FI59415C - FRAMEWORK FOR FOUNDATION AV CEFALEXIN - Google Patents

Description

F5Br * l M / id ^ ANNOUNCEMENT OF REPRESENTATION ςο WA IJ UTLÄGGNI NGSSKRIFT 0 ^ 415 C (45) Patent granted? tty JO 03 1031 ^ T ^ (51) Kv.ik.3 / Int.ci.3 C 07 D 501/22, 501/10 FINLAND — FINLAND £ 1) Patanttlhakumut - Patantanseknlng 792793 (22) HakamUptlvl - Anaeknlngadag 07 * 09.79 (23) Alkuptlvi — Glltlghaadag 13.07.71 (41) Tullut julklttkil - Bllvlt offantllg 07.09.79 '“•" “- j * r« ki «t« Hhallttu. M NfcMWr ». 1.1-M—« r ».-

Patent · och r * fl> t «rftyr« laan 7 Anaökan utlagd och utl.akrlften publfcerad 30.0U. Si (32) (33) (31) Pretty atuoikaua — Bagird prlorlt «05.10.70 USA (US) 78165 (71) Bristol-Nfyers Company, 3 ^ 5 Park Avenue, New York, NY, USA (US) (72) William Joseph Gottstein, Fayetteville, New York, Lee Cannon Cheney, Fayetteville, New York, USA (US) (7M 0y Kolster Ah (5M Process for the preparation of cefalexin) ) -Avdelad frän ansökan 1993/71 (patent 58133)

The invention relates to a new process for the preparation of cefalexin having the formula

Γ \? -S

y-CH - C - NH-f ^ I

- NH2 o J-CH3

COOH

or a pharmaceutically acceptable salt thereof.

The process according to the invention is characterized in that A) a compound of formula III 2 59415 rv 11 \ _ /? "~~ cx = / CV m --- I /" - j-f] Cfl3

11 ~ H J · --W - COOH

The CH3 ° cn3 3-imino group is protected by nitrosation or formylation to form an intermediate of formula / O / CH-CH,

\ - / I \. / 3 IV

I> —f— V — ci | 3 Z-N - C 1

| \ / -N - COOH

I CH 0 CH 3 wherein Z is nitroso or formyl, B) said intermediate or a salt thereof is oxidized with sodium metaperiodate at a pH below 5 to give a sulfoxide of formula

(J x) - CII-C | / 3 V

\ = T- / | N- z-N-c 'I ·

I ^ CU j --- N-— COOH

I J o /

CU

or a salt thereof, C) the resulting sulfoxide intermediate is heated in acetic acid anhydride or tetramethylurea in the presence of an acid catalyst having p-toluenesulfonic acid or acetic anhydride at a temperature of about 120-140 ° C for about 30-120 minutes to give a cephalosporin intermediate (r \ | VI1 ZN -Ct, ^, -C „3 ^

COOH

wherein Z is as defined above and D) the imino protecting group is cleaved by hydrolysis to form the product of formula

ch3 ch3 COOH

and E) hydrolyzing the resulting hetakefalexin of formula II to cefalexin of the formula "H - C - MH - T - Y">

^ ----- CH3 X

COOH

4,59415 or a non-toxic pharmaceutically acceptable salt thereof.

The final product, cefalexin, i.e. 7- (N-aminophenylacetamido) -3-methyl-3-cephem-4-carboxylic acid, is a known antibacterial agent. It can also be prepared from 7-deacetoxycephalosporanic acid (7-ADCA) and is described, for example, in J. Med. Chem., 12, 310-313 (1969), GB Patent 1,174,335, ZA Patent 67/1260 (Farmdoc 28,654), JA Patent 16871/66 (Farmdoc 23231), BE Patent 696,026 (Farmdoc 29494).

The intermediate hetakefalexin, i.e. 7- (2,2-dimethyl-5-oxo-4-phenyl-1-imidazolidinyl) -3-methyl-3-cephem-4-carboxylic acid, is a new compound.

The physical properties of hetakefalexin are shown in Example 3.

Conversion of the penicillin sulfoxide ester (by heating in the presence of a strong acid) to the corresponding ester of the corresponding N-acylated derivative of 7-ADCA is described in U.S. Patent 3,275,626 and J. Amer. Chem. Soc., 85, 1896 (1963) and 91 (6), 1401-1407 (1969). Variations of this method are disclosed in NL Patent Publications 68/06532 (Farmdoc 34,685) and 68/06533 (Farmdoc 34,686). In such patents, the side chain is usually the side chain of a fermentable penicillin, such as penicillin G or V (see column 7 in U.S. Patent 3,275,626), and the product is an ester that must be cleaved, e.g., by hydrogenation to form the active free acid of the final derivative of 7-ADCA. Example 3 of GB Patent 1,174,335 illustrates the application of this "sulfoxide rearrangement" to an ester of ampicillin sulfoxide in which the © C amino group is also protected, i. 6- [N- (2,2,2-Trichloroethylcarbonyl-D-amino-β-phenylacetamido) -penicillanic acid sulfoxide-2,2,2-trichloroethyl ester by heating and then using zinc and acetic acid for two protecting groups and to form cefalexin.

The prior art encompasses numerous other descriptions of penicillin sulfoxides and their preparations, such as those described in Chow et al., J. Org. Chem, 27, 1381 (1962), Gud-dal et al., Tetrahedron Letters no. 9, 381 (1962), Essery et al., J. Org. Chem., 30, 4388 (1965), which also includes ampicillin sulfoxide, and U.S. Patent 3,197,466.

The reaction product of acetone with cephaloglycine but not cefalexin is described in U.S. Patent 3,303,193. The reaction of certain ring-substituted cephalexins with acetone is described generally in U.S. Patents 3,489,750, 3,489,751 and 3,489,752.

Non-toxic pharmaceutically acceptable salts include, for example, (1) pharmaceutically acceptable salts of an acidic carboxylic acid group, such as sodium, potassium, calcium, aluminum and ammonium salts, and non-toxic substituted ammonium salts of amines, such as tri (lower) alkylamines, with benzylamine, N-benzyl-beta-phenylamine, 1-ephenamine, N, N'-dibenzylethylenediamine, dehydroabietylamine, N, N'-bis-dehydroabiethylethylenediamine, N- (lower) alkylpiperidines such as N-ethylpiperidine and other used to form salts of benzylpenicillins; and (2) non-toxic pharmaceutically acceptable acid addition salts (e.g., basic nitrogen salts) such as (a) mineral acid addition salts such as hydrochloride, hydrobromide, hydroiodide, sulfate, sulfamate, sulfonate, phosphate, etc. salts. and (b) organic acid addition salts such as maleate, citrate, tartrate, oxalate, succinate, benzoate, fumarate, malate, mandelate, ascorbate, β-naphthalene sulfonate, p -Toluenesulfonate and the like salts. Cefalexin can also exist as a free acid, which naturally occurs as an amphoteric ion.

In reaction step (A), the 3-imino group of the hetacillin starting material is protected by performing nitrosation or formylation.

The preferred method of nitrosation involves acidifying an aqueous suspension of hetacillin to a pH of about 2 with hydrochloric acid or dilute phosphoric acid until a solution is formed, cooling the solution to about 10 ° C and then adding sodium nitrite in small portions with stirring to form N-nitroso-hetacillin. N-formylhetacillin can be prepared, e.g., by reacting formic acid and hetacillin.

The imino-protected hetacillin or a salt thereof is oxidized by a known method, e.g., using the method described by Chow, Hall and Hoover in J. Org. Chem., 27, 1381 (1962) 6,59415 to form the corresponding sulfoxide V. Hetacillin is reacted with an oxidizing agent in an amount such that at least one atom of active oxygen is present per atom of thiazolidine sulfur. A suitable oxidizing agent is sodium metaperiodate. For best results, use sodium metaperiodate as an oxidizing agent at a pH below 5. The reaction mixture is maintained at a pH below 5 to prevent or minimize C-6 epimerization.

The sulfoxide intermediate obtained in step (B) is converted to the corresponding cephaloxporin intermediate by heating to elevated temperature in the presence of an acid catalyst. The acid catalysts used are p-toluenesulfonic acid and acetic anhydride.

Any organic liquid may be used as the reaction medium for regrouping, provided that it is substantially inert to the other reaction components under the conditions used. Preferred solvents are acetic anhydride and tetramethylurea, and most preferred is tetramethylurea. The reaction temperature should be in the range of about 100-140 ° C, preferably about 120-140 ° C. The reaction time is kept as short as possible to prevent the formation of unwanted by-products. The best results are obtained when the reaction is carried out for a period of from 30 minutes to about 120 minutes.

The nitroso or formyl protecting group is then removed by acid hydrolysis, e.g. using dry HCl in dioxane, by catalytic hydrogenation, e.g. using and Raney nickel, or by reducing zinc with acetic acid to give hetakefalexin (II). Hetakefalexin is converted to cefalexin by either acidic or basic hydrolysis. If it is desired to obtain hetakefalexin, the most preferred cleavage method is the use of dry HCl, e.g. using HCl in dioxane, in order to minimize hydrolysis to cefalexin.

An improved process for the preparation of the valuable known antibacterial agent cefalexin has been described above. The process described is an economically very useful process for the preparation of cefalexin and avoids several problems of the known processes.

7 59415

The following examples illustrate the present invention. All temperatures are in degrees Celsius. "Skellysolve B" is a petroleum ether fraction having a boiling point of 60-68 ° C and containing substantially n-hexane.

Example 1 6- (D-2,2-Dimethyl-3-nitroso-5-oxo-4-phenyl-1-imidazolidinyl) -penicillanic acid (N-nitrosoacetillin) To a suspension of 7.1 g (0.02 moles) of hetacillin in 350 ml of water, 6N hydrochloric acid is added dropwise at room temperature until all the hetacillin is dissolved. The solution is cooled in an ice bath at 10 ° C and 100 ml of ethyl acetate are poured on top. A total of 1.4 g (0.02 moles) of sodium nitrite dissolved in 25 liters of water is added in small portions over 5 minutes. The solution is stirred for 20 minutes at 10 ° C and the ethyl acetate layer is separated, washed with water and evaporated at 5 ° C under reduced pressure (15 mm) to give a yellow oil which crystallizes on stirring with ether. The product is recrystallized from methanol / water to give 4.5 g of crystals (54% yield); mp. 195 ° C (decomposes). Analysis: Calculated for c 19 H 24 N 4 O 5 S: C, 54.54; H, 5.30; N, 13.39. Found: C, 54.55; H, 5.58; N, 13.33.

O Ir (KBr) 2800-3600 cm (flybox OH), 1803 and 1790 (β-lactam C-N), 1750 and 1730 (flybox and imidazolidinone N), 700 (CgH5-); nmr (DMSO dg), 7.30 ppm cj (S, 5, CgH5-), 5.64 (S, 1,1-SH-N), 5.60 (d, 1.1, J = 4 cps, NCHCO) , 5.45 (d, 1.1, J = 4 cps, NCHS), 435 (S, 1, NCHCO), 2.00 (S, 6, CH 3 CH 3 CN), 1.48 (S, 6, ch 3 CH 3 cs).

Example 2 6- (D-2,2-Dimethyl-3-nitroso-5-oxo-4-phenyl-1-imidazolidinyl) -penicillanic acid sulfoxide (N-nitrosoacetillin sulfoxide)

A total of 10 g (0.024 mol) of 6- (D-2,2-dimethyl-3-nitroso-5-oxo-4-phenyl-1-imidazolidinyl) -penic acid is dissolved in 100 ml of water at pH 8 by dropwise addition of 10%. 59415 sodium hydroxide. After cooling to 0 [deg.] C., a solution of 6 g (0.025 mol) of sodium metaperiodate in 100 ml of water is added and stirring is continued for 3 hours. The pH of the solution is lowered to 2 using 1: 1 phosphoric acid. The product is extracted into ethyl acetate, washed with water and the solution is azeotroped to an oil which, when mixed with ether, crystallizes to give 6.5 g of white crystals; mp. ^ 160 ° C (decomposes slowly). The analytical sample is recrystallized from dimethyl / foramide and water.

Analysis for c 18 H 22 N 4 O 6 S:

Calculated: C, 52.52; H, 5.11; N, 12.92 Found: C, 52.66; H, 5.37; N, 13.45.

IR (KBr) 3540 cm -1 (hydrate OH), 2400-3400 (carboxy OH), 1804 (β-lactam-N). 1720-1750 (imidazolidinone

.0 L

C 1-7 and carboxyl (C 1-10), 1050 (S → O), 705 (C 1-6); nmr (DMSO d 6), 7.32 ppm δ (S, 5, C 9 H 8), 5.77 (S, 1,1-CH-N), 5.72 (d, 1, J = 4 , 5 cps NCHCO), 4.83 (d, 1, J = 4.5 cps, NCHS), 4.30 (S, 1, NCHCO), 2.12 and 2.05 (S, S, 6 CH 3 CH 3 CN) , 1.47 and 1.20 (S, S, 3.3, CH 3 CH 3 CS).

9 5941 5

Example 3

$ O

\. // o CH - C n I I / CH- ΤίίΛ (H *) ON-K 'N --------- r — 7 ------> / K LI____I CH; imi C \\ ζ XC11, O * X CO2H a y ,, y

cion2sW

(tl O

\ ^ CH-C · I I s

OH-H N - T-Γ O

<\ CH, O ^ Y'-CH,

CO a.H

c) 9h £ 0 '' N

H Cl dioxinji i N /. of / ° Il s # C / -Hr -CII-C - NH -! - ^ CH-C 6 5 (| 1 1 '> S ^ v. K112 hh H ----- rl 1 -h7ö ^ 0κ 1

> v _] T J * CO „H

ci.j c "; ch2

COgH

? Ci H21N; V

7- (D-2,2-dimethyl-3-nitroso-5-oxo-4-phenyl-1-imidazolidinyl) -3-methyl-3-cephem-4-carboxylic acid A solution of 21 g (0, 0487 moles) of 7- (D-2,2-dimethyl-3-nitroso-5-oxo-4-phenyl-1-imidazolidinyl) -penicillanic acid sulfoxide and 5 g of p-toluenesulfonic acid (anhydrous) (TSA) 500 ml in tetramethylurea (TMU) is heated at 135 ° C for 2 hours with stirring. The solvent is removed at 40 ° C / 0.1 mm to give an oil which is dissolved in 250 ml of ethyl acetate. The ethyl acetate is washed twice with 100 ml portions of water and extracted with dilute sodium bicarbonate solution. The final pH is 6.7. The aqueous layer is separated and mixed with 100 ml of ethyl acetate. The pH is adjusted to 2 using 1: 1 H 3 PO 4 and the aqueous solution is extracted twice with ethyl acetate. The ethyl acetate is washed with water and azeotroped into an oil at 35 ° C / 15 mm. The residue is mixed with "Skellysolfe B" to give an amorphous powder weighing 10.0 g, which is suspended in 150 ml of water and saturated sodium bicarbonate solution is added until all the substance is dissolved (final pH 7.5). containing 4 g (0.011 mol) of dibenzylethylenediamine diacetate in 75 ml of water and the mixture is stirred with 150 ml of 4-methyl-2-pentanone in a two-phase system, the mixture is stirred for 1/2 hour at room temperature and the crystalline salt is collected, washed with water and finally acetone and air dried to give 7.1 g, mp 150-152 ° C with decomposition.

Analysis for C54H60N10 ° 10S2 * 3H2O:

Calculated: C, 57.53; H, 5.90; N, 12.43 Found: C, 57.54; H, 6.21; N, 12.71.

57.40 6.38

IR (KBr) 3200-3600 cm-1 (NRH2 +); OH (water); 1760-1770 0 O

(Β-lactam-C-N); 1600 (C-O-); 755, 700 CgH5.

7.1 g of N, N-dibenzylethylenediammonium 7- (D-2,2-dimethyl-3-nitroso-5-oxo-4-phenyl-1-imidazolidinyl) -3-methyl-3-cephem-carboxylate are suspended To 50 ml of 1: 1 phosphoric acid and to 150 ml of water. The mixture is covered with 150 ml of ethyl acetate and shaken vigorously until all the salt is dissolved. The ethyl acetate is removed, washed with water and evaporated at 40 ° C (15 mm) to give a crystalline substance weighing 3.1 g, m.p. 175-180 ° C with decomposition. The IR and NMR spectra are identical to those of authentic N-nitrosoheta-dalexin.

5941 5 7- (D-2,2-dimethyl-5-oxo-4-phenyl-1-imidazolidinyl) -3-methyl-3-cephem-4-carboxylic acid (hetakefalexin) to 1 g (0.0025 mol) 7- (D-2,2-Dimethyl-3-nitroso-5-oxo-4-phenyl-1-imidazolidinyl) -3-methyl-3-cephem-4-carboxylic acid in 50 ml of dioxane (purifying by running the run) through an alumina column), dry hydrogen chloride is added for 5 minutes at room temperature. The solution is evaporated at 30 ° C (15 mm) to a fibrous substance which is slurried with ethyl acetate and collected. The material is then dissolved in water (50 mL) and basified to pH 4.8 with aqueous sodium bicarbonate solution. The mixture is filtered and the filtrate is evaporated at 30 ° C (15 mm) to a glass which is dried by azeotropic distillation with ethyl acetate. The yield of the sodium salt is 600 mg; the product is prescribed and is the sodium salt of 7- (D-2,2-dimethyl-5-oxo-4-phenyl-1-imidazolidinyl) -3-methyl-3-cephem-4-carboxylic acid (hetakefalexin); mp. 205 ° C with decomposition.

Analysis for C 19 H 20 N 3 O 4 SNa * H 2 O 2:

Calculated: C, 53.39; H, 5.19; N, 9.83 Found: C, 53.63; H, 5.00; N, 9.76 H 2 O, 4.30 (Karl Fischer) IR (KBr); 3600-2600 cm -1 (H 2 O, NH, CH 2, CH 3 and CH 3 S) 1760 cm -1 (β-lactam carbonyl), 1690 cm -1 (amide carbonyl), 1590 cm -1 (carboxylate carbonyl and C = C), 710 cm -1 mono-substituted phenyl.

NMR (100 MHz) 20 mg sample in 0.4 ml D 2 O with 2 drops of 0.1 NHCl; chemical shifts in ppm from TSP; 7.52 (S, 5H's monosubstituted phenyl), 5.71 (IS, 1H, CH-N), 5.28 (AB, 2H's / 3-lactam CH'S), 3.48 (AB, 2H's, S-CH2 ~ C =), 2.26 (S, 3H'S = C-CH3), 1.88 and 1.80 (two S's, 6H's C (CH3)).

This compound inhibits D.pneumoniae (+5% serum) at a concentration of 0.3 mcg / ml, Str.pyogenes (+5% serum) at a concentration of 0.3 mcg / ml, S.aureus Smith at a concentration of 0.6 mcg / ml, .enteritidis at a concentration of 4 mcg / ml and Pr.mirabilis at a concentration of 8 mcg / ml.

7- (D-N-aminophenylacetamido) -3-methyl-3-cephem-4-carboxylic acid (cefalexin)

Dissolve about 200 mg of hetaxalexin sodium in as little water as possible (about 1 ml), acidify to pH 4-5 with 12 5941 5 acetic acid and allow to stand at 5 ° C overnight. The crystalline material is collected, washed with water and finally with acetone to give, after drying at room temperature (15 minutes), using 22 ° crystalline cefalexin, m.p. At 195 ° C with decomposition, the NMR and IR spectra are identical to authentic cefalexin.

Example 4 7- (D-Of-aminophenylacetamido) -3-methyl-3-cephem-4-carboxylic acid by reduction of N-nitrosoacetalexalex with zinc and acetic acid to 500 mg of 7- (D-2,2-dimethyl-3-nitroso) -5-Oxo-4-phenyl-1-imidazolidinyl) -3-methyl-3-cephem-4-carboxylic acid dissolved in 20 ml of 90% acetic acid, 500 mg of zinc dust are added.

The mixture is stirred at 5 ° C in an ice bath for 3 hours. The zinc is collected by filtration and the filtrate is evaporated under pressure to give 0.1 mm of an oil which is mixed with ether to give 420 mg of a solid. The IR spectrum shows β-lactam at 1755 cm -1 for amide carbonyl at 1695 cm -1 and a strong carboxylate at 1580 cm -1 A bioautogram of this substance (against a B. subtilis seeded agar plate) on a paper strip of Miat. In the No. 1 system, n-butanol, ethanol, and water (4: 1: 5) show a biologically active spot corresponding to an R 1 value identical to that of authentic 7- (D- (Y-aminophenylacetamido) -3-methyl-3-cephem). With 4-carboxylic acid (cephalexin) Further purify 420 mg by dissolving it in 7 ml of water at pH 8 with NaSH, recovering the zinc sulphide and evaporating the filtrate to an oil which solidifies to give 31 mg of material. Example 5 7- (D-2,2-Dimethyl-3-nitroso-5-oxo-4-phenyl-1-imidazolidinyl) -3-methyl-3-cephem-4-k .hydrogenolysis of carboxylic acid

Using Raney nickel to obtain cefalexin

A suspension of 1 g (0.0024 mol) of 7- (D-2,2-dimethyl-3-nitroso-5-oxo-4-phenyl-1-imidazolidinyl-3-methyl-3-cephem -4-carboxylic acid and 1 g of wet Raney nickel No. 28, shake for 2 days under a hydrogen pressure of 3.5 kg / cm After 1 day, a further 2 g of catalyst are added in 25 ml of water. the equivalent of hydrogen has been consumed, the nickel is removed by filtration and the solution is adjusted to pH 2 with 2N hydrochloric acid, the mixture is filtered and the filtrate is adjusted to pH 4.7 with 10% sodium hydroxide and evaporated at 35 ° C / 0.1 mm, to give cefa-lexin as a white substance weighing 0.6 g. The paper chromatogram of this substance (against B. subtilis seeded agar plate; What No 1) developed with n-butanol, ethanol and water 4: 1: 5 shows a zone of inhibition corresponding exactly to the Rf value of the authentic cefalexin inhibition zone.

Example 6 7- (D- (X-Aminophenylacetamido) -3-methyl-3-cephem-4-carboxylic acid by cleavage with hydrogen chloride of N-nitro-sohetaxalexin

To a solution of 2 g (0.05 mol) of 7- (D-2,2-dimethyl-3-nitroso-5-oxo-4-phenyl-1-imidazolidinyl) -3-methyl-3-cephem-4- carboxylic acid dissolved in 50 ml of dioxane, bubble dry hydrogen chloride gas for 5 minutes. The solution is stirred for 5 minutes and the solvent is removed at 30 ° C / 15 mm. The residue is mixed with ethyl acetate to give 1.9 g of crude product. The material is dissolved in dilute hydrochloric acid at pH 2.5 and treated with charcoal (Darko KB) for 5 minutes and the filtrate is adjusted to pH 4 using 10% sodium hydroxide. The water is evaporated at 40 ° C / 15 mm to give 1.1 g of free amino acid.

The infrared spectrum is consistent with the authentic cefalexin spectrum. The residual NaCl is 38.34%. The bioassay is 350 K / mg. Chemical analysis is 365 f / mg.

Claims (3)

Claim: A process for the preparation of cefalexin having the formula _ 0 / \\ "- CU - C-NR --------- SS \! NH2 ^ ---------- N \ ^ CH3 For the preparation of COOH or a pharmaceutically acceptable salt thereof, characterized in that A) the 3-imino group of the compound of formula III // ~ V_ 11 N '/' N Γ ^ Η3 III H-fl -c J 111 | VC1I "C00H Cil3 3 is protected by nitrosation or formylation to form an intermediate of the formula c 1 -Nα, -1-, /> j WI N --- fS ^ C "3" 'a I ^ CH, -N - 00011 CH 3 j wherein Z is nitroso or formyl B) said intermediate or a salt thereof is oxidized with sodium metaperiodate at a pH below 5 to give a sulfoxide of formula 15 5941 5 Γ ~ \ «\) - CU-C τ CU. \ = / ι ___ ^ 3 Ζ The sulfoxide intermediate obtained in -,? minutes to obtain a cephalosporin intermediate of formula Co.V 11 ch: -c N --T '' "'I / IL 11' CH /, - CI13 VII CH 0 'I COOH itself the same as above, D) the imino-protecting group is cleaved by hydrolysis to form a product of formula II, CV ™ ___- e ··· J ------- CHi 11 / \ 'I CW3 CK3 COOH and E) the obtained formula II hetakefalexin is hydrolyzed to cefalexin of formula I. 16 5941 5 For the preparation of cefalexin.med form 0 '\\ ”y— CU— C— NII ------ I ~ NH ....... J <^ · - CH, 0 I COOH or a pharmaceutical company said at the same time, in the case of A) 3-iminogroups in the formulation // "\ Ϊ \ 'f-ll -Cv„ .CH, v- 7 .n Γ r —-Jcii, γιγ] 1 —N -I 3 111 | V ™ </ -C00H CH3 3 shielded genome by nitrosation or formation of an additional product with the formula c / \ .__ [.CH \ 7; H c \ / 3 iv \ —— / N-1-CIU X f I 3 | VCI1 SN - coo, 'Cll 3 υ color Z is nitroso or formyl,