D-Cycloserine

The first three amino acids of the pentapeptide chain of muramic acid are added sequentially, but the terminal d-alanyl-d-alanine is added as a dipeptide unit. To form this unit the natural form of the amino acid, l-alanine, is first racemized to d-alanine and two molecules are then joined by d-alanyl-d-alanine ligase. Both of these reactions are blocked by the antibiotic cycloserine, which is a structural analog of d-alanine.

D-cycloserine is a broad-spectrum polypeptide antibiotic produced by Streptomyces lavendulae and Streptomyces orchidaceus or synthesized by chemical methods. As white crystals with strong hygroscopic nature, it is soluble in water, slightly soluble in lower alcohols, acetone and dioxane, and hardly soluble in chloroform and petroleum ether. It is relatively stable in alkaline solution and decomposes rapidly in acidic or neutral solutions. As a broad-spectrum antibiotic, cycloserine is inhibitive against most Gram-positive and Gram-negative bacteria, rickettsia and some protozoa, with the exception of Mycobacterium tuberculosis., It is also effective on some of the Mycobacterium tuberculosis strains with tolerance to streptomycin, vinactane para-aminosalicylic acid, isoniazid and pyrazinamide. Cycloserine slightly synergizes with isoniazid in the inhibition of Mycobacterium tuberculosis H37RV, but it neither synergizes nor antagonizes against streptomycin. The product is a bacteriostatic agent, and thus won’t exert bactericidal effect even when increasing the dose or prolonging the action time with bacteria.
The Mechanism of D-cycloserine’s antibacterial action is to inhibit the biosynthesis of peptidoglycan of the cell wall. As it is a structural analog of D-alanine, D-cycloserine can competitively inhibit the activities of alanine racemase and D-alanyl-D-alanine synthetase, which are two important enzymes in peptidoglycan synthesis. D-cycloserine shows weak inhibitive activity against Mycobacterium tuberculosis which is only 1/10 to 1/20 that of streptomycin. The advantage of the product is that it is effective on drug-resistant Mycobacterium tuberculosis strains and less likely to induce drug resistance. The product can be used with other anti-tuberculosis drugs in the treatment of tuberculosis caused by drug-resistant Mycobacterium tuberculosis.
Cycloserine is a second-line anti-tuberculosis drug. It can inhibit the growth of Mycobacterium tuberculosis, but the effect is relatively weaker than that of the first-line drugs. Its efficacy in tuberculosis treatment is relatively low. Use the drug alone may produce drug resistance, but the resistance occurs slowly compared with that of other anti-tuberculosis drugs. No cross-resistance has been found between cycloserine and other anti-tuberculosis drugs. The mechanism of its antibacterial action is to inhibit the synthesis of peptidoglycan of bacterial cell wall, causing defective in cell wall architecture. The main structural component of the bacterial cell wall is peptidoglycan, which is composed of N-acetylglucosamine (GNAc) and N-acetylmuramic acid (MNAc). N-acetylmuramic acid is linked with pentapeptide and connects N-acetylglucosamine in a reduplicated and alternative manner. The formation of cytoplasmic peptidoglycan precursor may be hampered by cycloserine, as the latter can hinder the racemase and the synthetase of D-alanine, and thus blocks the formation of N-acetylmuramic acid.

Colorless needle or leafy crystals, or amorphous powder; melting point 155-156℃ (decomposition). Soluble in water; slightly soluble in methanol, ethanol, butanol, propylene glycol, isopropyl alcohol and acetone; and hardly soluble or insoluble in toluene, chloroform, ether, pyridine, benzene and carbon disulfide.

Used as an antibiotic medicine in the treatment of drug-resistant Mycobacterium tuberculosis infection.
Biochemical research

D-Cycloserine can be obtained through fermentation technique or through direct synthesis. The bacteria used in the fermentation is Actinomyces laven-dulae. The fermentation medium consists of dextrin, dextrose, starch, soybean powder, yeast powder, ammonium sulfate, ammonium nitrate, calcium carbonate, sodium chloride, magnesium sulfate and soybean oil. In the synthesis process, D-cycloserine is obtained from β-aminooxy alanine ethyl ester hydrochloride by reaction with potassium hydroxide in a cyclization reaction.

D-Cycloserine (68-41-7) is a partial agonist at the glycine modulatory site of NMDA glutamatergic receptors.1?Blocks kainate-induced seizures2?and displays anticonvulsant effects3?in rat models. D-Cycloserine facilitates synaptic plasticity but impairs glutamatergic neurotransmission in rat hippocampal slices.4?Second-line drug for the treatment of tuberculosis. ?Enhances activity-dependent plasticity in human adults.5

White to pale yellow cryst. powder

Oxamycin,Merck Sharp and Dohme,US,1956

antibacterial (tuberculostatic)

D-cycloserine has been used to inhibit serine hydroxymethyltransferase.

D-Cycloserine inhibits cell wall biosynthesis (D-Ala peptide bond formation). D-Cycloserine also prevents conversion of D-Ala to L-Ala. D-Cycloserine is an bacteriostatic. D-Cycloserine is an antibiot ic against Gram-negative bacteria.

ChEBI: D-cycloserine is a 4-amino-1,2-oxazolidin-3-one that has R configuration. It is an antibiotic produced by Streptomyces garyphalus or S. orchidaceus and is used as part of a multi-drug regimen for the treatment of tuberculosis when resistance to, or toxicity from, primary drugs has developed. An analogue of D-alanine, it interferes with bacterial cell wall synthesis in the cytoplasm by competitive inhibition of L-alanine racemase (which forms D-alanine from L-alanine) and D-alanine--D-alanine ligase (which incorporates D-alanine into the pentapeptide required for peptidoglycan formation and bacterial cell wall synthesis). It has a role as an antitubercular agent, an antiinfective agent, an antimetabolite, a metabolite and a NMDA receptor agonist. It is an organooxygen heterocyclic antibiotic, an organonitrogen heterocyclic antibiotic and a 4-amino-1,2-oxazolidin-3-one. It is a conjugate base of a D-cycloserine(1+). It is an enantiomer of a L-cycloserine. It is a tautomer of a D-cycloserine zwitterion.

Cycloserine is a broad-spectrum antibiotic produced by Streptomyces orchidaceus. It is structural analogue of Dalanine and acts through a competitive inhibition of the D-alanine that is involved in bacterial cell wall synthesis. Cycloserine is inhibitory to M. tuberculosis and active against Escherichia coli, S. aureus, and Enterococcus, Nocardia, and Chlamydia spp. It is used in the treatment of MDR tuberculosis and is useful in renal tuberculosis, since most of the drug is excreted unchanged in the urine.

Cycloserine may be made by a fermentation process or by direct synthesis. The fermentation process is described in US Patent 2,773,878. A fermentation medium containing the following proportions of ingredients was prepared:
Parts by Weight
Soybean meal
30.0
Cornstarch
5.0
Corn steep liquor
3.0
Sodium nitrate
3.0
This material was made up with distilled water to provide 41 g per liter, and the mixture was adjusted to pH 7.0 with potassium hydroxide solution. To the mixture were added per liter 5.0 g of calcium carbonate and 7.5 ml of soybean oil. 2,000 ml portions of this medium were then added to fermentation vessels, equipped with stirrers and aeration spargers, and sterilized at 121°C for 60 minutes. After cooling the flasks were inoculated with a suspension of strain No. ATCC 11924 of Streptomyces lavendulae,obtained from the surface of agar slants. The flasks were stirred for 4 days at 28°C at approximately 1,700 rpm. At the end of this period the broth was found to contain cycloserine in the amount of about 250 C.D.U./ml of broth. The mycelium was separated from the broth by filtration. The broth had a pH of about 7.5. Tests showed it to be highly active against a variety of microorganisms.
The direct synthetic process is described in US Patent 2,772,280. A solution of 73.3 g (0.332 mol) of β-aminoxyalanine ethyl ester dihydrochloride in 100 ml of water was stirred in a 500 ml 3-necked round-bottomed flask cooled in an ice-bath. To the above solution was added over a 30-minute period 65.6 g (1.17 mols) of potassium hydroxide dissolved in 100 ml of water, While the pH of the reaction mixture was 7 to 10.5, a red color appeared which disappeared when the pH reached 11 to 11.5. The light yellow solution was allowed to stand at room temperature for ? hour and then added to 1,800 ml of 1:1 ethanol-isopropanol. The reaction flask was washed twice with 10 ml portions of water and the washings added to the alcohol solution. The precipitated salts were filtered out of the alcohol solution and the filtrate cooled to 5°C in a 5 liter 3-necked round-bottomed flask. To the cold, well-stirred solution was added dropwise over a 35-minute period sufficient glacial acetic acid to bring the pH of the alcohol solution to 6.0. When the pH of the solution had reached 7 to 7.5, the solution was seeded and no further acetic acid added until crystallization of the oil already precipitated had definitely begun. The crystalline precipitate was collected on a filter, washed twice with 1:1 ethanolisopropanol and twice with ether. The yield of 4-amino-3-isoxazolidone was 22.7 g.

Seromycin (Lilly).

Antitubercular

Journal of the American Chemical Society, 79, p. 3236, 1957 DOI: 10.1021/ja01569a065

Chemical structure: amino acid derivatives

A fermentation product of Strep. orchidaceus and other related organisms now produced synthetically. Aqueous solutions are stable at pH 7.8 but the agent is rapidly destroyed in acid conditions. It is active against a wide range of Gram-negative and Grampositive bacteria, including Staphylococcus aureus, streptococci, including Enterococcus faecalis, various enterobacteria, Nocardia and Chlamydia spp. M. tuberculosis is inhibited by 8–16 mg/L. Some environmental mycobacteria, including M. avium, are also susceptible. Its action is specifically antagonized by d-alanine, from which media for in-vitro tests should be free. Its use is limited by neurological and psychiatric side effects. Primary resistance in M. tuberculosis is rare and develops only slowly in patients treated with cycloserine alone. Its inclusion in combinations deters the development of resistance to other drugs. There is no cross-resistance with other therapeutic antibiotics.
It is well absorbed when given orally, achieving a concentration of c. 10 mg/L 3–4 h after a 250 mg dose. Doubling the dose approximately doubles the plasma level. Some accumulation occurs over the first 3 or 4 days of treatment. In children receiving 20 mg/kg orally, plasma levels of 20–35 mg/L have been found. It is widely distributed throughout the body fluids, including the CSF. About 50% is excreted unchanged in the glomerular filtrate over 24 h and 65–70% over the subsequent 2 days. The remainder is metabolized. There is no tubular secretion and no effect of probenecid. Cycloserine accumulates in renal failure, reaching toxic levels if dosage is uncontrolled. It can be removed by hemodialysis.
Evidence of central nervous system toxicity, including headache, somnolence, vertigo, visual disturbances, confusion, depression, acute psychotic reactions and tremors, may develop over the first 2 weeks of treatment. The effects may be exacerbated by alcohol and can be reduced, to some extent, by administering pyridoxine. Treatment should be stopped promptly if any mental or neurological signs develop. Convulsions are said to occur in about 50% of patients when the plasma concentration exceeds 20–25 mg/L, but the relationship to dose is not particularly close. No permanent damage appears to be caused. Cycloserine inhibits mammalian transaminases and this and the convulsant effects of the drug have been attributed to a metabolite, amino-oxyalanine. Use of the drug should be avoided in patients with previous fits or other neurological or psychiatric abnormalities. Rare side effects include rashes, cardiac arrhythmia and deficiency in folate and vitamin B₁₂ leading to peripheral neuritis.
It is occasionally used in MDR tuberculosis (with other antituberculosis drugs) and other mycobacterioses (with appropriate additional drugs).

Mode of Action: Inhibits cell wall biosynthesis (D-Ala peptide bond formation). Also prevents conversion of D-Ala to L-Ala. Bacteriostatic.Partial agonist at the glycine modulatory site of NMDA glutamatergic receptors; antibiotic against Gram-negative bacteria.Mode of Resistance: D-Ala transport interference.

D-Cycloserine is considered to be the active form of the drug, having its action associated with the ability to inhibit two key enzymes, D-alanine racemase and D-alanine ligase. D-Alanine is an important component of the peptidoglycan portion of the mycobacterial cell wall. Mycobacterium are capable of utilizing natural occurring L-alanine and converting the L-alanine to D-alanine via the enzyme D-alanine racemase. The resulting D-alanine is coupled with itself to form a D-alanine–D-alanine complex under the influence of D-alanine ligase, and this complex is incorporated into the peptidoglycan of the mycobacterial cell wall . D-Cycloserine is a rigid analogue of D-alanine; therefore, it competitively inhibits the binding of D-alanine to both of these enzymes and its incorporation into the peptidoglycan. Resistance is associated with an over expression of D-alanine racemase.

Cycloserine is readily absorbed orally and distributes throughout body fluids including the cerebrospinal fluid. The concentrations of cycloserine in tissues, body fluids, and the cerebrospinal fluid are approximately equal to the plasma level. Cycloserine is partially metabolized, and 60 to 80% is excreted unchanged by the kidney.

D-(+)-4-Amino-3-isoxazolidinone (Seromycin) is an antibioticthat has been isolated from the fermentation beer of threedifferent Streptomyces species: S. orchidaceus, S. garyphalus,and S. lavendulus. It occurs as a white to pale yellow crystallinematerial that is very soluble in water. It is stable in alkaline,but unstable in acidic, solutions. The compoundslowly dimerizes to 2,5-bis(aminoxymethyl)-3,6-diketopiperazinein solution or standing.
The structure of cycloserine was reported simultaneouslyby Kuehl et al. and Hidy et al.81 to be D-( +)-4-amino-3-isoxazolidinone. It has been synthesized byStammer et al. and by Smart et al.83 Cycloserine is stereochemicallyrelated to D-serine. However, the L-form hassimilar antibiotic activity.
Although cycloserine exhibits antibiotic activity invitro against a wide spectrum of both Gram-negative andGram-positive organisms, its relatively weak potency andfrequent toxic reactions limit its use to the treatment of tuberculosis.It is recommended for patients who fail to respondto other tuberculostatic drugs or who are known tobe infected with organisms resistant to other agents. It isusually administered orally in combination with otherdrugs, commonly isoniazid.

Neurological symptoms, which tend to appear in the first week of therapy, consist of dizziness, confusion, irritability, psychotic behavioral changes, and even suicidal ideation. Cycloserine is contraindicated in patients with underlying psychiatric and seizure disorders.Other side effects include occasional peripheral neuropathy and low magnesium levels.

Cycloserine is readily absorbed after oral administration and is widely distributed, including the CNS. Unfortunately, D-cycloserine binds to neuronal N-methylasparate receptors and, in addition, affects synthesis and metabolism of γ-aminobutyric acid, leading to complex series of CNS effects. As a second-line agent, cycloserine should only be used when retreatment is necessary or when the organism is resistant to other drugs. Cycloserine should not be used as a single drug; it must be used in combination.

Cycloserine, 4-amino-3-isoxalidinone (34.1.19), can be synthesized both biosynthetically from the actinomycetes Streptomyces garyphalus, Streptomyces orchidaceus, and Streptomyces lavenduale as well as synthetically from the methyl ester of D-serine, the hydroxyl group of which is replaced with a chlorine atom when reacted with phosphorous pentachloride, and subsequent reaction of the resulting product (34.1.19) with hydroxylamine results in heterocyclization to the desired cycloserine (34.1.20).

Potentially hazardous interactions with other drugs
Alcohol: Increased risk of seizures.

Cycloserine is excreted largely unchanged by glomerular filtration. About 50% of a single 250 mg dose is excreted unchanged in the urine within 12 hours and about 70% is excreted within 72 hours. As negligible amounts of cycloserine appear in the faeces, it is assumed that the remainder of a dose is metabolised to unidentified metabolites.

Purify cycloserine by recrystallisation from aqueous EtOH or MeOH or aqueous NH3/EtOH or isoPrOH. Also recrystallise it from aqueous ammoniacal solution at pH 10.5 (100mg/mL) by diluting with 5 volumes of isopropanol and then adjusting to pH 6 with acetic acid. An aqueous solution, buffered to pH 10 with Na2CO3, can be stored in a refrigerator for 1week without decomposition. UV: max at 226nm (A1cm 1% 4.02). The tartrate salt has m 165-166o (dec), 166-168o (dec), and [] D 24 -41o (c 0.7, H2O). [Stammer et al. J Am Chem Soc 79 3236 1959, UV: Kuehl J Am Chem Soc 77 2344 1955, Beilstein 27 III/IV 5549.]

1) Watson?et al. (1990),?D-cycloserine acts as a partial agonist at the glycine modulatory site of the NMDA receptor expressed in Xenopus oocytes; Brain Res.,?510?158 2) Baran?et al. (1994),?The glycine/NMDA receptor partial agonist D-cycloserine blocks kainite-induced seizures in rats. Comparison with MK-801 and diazepam; Brain Res.,?652?195 3) L?scher?et al. (1994),?Anticonvulsant effects of the glycine/NMDA receptor ligands D-cycloserine and D-serine but not R(+)-HA-966 in amygdala-kindled rats; Br. J. Pharmacol., 112?97 4) Rouaud and Billard (2003),?D-cycloserine facilitates synaptic plasticity but impairs glutamatergic neurotransmission in rat hippocampal slices; Br. J. Pharmacol.,?140?1051 5) Forsyth?et al. (2015),?Augmenting NMDA receptor signaling boosts experience-dependent neuroplasticity in the adult human brain; Proc. Natl. Acad. Sci. USA,?112?15331