CN1158270C - Bipiperazine derivatives with calcium ion antagon action - Google Patents
Bipiperazine derivatives with calcium ion antagon action Download PDFInfo
- Publication number
- CN1158270C CN1158270C CNB011289007A CN01128900A CN1158270C CN 1158270 C CN1158270 C CN 1158270C CN B011289007 A CNB011289007 A CN B011289007A CN 01128900 A CN01128900 A CN 01128900A CN 1158270 C CN1158270 C CN 1158270C
- Authority
- CN
- China
- Prior art keywords
- piperazine
- compound
- derivatives
- present
- gram
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Abstract
The present invention relates to bipiperazine derivatives with calcium ion antagon action. The present invention can be used as a pharmaceutical compound used for preventing and curing angiocarpy and cerebral vessel diseases. The chemical name of the present invention is 1-substitute-4-[(4-(4-substitute phenyl)-1-piperazine) carbonyl methylene] piperazine. The structure of the present invention is shown in a formula (I), wherein in the formula, R1 can be alkyl, substitute benzoyl, or substitute cinnamon acyl, and R2 can be nitro, amino, or substitutive amino of alkyl or aryl.
Description
Technical field
What the present invention relates to is that a class has calcium antagonism, thereby can be used as the medicinal compound of two bridged piperazine derivatives classes of using in cardiovascular disease prevention.
Background technology
Two piperazine compounds are a kind of medicinal compounds of existing report in the literature.In " Acta Pharmaceutica Sinica " 1995,30 (1), reported relevant as, people such as Li Runtao to the synthetic of two piperazine bi-quaternary ammonium salt compounds and as the research of antitumor drug; " Acta Pharmaceutica Sinica " 1999,34 (9) reported people such as Li Runtao again to the synthetic of the two piperazine bi-quaternary ammonium salt analog derivatives of diacyl and in the research aspect the analgesic activities.These double piperazine quaternary ammonium salt compounds are except that showing antitumor and ease pain respectively aspect pharmacology active, its distinguishing feature on chemical structure is the structure of symmetric form exactly.
The modern medicine clinical practice shows that stenocardia, hypertension, irregular pulse, Peripheral blood vessel disease and other cardiovascular and cerebrovascular diseases have become common at present and HUMAN HEALTH constituted one of disease of bigger threat.The result of pathological study shows that its cause of disease is relevant with the metabolism of calcium ion.Study at present and can include as dihydropyridine compounds such as nifedipine, nicardipine, nitrendipines at the control medicine that uses with calcium antagonism mechanism, diltiazem compounds such as diltiazem , gram human relations sulphur , phenyl alkylamide compound such as verapamil, methoxyverapamil, two phenyl alkylamide compounds such as Fendiline, flunarizine.In the calcium ion channel blocker of this several types, stronger with the calcium blocking effect of dihydropyridines, also regulation is used it as positive control in new drug preclinical study governing principle compilation.
Summary of the invention
At above-mentioned situation, the present invention will provide a class can have even more ideal calcium antagonism, thereby can be applied to the medicinal compound of two bridged piperazine derivatives classes of cardiovascular disease prevention, can make doctor and/or patient in to the control of stenocardia, hypertension, irregular pulse, Peripheral blood vessel disease and other cardiovascular and cerebrovascular diseases, increase the selection and the scope of application at least to available medicine.
The said two bridged piperazine derivatives class medicinal compounds with calcium antagonism of the present invention, chemical name is 1-replacement-4-[(4-(4-substituted-phenyl)-1-piperazinyl) the carbonyl methylene radical] piperazine, structure is as shown in the formula (I):
Wherein, the R in the formula
1Can be alkyl such as methyl, ethyl, propyl group, can be 4-methoxyl group-benzoyl, 4-methyl-benzoyl, 2, substituted benzoyls such as 4-two chloro-benzoyls, also can be 4-methoxyl group-cinnamoyl, 4-methyl-cinnamoyl, (2, the 4-dichloro)-2-methyl-cinnamoyl, 2,4-dimethoxy-cinnamoyl, 4-bromo-cinnamoyl etc. replace cinnamoyl; R
2Can be nitro, amino, or as dimethylamino, diphenyl amino etc. contain alkyl or the substituent amino of aromatic base.
Can find out obviously that asymmetry and non-quaternary ammonium salt structure that above-mentioned each pair bridged piperazine derivatives of the present invention (I) is had are its different with aforementioned various pairs of diethylenediamine compounds on chemical structure distinguishing features.
According to substituent R in above-mentioned each pair bridged piperazine derivatives (I)
1And R
2The difference of structure type, its synthetic route can should have multiple choices mutually with method.For example, below recommend but what be not limited only to this is exactly one of alternative synthetic route.
Synthetic route A:
Work as R
1For the substituted benzoyl of aromatics or when replacing cinnamoyl, can adopt corresponding substituted aromatic base acyl chlorides, piperazine, chloroacetyl chloride and right-chloronitrobenzene is raw material, adopts following route to synthesize R
2Corresponding pair of diethylenediamine compound (I-A) for nitro.
Synthetic route B:
R
1During for the aliphatics substituting group, can be raw material with right-chloronitrobenzene, piperazine, chloroacetyl chloride and corresponding aliphatics substituted-piperazinyl, synthetic R
2Also be the corresponding pair of diethylenediamine compound (I-B) of nitro.
Synthetic route C:
With above-mentioned route A or resulting each R of route B
2For two bridged piperazine derivatives (I-A or I-B) of nitro are raw material,, can obtain R through the reduction reaction of conventional mode
2Each respective compound (I-C) for amino:
By above-mentioned various synthetic routes and method as can be seen, the synthetic method that obtains said pair of bridged piperazine derivatives of the present invention (I) is comparatively simple, and raw material is easy to get, and cost is also lower, and the yield of building-up reactions is higher.
With the above-mentioned positive object of reference of representative nifedipine as the strongest dihydropyridine compounds of Calcilytic effect, by
45Ca strides mobile Preliminary pharmacological test result's demonstration that the compound (I) of the above-mentioned different substituents form of the present invention is carried out in the film, and it can have the calcium barrier effect that is better than nifedipine.
What pharmacological testing adopted is radioactivity
45Ca strides flow assay method in the film, and two bridged piperazine derivatives (I) of the above-mentioned different substituents of research the present invention are to rat aorta voltage-dependent calcium channel (PDC) Ca
2+The influence of interior stream.
Test method operation: thoracic aorta is taken out in 9 rats stunnings, the PSS physiological liquid (sodium-chlor 137 mmoles/liter, calcium chloride 1.5 mmoles/liter, magnesium chloride 1.0 mmoles/liter, Repone K 4.6 mmoles/liter, HEPES hydroxyethyl piperazine ethanesulfonic acid 20 mmoles/liter, glucose 10 mmoles/liter) in get clean reticular tissue, every arterial cuts into 6 sections, puts into 37 ℃ of 9 ampoul tubes that fill 0.1 milliliter of PSS physiological liquid rapidly, constant temperature oxygen balance 400 minutes.Then arterial is transferred in 2 milliliters of PSS physiological liquids that are added with tested drug sample the logical oxygen of constant temperature 20 minutes rapidly.Again its immigration is contained
45Ca
2+With (tested drug sample is 20 microlitres) pre-treatment in 2 milliliters of PSS liquid of tested drug sample 10 minutes, allow
45Ca
2+Enter cell and gap.Be transferred to once more the high potassium PSS liquid that contains 20 microlitre samples (Repone K 100 mmoles/liter, sodium-chlor 41.6 mmoles/liter, all the other compositions are with the PSS physiological liquid) in handled 10 minutes, constant temperature leads to oxygen.Arterial all was transferred in 0 ℃ the EGTA liquid (except that no calcium chloride, all the other compositions are with PSS liquid) 60 minutes, with in every ampoule with 2 milliliters of EGTA washings 3 times, in the flush away intercellular substance
45Ca
2+After taking out arterial, blot, place weighing bottle, with the quality of ten thousand/balance with 54 arterial of loss of weight method weighing successively with filter paper.In 54 ampoules that arterial is housed, add 70% perchloric acid, 25 microlitres and 30% hydrogen peroxide, 50 microlitres then respectively successively, 70 ℃ of digestion of constant temperature 30 minutes.Add 3.5 milliliters of toluene scintillation solutions after the cooling respectively, lucifuge was placed 3 hours.With FJ-210P type liquid scintillation counter measurement
45Ca
2+Counting rate (cpm) adopts external-standard channels ratio method (ESCR) to the sample correction of quenching.Two bridged piperazine derivatives (I) of different substituents are as shown in table 1 to the test-results of calcium antagonism
Two bridged piperazine derivatives of table 1 different substituents are to the antagonistic action result of calcium ion
| Compound | Drug level (mg/ml) | Ca 2+Flow (μ mol/kg) (χ ± σ) | ||
| Two bridged piperazine derivatives (I) | R 1 | R 2 | ||
| 2,4-dichloro cinnamoyl | -NO 2 | 10 | 161.74±25.28 | |
| 4-methoxyl group cinnamoyl | -NO 2 | 10 | 156.13±15.20 | |
| -C 2H 5 | -NO 2 | 10 | 186.52±20.17 | |
| 4-methyl cinnamoyl | -NO 2 | 10 | 165.30±9.09 | |
| -C 2H 5 | -NH 2 | 10 | 180.80±14.04 | |
| Nifedipine (positive control) | / | / | 3 | 222.41±44.03 |
| / | / | 10 | 220.12±31 | |
| Distilled water (blank) | / | / | / | 248.96±50.55 |
Below again foregoing of the present invention is described in further detail by embodiment the synthetic example of some particular compound.But this should be interpreted as that the scope of the above-mentioned theme of the present invention only limits to following example.All technology that realizes based on foregoing of the present invention all belong to scope of the present invention.
Embodiment
Example 1:
The 1-[(4-methoxyl group) cinnamoyl]-4-[(4-(4-nitrophenyl)-1-piperazinyl) the carbonyl methylene radical] preparation of piperazine (1).
Adopt the method for said synthesis route A, 130 gram (0.67 mole) Uricidas are dissolved in 200 milliliters of toluene, and the reaction 10 hours of anhydrating adds 18 gram (0.11 mole) parachloronitrobenzenes, back flow reaction 7 hours, cooling is filtered, the washing solid, get compound (II), 21.5 gram, yield 94%, fusing point 128-129 ℃.Digest compound (II) with 5 and be dissolved in 60 milliliters of chloroforms, drip 3 milliliters of (0.038 mole) chloroacetyl chlorides under the room temperature, reacted 2 hours, suction filtration, the washing solid gets compound (III) 6.3 grams, yield 92.6%,
Fusing point 133-134 ℃.20 gram (0.1 mole) Uricidas are dissolved in 80 milliliters of toluene, divide water reaction 8 hours, add compound (III) 5 grams (0.018 mole), back flow reaction 8 hours, cooling is filtered, the washing solid, get compound (IV) 5.4 grams, yield 90%, fusing point 122-123 ℃.The compound (IV) of 1 gram (0.003 mole) is dissolved in the chloroform, add right-methoxyl group cinnamyl chloride 0.6 gram (0.003 mole) in batches, room temperature reaction 8 hours, boil off solvent, with dehydrated alcohol-chloroform (1: 1) recrystallization, obtain belonging to compound (1) product 1.02 grams in the route A product (I-A), yield 70%, mp.155-156 ℃.
Ultimate analysis: C:63.07 (calculated value 63.27), H:6.54 (calculated value 6.33),
N:14.01 (calculated value 14.19);
1H NMR：6.80-8.20(m，4H，p-NO
2-phH)，6.65-7.80(m，4H，p-CH
3O-phH)，
7.62(s，1H，CH＝)，6.90(s，1H，＝CH)，3.55(s，3H，CH
3O)，
3.30 (s, 2H, CH
2), 3.62-4.00 (m, 8H, piperazine H),
(2.20-2.90 m, 8H, piperazine H);
Mass spectrum: C
26H
31N
5O
5, 494.4 (M
+), 392.5,334.5,161.3,133.3,70.1.
The structure of products obtained therefrom compound (1) is;
Example 2:
The 1-[(4-methyl) cinnamoyl]-4-[(4-(4-nitrophenyl)-1-piperazinyl) the carbonyl methylene radical] preparation of piperazine (2).
Be similarly said synthesis route A, the preparation process of compound (IV) is with above-mentioned example 1.1 gram (0.003 mole) compound (IV) is dissolved in the chloroform, add right-methyl cinnamyl chloride 0.61 gram (0.0032 mole) in batches, room temperature reaction 6 hours, boil off solvent, with dehydrated alcohol-chloroform (1: 1) recrystallization, obtain belonging to compound (2) product 1.01 grams in the route A product (I-A), yield 71%, mp.205-207 ℃.
Ultimate analysis: C:65.09 (calculated value 65.39), H:6.75 (calculated value 6.54),
N:14.37 (calculated value 14.67);
1H NMR：6.80-8.20(m，4H，p-NO
2-phH)，7.20-7.40(m，4H，p-CH
3-phH)，
7.60(s，1H，CH＝)，6.90(s，1H，＝CH)，3.55(s，2H，CH
2)，
(3.51-4.00 m, 8H, piperazine H), 2.20-2.50 (m, 8H, piperazine H),
2.20(s，3H，CH
3)；
Mass spectrum: C
26H
31N
5O
4, 477.1 (M
+), 396.1,333.1,209.1,146.1,92.1.
The structure of products obtained therefrom compound (2) is;
Example 3:
1-[(2,4-dichloro)-2-methyl cinnamoyl]-4-[(4-(4-nitrophenyl)-1-piperazinyl) the carbonyl methylene radical] preparation of piperazine (3).
Adopt synthetic route A, the compound (IV) of 1 gram (0.003 mole) is dissolved in the chloroform, add 2 in batches, 4-two chloro-2-methyl cinnamyl chlorides 0.9 gram (0.0035 mole), room temperature reaction 6 hours boils off solvent, with dehydrated alcohol-chloroform (1: 1) recrystallization, obtain belonging to compound (3) product 1.11 grams in the route A product (I-A), yield 68%, mp.83-84 ℃.
Ultimate analysis: C:57.37 (calculated value 57.15), H:5.55 (calculated value 5.35),
Cl:12.68 (calculated value 12.98), N:12.69 (calculated value 12.82);
1H NMR：6.80-8.20(m，4H，p-NO
2-phH)，7.20-7.40(m，3H，2Cl-phH)，
6.50(s，1H，CH＝)，3.55(s，2H，CH
2)，2.0(s，3H，CH
3)，
(3.45-4.00 m, 8H, piperazine H), 2.60-2.80 (m, 8H, piperazine H);
Mass spectrum: C
26H
29Cl
2N
5O
4, 546.5 (M
+), 516.5,417.6,311.3,213.2,115.4.
The structure of products obtained therefrom compound (3) is:
Example 4:
The 1-[(4-methyl) cinnamoyl]-4-[(4-(4-aminophenyl)-1-piperazinyl) the carbonyl methylene radical] preparation of piperazine (4).
Adopt the method for said synthesis route C, the product compound (2) of above-mentioned example 2 gained of 1 gram (0.002 mole) is dissolved in 2 milliliters the 6N hydrochloric acid, adds zinc powder 0.8 gram in batches, reaction is 8 hours under the room temperature, add 100 milliliters of ammoniacal liquor, extract for 30 milliliters * 3 times with methylene dichloride, the extracting solution drying concentrates, refining with 60% ethanol, obtain belonging to compound (4) product 0.67 gram in the route C product (I-C), yield 75%, mp.182-183 ℃.
Ultimate analysis; C:67.44 (calculated value 69.77), H:7.23 (calculated value 7.43),
N:15.43 (calculated value 15.65);
1H NMR：7.20-7.50(m，4H，p-CH
3-phH)，6.60-6.90(m，4H，p-NH
2-phH)，
7.70(s，1H，CH＝)，6.95(s，1H，＝CH)，3.15(s，2H，CH
2)，
(3.10-3.90 m, 8H, piperazine H), 2.20-2.60 (m, 8H, piperazine H),
2.35(s，3H，CH
3)；
Mass spectrum: C
26H
33N
5O
2, 477 (M
+), 341.7,321.8,268.9,177.7,144.6.
The structure of products obtained therefrom compound (4) is:
Example 5:
1-ethyl-4-[(4-(4-nitrophenyl)-1-piperazinyl) carbonyl methylene radical] preparation of piperazine (5).
Adopt the method for said synthesis route B, the preparation process of compound (III) is with example 1.Compound (III) and 0.5 gram (0.0045 mole) ethyl piperazidine, 20 milliliters of chloroforms and 1 milliliter of triethylamine with 1 gram (0.004 mole), back flow reaction 5 hours, boil off solvent, with ethanol-chloroform (1: 1) recrystallization, obtain belonging to compound (5) product 0.9 gram in the route B product (I-B), yield 62%, mp.55-56 ℃.
Ultimate analysis: C:59.55 (calculated value 59.81), H:7.68 (calculated value 7.53),
N:19.12 (calculated value 19.38);
1H NMR：6.80-8.20(m，4H，p-NO
2-phH)，3.40(s，2H，COCH
2)，
(3.20-3.90 m, 8H, piperazine H), 2.00-2.80 (m, 8H, piperazine H),
2.6(m，2H，CH
2)，1.05(t，3H，CH
3)；
Mass spectrum: C
18H
27N
5O
3, 362.4 (M
+), 349.5,293.4,221.4,167.3,127.2.
The structure of products obtained therefrom compound (5) is:
Example 6:
1-ethyl-4-[(4-(4-aminophenyl)-1-piperazinyl) carbonyl methylene radical] preparation of piperazine (6).
Adopt the method for said synthesis route C, the product compound (5) of 1 gram (0.003 mole) above-mentioned example 5 gained is dissolved in 3 milliliters the 6N hydrochloric acid, adds zinc powder 1.2 grams in batches, reaction is 8 hours under the room temperature, add 100 milliliters of ammoniacal liquor, extract for 30 milliliters * 3 times with methylene dichloride, the extracting solution drying concentrates, refining with 60% ethanol, obtain belonging to compound (6) product 0.71 gram in the route C product (I-C), yield 71%, mp.94-95 ℃.
Ultimate analysis: C:65.45 (calculated value 65.23), H:8.95 (calculated value 8.82),
N:21.42 (calculated value 21.13);
1H NMR：6.60-6.90(m，4H，p-NH
2-phH)，3.55(s，2H，COCH
2)，
(3.00-3.80 m, 8H, piperazine H), 1.80-2.60 (m, 8H, piperazine H),
2.40(m，2H，CH
2)，1.05(t，3H，CH
3)；
Mass spectrum: C
18H
29N
5O, 332.7 (M
+), 288.3,247.4,191.1,128.1,85.1.
The structure of products obtained therefrom compound (6) is:
Claims (1)
1. have two piperazine derivatives of calcium antagonism, can be used as the medicinal compound that uses in cardiovascular disease prevention, chemical name is 1-replacement-4-[(4-(4-substituted-phenyl)-1-piperazinyl) the carbonyl methylene radical] piperazine, structure is as shown in the formula (I):
In the formula: R
1Can be alkyl, or the benzoyl or the cinnyl that replace by alkyl, alkoxyl group, halogen atom;
R
2Can be nitro, the amino that amino or alkyl or aromatic base replace.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB011289007A CN1158270C (en) | 2001-09-26 | 2001-09-26 | Bipiperazine derivatives with calcium ion antagon action |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB011289007A CN1158270C (en) | 2001-09-26 | 2001-09-26 | Bipiperazine derivatives with calcium ion antagon action |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1342649A CN1342649A (en) | 2002-04-03 |
| CN1158270C true CN1158270C (en) | 2004-07-21 |
Family
ID=4668710
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB011289007A Expired - Fee Related CN1158270C (en) | 2001-09-26 | 2001-09-26 | Bipiperazine derivatives with calcium ion antagon action |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1158270C (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7851474B2 (en) * | 2005-08-02 | 2010-12-14 | Neurogen Corporation | Dipiperazinyl ketones and related analogues |
| WO2024043334A1 (en) * | 2022-08-26 | 2024-02-29 | 富士フイルム株式会社 | Novel piperazine derivative or salt thereof and pharmaceutical composition |
-
2001
- 2001-09-26 CN CNB011289007A patent/CN1158270C/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN1342649A (en) | 2002-04-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1035996C (en) | Stilbene derivatives and carcinostatics containing them | |
| CN1535146A (en) | 5-halo-tryptamine derivatives used as ligands of 5-HT 6 and/or 5-HT7 serotonin receptors | |
| CN103980184A (en) | Piperidine compounds, pharmaceutical composition comprising the same and its use | |
| CN1152304A (en) | Substituted benzamidines, their production and their use as medicaments | |
| US9120728B2 (en) | Modulators of cortical dopaminergic- and NMDA-receptor-mediated glutamatergic neurotransmission | |
| CN1158270C (en) | Bipiperazine derivatives with calcium ion antagon action | |
| CN1944448A (en) | Puerarin derivative and its medicinal use | |
| CN1990470A (en) | Phthiobuzonum derivative, its manufacturing process, pharmaceutical combination and uses thereof | |
| CN1166623C (en) | 2-phenoxyaniline derivatives | |
| CN1763030A (en) | Puerarin derivate and medical usage thereof | |
| CN1037266C (en) | Quinoline derivative fumarates | |
| CN1775762A (en) | Liyustrazine acyl piperazine derivative, and its preparing method and medicinal composition and use | |
| CN1185214C (en) | Novel indeno indolone compound, its preparation method and drug composition comprising the same | |
| WO2023081897A1 (en) | Isotopically enriched 3,4-methylenedioxy-n-ethylamphetamine (mde) and stereoisomers thereof | |
| CN1041758A (en) | The preparation of 4-aryl dihydropyridine acid amides | |
| CN1146537C (en) | Dofetilide polymorphs | |
| CN1046281C (en) | Pharmacologically active enantiomers | |
| CN1312809A (en) | N-substituted azabicycloheptane deriratives, production and use thereof | |
| CN1077890C (en) | 4-aminoethyoxy-indolone derivatives as dopamine D2 agonists | |
| CN1022632C (en) | Carboxamide compounds, process for preparing same and pharmaceutical composition containing same | |
| CN1545505A (en) | Polymorph salt of a pyridazinone derivative for the treatment of arrythmia | |
| CN1660833A (en) | Compound containing structure of o-naphthaquinone and application | |
| CN1366519A (en) | Phenoxazine compounds, its pharmaceutical compositions and medical use | |
| CN1403456A (en) | Chiral isoquinoline compound with cardiac vascular activity and its synthesis process | |
| CN1563005A (en) | Ramification in albocarbon category and application in anoxia system |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C06 | Publication | ||
| PB01 | Publication | ||
| ASS | Succession or assignment of patent right |
Owner name: SUN ZEQUN Effective date: 20030418 Owner name: SICHUAN UNIVERSITY Free format text: FORMER OWNER: SHANGHAI ZHONGPU BIOLOGICAL SCIENCE +. TECHNOLOGY CO., LTD. |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20030418 Address after: 17 West China College of pharmacy, No. three, Renmin South Road, Sichuan, Chengdu, 610041 Applicant after: Sichuan University Co-applicant after: Sun Zequn Address before: 201318 No. 186, Lane 401, Yue Yue Road, Pudong, Shanghai, 14, China, No. 2, Zhoupu Applicant before: Zhongpu Bio-Technology Co Ltd, Shanghai |
|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C19 | Lapse of patent right due to non-payment of the annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |