-4), 4.97 (d, 1H, J 3.8 Hz, H-1), 4.82 (d, 1H, J 12.1 Hz, PhCH2), 4.70 (d, 1H, J 11.5 Hz, PhCH2), 4.64 (d, 1H, J 11.6 Hz, PhCH2), 4.57 (d, 1H, J 11.5 Hz, PhCH2), 4.33– 4.38 (m, 1H, Hsn-2), 4.03–4.09 (m, 2H, Hsn-3, H-5), 3.94 (dd, 1H, J 9.9, 3.3 Hz, H-3), 3.78 (dd, 1H, J 10.4, 3.8 Hz, H-2), 3.75 (dd, 1H, J 8.2, 6.0 Hz, Hsn-30 ), 3.69 (dd, 1H, J 11.0, 5.0 Hz, Hsn-1), 3.60 (dd, 1H, J 10.4, 5.5 Hz, Hsn-10 ), 3.40 (dd, 1H, J 13.2, 8.2 Hz, H-6), 3.13 (dd, 1H, J 12.6, 4.4 Hz, H-60 ), 2.14 (s, 3H, –COCH3), 1.40 (s, 3H, – CH3), 1.36 (s, 3H, –CH3); 13C NMR (150 MHz, CDCl3): d 21.1, 25.8, 27.0, 51.3, 66.8, 68.6, 68.8, 69.2, 72.5, 73.6, 74.9, 75.6, 75.8, 98.2, 109.8, 127.9–128.6 (resonance overlap), 138.1, 138.6, 170.6; LRESI-MS m/z calcd for [M+Na]+ 564.6, found 564.0; HR-ESI-MS m/z calcd for C28H35O8N3 Na 564.2316 [M+Na]+, found 564.2325. 4.2.12. 3-O-(60 -Azido-20 ,30 ,40 -tri-O-benzyl-60 -deoxy-a-Dgalactosyl)-1,2-isopropylidene-sn-glycerol (16) To a solution of 15a (1.1 g, 2.0 mmol) in MeOH (30 mL) was added catalytic NaOMe (11 mg, 0.2 mmol). The reaction mixture was stirred for 30 min, and then was neutralized with Amberlite IR120 resin (H+). After filtration, the filtrate was concentrated in vacuo to get a residue. To a solution of the residue in DMF (10 mL) was added BnBr (0.36 mL, 3.0 mmol). The reaction solution was cooled to 0 °C, and then NaH (160 mg, 4 mmol) was slowly added in 10 min. After stirring for 2 h, the reaction mixture was diluted with water (30 mL) and extracted with CH2Cl2 (30 mL 3). All of the organic extracts were combined, dried over
Na2SO4 and concentrated. The residue was chromatographed on silica gel (1:8 EtOAc–petroleum ether) to give 16 (0.98 g, 82% over 1 two steps) as a thick oil. ½a22 H NMR D +20.6 (c 1.9, CHCl3); (600 MHz, CDCl3): d 7.27–7.40 (m, 15H, Ar), 4.94 (d, 1H, J 3.3 Hz, H-1), 4.58–5.01 (m, 6H, 3 PhCH2), 4.33–4.37 (m, 1H, Hsn-2), 4.01–4.07 (m, 2H, H-3, H-5), 3.91 (dd, 1H, J 9.9, 3.3 Hz, H-2), 3.84 (dd, 1H, J 8.8, 5.5 Hz, Hsn-3), 3.79 (d, 1H, J 2.2 Hz, H4), 3.74 (dd, 1H, J 8.8, 6.6 Hz, Hsn-30 ), 3.65 (dd, 1H, J 11.0, 5.5 Hz, Hsn-1), 3.58 (dd, 1H, J 11.0, 5.5 Hz, Hsn-10 ), 3.49 (dd, 1H, J 12.1, 7.7 Hz, H-6), 2.96 (dd, 1H, J 12.1, 4.4 Hz, H-60 ), 1.40 (s, 3H, – CH3), 1.37 (s, 3H, –CH3); 13C NMR (150 MHz, CDCl3): d 25.8, 27.0, 51.6, 66.9, 69.2, 70.2, 73.5, 73.8, 74.9 (2C), 75.3, 76.6, 98.2, 109.7, 127.8–128.1, 128.6–128.8 (resonance overlap), 138.3, 138.7, 138.9; LR-ESI-MS m/z calcd for [M+Na]+ 612.3, found 612.4. HR-ESI-MS m/z calcd for C33H39O7N3 Na 612.2680 [M+Na]+, found 612.2684. 4.2.13. 3-O-(60 -Amino-20 ,30 ,40 -tri-O-benzyl-60 -deoxy-a-Dgalactosyl)-sn-glycerol (17) TsOH (320 mg, 1.7 mmol) was added to a stirred solution of 16 (500 mg, 0.85 mmol) in MeOH (20 mL). After stirring for 2 h at ambient temperature, the solution was concentrated and dissolved in CH2Cl2. The organic layer was washed sequentially with satd aq NaHCO3 and water, dried over Na2SO4, filtered and concentrated to get a crude product. To a solution of the crude product in THF (25 mL) and water (0.2 mL) was added PPh3 (450 mg, 3.4 mmol). The reaction mixture was stirred at 50 °C for 5 h. The mixture was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (CH2Cl2/MeOH) to give 17 (390 mg, 88% over two steps) as a colorless waxy substance; 1 ½a22 D +27.0 (c 1.2, CHCl3); H NMR (CDCl3, 600 MHz): d 7.26–7.40 (m, 15H, Ar), 4.59–4.95 (m, 6H, 3 PhCH2), 4.77 (d, 1H, J 3.3 Hz, H-1), 4.04 (dd, 1H, J 9.9, 4.4 Hz, H-3), 3.86–3.91 (m, 2H, H-5, Hsn2), 3.77–3.81 (m, 2H, Hsn-3, Hsn-30 ), 3.68 (dd, 1H, J 9.9, 5.5 Hz, Hsn1), 3.61 (d, 1H, J 4.4 Hz, H-4), 3.50 (dd, 1H, J 11.0, 6.6 Hz, Hsn-10 ), 3.05–3.10 (m, 3H, H-2, –NH2), 2.95 (dd, 1H, J 12.1, 8.8 Hz, H-6), 2.42 (dd, 1H, J 12.1, 3.3 Hz, H-60 ); LR-ESI-MS m/z calcd for [M+H]+ 524.3; found 524.0. HR-ESI-MS m/z calcd for C30H38O7N H 524.2643 [M+H]+, found 524.2649. 4.2.14. General procedure for 18a–g formation To a solution of compound 17 (80 mg, 0.15 mmol) in dry pyridine (4 mL), catalytic DMAP (2 mg, 0.015 mmol) and acyl chloride (6 equiv) were added. The reaction mixture was stirred at room temperature for 4 h, and then was diluted with EtOAc (15 mL) and washed with satd aq NH4Cl (15 mL). The organic phase was dried over Na2SO4, filtered and concentrated. Purification by flash chromatography (petroleum ether–EtOAc) yielded the corresponding compound 18a–g (67–90%) as a colorless syrup or white solid. 4.2.14.1. 1,2-Dihexanoyl-3-(N-hexanoyl-60 -amino-20 ,30 ,40 -tri-Obenzyl-60 -deoxy-a-D-galactosyl)-sn-glycerol (18a, 1 78%). ½a22 +23.8 (c 0.35, CHCl ); H NMR (CDCl , 600 MHz): 3 3 D d 7.26–7.40 (m, 15H, Ar), 5.37 (dd, 1H, J 6.6, 3.8 Hz, –NHCO–), 5.22–5.24 (m, 1H, Hsn-2), 4.63–4.99 (m, 6H, 3 PhCH2), 4.80 (d, 1H, J 3.3 Hz, H-1), 4.35 (dd, 1H, J 12.1, 4.4 Hz, Hsn-1), 4.16 (dd, 1H, J 12.1, 6.6 Hz, Hsn-10 ), 4.02 (dd, 1H, J 9.9, 3.3 Hz, H-3), 3.89 (dd, 1H, J 9.9, 2.2 Hz, H-2), 3.80 (br s, 1H, H-4), 3.73–3.75 (m, 1H, H5), 3.66 (dd, 1H, J 11.0, 5.5 Hz, Hsn-3), 3.56 (dd, 1H, J 11.0, 5.5 Hz, Hsn-30 ), 3.38–3.42 (m, 1H, H-6), 3.19–3.24 (m, 1H, H-60 ), 2.24–2.32 (m, 4H, 2 –CH2–COO–), 1.95 (t, 2H, J 7.7 Hz, –NHCO–CH2–), 1.48–1.65 (m, 6H, 3 –CO–CH2–CH2–), 1.22–1.32 (m, 12H, 3 – CH2–CH2(CH2)2–CH3), 0.87–0.89 (m, 9H, 3 –CH3); HR-ESI-MS m/z calcd for C48H68O10N 818.4838 [M+H]+, C48H67O10N Na 840.4657 [M+Na]+, found 818.4854 [M+H]+, 840.4671 [M+Na]+.
C. Li et al. / Carbohydrate Research 376 (2013) 15–23
4.2.14.2. 1,2-Dioctanoyl-3-(N-octanoyl-60 -amino-20 ,30 ,40 -tri-Obenzyl-60 -deoxy-a-D-galactosyl)-sn-glycerol (18b, 1 87%). ½a22 D +20.8 (c 0.37, CHCl3); H NMR (CDCl3, 600 MHz): d 7.26–7.40 (m, 15H, Ar), 5.37 (dd, 1H, J 7.2, 4.2 Hz, –NHCO–), 5.22–5.24 (m, 1H, Hsn-2), 4.63–4.98 (m, 6H, 3 PhCH2), 4.80 (d, 1H, J 3.3 Hz, H-1), 4.35 (dd, 1H, J 12.1, 3.3 Hz, Hsn-1), 4.16 (dd, 1H, J 12.1, 6.6 Hz, Hsn-10 ), 4.02 (dd, 1H, J 9.9, 4.4 Hz, H-3), 3.89 (dd, 1H, J 9.9, 3.3 Hz, H-2), 3.79 (br s, 1H, H-4), 3.73–3.75 (m, 1H, H5), 3.65 (dd, 1H, J 11.0, 5.5 Hz, Hsn-3), 3.56 (dd, 1H, J 11.0, 5.5 Hz, Hsn-30 ), 3.38–3.42 (m, 1H, H-6), 3.21–3.24 (m, 1H, H-60 ), 2.26–2.34 (m, 4H, 2 –CH2–COO–), 1.95 (t, 2H, –NHCO–CH2–, J 7.7 Hz), 1.48–1.62 (m, 6H, 3 –CO–CH2–CH2–), 1.20–1.30 (m, 24H, 3 – CH2–CH2(CH2)4–CH3), 0.86–0.88 (m, 9H, 3 –CH3); LR-ESI-MS m/ z 924.6 [M+Na]+; HR-ESI-MS m/z calcd for C54H79O10N Na 924.5596 [M+Na]+, found 924.5605. 0
0
0
0
4.2.14.3. 1,2-Dilauroyl-3-(N-lauroyl-6 -amino-2 ,3 ,4 -tri-O-benzyl-60 -deoxy-a-D-galactosyl)-sn-glycerol (18c, 90%). ½a22 D 1 +10.6 (c 0.20, CHCl3); H NMR (CDCl3, 600 MHz): d 7.27–7.40 (m, 15H, Ar), 5.35 (dd, 1H, J 7.1, 4.4 Hz, –NHCO–), 5.22–5.23 (m, 1H, Hsn-2), 4.63–4.98 (m, 6H, 3 PhCH2), 4.80 (d, 1H, J 3.3 Hz, H-1), 4.35 (dd, 1H, J 12.1, 3.3 Hz, Hsn-1), 4.16 (dd, 1H, J 12.1, 6.6 Hz, Hsn-10 ), 4.02 (dd, 1H, J 9.9, 3.3 Hz, H-3), 3.88 (dd, 1H, J 9.9, 3.3 Hz, H-2), 3.80–3.81 (br s, 1H, H-4), 3.73–3.75 (m, 1H, H-5), 3.65 (dd, 1H, J 11.0, 5.5 Hz, Hsn-3), 3.55 (dd, 1H, J 11.0, 5.5 Hz, Hsn-30 ), 3.38– 3.43 (m, 1H, H-6), 3.19–3.24 (m, 1H, H-60 ), 2.26–2.34 (m, 4H, 2 –CH2–COO–), 1.95 (t, 2H, –NHCO–CH2–, J 6.6 Hz), 1.48–1.65 (m, 6H, 3 –CO–CH2–CH2–), 1.20–1.31 (m, 48H, 3 –CH2– CH2(CH2)8–CH3), 0.88 (t, 9H, J 6.6 Hz, 3 –CH3); HR-ESI-MS m/z calcd for C66H103O10N Na 1092.7474 [M+Na]+, found 1092.7458. 4.2.14.4. 1,2-Dimyristoyl-3-(N-myristoyl-60 -amino-20 ,30 ,40 -tri-Obenzyl-60 -deoxy-a-D-galactosyl)-sn-glycerol (18d, 1 83%). ½a22 D +10.8 (c 0.35, CHCl3); H NMR (CDCl3, 600 MHz): d 7.26–7.40 (m, 15H, Ar), 5.36 (dd, 1H, J 7.1, 3.8 Hz, –NHCO–), 5.22–5.23 (m, 1H, Hsn-2), 4.62–4.98 (m, 6H, 3 PhCH2), 4.80 (d, 1H, J 3.3 Hz, H-1), 4.35 (dd, 1H, J 12.1, 3.3 Hz, Hsn-1), 4.16 (dd, 1H, J 12.1, 5.5 Hz, Hsn-10 ), 4.02 (dd, 1H, J 9.9, 3.3 Hz, H-3), 3.88 (dd, 1H, J 9.9, 3.3 Hz, H-2), 3.80 (br s, 1H, H-4), 3.73–3.75 (m, 1H, H5), 3.65 (dd, 1H, J 11.0, 5.5 Hz, Hsn-3), 3.55 (dd, 1H, J 11.0, 5.5 Hz, Hsn-30 ), 3.38–3.41 (m, 1H, H-6), 3.21–3.23 (m, 1H, H-60 ), 2.26–2.35 (m, 4H, 2 –CH2–COO–), 1.94 (t, 2H, J 7.7 Hz, –NHCO–CH2–), 1.47–1.63 (m, 6H, 3 –CO–CH2–CH2–), 1.18–1.31 (m, 60H, 3 – CH2–CH2(CH2)10–CH3), 0.87 (t, 9H, J 7.6 Hz, 3 –CH3); LR-ESI-MS m/z 1176.9 [M+Na]+; HR-ESI-MS m/z calcd for C72H116O10N 1154.8594 [M+H]+, C72H115O10N Na 1176.8413 [M+Na]+, found 1154.8571 [M+H]+, 1176.8405 [M+Na]+. 4.2.14.5. 1,2-Dipalmitoyl-3-(N-palmitoyl-60 -amino-20 ,30 ,40 -tri-Obenzyl-60 -deoxy-a-D-galactosyl)-sn-glycerol (18e, 1 74%). ½a22 D +12.0 (c 0.60, CHCl3); H NMR (CDCl3, 600 MHz): d 7.26–7.40 (m, 15H, Ar), 5.37 (dd, 1H, J 7.2, 4.2 Hz, –NHCO–), 5.21–5.23 (m, 1H, Hsn-2), 4.62–4.98 (m, 6H, 3 PhCH2), 4.80 (d, 1H, J 3.3 Hz, H-1), 4.35 (dd, 1H, J 12.1, 3.3 Hz, Hsn-1), 4.16 (dd, 1H, J 12.1, 6.6 Hz, Hsn-10 ), 4.02 (dd, 1H, J 9.9, 4.4 Hz, H-3), 3.88 (dd, 1H, J 9.9, 3.3 Hz, H-2), 3.80 (br s, 1H, H-4), 3.73–3.75 (m, 1H, H5), 3.65 (dd, 1H, J 11.0, 5.5 Hz, Hsn-3), 3.55 (dd, 1H, J 11.0, 5.5 Hz, Hsn-30 ), 3.38–3.42 (m, 1H, H-6), 3.19–3.23 (m, 1H, H-60 ), 2.25–2.35 (m, 4H, 2 –CH2COO–), 1.95 (t, 2H, J 6.6 Hz, –NHCO–CH2–), 1.48–1.64 (m, 6H, 3 –CO–CH2–CH2–), 1.20–1.31 (m, 72H, 3 – CH2–CH2(CH2)12–CH3), 0.87 (t, 9H, J 6.6 Hz, 3 –CH3); LR-ESI-MS m/z 1260.9 [M+Na]+; HR-ESI-MS m/z calcd for C78H128O10N 1238.9533 [M+H]+, C78H127O10N Na 1260.9352 [M+Na]+, found 1238.9545 [M+H]+, 1260.9370 [M+Na]+.
21
4.2.14.6. 1,2-Distearoyl-3-(N-stearoyl-60 -amino-20 ,30 ,40 -tri-O0 benzyl-6 -deoxy-a-D-galactosyl)-sn-glycerol (18f, 1 67%). ½a22 D +5.4 (c 0.10, CHCl3); H NMR (CDCl3, 600 MHz): d 7.26–7.40 (m, 15H, Ar), 5.36 (dd, 1H, J 7.7, 4.4 Hz, –NHCO–), 5.20–5.23 (m, 1H, Hsn-2), 4.62–4.98 (m, 6H, 3 PhCH2), 4.80 (d, 1H, J 3.3 Hz, H-1), 4.34 (dd, 1H, J 12.1, 3.3 Hz, Hsn-1), 4.16 (dd, 1H, J 12.1, 6.6 Hz, Hsn-10 ), 4.02 (dd, 1H, J 9.9, 4.4 Hz, H-3), 3.88 (dd, 1H, J 9.9, 2.2 Hz, H-2), 3.80 (br s, 1H, H-4), 3.73–3.75 (m, 1H, H5), 3.65 (dd, 1H, J 11.0, 5.5 Hz, Hsn-3), 3.56 (dd, 1H, J 11.0, 6.6 Hz, Hsn-30 ), 3.38–3.42 (m, 1H, H-6), 3.19–3.23 (m, 1H, H-60 ), 2.26–2.34 (m, 4H, 2 –CH2–COO–), 1.94 (t, 2H, J 7.7 Hz, –NHCO–CH2–), 1.49–1.62 (m, 6H, 3 –CO–CH2–CH2–), 1.18–1.31 (m, 84H, 3 – CH2–CH2(CH2)14–CH3), 0.87 (t, 9H, J 6.6 Hz, 3 –CH3); LR-ESI-MS m/z calcd for [M+Na]+ 1345.0, found 1345.0. 4.2.14.7. 1,2-Diisovaleryl-3-(N-isovaleryl-60 -amino-20 ,30 ,40 -tri-Obenzyl-60 -deoxy-a-D-galactosyl)-sn-glycerol (18g, 1 82%). ½a22 D +23.8 (c 0.65, CHCl3); H NMR (CDCl3, 600 MHz): d 7.26–7.40 (m, 15H, Ar), 5.35 (dd, 1H, J 7.2, 4.2 Hz, –NHCO–), 5.21–5.24 (m, 1H, Hsn-2), 4.63–4.99 (m, 6H, 3 PhCH2), 4.79 (d, 1H, J 2.8 Hz, H-1), 4.35 (dd, 1H, J 12.1, 3.3 Hz, Hsn-1), 4.15 (dd, 1H, J 12.1, 6.6 Hz, Hsn-10 ), 4.02 (dd, 1H, J 9.9, 3.3 Hz, H-3), 3.88 (dd, 1H, J 9.9, 2.2 Hz, H-2), 3.79 (br s, 1H, H-4), 3.72–3.74 (m, 1H, H5), 3.66 (dd, 1H, J 11.0, 5.5 Hz, Hsn-3), 3.57 (dd, 1H, J 11.0, 6.6 Hz, Hsn-30 ), 3.40–3.44 (m, 1H, H-6), 3.20–3.24 (m, 1H, H-60 ), 2.16–2.22 (m, 4H, 2 –CH2–COO–), 1.81–2.09 (m, 5H, –NHCO–CH2–, 3 – CH(CH3)2), 0.85–0.95 (m, 18H, 6 –CH3). LR-ESI-MS m/z 776.4 [M+H]+, 798.4 [M+Na]+; HR-ESI-MS m/z calcd for C45H62O10N 776.4368 [M+H], C45H61O10N Na 798.4188 [M+Na], found 776.4375 [M+H]+, 798.4194 [M+Na]+. 4.2.15. General procedure for 2a–g formation A solution of 18a–g (100 mg) in THF/i-PrOH (9:1, 20 mL) was treated with 10% palladium(II) hydroxide (100 mg) and stirred at ambient temperature under a hydrogen atmosphere for 8 h. After filtration, the solvent was evaporated and the residue was purified by column chromatography (CH2Cl2–MeOH) to afford 2a–g (82– 90%) as a colorless syrup or waxy solid. 4.2.15.1. 1,2-Dihexanoyl-3-(N-hexanoyl-60 -amino-60 -deoxy-a-Dgalactosyl)-sn-glycerol (2a, 82%). ½a22 D +109.2 (c 0.5, CHCl3); 1 H NMR (CDCl3, 600 MHz): d 6.46 (dd, 1H, J 12.1, 6.6 Hz, –NHCO–), 5.24–5.26 (m, 1H, Hsn-2), 4.88 (d, 1H, J 3.3 Hz, H-1), 4.35 (dd, 1H, J 12.1, 3.3 Hz, Hsn-1), 4.14 (dd, 1H, J 12.1, 6.6 Hz, Hsn-10 ), 3.66–3.90 (m, 7H, H-2, H-3, H-4, H-5, H-6, Hsn-3, Hsn-30 ), 3.26–3.28 (m, 1H, H-60 ), 2.30–2.34 (m, 4H, 2 –CH2–COO–), 2.20 (t, 2H, J 7.7 Hz, – NHCO–CH2–), 1.59–1.62 (m, 6H, 3 –CO–CH2–CH2–), 1.25–1.34 (m, 12H, 3 –CH2–CH2(CH2)2CH3), 0.86–0.90 (m, 9H, 3 –CH3); 13 C NMR (150 MHz, CDCl3): d 14.1 (3CH3), 22.5 (2CH2), 24.7, 24.8, 25.6, 29.9, 31.4, 31.6, 31.7, 34.3, 34.5, 36.7, 39.5 (C-60 ), 62.7, 67.5, 69.0, 69.1, 69.4, 70.3, 70.4, 100.1 (C-10 ), 173.7, 174.0, 175.2; LRESI-MS m/z 548.2 [M+H]+, 570.2 [M+Na]+; HR-ESI-MS m/z calcd for C27H50O10N 548.3429 [M+H]+, C27H49O10N Na 570.3249 [M+Na]+, found 548.3436 [M+H]+, 570.3254 [M+Na]+. 4.2.15.2. 1,2-Dioctanoyl-3-(N-octanoyl-60 -amino-60 -deoxy-a-Dgalactosyl)-sn-glycerol (2b, 88%). ½a22 D +69.7 (c 1.4, CHCl3); 1 H NMR (CDCl3, 600 MHz): d 6.29 (dd, 1H, –NHCO–, J 12.1, 6.6 Hz), 5.24–5.26 (m, 1H, Hsn-2), 4.88 (d, 1H, J 3.3 Hz, H-1), 4.33 (dd, 1H, J 12.1, 3.3 Hz, Hsn-1), 4.14 (dd, 1H, J 12.1, 6.6 Hz, Hsn-10 ), 3.66–3.84 (m, 7H, H-2, H-3, H-4, H-5, H-6, Hsn-3, Hsn-30 ), 3.20– 3.22 (m, 1H, H-60 ), 2.30–2.36 (m, 4H, 2 –CH2–COO–), 2.20 (t, 2H, J 7.7 Hz, –NHCO–CH2–), 1.58–1.62 (m, 6H, 3 –CO–CH2– CH2–), 1.22–1.32 (m, 24H, 3 –CH2–CH2(CH2)4CH3), 0.88 (t, 9H, J 6.6 Hz, 3 –CH3); 13C NMR (150 MHz, CDCl3): d 14.3 (3CH3), 22.8, 25.1, 25.2, 25.9, 29.2–29.5 (resonance overlap), 31.9, 34.3,
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C. Li et al. / Carbohydrate Research 376 (2013) 15–23
34.5, 36.8, 39.7 (C-60 ), 62.6, 67.4, 68.9, 69.3, 69.5, 70.3, 70.4, 100.1 (C-10 ), 173.7, 173.9, 175.0; LR-ESI-MS m/z 632.5 [M+H]+, 654.5 [M+Na]+; HR-ESI-MS m/z calcd for C33H62O10N 632.4368 [M+H]+, C33H61O10N Na 654.4188 [M+Na]+, found 632.4376 [M+H]+, 654.4194 [M+Na]+. 4.2.15.3. 1,2-Dilauroyl-3-(N-lauroyl-60 -amino-60 -deoxy-a-Dgalactosyl)-sn-glycerol (2c, 90%). ½a22 D +56.2 (c 1.9, CHCl3); 1 H NMR (CDCl3, 600 MHz): d 6.35 (dd, 1H, J 12.1, 6.6 Hz, –NHCO– ), 5.24–5.26 (m, 1H, Hsn-2), 4.87 (d, 1H, J 3.3 Hz, H-1), 4.34 (dd, 1H, J 11.0, 3.3 Hz, Hsn-1), 4.14 (dd, 1H, J 12.1, 5.5 Hz, H sn-10 ), 3.65–3.85 (m, 7H, H-2, H-3, H-4, H-5, H-6, Hsn-3, Hsn-30 ), 3.21– 3.23 (m, 1H, H-60 ), 2.30–2.34 (m, 4H, 2 –CH2–COO–), 2.19 (t, 2H, J 7.4 Hz, –NHCO–CH2–), 1.59–1.62 (m, 6H, 3 –CO–CH2– CH2–), 1.22–1.32 (m, 48H, 3 –CH2–CH2(CH2)8CH3), 0.88 (t, 9H, J 6.6 Hz, 3 –CH3); 13C NMR13C NMR (150 MHz, CDCl3): d 14.3 (3CH3), 22.9, 25.1, 25.2, 25.9, 29.4–30.0 (resonance overlap), 32.1, 34.3, 34.5, 36.8, 39.7 (C-60 ), 62.7, 67.3, 69.1, 69.3, 69.4, 70.3, 70.4, 100.1 (C-10 ), 173.6, 173.9, 175.0; LR-ESI-MS m/z 800.4 [M+H]+; HR-ESI-MS m/z calcd for C45H86O10N 800.6246 [M+H]+, found 800.6253. 4.2.15.4. 1,2-Dimyristoyl-3-(N-myristoyl-60 -amino-60 -deoxy-aa-D-galactosyl)-sn-glycerol (2d, 85%). ½a22 D +46.0 (c 1.0, 1 CHCl3); H NMR (CDCl3, 600 MHz): d 6.08 (dd, 1H, J 12.1, 6.6 Hz, –NHCO–), 5.25–5.27 (m, 1H, Hsn-2), 4.88 (d, 1H, J 3.3 Hz, H-1), 4.32 (dd, 1H, J 12.1, 3.8 Hz, Hsn-1), 4.13 (dd, 1H, J 12.1, 6.1 Hz, Hsn-10 ), 3.67–3.81 (m, 7H, H-2, H-3, H-4, H-5, H-6, Hsn-3, Hsn-30 ), 3.13–3.15 (m, 1H, H-60 ), 2.30–2.34 (m, 4H, 2 –CH2–COO–), 2.20 (t, 2H, J 7.7 Hz –NHCO–CH2–), 1.59–1.62 (m, 6H, 3 –CO–CH2– CH2–), 1.21–1.31 (m, 60H, 3 –CH2–CH2(CH2)10–CH3), 0.87 (t, 9H, J 7.1 Hz, 3 –CH3); 13C NMR (150 MHz, CDCl3): d 14.4 (3CH3), 22.9, 25.1, 25.2, 25.9, 29.4–30.0 (resonance overlap), 32.1, 34.3, 34.5, 36.8, 39.6 (C-60 ), 62.5, 67.7, 68.7, 69.4, 69.8, 70.3, 70.4, 100.1 (C-10 ), 173.6, 173.9, 175.2; LR-ESI-MS m/z 884.8 [M+H]+; HR-ESI-MS m/z calcd for C51H98O10N 884.7185 [M+H]+, found 884.7198. D–
4.2.15.5. 1,2-Dipalmitoyl-3-(N-palmitoyl-60 -amino-60 -deoxy-a½a22 +63.2 (c 1.0, D (dd, 1H, J 12.1, 6.6 Hz, –NHCO–), 5.24–5.26 (m, 1H, Hsn-2), 4.87 (d, 1H, J 3.3 Hz, H-1), 4.32 (dd, 1H, J 12.1, 3.8 Hz, Hsn-1), 4.13 (dd, 1H, J 12.1, 6.1 Hz, Hsn-10 ), 3.66–3.82 (m, 7H, H-2, H-3, H-4, H-5, H-6, Hsn-3, Hsn-30 ), 3.15–3.18 (m, 1H, H-60 ), 2.30–2.34 (m, 4H, 2 –CH2–COO–), 2.20 (t, 2H, J 7.7 Hz, –NHCO–CH2–), 1.59–1.62 (m, 6H, 3 –CO–CH2– CH2–), 1.21–1.31 (m, 72H, 3 –CH2–CH2(CH2)12–CH3), 0.87 (t, 9H, J 6.6 Hz, 3 –CH3); 13C NMR (150 MHz, CDCl3): d 14.3 (3CH3), 22.9, 25.1, 25.2, 25.9, 29.6–30.0 (resonance overlap), 32.2, 34.3, 34.6, 36.8, 39.7 (C-60 ), 62.6, 67.7, 68.8, 69.4, 69.8, 70.4, 70.5, 100.2 (C-10 ), 173.6, 173.8, 175.1; HR-MALDI-MS m/z calcd for C57H109O10NNa 990.7949 [M+Na]+, found 990.7919. 17 D-galactosyl)-sn-glycerol (2e, 85%) . 1 CHCl3); H NMR (CDCl3, 600 MHz): d 6.08
0
0
4.2.15.6. 1,2-Distearoyl-3-(N-stearoyl-6 -amino-6 -deoxy-a-Dgalactosyl)-sn-glycerol (2f, 82%). ½a22 D +31.5 (c 2.6, CHCl3); 1 H NMR (CDCl3, 600 MHz): d 6.09 (dd, 1H, J 11.0,5.5 Hz, –NHCO– ), 5.24–5.26 (m, 1H, Hsn-2), 4.88 (d, 1H, J 3.3 Hz, H-1), 4.32 (dd, 1H, J 11.0, 3.3 Hz, Hsn-1), 4.13 (dd, 1H, J 12.1, 6.1 Hz, Hsn-10 ), 3.67– 3.86 (m, 7H, H-2, H-3, H-4, H-5, H-6, Hsn-3, Hsn-30 ), 3.13–3.17 (m, 1H, H-60 ), 2.30–2.34 (m, 4H, 2 –CH2–COO–), 2.19 (t, 2H, J 7.7 Hz, –NHCO–CH2–), 1.57–1.64 (m, 6H, 3 –CO–CH2–CH2–), 1.15–1.35 (m, 84H, 3 –CH2–CH2(CH2)14–CH3), 0.88 (t, 9H, J 6.6 Hz, 3 –CH3); 13C NMR (150 MHz, CDCl3): d 14.2 (3CH3), 22.9, 25.1, 25.2, 25.9, 29.6–29.9 (resonace overlap), 32.2, 34.3, 34.5, 36.8, 39.6 (C-60 ), 62.6, 67.6, 68.9, 69.3, 69.7, 70.3, 70.4,
100.1 (C-10 ), 173.6, 173.8, 175.1; HR-MALDI-MS m/z calcd for C63H121O10NNa 1074.8888 [M+Na]+, found 1074.8886. 4.2.15.7. 1,2-Diisovaleryl-3-(N-isovaleryl-60 -amino-60 -deoxy-a½a22 D +75.0 (c 2.2, CHCl3); 1H, J 12.1, 6.6 Hz, –NHCO–), 5.25–5.27 (m, 1H, Hsn-2), 4.88 (d, 1H, J 2.8 Hz, H-1), 4.36 (dd, 1H, J 12.1, 2.2 Hz, Hsn-1), 4.14 (dd, 1H, J 11.5, 6.1 Hz, Hsn-10 ), 3.65–3.88 (m, 7H, H-2, H-3, H-4, H-5, H-6, Hsn-3, Hsn-30 ), 3.27–3.30 (m, 1H, H-60 ), 2.17–2.22 (m, 4H, 2 –CH2–COO–), 2.05–2.10 (m, 5H, – NHCO–CH2–, 3 –CH(CH3)2), 0.92–0.97 (m, 18H, 6 –CH3); 13C NMR (150 MHz, CDCl3): d 22.3–22.7 (resonace overlap), 25.8, 25.9, 26.2, 39.8 (C-60 ), 43.3, 43.5, 45.9, 62.7, 67.1, 69.2, 69.4, 70.1, 70.2, 70.3, 100.1 (C-10 ), 172.9, 173.2, 174.1; LR-ESI-MS m/z 506.2 [M+H]+, 528.2 [M+Na]+; HR-ESI-MS m/z calcd for C24H44O10N 506.2960 [M+H]+, found 506.2959. D-galactosyl)-sn-glycerol (2g, 83%). 1 H NMR (CDCl3, 600 MHz): d 6.57 (dd,
4.3. Bioassay methods The Myt1 assay was performed using the ADPGlo™ assay kit (Promega) which measures the generation of ADP by Myt1. Generation of ADP by the Myt1 reaction leads to an increase in the luminescence signal in the presence of™ assay kit. The Myt1 assay was performed at 30 °C for 30 min in a final volume of 25 lL according to the following assay reaction recipe: 5 lL of diluted active Myt1, 5 lL of stock solution of peptide substrate, 5 lL kinase assay buffer, 5 lL of compound or 10% DMSO, and 5 lL of 50 mM ATP stock solution. The assay was started by incubating the reaction mixture in a 96-well plate at 30 °C for 30 min. After a 30 min’ incubation period, the assay was terminated by the addition of 25 mL of ADPGlo™ reagent (Promega). The 96-well plate was shaken and then incubated for 40 min at ambient temperature. Then 50 mL of Kinase Detection Reagent was added, the 96-well plate shaken and then incubated for a further 30 min at ambient temperature. The 96-well reaction plate was then read using the ADPGlo™ Luminescence Protocol on a GloMax plate reader (Promega; Catalog #E7031). Blank control was set up that included all the assay components except the addition of the substrate (replace with equal volume of kinase assay buffer). The corrected activity for Myt1 target was determined by removing the blank control value. Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 81072574), program for New Century Excellent Talents in University (NCET-08-0506), Special Fund for Marine Scientific Research in the Public Interest (No. 201005024) and PCSIRT (No. IRT09444). Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.carres.2013.02.008. References 1. Liu, F.; Stanton, J. J.; Wu, Z.; Piwnica-Worms, H. Mol. Cell. Biol. 1997, 17, 571– 583. 2. Liu, F.; Rothblum-Oviatt, C.; Ryan, Ch. E.; Piwnica-Worms, H. Mol. Cell. Biol. 1999, 5113–5123. 3. Mueller, P. R.; Coleman, T. R.; Kumagai, A.; Dunphy, W. G. Science 1995, 270, 86–89. 4. Lago, M. A. Myt1 Kinase Inhibitors. U.S. Patent No. US 6,391,894, May 21, 2002. 5. Zhou, B.; Tang, S.; Johnson, R. K.; Mattern, M. P.; Lazo, J. S.; Sharlow, E. R.; Harich, K.; Kingston, D. G. I. Tetrahedron 2005, 61, 883–887. 6. Wu, H. J.; Li, C. X.; Song, G. P.; Li, Y. X. Chin. J. Chem. 2008, 26, 1641–1646.
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