AU2021202066A1 - Use of iminosugar compound in preparation of anti-sars-cov-2 virus drug - Google Patents
Use of iminosugar compound in preparation of anti-sars-cov-2 virus drug Download PDFInfo
- Publication number
- AU2021202066A1 AU2021202066A1 AU2021202066A AU2021202066A AU2021202066A1 AU 2021202066 A1 AU2021202066 A1 AU 2021202066A1 AU 2021202066 A AU2021202066 A AU 2021202066A AU 2021202066 A AU2021202066 A AU 2021202066A AU 2021202066 A1 AU2021202066 A1 AU 2021202066A1
- Authority
- AU
- Australia
- Prior art keywords
- compound
- dnj
- acid
- cov
- sars
- 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.)
- Abandoned
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
- A61K31/445—Non condensed piperidines, e.g. piperocaine
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
Abstract
The present invention discloses use of an iminosugar compound in preparation of an
anti-coronavirus drug, and particularly use of NB-DNJ (Miglustat) in preparation of an
anti-SARS-CoV-2 drug. The iminosugar compound can be used to prepare a broad-spectrum
antiviral drug, and has the characteristics of low toxicity and good activity.
Description
USE OF IMINOSUGAR COMPOUND IN PREPARATION OF ANTI-SARS-COV-2 VIRUS DRUG
The present invention relates to the field of chemical pharmacy, and particularly to use of an iminosugar compound in preparation of an antiviral drug.
Molecules of an iminosugar (DNJ) drug were first discovered and extracted from mulberry leaves in 1987 as natural products. A structure of the iminosugar simulates a cyclic structure of sugar, in which oxygen in a ring of cyclic glucose is replaced by nitrogen.
The structural similarity with sugar molecules means that many iminosugar molecules are competitive inhibitors for enzyme that uses sugar as a substrate. These compounds have shown excellent therapeutic potential for a variety of diseases, including glycosylated fat storage disorders (Gaucher) taking glucosylceramide synthase as a target and Type II diabetes taking intestinal a-glucosidase as a target. An action mechanism of inhibiting the virus is to inhibit activity of endoplasmic reticulum a-glucosidase (ER-D-glucosidase /11), thereby disrupting normal folding of the virus-coated glycosylated protein and interfering with generation of a new virus in a host cell, and hence destroying the virus. This virus inhibition mechanism has also been fully verified in clinic. Two patients with glucosidase gene mutation were not infected by the virus due to lack of this enzyme activity, and the virus also cannot be effectively copied in cells isolated from bodies of these two patients, As a brand-new virus-inhibiting strategy, this compound is aimed at a endoplasmic reticulum a-glucosidase target of the host cell, does not depend on the morphology of the virus itself, can effectively inhibit various "coated viruses" and their variants to achieve a broad spectrum antiviral effect.
The main technical problem to be solved by the present invention is to provide an antiviral compound, preferably a drug for treating a coronavirus, and more preferably a drug for treating a SARS-CoV-2 coronavirus.
In order to solve the above technical problems,
The present invention first provides an imine compound, including a tautomer, an optical isomer, a solvate, a polymorph, a pharmaceutically acceptable salt, an ester, a pharmaceutically acceptable prodrug or a derivative. An imine sugar compound of the present invention refers to a sugar analogue substituted with a nitrogen atom at a position of an inner epoxy atom. In a specific embodiment, the imino sugar compound is NB-DNJ or N-7-oxadecyl-DNJ. Preferably, the imino sugar compound is formula 1 of NB-DNJ.
~OH
H3 C
Formula 1
Further, the present invention provides an effect and a method of an imine sugar compound for treating viral infection. The compound is used for the treatment of viral infection, including infection by a coronavirus, especially infection by a SARS-CoV-2 coronavirus.
The iminosugar compound of the present invention, especially the compounds of NB-DNJ and N-7-oxadecyl-DNJ, has significant anti-SARS-CoV-2 activity, low cytotoxicity, and good drug safety.
Any pharmaceutically acceptable salt of the imine sugar compound of the present invention can be used for the objective of the present invention. "A pharmaceutical salt" refers to a pharmaceutically acceptable non-toxic acid salt which is suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions or other problems or complications within the scope of medical judgment, corresponding to a reasonable benefit/risk ratio. Examples of the pharmaceutical salt include, but are not limited to: inorganic or organic acid addition salts of basic residues such as amines; alkali or organic addition salts of acidic residues such as carboxylic acid, etc., and include combinations of one or more the forgoing salts. The pharmaceutical salt includes non-toxic salt and quaternary ammonium salt such as a parent compound formed from non-toxic inorganic acid or non-toxic organic acid. For example, the non-toxic acid salt includes salts derived from inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, etc.; other acceptable inorganic salts including metal salts such as sodium salt, potassium salt, cesium salt, etc.; alkaline earth metal salts such as calcium salt, magnesium salt, etc., and combinations of one or more of the forgoing salts.
The organic salts of the compound include salts prepared from organic acids such as acetic acid, trifluoroacetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-aminobenzenesulfonic acid, 2-acetylbenzoic acid, fumaric acid, p-toluenesulfonic acid,methane sulfonic acid, ethane disulfonic acid,oxalic acid, isethionic acid, HOOC-(CH2)n-COOH (where n is 0 to 4); organic amine salts, such as: triethylamine salt
, pyridine salt, picoline salt, ethanolamine salt, triethanolamine salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, etc.; and amino acid salts, such as arginine salt, aspartate, glutamate, etc., and combinations of one or more of the foregoing salts.
A plurality of the compounds of the present invention can have a chiral center where various stereoisomers can be obtained. The term "stereoisomer" refers to all isomers of individual compounds that differ only in orientation of atoms of the compounds in space. The term stereoisomer includes mirror image isomer (enantiomer) and mixtures of the mirror image isomer (racemic modification and racemic mixtures). The present invention relates to each of these stereoisomers, and mixtures thereof. The enantiomer can be isolated by conventional techniques such as chromatography or fractional crystallization. Optical isomer can be resolved by conventional techniques of optical resolution to obtain optically pure isomer. This resolution can be performed on any chiral synthetic intermediate or on the compound of the present invention. The optically pure isomer can also be obtained individually using enantiomer-specific synthesis.
"A pharmaceutical excipient or carrier" refers to a pharmaceutically acceptable material, a composition or a vehicle. Each component should be pharmaceutically acceptable in the sense of being compatible with other ingredients of a pharmaceutical composition. Each component should also be suitable for use in contact with human and animal tissues or organs without excessive toxicity, irritation, allergic reactions, immunogenicity or other problems or complications, corresponding to a reasonable benefit/risk ratio.
Preferably, an effective amount of the iminosugar compound of the present invention or a
pharmaceutical salt thereof is used according to a usual route of administration and according to
methods known in the art in a conventional pharmaceutical composition (the pharmaceutical composition contains an effective amount of active ingredients and a suitable pharmaceutical carrier)
and a dosage form and is administered to patients in need of such treatment.
"Therapeutically effective amount" refers to an amount of active ingredients, that is, when administered, the active ingredients are sufficient to prevent the development of one or more
symptoms of targeted disease, or alleviate the one or more symptoms to some extent. The specific dosage of the compound administered according to the present invention is determined by the specific circumstances surrounding a case, including the compound administered, an administration route, specific conditions to be treated, and similar considerations. Particularly, a "therapeutically effective amount of a compound" refers to an amount of a compound sufficient to prevent or alleviate diseases caused by one or more viruses to some extent.
Depending on a type and severity of diseases infected by viruses to be treated and response of
specific patients to drug therapy, a single dose and a daily dose are different. Therefore, the exact
single dose is determined according to standard medical principles under guidance of a doctor. The compound of the present invention or the pharmaceutically acceptable salt thereof can be
used alone or in combination with other active therapeutic agents.
The other therapeutic agents include other an antiviral drug, an antimicrobial drug, an immune enhancer, a sterol compounds, a biological agent, and the like.
In one embodiment of the present invention, the compound used can dosage unit formulations containing conventional pharmaceutical carriers that are administered orally, topically, parenterally, by inhalation or spray, sublingually, transdermally, by an oral cavity, rectally, intravenously, intramuscularly or by other means. The pharmaceutical composition can be formulated into any pharmaceutical form, such as: a tablet, a granule, an injection, a gel, a pill, a capsule, a suppository, an implant, a slow release, a controlled release, a pellet and combinations thereof. A plurality of dosage forms such as the tablet and the capsule can be subdivided into appropriate dosage unit dosage forms containing appropriate amounts of active ingredients such as effective amounts for desired objectives.
A pharmaceutical carrier and an excipient suitable for use in the oral formulations disclosed
herein include, but are not limited to, a diluent such as a filler and a bulking agent, a binder, a
lubricant, an anti-caking agent, a disintegrating agent, a sweetener, a buffer, a preservative, a solubilizer, an isotonic agent, suspending agent and a dispersing agent, a wetting agent or an
emulsifying agent, a flavoring agent and an aromatic agent, a thickening agent and a vehicle.
In another embodiment, the compound used in a use of the present invention is an injection preparation to be administered to the patient to be treated. The injection suitable for the present invention refers to a sterile solution or a bacteria-free solution as well as an emulsion solution or a
suspension solution for injection into the human body that are prepared by the drug and a suitable
solvent or a dispersion medium, and a powdered sterile preparation that is formulated into a solution or a suspension solution before use. The injections include an injection (a large-volume injection for intravenous infusion is also called an intravenous infusion), a sterile powder for the injection, and a concentrated solution for the injection.
In this specification and claims, the term "including or containing" and their variants are not intended to exclude other technical features, additives, components or steps. The term includes the
wording "consisting of' and its variants. Additional objectives, advantages, and features of the
present invention become apparent to the person skilled in the art after checking specification of the
present invention, or can be understood through the practice of the present invention. The following embodiments are provided by way of illustration, but are not intended to limit the present invention.
Furthermore, the present invention covers all possible combinations of the particular and preferred
embodiments described herein. Terminology convention: "A stereoisomer" or "a stereoisomer" are compounds that have the same chemical composition
but differ in arrangement of atoms or groups in space. The stereoisomer includes "a diastereomer"
and "an enantiomer" "The diastereomer" is the stereoisomer that have two or more chiral centers and whose
molecules are not mirror images for each other. The diastereomer has different physical properties,
such as a melting point, a boiling point, a spectral feature and reactivity. In the presence of a resolving agent or chromatography, a chiral HPLC column can be used to separate a mixture of
diastereomers under high-resolution analysis steps such as electrophoresis and crystallization.
"The enantiomer" refers to two stereoisomers of a compound without overlapping mirror images for each other. A 50:50 mixture of the enantiomers is called a racemic mixture or a racemate,
which can appear in a chemical reaction or treatment process when there is no stereoselectivity or
stereospecificity.
"The prodrug" refers to a compound that is metabolized in a host,for example, hydrolyzed or oxidized to produce the compound of the present invention. Typical examples of the prodrug include compounds having a biolabile protective group on a functional group of an active compound. The prodrug includes a compound that can be oxidized, reduced, aminated, deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrated, alkylated, dealkylated, acylated, deacylated, phosphorylated, and dephosphorylated to produce an active compound. "The derivative" refers to more complex products derived from substitution of hydrogen atoms or atomic groups of compounds by other atoms or atomic groups.
FIG.1 shows 1H NMR spectrum and a compound structure of NB-DNJ.
FIG. 2 shows 1H NMR spectrum and a compound structure of N-7-oxadecyl-DNJ.
FIG. 3 shows 1H NMR spectrum and a compound structure of MON-DNJ.
FIG. 4 shows a cytotoxicity test of an iminosugar compound.
FIG. 5 shows anti-SARS-CoV-2 virus infection activity of an iminosugar compound.
FIG. 6 shows that detection of a copy number of N protein gene shows anti-SARS-CoV-2 virus activity of an iminosugar compound.
FIG. 7 shows that detection of a copy number of ORF gene shows anti-SARS-CoV-2 virus activity of an iminosugar compound.
The preferred embodiments of the present invention are described in detail below with reference to the drawings, so that the advantages and features of the present invention can be more easily understood by the person skilled in the art, so as to make a clearer definition of the protection scope of the present invention.
1) A cytotoxicity test of an iminosugar compound
Experimental steps
1. Vero-E6 cells were paved and planted in a 96-pore plate to reach a full confluence state after 1 day.
2. Each iminosugar compound (NB-DNJ, N-7-oxadecyl-DNJ and MON-DNJ, all obtained from Oxford Glycobiology Institute) took 1000 pM as a starting concentration and was diluted using a MEM medium based on a gradient dilution.
3. The medium was sucked out in the 96-pore plate and cells were washed with Ix PBS.
4. A PBS cleaning solution was removed, and 200 pl of a diluent of the iminosugar compound was added.
5. The solution was incubated at a temperature of 37 °C under 5% CO 2 for 48 h.
6 After incubation was ended, 100ul of medium was removed and 20ul of Promega CellTiter
96@ AQueous One Solution reagent was added.
7. A pore plate was incubated at a temperature of 37 °C under 5% CO 2 for 1-4 h.
8. An absorbance value at 490nm of a wavelength was measured in a 96-pore plate microplate reader.
Test results
Compounds of the NB-DNJ, the N-7-oxadecyl-DNJ and the MON-DNJ (see FIGS 1, 2 and 3 for specific compound structures) had low cytotoxicity, and cell survival rates were all above 80% at a drug concentration as high as ImM (FIG. 4). The results showed that the safety of the compounds of the NB-DNJ, the N-7-oxadecyl-DNJ and the MON-DNJ was good.
2) A test of anti-SARS-CoV-2 virus activity of the iminosugar compound.
A stock solution of a drug was configured.
Iminosugar drugs (the NB-DNJ, the N-7-oxadecyl-DNJ and the MON-DNJ) were dissolved with MilliQ ultrapure water. A 100mM stock solution was configured, filtered with a 0.22tm filter, and stored in a -20°C refrigerator. The stock solution of the drug was diluted with MEM according to a gradient. 200ul of the diluted drug was added to each pore.
Experimental steps
1. Vero-E6 cells were planted in a 96-pore plate, and a density of the cells reaches 90-100% confluence after 24 h.
2. The MEM medium was sucked out and the cells were soaked with PBS.
3. The SARS-CoV-2 virus was diluted with the MEM. MOI was adjusted to lpfu per cell with a final volume of 100ul/well.
4. The PBS in the pore was sucked out and the virus/MEM mixture was added. The PBS was incubated at a room temperature for 90 min with gentle shaking.
5. At the end of virus incubation, a pathogen was removed. The cells were washed twice with the PBS, and the diluted drug was added to each pore.
6. Incubation was performed in a 37 °C, 5% CO 2 incubator for 48 h.
7. The medium was sucked out. The cells were labeled with an anti-S protein antibody, and a ratio of S protein positive cells (S+ cells) was analyzed by a flow cytometry (FACS).
8. An RNA of the cells was extracted. A RT-PCR quantitative test was performed on N protein and
ORFI gene of the drug-treated cells, and the copy number of the genes was detected to reflect a viral load.
Test results
FACS analysis results were shown in FIG. 5. The compounds of the NB-DNJ, the N-7-oxadecyl-DNJ and the MON-DNJ could effectively reduce a ratio of S protein positive cells, indicating that these compounds could inhibit infection or copy of a SARS-CoV-2 virus. Inhibitory activity of N-7-oxadecyl-DNJ at a cell level was relatively preferred.
RT-PCR quantitative test results were shown in FIG. 6 and FIG. 7. The compounds of the NB-DNJ, the N-7-oxadecyl-DNJ and the MON-DNJ could effectively reduce a copy number of N protein and ORF1 gene. The compounds of the NB- DNJ and the N-7-oxadecyl-DNJ could reduce a copy number of viral genes by nearly 1000 times compared with a drug-free treatment group at a level of 4uM, showing very good antiviral activity. Antiviral activity of the compounds of the MON-DNJ at a gene level was at least 10 times lower than that of the NB-DNJ and the N-7-oxadecyl-DNJ, indicating that the iminosugar compound had a structure-activity relationship against the SARS-CoV-2 virus.
The NB-DNJ, also known as Miglustat, was an active ingredient of the prescription drug of Zavesca, and had been on the market for many years for the treatment of Gaucher disease and type II diabetes, and the MON-DNJ had completed phase 2a clinical trials for an anti-dengue fever virus. Although there have been many reports on the antiviral activity of the iminosugar compound, there has not been any research on anti-SARS-CoV-2 activity of this compound. Moreover, it could be known from the test results of the present invention that the anti-SARS-CoV-2 virus activity of different iminosugar compounds showed no obvious difference. The present invention unexpectedly discovered and confirmed that the NB-DNJ could effectively resist the SARS-CoV-2 virus. Comprehensively considering a path of clinical development, the NB-DNJ was expected to be developed into a first-line preventive and therapeutic drug for a new coronavirus.
The forgoing are only the embodiments of the present invention and do not limit the patent scope of the present invention. All equivalent transformations made using the contents of the description and drawings of the present invention, or directly or indirectly applied to other related technical fields are also included in the scope of patent protection of the present invention.
Claims (1)
1, Use of an iminosugar compound in preparation of an antiviral drug.
2, The use according to claim 1, wherein a coronavirus is SARS-CoV-2.
3, The use according to claim 1, wherein the iminosugar compound is NB-DNJ or N-7-oxadecyl-DNJ.
4, The use according to claim 1, wherein the iminosugar compound is the NB-DNJ.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010260813.X | 2020-04-03 | ||
| CN202010260813 | 2020-04-03 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| AU2021202066A1 true AU2021202066A1 (en) | 2021-10-21 |
Family
ID=78177114
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| AU2021202066A Abandoned AU2021202066A1 (en) | 2020-04-03 | 2021-04-01 | Use of iminosugar compound in preparation of anti-sars-cov-2 virus drug |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN113425720A (en) |
| AU (1) | AU2021202066A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022040741A1 (en) * | 2020-08-26 | 2022-03-03 | COVIRIX Medical Pty Ltd | Glucosidase inhibitors for the treatment and prevention of pulmonary infections |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8450345B2 (en) * | 2009-02-23 | 2013-05-28 | The Chancellor, Masters And Scholars Of The University Of Oxford | Iminosugars and methods of treating viral diseases |
| JP2015518896A (en) * | 2012-06-06 | 2015-07-06 | ユニザー ヴィロロジー，エルエルシー | Novel imino sugars and their uses |
-
2021
- 2021-03-31 CN CN202110353666.5A patent/CN113425720A/en active Pending
- 2021-04-01 AU AU2021202066A patent/AU2021202066A1/en not_active Abandoned
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022040741A1 (en) * | 2020-08-26 | 2022-03-03 | COVIRIX Medical Pty Ltd | Glucosidase inhibitors for the treatment and prevention of pulmonary infections |
| AU2021332085B2 (en) * | 2020-08-26 | 2022-12-08 | COVIRIX Medical Pty Ltd | Glucosidase inhibitors for the treatment and prevention of pulmonary infections |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113425720A8 (en) | 2022-08-19 |
| CN113425720A (en) | 2021-09-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| AU2017214668A1 (en) | Pharmaceutical compositions and methods for countering chemotherapy induced cardiotoxicity | |
| EP3970712A2 (en) | Solid oral dosage forms of 2r,6r-hydroxynorketamine or derivatives thereof | |
| JP2010507672A (en) | Methods and combination therapies for treating Alzheimer's disease | |
| JP5925065B2 (en) | Pharmaceutical composition | |
| US9839627B2 (en) | Methods of treating fragile X associated disorders, ADHD, and autism spectrum disorder | |
| US20110070319A1 (en) | Bifeprunox doses for treating schizophrenia | |
| CN112638866B (en) | Co-crystals of sorafenib derivatives and process for preparing same | |
| US20220062296A1 (en) | Combinations of gabaa alpha 5 agonists and sv2a inhibitors and methods of using in the treatment of cognitive impairment | |
| CA2757574C (en) | Inhibitors of bacterial type iii secretion system | |
| US20210077487A1 (en) | Pharmaceutical composition containing hdac6 inhibitor as active ingredient for prevention or treatment of itching | |
| CA2641659A1 (en) | 4-acylaminopyridine derivative mediated neurogenesis | |
| AU2021202066A1 (en) | Use of iminosugar compound in preparation of anti-sars-cov-2 virus drug | |
| TWI335220B (en) | Use of caffeic acid phenethyl ester for manufacturing a medicament for treating neurodegenerative and cardiovascular disorders | |
| JP2006516629A (en) | Compositions and methods containing substituted quinolines and substituted diphenyl sulfones | |
| JP5105818B2 (en) | Pharmaceutical composition | |
| US20050096318A1 (en) | Pharmaceutically active morpholinol | |
| US7098206B2 (en) | Pharmaceutically active morpholinol | |
| US10064843B2 (en) | Bis-amide derivative and use thereof | |
| JP2021167279A (en) | Pharmaceutical composition for preventing or treating mitochondrial disease | |
| WO2020059841A1 (en) | Therapeutic agent for prion diseases | |
| US20230181516A1 (en) | Use of aminoacetonitrile compounds for the treatment of infection and disease | |
| US10870633B2 (en) | Types of C-3 substituted kynurenic acid derivatives with improved neuroprotective activity | |
| JP3681770B2 (en) | Treatment for senile dementia or Alzheimer's disease | |
| WO2005118521A1 (en) | Monomethylphytosphingosine and the composition for anti-cancer containing the same | |
| CN117836284A (en) | Methods of treating nerve agent-induced seizures |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| MK5 | Application lapsed section 142(2)(e) - patent request and compl. specification not accepted |