COMPLEXES

The present invention concerns a novel process for preparing isothiocyanato fuctionalized metal complexes. The complexes formed are bifunctional compounds which are useful as various therapeutic and/or diagnostic agents.

Functionalized chelants, or bifunctional coordinators, are known to be capable of being covalently attached to an antibody having specificity for cancer or tumor cell epitopes or antigens. Radionuclides complexes of such antibody/chelant conjugates are useful in diagnostic and/or therapeutic applications as a means of conveying the radionuclide to a cancer or tumor cell. See, for example, Meares et al., Anal. Biochem. JJ42, 68-78 (1984); and Krejcarek et al., Biochem. and Biophys. Res. Comm. J_χ, 581-585 (1977).

The present invention concerns a process for preparing isothiocyanto fuctionalized metal complexes. A radionuclide can be used, and is prefered, in these complexes.

Isothiocyanto functionalized ligands are reported in the literature and are being used to

conjugate radioactive isotopes to antibodies. For example see Gansow et al., Inorg. Che . 25, 2772-81 (1986); Meares et al., Analytical Biochem. 142, 68-78 (1984); U. S. Patent 4,454,106.

The methodology taught in the art to prepare such complexes involves treatment of an antibod /chelant conjugate with the radionuclide to form a complex followed by purification of the complex. A major disadvantage of such methodology is that the radionuclide (a lanthanide or transition metal) must be kinetically labile in order to be rapidly sequestered by the antibody/chelant conjugate.

Another disadvantage associated with the use of labile radionuclides for antibody labelling is that substitutionally labile trace metals (which are not radioactive) are frequently incorporated into the chelate. Competition for such non-active trace metals diminishes the biological efficacy of the antibody/chelate complex since a lower quantity of radionuclide is delivered to the target site. •

Mikoler et al., European published application 139,675, teach the preparation of isothiocyanate fuctionalized chelates which can subsequently be conjugated to bio-organic molecules, e.g. haptens, antigens and antibodies. These complexes are prepared by chelating the isothiocyanate functionalized ligand.

In contrast, the present invention is directed to a novel process to prepare isothiocyanate compounds which comprises reacting a amino fuctionalized chelate with thiophosgene. The present process concerns the preparation of the isothiocyanto function on a ligand

after the metal has been chelated with the ligand. The formation of the isothiocyanto moiety on the ligand after the complex has been formed is important for several reasons: (1) when the complex requires heating or severe extremes in pH to form the complex, such as with rhodium or lanthanide macrocycles, the present process avoids the destruction of the isothiocyanate fuctionality during chelation; (2) when there are other primary or secondary amines present in the ligand, formation of the isothiocyanate fuctionalized ligand prior to chelation is impractical due to side reactions; (3) by forming the complex at as early a stage in the reaction as possible, complexation of undesired metals is reduced, and thus purity of the final product is enhanced; and (4) by forming the complex prior to the introduction of the isothiocyanate, purification of the complex, such as by ion exchange chromatography, is simplified and the added complication of the hydrolysis of the isothiocyanate during purification is also reduced.

Suprisingly, the present process provides a process to prepare isothiocyanates by thiophosgenation of amino fuctionalized chelates which results in a process that is rapid with high yield and provides a product having low metal contamination present. As there are a fewer number of reactions required overall by the present process on the ligand prior to chelation, the amount of undesired metal contamination is reduced.

The ligands of interest are generally strong chelators for many different metals which may be present in the reagents or containers used to store or transfer the ligand. Although amino fuctionalized chelates of many types can be used, the chelates formed from ligands

hat are aminocarboxylic acid cheiants, aminophosphonic acid cheiants or polyaza chelan.s are particularly preferred.

One of the possible classes of ligands useful . in .his process are aminocarboxylic acid cheiants.

Examples of some of the possible aminocarboxylic acid cheiants are given in Table I following and which are named as follows:

I A is p-aminobenzyl ethylenediamine- tetraacetic acid, the preparation of which is given in ϋ. S. Patent 4,622,420 and J. Med. Chem. V7(4), 1304 (•974); .

I B is p-aminobenzyl hydroxyethylethyiene- diaminetriacetic acid, the preparation of which is given in ϋ. S. Patent 4,622,420;

I C is p-aminobenzyl diethylenetriamine- oentaacetic acid, the preparation of which is given in U. S. Patents 4.622,420 and 4,647,447:

I D is N'-p-aminobenzyl diethylenetriamine- .N,N",N"-tetraacetic acid, the preparation of which is given in J. Radioanalytical Chem. 57_(12) , 553 (1980);

I E is 6-(p-aminobenzyl)-1,4,3, 11- tetraazacyclotetradecane-l ,4,8, 11-tetraacetic acid, the preparation of which is given in Analytical Biochem. _48_, 2^9-253 (1985);

I ? is 1-[2-(^-amiπophenyi)ethyl]-1,4,7,10- tεtraazacycIododecane-4,7, 10-triacetic acid, the preparation of which is given in Saudi Arabian Patent; 1211k, issued October 10, ¹ 989:

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I G is α-[2-(4-aminophenyl)ethyl]-1.4,7.10- tetraazacyclododecane-1 ,4,7, 10-tetraacetic acid, the preparation of which is given in Saudi Arabian Patent 3277A, issued October 10, 1989;

I H is 1-(5-amino-2-methoxybenzyi)-1 ,4,7, 10- tetraazacyclododecane-4,7, 10-triacetic acid, the preparation of which is given in Saudi Arabian Patent 3277A, issued October 10, 1989;

I I is 1-(5-amino-2-hydroxybenzyl)-1 ,4,7, 10- tetraazacyciododecane-4,7, 10-triacetic acid, the preparation of which is given in Saudi Arabian Patent 3277A, issued October 10, 1989;

I J is 2-[(2-{[bis(carboxymethyl)]amino}ethyl)-

(carboxymethyl)amino]-2-[5-amino-2-(carboxymethyloxy)- phenyljethanoic acid, the preparation of which is given below;

I is 2-[(2-{[bis(carboxymethyl)]amino}ethyl)-

(ca boxyme hyl) mino]-2-(5-amino-2-hydroxyphenyl)- ethanoic acid, the preparation of which is given below;

I L is 2,6-bis{[(2-{[bis(carboxymethyl)]amino}- ethyl) (carboxymethyl)]aminomethyl}-4-(amino)phenol, the preparation of which is given below;

I M is α-(4-aminophenyl)-1,4,7, 10- tetraazacyciododecane-1 ,4,7, 10-tetraacetic acid, the preparation of which is given in Saudi Arabian Patent 3277A, i ^' ssued October 10, 1989;

I N is α-(4-aminophenyl)-1 ,4,7. 0- tetraazacvcioάoGecane-". , 4,7-triacetic acid, -he

-o-

prεoaration of which is given in Saudi Arabian Paten 32 ⁷⁷ A. issued October 10. 1989: and

I 0 is α-[2-(4-aminophenyl)ethyl]-1 ,4,7.10- tetraazacyclGdodecane-l-(R,S)-acetic-4,7.10-t is-(.R- methyiace ic) acid, the preparation of which is giver, in Saudi Arabian Patent 3277A, issued October 10, 1989.

The compounds of Examples I J, I K and I L above can be prepared in ways well known to the art. Thus, for example, see Cheiating Agents and Metal Chelates, Dwyer & Mellor, Academic Press (1964), Chapter 7. See also methods for making amino acids in Synthetic Production and Utilization of Amino Acids, (edited by Ka eko, st al.) John Wiley 3. Sons (1974). For example, in a compound corresponding to Example I J but having an aceta.iido group present in place of the amino group, the acetamido group is hydrolyzed with NaOH in H ₂ 0 to provide the compound of Example I J. To prepare a compound corresponding to Example I K, a 5-amino-2- hydroxvphenyl compound is reacted with the appropriate linear or branched amine or polyalkvlene amine and an aldehyde or aldehyde precurser equivalent in the presence of caustic and a suitable solvent, at a temperature of 20°C or less, followed by heating and separating the desired product, then reacting the product obtained with glycolonitrile, in caustic, at a pH of 9 or higher, at a temperature of 20°C or less, followed by hydrolysis of the cyano group with HCI in H^0, to provide the product of Example I K. To provide the compound of Example I L. the compound corresponding to Example I N but having an acetamido group present in tiace of the amino srouo, the acetamido grouo is

hydrolyzed with DC1 in D2O, with heating, to provide the product of Example I L,

The above reaction conditions and reagents for the various steps above are as follows. When the temperature is "20°C or less" this is usually accomplished by use of an ice/water bath. "Heating" is done either at reflux or above room temperature. The preferred "caustic" is sodium hydroxide, but any suitable base the is able to maintain the desired pH without adverse effect on the product formed from the reaction is acceptable. A "suitable solvent" is inert and provides solubility for the reactants, examples of such solvents are water, and alcohols such as methanoi. The desired product may be seperated by any conventional methods, for example precipitation from a solvent such as acetone..

TABLE I

H -

COOK

ID

H,X

TABLE I CONT'D

TABLE I CONT'D

TABLE I CONT'D

OH

H. _. N

TABLE I CONT'D

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Another one of the possible classes of ligands useful in this process are aminop osphonic acid cheiants. Examples of some of the possible aminophosphonic acid cheiants are given in Table II following and which are named as follows:

• -~~-

II A is p-amminobenzyl ethylenediamine- tetramethylenephosphonic acid , the preparation of which is given below ;

10 II B is 6-( p-aminobenzyl ) - 1 , 4 , 8 , 1 1 - tetraazacyclotetradecane-1,4,8,11-tetramethylene¬ phosphonic acid, the preparation of which is given below; and

5 II C is 1-[2-(4-aminophenyl)ethyl]-1,4,7,10- tetraazacyclododecane-4,7,10-trimethylenephosphonic acid, the preparation of which is given below. -

Aminophosphonic acids can be prepared by a 0 number of known synthetic techniques. Of particular importance is the reaction of a compound containing at least one reactive amine hydrogen with a carbonyl compound (aldehyde or ketone) and phosphorous acid or derivative thereof. [See the procedure of Moeoritzer 5 and Irani, J. Org. Chem. 1~ 603 (1966).] For example, p-nitrobenzyi ethylenediamine reacted with formaldehyde and phosphorous acid can be converted to the p-nitrobenzyl ethylenediaminetetramethylene- phosphonic acid. Reduction of the nitro group would yield p-aminobenzyl ethylenediaminetetramethylene- ohosDhonic acid.

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TABLE II

A futher suitaoie class of ligands which may be _seα in the process of this invention are poiyaza cheiants. Examples of some of these poiyaza cheiants are given in Table III and are named as follows:

III A is 3-[(4-aminophenyi)methyI]-1 ,5.8, 12- tetraazacyclotetradecane, the preparation of which is given in European published Application 296,522. published December 28, T988;

III 3 is 6-[(4-aminophenyi)methyl]-1.4,8, 11- tetraazaundecane, the preparation of which is given in European published Application 296.522. published December 28, 1988:

III C is 1.4,7, 10-tetraaza-1-[(4-aminophenyl)- methyl]cyclododecane, the preparation of which is given in European published Application 296,522, published December 28, 1988; and

III D is 6-(3-aminopropyl)-1 ,4,7.11- tetraazaundecane. the preparation of which is given in European published Application 296,522, published December 23, 1988.

T BLE III

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The thiophosgene is added in excess to the mixture. The amount of excess used depends on the concentration of the starting amino fuctionalized chelate. The lower the concentration of chelate, the larger the excess of thiophosgene, to insure the rapid and complete conversion of amine to the isothiocyanate. For example, if the concentration of chelate is 10~3M, the ratio of thiophosgene to chelate is 5-20:1; if the concentration of chelate is 10~°M, the ratio of thiophosgene to chelate is several thousand times higher (i.e. 10^:1). The excess thiophosgene is removed by conventional techniques such as evaporation, cnro atrography or extraction.

The process is run in a polar solvent, especially water or polar organic solvents in which the complexes are soluble, for example ethanol, acetonitrile, dimethylformamide, tetrohydrofuran or dioxane. Mixtures of solvents such as water and a non- reactive solvent are especially preferred, such as, for example, water/acetonitrile, water/dimethyl orma ide, water/chloroform, water/tetrohydrofuran, water/methylene chloride, water/ethanol, water/dioxane and water/butanol. The solvent can be a single phase or two phase system, but it is desirable that the complex be in solution.

The pH of the reaction may be from 2 to 10, preferrabiy from 6 to 3. The pH stability of the complex may restrict the operable pH range. Some complexes, such as ethylenediamine-tetraacetic acid cnelates of lanthanides. are not very stable at pH 2. Additional base can oe used to maintain the pH in the desired range or conventional buffers can oe used.

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The time of reaction when carried out with excess thiophosgene is very fast and usually complete after 5 to 10 minutes at room temperature (15 to 25°C). Higher or lower temperatures can be used (e.g. 0 to 50°C) but room temperature is preferred. Ambient pressure is used although higher or lower pressures can be employed. Pressure is not a critical feature of the present process.

The yield for the process is at least 50percent by weight.

Although any metal, whether a radioactive metal or not. can be used which complexes with the amino fuctionalized chelant. The complexes formed should have reasonable stability such that the metal complex is not readily dissociated. Complexes with stability constants of , Q° should be suitable. The radionuclides are prefered because of the use of the resulting products in a pharmaceutical drug for therapy and/or diagnosis. Especially preferred radioactive isotopes are those of samarium (Sm-153), holmium (Ho-166), ytterbium (Yb-175), lutetium (Lu-177), gadolinium (Gd-159), yttrium (Y-90), rhodium (Rh-105), indium (In-111), and technecium (Tc- 99m) .

Preparation of Starting Materials

Some of the chemicals used were obtained commercially from various sources, such as thiophosgene was from Aldrich Chemicals.

The preparation of many of the starting naterials for this process can be found in the literature. 1-(4-aminobenzyi)diethylenetriamine- pentaacetic acid was prepared according to the procedure

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of M . W . 3rechbiel , et al . . Inorg. Chem. 25_, 2772-278 1 1986 ) -

The preparation of α-(4-aminobenzyl)-1 , , , 10- tetraazacyciododecane-1 ,4,7, 10-tetraacetic acid., samarium(III) complex, α-(4-aminobenzyl)-1. , , 10

-tetraazacyclododecane-1,4, , 10-tetraacetic acid, yttrium complex, and 1-[2-(4-aminophenyl)ethyl]-1 ,4,7, 10- tetraazacyciododecane-4,7, 10-triacetic acid are shown in

Saudi Arabian Patent 3277A, issued October 10, 1989.

Radionuclides can be produced in several ways.

In a nuclear reactor a nuclide is bombarded with neutrons to obtain a radionuclide, e.g.

Sm-152 + neutron •* ^■ 3m-153 + gamma

Another method of obtaining radionuclides is to bombard nuclides with particles produced by a linear accelerator or a cyclotron. Yet another way is to isolate the radionuclide from a mixture of fission products. The method of obtaining the nuclides employed in the present invention is not critical thereto.

The present process has been used to make valuable synthetic precursors for radioactive pharmaceuticals. ΪSee Saudi Arabian Patent 3277A, issued October 10, 1989, and European published Application 296,522, published December 28, 1988.]

In the following examples, the following ^' erms and conditions were used unless otherwise soecified.

Glossary

3A-2, 3 , 2-tet means 6-[( 4-aminophenyI )methyI]- 1 , 4.8 , 1 1 -tetraazaundecane ;

_ _/. ^" 3ITC-2 , 3 , 2-tet means 6-[(4-isothiocyanato- phenyi)methyl}-1 ,4,8,11-tetraazaundecane;

HEPES means ^-2-hydroxyethylpiperazine-N'-2- ethane sulfonic acid: and

"0 TLC means thin layer chromotography.

General Experimental

Mass spectra were obtained on a 7G A3-MS high 15 resolution mass spectrometer (fast atom bombardment with xenon, using 3:1 dithiothreitol:dithioerythritol) .

R - values are reported using these solvent systems and commercially available, normal phase, silica 20 TLC plates (GHLF 250 micron, Anaitek™ Inc.).

The following HPLC system was used for ^" analyses and sample separations:

_?c - System I consisted of LKB 2150 pump, and 2152 controller, a ϋV detector - LKB 2238 UV Cord, a Berthold™ L3 506 A HPLC Radioactivity Monitor (of the International Berthold Group) and a Gilson ^" Fraction collector 201-202 (Gilson ^*M International. Middleton,

30 I).

All percentages are by weight unless stated otherwise.

5amarium- ^' 53 was produced by the Reserarch Reactor, University of Missouri (Columbia, M0). It was

-21 -

suppiied as a solution of 0.2 to 0.3 miilimolar (mmole) concentration of samarium in 0.1N hydrochloric acid (HC1).

The invention will be further clarified by consideration of the following examples, which are intended to be purely exemplary of the use of this invention.

Process of the Invention

Example 1

Preparation of [ 1 ^ι 0 ^υ 5 ³ Rh(BITC-2,3.2-tet)Cl ₂ ] ⁺ .

[ ¹⁰⁵ Rh(BA-2,3,2-tet)Cl ₂ ] ⁺ was converted to the reactive [ ¹⁰⁵ Rh(BITC-2,3,2-tet)Cl ₂ ] ⁺ derivative by mixing 2 ml of the [ ¹⁰⁵ Rh(BA-2,3,2-tet)Cl ₂ ] ⁺ (approximately 5 Ci/ml, 1 X 10~ ⁴ M) with 0.002 ml of thiophosgene. The reaction was allowed to proceed 15 minutes at room temperature. The product was isolated by passing solution through a Hamilton PRP-1 Chrompak. The ^r . ¹⁰⁵ Rh(BITC-2,3,2-tet)Cl ₂ ] ⁺ was eluted with 2 mL of acetonitrile. The product was characterized by comparison to κnown standards using cation exchange and reverse phase chromatography. Using this procedure yields of between 50 to 85 percent were obtained.

Example 2

Preparation of [Rh(BITC-2,3.2-tet)C1 ] ⁺ . ^•

[Rh(BA-2,Ξ,2-tet)Cl ₂ ] ⁺ (10 mg) was dissolved in a mixture of 5 ml of pH 7 phosphate buffer (0.3M). 0.5 mi of acetonitrile. and 1 g of sodium chloride. The reaction mixture was stirred at room temperature (about 22°C) and 10μl of thiophosgene was added. After ^' 5

minutes the hazy mixture was cent ifuged, the yellow solid was washed with acetonitrile and centrifuged. The acetonitrile solution was stripped at reduced pressure to yield 3.1 mg of [Rh(BITC-2,3,2-tet)Cl ₂ ] ⁺ ; The aqueous phase of the mixture, after centrifugation, was loaded on to a Chrom-Prep column washed with saturated sodium chloride, then water and eluted with acetonitrile. The acetonitrile fraction was then concentrated at reduced pressure to yield 5.6 mg of the desired product, overall yield is 80 percent.

Example 3

■ Preparation of [ ¹⁰⁵ Rh(BITC-2,3,2-tet)Cl ₂ ] ⁺ .

Ten μl of freshly made thiophosgene solution

(10 μl of thiophosgene in 5 ml of 90percent acetonitrile) was added to 400 μl of a solution of [ ¹⁰⁵ Rh(BA-2,3 _> 2-tet)Cl ₂ ] ⁺ in 90 percent acetonitrile. The solution was mixed immediately and then allowed to stand at room temperature (about 22°C) for 20 minutes. The reaction mixture was then placed in a heating block (about 37°C). Excess unreacted thiophosgene as well as the solvent were evaporated by a gentle jet of N ₂ for one hour. The dry [ ¹⁰⁵ Rh(BITC-2,3,2-tet)Cl ₂ ] ⁺ , yield >95percent,is free from any unreacted thiophosgene.

Example 4

Preparation of α-(4-isothiocyanatobenzyl) -1,4,7,1O^tetraazacyclodoάecane-1,4,7,10-tetraacetic acid, samarium(III) complex.

A small sample, 7 mg, (10.8 μmole) of α-(ϋ -amlnobenzyI)-1,4,7, 10-tetraazacyclododecane-1 , ,7.10 -tetraacetic acid, samarium(III) complex was dissolved

in 400 μi water. Excess thiophosgene (50 μl) was added, followed by 00 μl CHCI ₃ and the two-phase reaction stirred vigorously for 30 minutes. At the end of this time, the water layer was extracted with 500 μl CHCI ₃ , four times, and the water layer then was lyophilized to give the desired titled product in quantitative yield.

The UV showed this compound to have a band at 272 and 282 nm. The TLC, silica gel developed by 75:25 V:V CH CN:H ₂ 0, gave R = 0.38. The starting material has an Rf = 0.19. The IR (KBr pellet) showed -SCN stretch at 2100 cm" ¹ ); fast atom bombardment mass spectrum [M+H] ⁺ = 687.

Example 5

Preparation of α-(4-isothiocyanatobenzyl) -1,4,7, 10-tetraazacyclododecane-1 ,4,7, 10-tetraacetic acid, sodium salt, yttrium(III) complex.

A small sample of the α-(4-aminobenzyl)-

1,4,7,10-tetraazacyclododecane-1 ,4,7, 10-tetraacetic acid, yttrium complex (10 mg, 17 μmole) was dissolved in 400 μl H2O. To this solution was added 64 μi thiophosgene (500 μmoie) and 400 μl CHCI ₃ and the resulting mixture stirred vigorously for 40 minutes. During this time several small additions of solid NaHCθ3 were made to keep the pH at about 8. At the end of the reaction, the water layer was separated and extracted with 1 ml of CHCI ₃ , four times, and lyophilized. ^' The title product was characterized by TLC and UV spectroscopy.

Sxample 6

Preparation of α-[2-(--isothiocyanatophenyD- ethyl]-1,4,7-l0-tetraazacyclododecane-1 ,4,7, 10- tetraacetic acid, samarium-153 complex.

To a solution of α-[2-(4-aminophenyl)-ethylj- 1,4,7,10-tetraazacyclododecane-1,4,7, 10-tetraacetic acid, samarium-153 complex prepared from 150 μl of ¹ "g _m solution in 0.1N HC1 (about 4.6 mCi) was added 2 μl of

10 HEPES buffer (0.5M, pH 8.9), 2 μl of thiophosgene and 200 μl of chloroform. The mixture was vortexed vigorously 2 or 3 times for a few seconds each time. The chloroform layer was discarded and the aqueous layer which contained mainly the desired product was saved and ^l5 - further purified. The yield of α-[2-(4- isothiocyanatophenyl)ethyl]-1,4,7-10-tetraazacyclo- dodecane-1,4,7, 10-tetraacetic acid, samarium-153 complex, based on '"g _m activity measurement by HPLC on

_?n GF-250 column using System I, was around 85 to 90 percent. To purify, the aqueous layer was passed through a Sep-Pak™ C-18 cartridge and eluted with 90 percent acetonitrile in water. The first 300 μl of effluent was discarded, and the SCN-derivative which

25 came off in the next 900 μl was characterized by HPLC on GF-250. The recovery of the ^"1 "sm activity was in general better than 90 percent. The bulk of the solvent was then evaporated off in a Speed Vac™ concentrator over a period of 1.5 to 2 hours.

30 αxamp e

Preparation of --(^-isothiocyanatophenyi)- ,-,7, lO-tetraazacycIcdodecane-l .4,7. "0-tetraacεtic ici≤, samarium-1 complex.

To a solution of α-(4-amiπopnenyl)-1 ,4.7, 10- tetraazacyclododecane-1 ,4,7, 10-tetraacetic acid, samarium-153 complex prepared from 220 μl of ^sm solution in 0.1N HC1 were added 2 μl of HEPES buffer (0.5M, pH 8.9), 2 μl of thiophosgen and 200 μl ^" of chloroform. It was vortexed vigorously 2 or 3 times for a few seconds each time. The chloroform layer was discarded and the aqueous layer which contained mainly the desired product was saved and further purified. The

10 yield of α-(4-isothiocyanatophenyl)-1 ,4,7, 10- tetraazacyclododecane-1 ,4,7, 10-tetraacetic acid, samarium-153 complex, as analyzed by HPLC on GF-250 column based on the '"sm activity using the HPLC System I was usually over 90 percent. To purify, the aqueous 15. layer was passed through a Sep-Pak ^™ C-18 cartridge and eluted with 90 percent acetonitrile in water. The first 300 μl of effluent was discarded, and the desired product came _. off in the next 1200 μl, with 86 to 93 percent recovery. The bulk of the solvent was then 0 evaporated off in a Speed Vac ^"M concentrator over a period of about 2 hours.

Other embodiments of the invention will be apparent to those skilled in the art from a 5 consideration of this specification or practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope and spirit of the invention being indicated by the following claims: