Title of Invention

CYSTEINE PROTEASE INHIBITORS

Abstract The invention discloses a compound of the formula II wherein R1 ,R2 ,R3 ,R4 and R6 are as defined in the specification. The invention is also for a pharmaceutical composition comprising said compound.
Full Text Field of the invention.
This invention relates to inhibitors of cysteine proteases, especially those of the papain
superfamily. The invention provides novel compounds useful in the prophylaxis or
treatment of disorders stemming from misbalance of physiological proteases such as
cathepsin K.
Description of the related art.
The papain superfamily of cysteine proteases is widely distributed in diverse species
including mammals, invertebrates, protozoa, plants and bacteria. A number of
mammalian cathepsin enzymes, including cathepsins B, F, H, K, L, O and S, have been
ascribed to this superfamily, and Inappropriate regulation of their activity has been
implicated in a number of metabolic disorders including arthritis, muscular dystrophy,
inflammation, glomerulonephritis and tumour invasion. Pathogenic cathepsin like
enzymes include the bacterial gingipains, the malarial falcipains I, II, III et seq and
cysteine proteases from Pneumocystis carinii, Trypanosoma cruzei and brucei, Crithidia
fusiculata, Schistosoma spp.
The inappropriate regulation of cathepsin K has been implicated in a number of
disorders including osteoporosis, gingival diseases such as gingivitis and periodontitis,
Paget's disease, hypercalcaemia of malignancy and metabolic bone disease. In view of
its elevated levels in chondroclasts of osteoarthritis synovium, cathepsin K is implicated
in diseases characterised by excessive cartilege or matrix degradation, such as
osteoarthritis and rheumatoid arthritis. Metastatic neoplastic cells typically express high
levels of proteolytic enzymes that degrade the surrounding matrix and Inhibition of
cathepsin K may thus assist in treating neoplasias.
International patent application no WO02057270 discloses compounds of the formula I


where UVWXY broadly corresponds to the P3 and P2 of dipeptide cysteine protease
inhibitors, Z is inter alia O, S, methylene or -NR-, R1 is alkyl, alkylaryl etc and P1 and Q1
are each methylene, optionally substituted with various carbon chains and cyclic
groups. The compounds are alleged to be useful for the treatment of protozoal
infections such as trypanosomes.
We have now discovered that Introduction of a halogen atom at a particular ring position
produces an order of magnitude increase in potency against cathepsin K.
Brief description of the invention
In accordance with the invention, there is provided compounds of the formula II:

wherein
one of R1 and R2 is halo and the other is H or halo;
R3 is C1-C5 straight or branched chain, optionally fluorinated, alkyl;
R4 is H; or
R3 together with R4 and the adjacent backbone carbon atom defines
a spiro-C5-C7 cycloalkyl, optionally substituted with 1 to 3 substituents selected
from halo, hydroxyl, C1-C4 alkyl or C1-C4 haloalkyl; or optionally bridged with a
methylene group; or
a C4-C6 saturated heterocycle having a hetero atom selected from
O, NRa, S, S(=O)2; where Ra is H, C1-C4 alkyl or CH3C(=O)-;
R5 is independently selected from H or methyl;
E is -C(=O)-, -S(=O)m-, -NR5S(=O)m-, -NR5C(=O)-, -0C(=O)-;
R6 is a stable, optionally substituted, monocyclic or bicyclic, carbocycle or hetorocycle
wherein the or each ring has 4, 5 or 6 ring atoms and 0 to 3 hetero atoms selected from

S, O and N and wherein the optional substituents comprise 1 to 3 members selected
from R7;
R7 is independently selected from halo, oxo, nitrile, nitro, C1-C4 alkyl, -NRaRb, -
XNRbR9, -NRbC1-C4alkylR9, NH2CO-, X-R9, X-O-R9, O-X-R9, X-C(=O)R9, X-
(C=O)NRaR9, X-NRbC(=O)R9, X-NHSOmR9, X-S(=O)mR9, X-C(=O)OR9, X-
NRbC(=O)OR9;
R9 is independently H, C1-C4 alkyl, C3-C6 cycloalkyl, pyrrolidinyl, piperidinyl, morpholinyl,
thiomorpholinyl, piperazinyl, indolinyl, pyranyl, thiopyranyl, furanyl, thienyl, pyrrolyl,
oxazolyl, isoxazolyl, thiazolyl, imidazolyl, pyridinyl, pyrimidinyl, pyrazinyl, indolyl, phenyl,
any of which is optionally substituted with R10;
R10 is independently selected from hydroxy, -X-R9, -XNRaRb, -XNRbR9, -NRbC1-
C4alkylR9, nitro, cyano, carboxy, oxo, C1-C4 alkyl, C1-C4-alkoxy, C1-C4 alkanoyl,
carbamoyl;
X is independently a bond or C1-C4 alkyl;
Rb is selected from H, C1-C4 alkyl
m is independently 0,1 or 2;
and pharmaceutically acceptable salts thereof.
Without in any way wishing to be bound by theory, or the ascription of tentative binding
modes for specific variables, P1, P2 and P3 as used herein are provided for
convenience only and have their conventional meanings and denote those portions of
the inhibitor believed to fill the S1, S2 and S3 subsites respectively of the enzyme,
where S1 is adjacent the cleavage site and S3 remote from the cleavage site.
Preferably the stereochemistry of the P1 group is as depicted in the partial structure
below:

Preferably the halogen of R1 and/or R2 is chlorine and most preferably fluorine. It is
currently preferred that R2 is halo, especially fluorine and R1 is H, but the invention

extends to compounds wherein R1 is halo, especially F and R2 is H or R1 and R2 are
eachF.
It will be appreciated that the P1 group may exist in alternative forms, such as

and the invention extends to all such alternative forms.
Preferably the stereochemistry of the P2 group corresponds to an L-amino acid as
depicted in the partial structure below:

but the invention also extends to D-isomers.
The invention also includes all isomers and enantiomers at other chiral centres.
Currently preferred P2 groups include those wherein R4 is H and wherein R3 is iso-butyl.
A further preferred P2 group is homo-t-butyl, that is -CH2C(CH3)3
Alternative preferred P2 groups included those wherein R3 and R4 together define
spirocycloalkyl, such as cyclopentyl, cycloheptyl and especially cyclohexyl.
If a P2 cycloalkyl is substituted, the substitution is typically para to the linkage to the
backbone. Representative substituents include monofluoro, difluoro, monohydroxy,
geminal hydroxyl & methyl substituents, monomethyl or geminal methyl.
Alternative P2 groups include those wherein R3 and R4 together

define a 6 membered, saturated heterocycle, wherein a hetero atom selected from O, S,
S(=O)2 or NRx where X is H or methyl, situated at the position corresponding to para or
meta to the point of attachment to the backbone.
Representative P2 groups in accordance with the two paragraphs immendiateiy above
include



It is currently preferred that R5 is H.
Preferred E groups include -S(=O)m- especially -S(=O)2-, and most preferably -C(=O)-.
Typically Re is a monocyclic ring with 5 or especially 6 ring atoms, or a bicyclic ring
structure comprising a 6 membered ring fused to a 4,5 or 6 membered ring.
Typical R6 groups include saturated or unsaturated heterocycles or saturated or
unsaturated carbocycles, any of which are optionally substituted as described above.
Illustrative variants include C3-8 cycloalkyl, phenyl, benzyl, tetrahydronaphthyl, indenyl,
indanyl, heterocyciyl such as from azepanyl, azocanyl, pyrrolidinyl, piperidinyl,
morpholinyl, thiomorpholinyl, piperazinyl, indolinyl, pyranyl, tetrahydropyranyl,
tetrahydrothlopyranyl, thiopyranyl, furanyl, tetrahydrofuranyl, thienyl, pyrrolyl, oxazolyl,
isoxazolyl, thiazolyl, imidazotyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, tetrazolyl,
pyrazolyl, indolyl, benzofuranyl, benzothienyl, benzimidazolyl, benzthiazolyl,
benzoxazolyl, benzisoxazolyl, quinolinyl, tetrahydroquinolinyl, isoquinolinyl,
tetrahydroisoquinolinyl, quinazolinyl, tetrahydroqulnazolinyl and quinoxalinyl, any of
which may be substituted as described above.
The saturated heterocycle thus includes radicals such as pyrrolinyl, pyrrolidinyl,
pyrazolinyl, pyrazolidinyl, piperidinyl, morpholinyl, thiomorpholinyl, pyranyl, thiopyranyl,
piperazinyl, indolinyl, azetidinyl, tetrahydropyranyl, tetrahydrothiopyranyl,
tetrahydrofuranyl, hexahydropyrimidinyl, hexahydropyridazinyl, 1,4,5,6-
tetrahydropyrimidinylamine, dihydro-oxazolyl, 1,2-thiazinanyl-1,1-dioxide, 1,2,6-
thladlazinanyl-1,1-dioxide, isothiazolidinyl-1,1 -dioxide and imidazolidinyl-2,4-dione,
whereas the unsaturated heterocycle include radicals such as furanyl, thienyl, pyrrolyl,
oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl,
tetrazolyl, thiadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, indolyl,

isoindolyl. In each case the heterocycle may be condensed with a phenyl ring to form a
bicyclic ring system.
Preferred monocyclic R6 groups include substituted pyridyl, substituted pyrimidyl,
substituted phenyl, particularly phenyl substituted with a cyclic group such as
pyrrolidine-1-yl, piperidine-1-yl, 4-methylpiperidin-1-yl, 4-(piperidirv-3-ylmethyl)-piperidin-
1-yl, morpholin-4-yl, 4-methylpiperazin-1-yl, 2-morpholin-4-yl-ethylamino, 2-morpholin-
4-yl-ethyloxy, l-pyrid-2-ylmethylamino, piperazin-1-yl, piperid-4-yl or N-piperazinyl, N-
substituted with Ra or piperidin-1-yl which is 4-substituted with -NRaRb. A phenyl R6 is
conveniently substituted at the 3 or 4 position (para or meta), for example with such a
cyclic group.
Alternative cyclic substituents to a monocyclic R8 (such as phenyl) include aryl groups
such as phenyl or a 5 or 6 membered heteroaryl group such as thiophene, furyl,
triazole, thiazole, diazole, pyrazole or pyrrolidine. Favoured cyclic substituents In this
context include thiazol-2-yl, pyrid-3-yl and especially pyrid-2-yl, thien-2-yl orthiazol-5-yl.
This cyclic substituent (ie R7) is typically bonded direct to such R8 species (ie X is a
bond), but may also for example comprise an amine spacer such as -NH-, -N(Me), -
CH2NH, -CH2N(Me)-, a C1-C3alkyl spacer such as -CH2- or a C1-C3-alkyloxy spacer
such as ethyloxy
Any of the cyclic substituents to R6 in the immediately preceding paragraph may be
substituted as described above with R10. For example a heterocycle R7 group such as
thiazolyl can be substituted with C1-C4 alkyl such as methyl.
Preferably, any of the cyclic substituents to R6 in the two immediately preceding
paragraphs may itself be substituted with a cyclic group (that is R7 comprises an R9
moiety) typically a saturated heterocyclic group such as piperidine, piperazine or
morphoiine, which saturated cyclic group is optionally substituted, for example with C1-
C3 alkyl, fluoro, difiouro, C1-C3alkyloxy or C1-C3alkyloxyC1-C3alkyl. As provided In the
definition of R7, this saturated cyclic group (ie R9) may be spaced from the R6 group by
X (eg C1-C3alkyl), amine (eg -NH-), amide, sulphonamide etc, but is typically bonded
directly or via methylene.

Representative R8 groups in accordance with the immediately preceding paragraph
include heterocycles such as pyrrolidine-1-yl, piperidine-1-yl, 4-methylpiperidin-l-yl, 4-
(piperidin-3-ylmethyl)-piperidin-1-yl, morpholin-4-yl, 4-methylpiperazin-1-yl, 2-
morpholin-4-yl-ethylamino, 2-morpholin-4-yl-ethyloxy, 1-pyrid-2-ylmethylamino,
piperazin-1-yl, piperid-4-yl or N-piperazinyl, N-substituted with Ra or piperidin-1-yl which
is 4-substituted with -NRaRb,
Currently preferred R9 substituents include 4-substituted piperazin-4-yl, such as 4-
methyl-piperazin-4-yl or 4-methyloxyethyl-piperazin-4-yl, piperid-1-ylmethyl which is
optionally 4-substituted with fluoro or diflouro or morpholinylmethyl.
Alternative preferred substituents to a monocyclic R8 (such as phenyl) include -NRaRb,
-CH2NRaRb, C1-C4 straight or branched alkyl or -O-R9.

where Rq and Rq' are independently selected from H, C1-C4 alkyl or C1-C4alkanoyl or
together define an unsaturated 5-7 membered ring, such as piperidine, piperazine or

morphdine, which may in turn be substituted with groups corresponding to R10,
particularly C1-C4 alkyl, fluoro or difluoro.

Representative bicyclio-groups for R6 include naphthylenyl, especially naphthylen-2-yl;
benzo[1,3]dioxolyl, especially benzo[1,3]dioxol-5-yl,
benzofuranyl, especially benzofuran-2-yl, and especially C1-C6 alkoxy substituted
benzofuranyl, more especially 5-(2-piperazin-4-carboxylic acid tert-butyl ester- ethoxy)
benzofuran-2-yl,6-(2-morpholino-4-yl-ethoxy)-benzofuran-2-yl, 5-(2-plperazln-1-yl-
ethoxy)benzofuran-2-yl, 5-(2-cyclohexyl-ethoxy)-benzofuran-2-yl;
7-methoxy-benzofuran-2-yl, 5-methoxy-benzofuran-2-yl, 5,6-dimethoxy-benzofuran-2-yl,
especially halogen substituted benzofuranyl, more especially 5-fluoro-benzofuran-2-yl,
5,6-difluoro-benzofuran-2-yl, especially C1-C6alkyl substituted benzofuranyl, most
especially 3-methyl-benzofuran-2-yl; benzo[b]thiophenyl, especially
benzo[blthiophen-2-yl; especially C1-C6alkoxy substituted benzo[b]thiopheny], more
especially 5,6-dimethoxy- benzo[b]thiophen-2-yl, quinolinyl, especially quinolin-2-yl,
qulnolin-3-yl, quinolin-4-yl, quinolin-6-yl, and quinolin-S-yl; quinoxalinyl, especially
quinoxalin-2-yl; 1,8-naphthyridinyl, especially 1,8- naphthyridin-2-yl; indolyl, especially
lndol-2-yl, especially indol-6-yl, indol-5-yl, especially C1-C6alkyl substituted indolyl, more

especially N-methyllndol-2-yl; furop^-blpyridinyl, especially furo[3,2-b]pyriclin-2-yl, and
Q-Co-alkyl substituted furo[3,2-b]pyridinyl, especially 3-methyl-furo[3,2-blpyridin-2-yl;
thieno[3,2-b]thiophene, especially thieno[3,2-b]thiophene-2-yl, mora especially d-
Cealkyl substituted thleno[3,2-b]thiophene-2-yl, more especially
S-tert-buiyl-a-methylthienoIS^-blthiophene^-yl.
Favoured R6 groups include bicyclic rings such as napthyl, quinoloyt, benzofuranyl,
benzothienyl, Indolyl and indolinyl, particularly where the linkage is to the 2 position of
the ring. Favoured substituents to a bicyclic R6 group include pyrrolldine-1-yl,
piperidine-1-yl, 4-methylpiperidin-1-yl, 4-(piperidin-3-ylmethyl)-piperidin-1-yl, morpholin-
4-yl, 4-methylpiperazin-1-yl, 2-morpholin-4-yl-ethylamino, 2-morpholin-4-yl-ethyloxy, 1-
pyrid-2-ylmethylamino, piperazin-1-yl, piperid-4-yl or N-piperazinyl, N-substituted with
Ra or piperidin-1-yi which is 4-substituted with -NRaRb. Especially preferred
substituents, particularly in conjunction with benzofuranyl include 2-morpholin-4-yl-
ethyloxy and N-methyl-piperidin-4-yloxy and those defined below.
A currently favoured bicyclic R6 group is optionally substituted benzothlazol or
benzofuryl or benzoxazolyl, including those wherein the substituent is -OR9 or-NRbR9.
For example, favoured R6 groups include benzofur-2-yl, unsubstituted or substituted in
the 5 position with a saturated heterocycle such as piperidine, piperazlne or morpholine,
which is optionally substituted with C1-C3 alkyl and/or spaced from the benzofuryl by
oxy, methyloxy orethyloxy. Particularly favoured benzofuryl R6 groups thus include:

Returning to formula II in general:
X is typically methylene or especially a bond.

Cy-Cn alkyl, where n is 4, on its own or within compound expressions such as C1-C4
alkoxy, includes methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, t-butyl,
cyclopropyl, methylcyclopropyl and the like, extended in a likewise fashion for other
values of n. For example C5 alkyl includes homo-t-butyl (-CH^C{CHz)z).
Halogen or halo includes bromo, chloro and especially fiuoro.
Haloalkyl means an alkyl group as defined above where at least one carbon atom bears
1 to 3 halogen atoms, preferably fluorine atoms. Representative haloalkyl group
includes fluoromethyl, difluoromethyl, trifluoromethyl, 2, fluoroethyl, 2,2difluorethyl, 2,2,2
trifluorethyl and the like.
The P1 building block employed in the present invention represent novel compounds
and forms an additional aspect of the invention. Accordingly, this further aspect of the
invention provides compounds of the formula

where R1 and R2 are as defined above and PG is a nitrogen protecting group as
defined below, especially formyl, acetyl, benzoyl, pivaloyl, t-butylacetyl, phenylsuffbnyl,
benzyl, Fmoc, t-butoxycarbonyl (BOC) and benzyloxycarbonyl (Cbz). This aspect of the
invention further includes the correspdonding unprotected amines (ie PG=H).
The preferred embodiment of this aspect of the invention comprises compounds of the
formula

where PG is as described above.

The invention further embraces various novel P3 building blocks as illustrated in the
examples below, as the acid or protected with a carboxy protecting group.
Favoured compounds of the invention include those permutations formed by
independent selection of a P3, P2 and P1 member from each of Tables A, B and C:







Additional aspects of the invention include a pharmaceutical composition comprising a
compound as defined above and a pharmaceutically acceptable carrier or diluent
therefor.
A further aspect of the invention is the use of a compound as defined above in the
manufacture of a medicament for the treatment of disorders mediated by cathepsin K,
such as:
osteoporosis,
gingival diseases such as gingivitis and periodontitis,
Paget's disease,
hypercalcaemla of malignancy
metabolic bone disease
diseases characterised by excessive cartilege or matrix degradation, such as
osteoarthritis and rheumatoid arthritis,
bone cancers including neoplasia,
pain.
The compounds of the invention can form salts which form an additional aspect of the
invention. Appropriate pharmaceutically acceptable salts of the compounds of Formula
II include salts of organic acids, especially carboxylic acids, including but not limited to
acetate, trifluoroacetate, lactate, gluconate, citrate, tartrate, maleate, malate,
pantothenate, isethionate, adipate, alginate, aspartate, benzoate, butyrate, digluconate,
cyclopentanate, glucoheptanate, glycerophosphate, oxalate, heptanoate, hexanoate,
fumarate, nicotinate, palmoate, pectinate, 3-phenylpropionate, picrate, pivalate,
proprionate, tartrate, lactobionate, pivolate, camphorate, undecanoate and succinate,
organic sulphonic acids such as methanesulphonate, ethanesulphonate,
2-hydroxyethane sulphonate, camphorsulphonate, 2-napthalenesulphonate,

benzenesulphonate, p-chlorobenzenesulphonate and p-toluenesulphonate; and
inorganic acids such as hydrochloride, hydrobromide, hydroiodide, sulphate,
bisulphate, hemisulphate, thiocyanate, persulphate, phosphoric and sulphonic acids.
The compounds of Formula li may in some cases be isolated as the hydrate.
(twill be appreciated that the invention extends to prodrugs, solvates, complexes and
other forms releasing a compound of formula II in vivo.
While H is possible for the active agent to be administered alone, it is preferable to
present it as part of a pharmaceutical formulation. Such a formulation will comprise the
above defined active agent together with one or more acceptable carriers/excipients
and optionally other therapeutic ingredients. The carriers) must be acceptable in the
sense of being compatible with the other ingredients of the formulation and not
deleterious to the recipient.
The formulations include those suitable for rectal, nasal, topical (including buccal and
sublingual), vaginal or parenteral (including subcutaneous, intramuscular, Intravenous
and intradermal) administration, but preferably the formulation is an orally administered
formulation. The formulations may conveniently be presented in unit dosage form, e.g.
tablets and sustained release capsules, and may be prepared by any methods well
known in the art of pharmacy.
Such methods include the step of bringing into association the above defined active
agent with the carrier. In general, the formulations are prepared by uniformly and
intimately bringing into association the active agent with liquid carriers or finely divided
solid carriers or both, and then if necessary shaping the product The invention extends
to methods for preparing a pharmaceutical composition comprising bringing a
compound of Formula II or its pharmaceutically acceptable salt in conjunction or
association with a pharmaceutically acceptable carrier or vehicle. If the manufacture of
pharmaceutical formulations involves intimate mixing of pharmaceutical excipients and
the active ingredient in salt form, then it is often preferred to use excipients which are
non-basic In nature, i.e. either acidic or neutral.

Formulations for oral administration in the present invention may be presented as
discrete units such as capsules, cachets or tablets each containing a predetermined
amount of the active agent; as a powder or granules; as a solution or a suspension of
the active agent in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid
emulsion or a water in oil liquid emulsion and as a bolus etc.
With regard to compositions for oral administration (e.g. tablets and capsules), the term
suitable carrier includes vehicles such as common excipients e.g. binding agents, for
example syrup, acacia, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone (Povidone),
methylcellulose, ethylcellulose, sodium carboxymethylcellulose,
hydroxypropylmethylcellulose, sucrose and starch; fillers and carriers, for example com
starch, gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium
phosphate, sodium chloride and alginic acid; and lubricants such as magnesium
stearate, sodium stearate and other metallic stearates, glycerol stearate stearic acid,
silicone fluid, talc waxes, oils and colloidal silica. Flavouring agents such as peppermint,
oil of wintergreen, cherry flavouring or the like can also be used. It may be desirable to
add a colouring agent to make the dosage form readily identifiable. Tablets may also be
coated by methods well known in the art.
A tablet may be made by compression or moulding, optionally with one or more
accessory ingredients. Compressed tablets may be prepared by compressing in a
suitable machine the active agent in a free flowing form such as a powder or granules,
optionally mixed with a binder, lubricant, inert diluent, preservative, surface-active or
dispersing agent Moulded tablets may be made by moulding in a suitable machine a
mixture of the powdered compound moistened with an inert liquid diluent The tablets
may be optionally be coated or scored and may be formulated so as to provide slow or
controlled release of the active agent.
Other formulations suitable for oral administration include lozenges comprising the
active agent in a flavoured base, usually sucrose and acacia or tragacanth; pastilles
comprising the active agent in an inert base such as gelatin and glycerin, or sucrose
and acacia; and mouthwashes comprising the active agent in a suitable liquid carrier.

The appropriate dosage for the compounds or formulations of the invention will depend
upon the indication and the patient and is readily determined by conventional animal
trials. Dosages providing intracellular (for inhibition of physiological proteases of the
papain superamily) concentrations of the order 0.01-100 \M, more preferably 0.01-10
\iM, such as 0.1-25uJM are typically desirable and achievable.
Compounds of the invention are prepared by a variety of solution and solid phase
chemistries.
The compounds are typically prepared as building blocks reflecting the P1, P2 and P3
moieties of the end product inhibitor. Without in any way wishing to be bound by theory,
or the ascription of tentative binding modes for specific variables, the notional concepts
P1, P2 and P3 as used herein are provided for convenience only and have substantially
their conventional Schlecter & Berger meanings and denote those portions of the
inhibitor believed to fill the S1, S2, and S3 subsites respectively of the enzyme, where
S1 is adjacent the cleavage site and S3 remote from the cleavage site. Compounds
defined by Formula I are intended to be within the scope of the invention, regardless of
binding mode.
Broadly speaking the P1 building block will be an N-protected- 6-fluoro-3-oxo-
hexahydro-furo[3,2-b]pyrrole, P2 will be an N-protected amino acid, whereas P3
typically comprises a capping group such as a substituted, heteroaroyl or aroyl moiety.
The suitably protected Individual building blocks can first be prepared and subsequently
coupled together i.e. P2+P1-> P2-P1. Alternatively, precursors of the building blocks
can be coupled together and modified at a later stage of the synthesis of the inhibitor
sequence. Further building blocks, precursors of building blocks or prefabricated bigger
fragments of the desired structure, can then be coupled to the growing chain, e.g. R3-E-
P2*+ P1-> R3-E-P2-P1 or R3-E*+P2-P1 -> R3-E-P2-P1, where * denotes an activated
form.
Coupling between two amino acids, an amino acid and a peptide, or two peptide
fragments can be carried out using standard coupling procedures such as the azide

method, mixed carbonic-carboxylic acid anhydride (isobutyl chloroformate) method,
carixxJiimide (dicyclohexylcarbodiimide, diisopropylcarbodiimide, or water-soluble
carbodiimide) method, active ester (pnitrophenyl ester, N-hydroxysuccinic imido ester)
method, Woodward reagent K-method, carbonyldiimidazole method, phosphorus
reagents or oxidation-reduction methods. Some of these methods (especially the
carbodiimide method) can be enhanced by adding 1-hydroxybenzotriazole or 4-DMAP.
These coupling reactions can be performed in either solution (liquid phase) or solid
phase.
More explicitly, the coupling step involves the dehydrative coupling of a free carboxyl of
one reactant with the free amino group of the other reactant in the present of a coupling
agent to form a linking amide bond. Descriptions of such coupling agents are found in
general textbooks on peptide chemistry, for example, M. Bodanszky, "Peptide
Chemistry", 2nd rev ed., Springer-Verlag, Berlin, Germany, (1993) hereafter simply
referred to as Bodanszky, the contents of which are hereby incorporated by reference.
Examples of suitable coupling agents are N,N'-dicyclohexylcarbodiimide, 1-
hydroxybenzotriazole in the presence of N,N'- dicyclohexylcarbodiimide or N-ethyl-hT- [
(3dimethylamino) propyl] carbodiimide. A practical and useful coupling agent is the
commercially available (benzotriazol-1-yloxy) tris- (dimethylamino) phosphonium
hexafluorophosphate, either by itself or in the present of 1 -hydroxybenzotriazole or 4-
DMAP. Another practical and useful coupling agent is commercially available 2-(IH-
benzotrlazol-1-yl)-N, N, N'.N'- tetramethyluronium tetrafluoroborate. Still another
practical and useful coupling agent is commercially available 0-(7-azabenzotrizol-1-yl)-
N, N,N\ N'-tetramethyluronium hexafluorophosphate.
The coupling reaction is conducted in an inert solvent, e. g. dichloromethane,
acetonitrile or dimethylformamide. An excess of a tertiary amine, e. g.
dilsopropylethylamine, N-methylmorpholine, N-methylpyrrolidine or 4-DMAP is added to
maintain the reaction mixture at a pH of about 8. The reaction temperature usually
ranges between 0 °C and 50 °C and the reaction time usually ranges between 15 min
and 24 h.
The functional groups of the constituent non-natural amino acids generally must be
protected during the coupling reactions to avoid formation of undesired bonds. The

protecting groups that can be used are listed in Greene, "Protective Groups in Organic
Chemistry", John Wiley & Sons, New York (1981) and The Peptides: Analysis,
Synthesis, Biology", Vol. 3, Academic Press, New York (1981), hereafter referred to
simply as Greene, the disclosures of which are hereby incorporated by reference.
The alpha-carboxyl group of the C-terminal residue is usually protected as an ester that
can be cleaved to give the carboxylic acid. Protecting groups that can be used include
1) alkyl esters such as methyl, trimethylsilyl and tbutyl, 2) araikyl esters such as benzyl
and substituted benzyl, or 3) esters that can be cleaved by mild base or mild reductive
means such as trichloroethyl and phenacyl esters.
The alpha-amino group of each amino acid to be coupled is typically N- protected. Any
protecting group known in the art can be used. Examples of such groups include: 1)
acyl groups such as formyl, trifluoroacetyl, phthalyl, and p-toluenesulfonyl; 2) aromatic
carbamate groups such as benzyloxycarbonyl (Cbz or Z) and substituted
bensyloxycarbonyls, and 9-fluorenyImethyloxycarbonyl (Fmoc); 3) aliphatic carbamate
groups such as tertbutyloxycarbonyl (Boc), ethoxycarbonyl,
diisopropylmethoxycarbonyl, and allyloxycarbonyl; 4) cyclic alkyl carbamate groups
such as cyclopentyloxycarbonyl and adamantyloxycarbonyl; 5) alkyl groups such as
triphenylmethyl and benzyl; 6) trialkylsilyl such as trimethylsilyl; and 7) thiol containing
groups such asphenylthiocarbonyl anddithiasuccinoyl. The preferred alpha-amino
protecting group is either Boc or Fmoc. Many amino acid derivatives suitably protected
for peptide synthesis are commercially available.
The alpha-amino protecting group is typically cleaved prior to the next coupling step.
When the Boc group is used, the methods of choice are trifluoroacetic acid, neat or in
dichloromethane, or HCI in dioxane or in ethyl acetate. The resulting ammonium salt is
then neutralized either prior to the coupling or in situ with basic solutions such as
aqueous buffers, or tertiary amines in dichloromethane or acetonitrile or
dimethylforrnarnide. When the Fmoc group is used, the reagents of choice are
plperidine or substituted piperidine in dimethylforrnarnide, but any secondary amine can
be used. The deprotection is carried out at a temperature between 0 °C and room
temperature usually 20-22 °C.

Any of the natural or non-natural amino acids having side chain functionalities will
typically be protected during the preparation of the peptide using any of the above
described groups. Those skilled in the art will appreciate that the selection and use of
appropriate protecting groups for these side chain functionalities depend upon the
amino acid and presence of other protecting groups in the peptide. In the selection of
such protecting groups it is desirable that the group is not removed during the
deprotection and coupling of the alpha-amino group.
For example, when Boc is used as the alpha-amino protecting group, the following side
chain protecting groups are suitable: p-toluenesulfonyl (tosyl) moieties can be used to
protect the amino side chain of amino acids such as Lys and Arg; acetamidomethyl,
benzyl (Bn), or tert-butylsulfonyl moities can be used to protect the sulfide containing
side chain of cysteine; benzyl (Bn) ethers can be used to protect the hydroxy containing
side chains of serine, threonine or hydroxyproline; and benzyl esters can be used to
protect the carboxy containing side chains of aspartic acid and glutamic acid.
When Fmoc is chosen for the alpha-amine protection, usually tert. butyl based
protecting groups are acceptable. For instance, Boc can be used for lysine and arginine,
tertbutyl ether for serine, threonine and hydroxyproline, and tert-butyl ester for aspartic
acid and glutamic acid. Triphenylmethyl (Trityl) moiety can be used to protect the sulfide
containing side chain of cysteine.
Once the inhibitor sequence is completed any protecting groups are removed in
whatever manner is dictated by the choice of protecting groups. These procedures are
well known to those skilled in the art.
The first stage in a synthesis of compounds of the general formula II is typically the
preparation In solution of a functionalized P1 building block. Different nomenclature of
compounds according to the present invention can be used. For convenience the
carbohydrate nomenclature will generally be used herein. A typical scheme towards a
bicyclfc P1 group starts with the ring closure of a suitably protected intermediate which
is available in 4 steps from 1,2:5,6-di-0-isopropylidene-D-allofuranose, described by
Mayer zum Reckendorf, Chem. Ber. 101 (1968), 3802-3807, giving a precursor of 3S,
4R stereochemistry.


In Scheme 1 the azide group of derivative 1 is reduced for example by catalytic
hydrogenation using palladium on charcoal or other catalysts suitable, In a suitable
solvent such as an alcohol, like ethanol or methanol into the free amine. The obtained
nucleophilic nitrogen reacts spontaneously, or optionally in the presence of a suitable
base like such as triethyl amine or sodium acetate, with the C-6 position forming a 5,5-
bicycle. The leaving group at C-6 is not limited to sulfonate esters, but also other leaving
groups such as halogen could be used throughout the synthesis of compounds
according to the present invention. The reduction of the azide residue into an amine
could also be performed by other methods known from literature, such as treating the
azide derivative with a trialkyl- or triarylphosphine followed by hydrolysis of the formed
imine derivative. After the ring closure the amine may be N-protected with a suitable
protecting group such as a carbamate, like benzyl carbamate of compound 3 or any
other similar protecting group which is normally not cleaved with acid. Suitable
protecting groups which can be found in: Protective groups in organic chemistry, 3rt
edition, 1999, Theodora W. Greene and Peter G. M. Wuts (Wiley&sons).
For a 3R, 4S bicycle a similar approach could be used starting from 3-azido-3-deoxy-
1,2:5,6-di-0-isopropylidene-D-gulofuranose which can be prepared as described in
Tetrahedron Asymmetry, 10 (1999) 1855-1859. This intermediate can then be treated
as described in Scheme 2.


Compound 4 can be treated with a mild acid, such as diluted acetic acid or similar,
which can selectively hydrolyze the 5,6-acetal of compound 4, to obtain a diol. The
primary alcohol can be selectively reacted with an alkyl- or arylsulfonyl chloride like p-
toluenesulfonyl chloride to give compound 5. The azide group of derivative 5 is reduced
for example by catalytic hydrogenation using palladium on charcoal or other catalysts
suitable, in a suitable solvent such as an alcohol, like ethanol or methanol into the free
amine. The obtained nucleophilic nitrogen reacts spontaneously, or optionally in the
presence of a suitable base like such as triethyl amine or sodium acetate, with the C-6
position forming a 5,5-bicycIe which can be N-protected with a suitable protecting group
such as its benzyl carbamate (Cbz) to give compound 6.
Alternatively 3-azido-3-deoxy-1,2:5,6-di-0-isopropylidene-D-idofuranose (Bull. Chem.
Soc. Japan, 57, f(1984), 237-241) could be a suitable starting material for the 3R, 4S
bicycle according to Scheme 3.


Compound 6 can be treated with a mild acid, such as diluted acetic acid or similar,
which can selectively hydrolyze the 5,6-acetal of compound 6, to obtain a diol. The
primary alcohol can be selectively reacted with an alkyl- or arylsulfonyl chloride like p-
toluenesulfonyl chloride to give compound 7. The azide group of derivative 7 is reduced
for example by catalytic hydrogenation using palladium on charcoal or other catalysts
suitable, in a suitable solvent such as an alcohol, like ethanol or methanol into the free
amine. The obtained nucleophilic nitrogen reacts spontaneously, or optionally in the
presence of a suitable base like such as triethyl amine or sodium acetate, with the C-6
position forming a 5,5-bicycle which can be N-protected with a suitable protecting group
such as its benzyl carbamate (Cbz) to give compound 8.
The ring closure is not limited to the substrates shown above but could also be applied
to derivatives as depicted in Scheme 4.


Rx in Scheme 4 may be chosen from methyl, trifluoromethyl, p-methylphenyl or similar
residues present in readily available alkylsulfonylhalides, preferably a bulky Rx suitable
for regioselective reaction on the primary alcohol of a diol as described in Chem. Ber.
101 (1968), 3802-3807. R1' and R2" are R1 and R2 as defined. Pg could be a suitable
protecting group such as a carbamate, like benzyl carbamate or any similar protecting
group which is not normally cleaved with acid.
Further substrates for the ring closure reaction could be compounds depicted in
Scheme 5. .


Rx in Scheme 5 can be chosen from methyl, trifluoromethyl, p-methylphenyl or similar
residues present in readily available alkylsulfonylhalides, preferably a bulky Rx suitable
fonregloselective reaction on the primary alcohol of a diol as described in Chem. Ber.
101 (1968), 3802-3807. Rr and R2' are R1 and R2 as defined above. Ry can be
hydrogen or a hydroxyl protective group, preferably an ether type protective group.
Preferably Ry is hydrogen. PG could be a suitable N-protecting group such as a
carbamate, for derivatives in Scheme 5, Ry is typically hydrogen.
Other methodologies to obtain a 5,5-bicycle is disclosed by G. Lin and Z. Shi,
Tetrahedron, 53, 4,1369-1382,1997.
Further modification of the 5,5-bicyclic compound obtained in scheme 1 is outlined in
Scheme 6.


Compound 9 is protected with a suitable acid stable protecting group such as
substituted methyl ether, in particular a benzyl ether, by treating the mono-ol 9 with a
base such as sodium hydride or sodium hydroxide in an aprotic solvent such as N,N-
dimethylformamide (DMF) in the presence of the desired alkylating agent such as the
benzyl halide, in particular benzyl bromide. The obtained material can then be reduced
into compound 10 according to methods described by G. J. Ewing and M. J. Robins,
v> Org. Lett. 1, 4,1999, 635-636, or by references therein. Preferably the reduction is
performed with excess boron trifluoride etherate in the presence of a reducing agent
such as trialkylsilane, in particular with excess triethylsilane in a suitable non-protic
solvent such as dichloromethane. Catalytic hydrogenation of compound 10 using for
example pailadium-on-charcoal in a suitable solvent or solvent mixture such as ethyl
acetate-ethanol in a hydrogen atmosphere, in the presence of di-tert-bufyi dicarbonate
• followed by treatment of the product with acetic anhydride in pyridine gives Intermediate
11. By repeated catalytic hydrogenation, as described above, the mono-ol 12 is
obtained.
A fluorine can be introduced on compound 12, and the bicyclic compound then N-
deprotected according to Scheme 7.


Compound 13 can be treated with a fluorinating agent such as [bis-(2-
methoxyethyl)aminosulfur trifluoride] (Deoxo-Fluor®) or with similar fluorinating agents
such as diethylaminosulfur trifluoride (DAST) which gives the product 14 with inversion
of configuration at C-5. Compound 14 is then deacetylated by treatment for example
with methanolic sodium methoxide, or any similar alkaline solutions with an inorganic
base such as sodium hydroxide or sodium carbonate, followed by N-deprotection using
acidic conditions such as dichloromethane-trifluoroacetic acid solutions or other , -,
methods which could be found in: Protective Groups in Organic Chemistry, 3rd edition,
1999, Theodora W. Greene and Peter G. M. Wuts (Wiley & Sons).
Alternatively the epimeric fluorine can be obtained by treating derivative 9 above
according to Scheme 8.


Inversion of configuration at C-5 can be accomplished by reacting compound 16 under
Mitsunobo conditions which gives a benzoate ester. Ester hydrolysis with methanolic
sodium methoxide followed by treatment of the mono-ol with benzyl bromide provides
benzyl protected epimer 17. Reaction steps d-j in Scheme 8 are as described for
Schemes 6 and 7.
A further route to a "difluoro derivative" wherein R1 and R2 are fluoro is shown in
Scheme 9.
i



The synthesis of the P1 building block can be started from compound 21 (3-azido-3-
deoxy-1,2-0-isopropylidene-D-allofuranose) which is described by Mayer zum
Reckendorf, Chem. Ber. 101 (1968), 3802-3807. Treatment of compound 21 with a
benzylating agent like benzyl bromide or benzyl chloride in the presence of a base, such
as sodium hydride or sodium hydroxide in a aprotic polar solvent, such as N.N-
dimethylformamide gives derivative 22. Compound 22 is then treated with a trialkyl
silane, such as triethyl silane, with an excess of a Lewis acid such as boron trifluoride
etherate or trimethylsilyl trifluoromethanesulfonate, in a aprotic solvent such as
dichloromethane. The resulting azide can then be selectively reduced by catalytic
hydrogenation using for example Palladium on charcoal in the presence of di-tert-bulyl
carbonate to obtain compound 23. Alternatively the azide could be reduced with other
methods known from literature such as triphenylphosphine-water, followed by protection
giving a suitable carbamate. In order to avoid problems with regioselectivity in the
following steps, compound 23 could be treated with an acylating agent such as an acyl
chloride or acid anhydride, such as benzoyl chloride, in neat organic base such as
pyridine or triethyl amine, or in a mixture of an aprotic solvent such as dichloromethane
and a base to give compound 24. Catalytic hydrogenation of compound 24 as described
above gives diol 25. Selective benzylation at the primary alcohol of compound 25 can
be accomplished by several methods known from the literature. In Scheme 9 the diol is
refluxed with dibutyl tin oxide in a suitable solvent such as toluene to form a tin acetal.
The tin acetal can then be reacted with a small excess of benzyl bromide and cesium
fluoride in DMF giving the desired compound 26. Oxidation of 26 with a suitable
oxidizing agent such as Dess-Martin periodinane in dichloromethane converts the
secondary alcohol into the keto compound 27 suitable to convert into the d'rfluoride 28.
This can be accomplished by treating compound 27 with an excess fluorinating agent
such as Deoxo-Fluor®, or with diethylaminosulfur trifluoride (DAST), in an aprotic
solvent such as dichloromethane or 1,2-dichloroethane. The benzoate ester of
compound 28 can be cleaved with alkali such as methanolic sodium methoxide,
followed by debenzylation using catalytic hydrogenation to obtain diol 29. Selective
introduction of a sulfonate ester at the primary alcohol can be accomplished by treating
the compound 29 with a small excess of alky!- or arylsulfonyl chloride in the presence of
a base such as pyridine in suitable solvent such as dichloromethane, adding the
sufonylating agent at reduced temperature and slowly increase up to room temperature,

which gives mono-ol 30. Treatment of compound 30 under acidic conditions such as
mixtures of dichlormethane-trifluoroacetic acid liberates the amine, and treating the
product with a base such as triethyl amine promotes the internal ring closure which
gives building block 31.
Alternative routes to 5,5-bicycles are shown in Schemes 10 and 11.

In Scheme 10 a derivative such as compound 32 (available as described above or with
methods well known in the art) with the substituents at C-3 and C-4 in cis relationship,
Lg being a leaving group such as halogen or a sulfonate ester, and with R equal to an
azide or a nitrogen protected with a suitable N-protecting group, can be treated with a
fluorinating agent such as mentioned above, producing compound 33. Upon liberating
the masked amine with either reduction of the azide or by a suitable deprotection
method, the amine could perform an intramolecular attack at C-6 producing a 5,5-
bicycle with structure 34, which could optionally be N-protected (Pg = protecting group
or hydrogen). Reduction of C-1 with a suitable reducing agent such as described above
or with a similar reducing agent would give building block 35.
In Scheme 11 an alternative route to a difluoro-5,5-bicycle is depicted.


In Scheme 11 compound 36 (available as described above or with methods well known
in the art) with the substituents at C-3 and C-4 in cis relationship, Lg being a leaving
group such as halogen or a sulfonate ester, and with R equal to an azide or a nitrogen
protected with a suitable protecting group, can be oxidized with a Swern-type reaction
or other suitable methods which can give compound 37. Treatment of compound 37
according to Scheme 11 with an excess of fluorinating agent such as mentioned above,
gives compound 38. Upon liberating the masked amine of 38 with either reduction of the
azide or by a suitable deprotection method, the amine could perform an intramolecular
attack at C-6 producing a 5,5-bicycle with structure 39, which could optionally be N-
protected (Pg = protecting group or hydrogen). Reduction of C-1 with a suitable
reducing agent such as described above or with a similar reducing agent gives building
block 40.
A convenient route to compounds wherein R1 or R2 is a halogen such as chloro is
depicted in Scheme 12


The P1 building block is then elongated with the natural or non natural P2 amino acid
and the P3 group by conventional solution or solid phase chemistries, such as those
outlined or exemplified below, or disclosed in WO00/69855 or WO02/057270. P2 and
P3 groups are either commercially available as enantiomers or resolvable from the
racemate or obtainable using simple chemical transformations known to one skilled in
the art. For example, 4-(methyl-piperazine-1-yl)-benzoic acid can be obtained using
Buchwald chemistry (S. L. Buchwald & J. P. Wolfe, Journal of Organic Chemistry, 2000,
65,1144) and subsequently elaborated. Other P3 cores such as 4-(1-piperidin-4-yl)-
benzoic acid are prepared from 1 -(4-phenyl-piperidine-1-yl)-ethanone using a Friedel-
Crafts acylation reaction and subsequently elaborated using standard chemical
transformations known to one skilled in the art. Alternatively, other P3 moieties, such as
5-[2-(4-morpholinyl)ethoxy]-2-benzofuran-2-carboxylic acid, are prepared using
M'rtsunobu reactions on solid phase as detailed by L. S. Richter & T. R. Gadek in
Tetrahedron Lett., 1994, 35,4705.


Urethane compounds i.e. E is -OC(=O)- can be formed for example by reaction of an
Re alcohol with the isocyanate of the P2 amino acid. The isocyanate, or equivalent
reactive intermediate, can be formed by reaction of the amino group of the P2-amino
acid with phosgene, or with dinitrophenylcarbonate in the presence of a suitable base,
e.g. triethylamine. Alternatively they can be formed by reaction of the amino group of
the P2 amino acid with a suitable chloroformate, e.g. benzylchloroformate.
Sulphonamide derivatives i.e. E = S(=O)2- can be prepared by reaction of the amino
group of the P2 amino acid with a suitable sulfonyl chloride in a solvent such as
dichloromethane in the presence of a suitable base such as triethylamine or
dimethylaminopyridine.
Sulphamide derivatives i.e. E = NRaS(=O)2- can be prepared by reacting a suitable Re
amine in a sulphonyl chloride solvent followed by reaction of the formed sulfamoyl
chloride derivative with the amino group of the above mentioned R4 amino acid in a

solvent such as dichloromethane in the presence of a suitable base such as
triethylamine.
Alternatively the P1 building block as the hydroxyl may be elongated and subsequently
oxidised as shown in Scheme 14 and the Examples.

The term "N-protecting group" or "N-protected" as used herein refers to those groups
intended to protect the N-terminus of an amino acid or peptide or to protect an amino
group against undesirable reactions during synthetic procedures. Commonly used N-

protecting groups are disclosed in Greene, "Protective Groups in Organic Synthesis"
(John Wiley & Sons, New York, 1981), which is hereby incorporated by reference. N-
protecting groups include acyl groups such as formyi, acetyl, propionyl, pivaloyl, t-
butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoracetyl, trichloroacetyl, phthalyl, o-
nitrophenoxyacetyl, a-chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, 4-
nitrobenzoyl, and the like; sulfonyl groups such as benzenesulfonyl, p-toluenesuHbnyl,
and the like, carbamate forming groups such as benzyloxycarbonyl, p-
chlorobenzyloxycarbonyl, p-methoxybenzyioxycarbonyl, p-nttrobenzyloxycarbonyl,
2-nltrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl,
4-methoxybenzyloxycarbonyl>2-nitro-4,5-dimethoxybenzyloxycarbonyl>
3,4,5-trimethoxybenzyloxycarbonyl, 1 -(p-biphenylyl)-l -methylethoxycarbonyl,
a,o>dimethyl-3l5-dimethoxybenzyloxycarbonyl, benzhydryloxycarbonyl,
t-butoxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl,
methoxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, phenoxycarbonyl, 4-
nitrophenoxycarbonyl, fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl,
adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl, and the like; alkyl
gropus such as benzyl, triphenyl methyl, benzyloxymethyl and the like; and silyl groups
such as trimethylsilyl and the like. Favoured N-protecting groups include fbrmyl, acetyl,
benzoyl, pivaloyl, t-butylacetyl, phenylsulfonyl, benzyl, t-butoxycarbonyl (BOC) and
benzyloxycarbonyl (Cbz).
Hydroxy and/or carboxy protecting groups are also extensively reviewed in Greene ibid
and include ethers such as methyl, substituted methyl ethers such as methoxymethyl,
methylthlomethyl, benzyloxymethyl, t-butoxymethyl, 2-methoxyethoxymethyl and the
like, silyl ethers such as trimethylsilyl (TMS), t-butyldimethylsilyl (TBDMS) tribenzylsilyl,
triphenylsilyl, t-butyldiphenylsilyl triisopropyl silyl and the like, substituted ethyl ethers
such as 1-ethoxymethyl, 1-methyl-1-methoxyethyl, t-butyl, allyl, benzyl, p-
methoxybenzyl, dipehenylmethyl, triphenylmethyl and the like, aralkyl groups such as
trityl, and pixyl (9-hydroxy-9-phenylxanthene derivatives, especially the chloride). Ester
hydroxy protecting groups include esters such as formate, benzylformate,
chloraacetate, methoxyacetate, phenoxyacetate, pivaloate, adamantoate, mesitoate,
benzoate and the like. Carbonate hydroxy protecting groups include methyl vinyl, allyl,
cinnamyl, benzyl and the like.

Detailed Description of the Embodiments
Various embodiments of the invention will now be described byway of illustration only
with reference to the following Examples.

A mixture of 54 (5.2 g, 13.0 mmol), palladium-on-carbon (10%, Acros, 0.66 g) in
methanol was hydrogenated at slight positive pressure. The hydrogen was changed 3
times over a period of 1 h, after TLC (petroleum ether-ethyl acetate 7:3 and
dichloromethane-methanol 9:1, staining with ammonium molybdate-cerium sulfate)
indicated complete conversion of the starting material into a major non-UV active spot
which colours AMC, and some weaker higher moving spots (dichloromethane-methanol
9:1). The reaction mixture was then filtered through Celite and concentrated which gave
crude compound 55.

To a suspension of the residue in dichloromethane (60 ml) and pyridine (3.2 ml, 40
mmol) at 0 °C was added benzylchloroformate (0.93 ml, 6.5 mmol). The reaction
mixture was stirred at rroom temperature for 2 h after which additional pyridine (3 ml)
and benzylchloroformate (0.8 ml) was added at 0 °C. The reaction mixture was then
stirred at room temperature overnight, then diluted with dichloromethane (100 ml),
washed successively with 1M aq. sulfuric acid (2 x 50 ml) and 1M aq. sodium hydrogen
carbonate (1 x 50 ml), then dried (sodium sulfate), filtered and concentrated onto silica.
Flash chromatography (diameter: 4 cm, YMC-gel: 50 g, packing eluent: ethyl acetate in
petroleum ether 1:4) of the residue using ethyl acetate in petroleum ether 1:4 (350 ml),
2:3 (250 ml), 1:1 (250 ml), 3:2 (250 ml) and 3:1 (150 ml) gave compound 56 as a foamy
syrup (2.71 g, 8.1 mmol, 62% over 2 steps) after drying in vacuum overnight
NMRdata (400 MHz, CDCI3): 1H, 1.33,1.52 (2 s, 6H, C(CH3)2), 2.34 (2 d, 1H, -OH),
3.04 (m, 1H, H-6a), 3.97 (m, 1H, H-6b), 4.19 (m, 1H, H-5), 4.33 (m, 1H, H-3), 4.68,4.84
(2d, 1H, H-2),4.79 (t, 1H, H-4), 5.08-5.24 (m, 2H, CH2Ph), 5.86 (brs, 1H, H-1),7.30-
7.42 (m, 5H, Ar-H).

To a stirred suspension of sodium hydride (60% in mineral oil, Aldrich, 0.34 g, 8.4
mmol) and compound 56 (2.17 g, 6.47 mmol) in dimethylformamide (30 ml) was added
benzyl bromide (0.81 mmol, 6.8 mmol) during 5 minutes. After stirring 1 h (TLC: ethyl
acetate in petroleum ether 2:3), methanol (approx 2 ml) was added to destroy excess
reagent, then immediately partitioned between ethyl acetate (180 ml) and water (150
ml). The organic layer was washed with water (3 x 100 ml), then dried (sodium sulfate),
filtered and concentrated onto silica. Flash chromatography (diameter 4 cm, YMC-gel:
40 g, packing eluent: ethyl acetate in petroleum ether 1:4) of the residue using ethyl

acetate in petroleum ether 1:4 (100 ml), 3:7 (250 ml) and 2:3 (250 ml) gave a colourless
syrup (2.7 g, 6.35 mmol, 98%) after drying in vacuum overnight.
NMRdata (400 MHz, CDCI3): 1H, 1.31 (s, 3H, C(CH3)(CH3)), 1.51 (d, 3H, C(CH3)(C^)),
3.29 (m, 1H, H-6a), 3.78-3.96 (m, 2H, H-5 and H-6b), 4.22 (dd, 1H, H-3), 4.64,4.84 (2
M, 4H, H-2, H-4 and CH2Ph), 5.07-5.22 (m, 1H, CH2Ph), 5.94 (m, 1H, H-1), 7.28-7.39
(m, 10H,Ar-H).

To a stirred solution of compound 7 (2.635 g, 6.19 mmol) in dichloromethane (28 ml)
and triethyl silane (9.9 ml, 61.9 mmol) at 0 °C was added borontrifluoride etherate (7.9
ml, 61.9 mmol) in one portion. The reaction mixture was then stirred at rt for 24 h (TLC:
petroleum ether-ethyl acetate 4:1 and ethyl acetate-toluene 3:2), then 1M aq. sodium
hydrogen carbonate (40 ml) and some solid sodium hydrogen carbonate was carefully
added until bubbling stopped. The resulting mixture was partitioned between
dichloromethane (100 ml) and water (100 ml). The organic layer was washed with 1M
aq. sodium hydrogen carbonate (1 x 100 ml) and brine (1 x 100 ml), then dried (sodium
sulfate), filtered and concentrated onto silica. Flash chromatography (diameter 4 cm,
YMC-gel: 48 g, packing eluent: ethyl acetate-toluene 3:2) of the residue using ethyl
acetate In toluene 3:2 (750 ml) gave a colorless hard syrup (1.38 g, 3.74 mmol, 60%) of
about 85-90% purity according to TLC. LR-MS: Calcd for C21H24NO5: 370.2. Found:
370.0 [M+H].
1


A mixture of compound 58 (1.38 g, 3.74 mmol), palladium-on-carbon (Acros, 10%, 0.12
g) and di-tert-butyl-dicarbonate (0.82 g, 3.7 mmol) in ethyl acetate (50 ml) was
hydrogenated at slight overpressure. The hydrogen was changed 2 times over a period
of 1 h and the reaction was monitored by LC-MS. After 1 h, additional palladium-on-
carbon (0.1 g) was added and the reaction mixture was treated with hydrogen for 1
more hour. The reaction mixture was then filtered through Celite and concentrated. The
residue was treated with 2:1 pyridine-acetic anhydride (18 ml) overnight, and then
concentrated. The residue was redissolved in dichloromethane (60 ml) and was washed
successively with 1M aq. sulfuric acid (2 x 40 ml) and 1M aq. sodium hydrogen
carbonate (1 x 40 ml), and then dried (sodium sulfate) filtered and concentrated. Rash
chromatography (diameter: 3 cm, YMC-gel: 20 g, packing eluent: ethyl acetate In
toluene 1:4) of the residue (dissolved in toluene-ethyl acetate 4:1) using ethyl acetate in
toluene 1:4 (200 ml) and 1:3 (150 ml) gave a colourless syrup (1.13 g, 3.0 mmol, 80%)
after drying in vacuum overnight.
NMRdate (400 MHz, CDCI3): 1H, 1.45 (s, 9H, C(CH3)3), 2.08 (s, 3H, COCH3), 3.10 (m,
1H, H-6a), 3.74-3.99 (m, 3H, H-1a, H-5 and H-6b), 4.11 (m, 1H, H-1b), 4.16-4.74 (m, 4H
H-3, H-4 and CH2Ph), 5.31 (m, 1H, H-2), 7.28-7.40 (m, 5H, Ar-H).

A mixture of compound 60 (1.08 g, 2.86 mmol) and palladium-on-carbon (10%, 0.15 g)
in ethyl acetate (30 ml) was hydrogenated at slight over pressure for 2 h (TLC: toluene-

ethyl acetate 4:1 and 1:1), then filtered through Celite and concentrated. The mixture
was concentrated from dichloromethane (3x10 ml), then dissolved in dichloromethane
and to the solution was added bis-(2-meihoxyethyl)aminosulphur trifluoride (50% in
THF, 2.12 ml, 2 eq.) at 0 °C. After stirring at rt overnight additional bis(2-
methoxyethyl)aminosulphur trifluoride (50% in THF, 2 ml) was added and the reaction
mixture was stirred at rt for another night (TLC: toluene-ethyl acetate 1:1, ninhydrine
staining), then 1M aq. sodium hydrogen carbonate was added carefully until bubbling
stopped. The resulting mixture was diluted with dichloromethane (50 ml), and the
organic layer was washed once with 1M aq. sodium hydrogen carbonate (40 ml), then
dried (sodium sulfate), filtered and concentrated. Flash chromatography (diameter 3
cm, Silica: 25 g, packing eluent: toluene-ethyl acetate 4:1) of the residue (dissolved in
toluene-ethyl acetate 4:1) using toluene-ethyl acetate 4:1 gave compound 62 (0.49 g,
1.7 mmol, 59 %) as a colourless syrup after drying in vacuum overnight. Some starting
material and sulphur intermediate could be recovered from the reaction mixture.
LR-MS: Calcd for C9H13FN05:234.1. Found: 234.0 [M+2H-f-Butyl].

To a solution of compound 62 (0.49 g, 1.7 mmol) in methanol (9.5 ml) was added 0.5 M
methanolic sodium methoxide (1 ml), then stirred at rt for 30 min (TLC: Toluene-ethyl
acetate 3:2, ninhydrine staining). Methanol washed Dowex W X 8 (50-100 mesh, H+-
form) was carefully added (pH was monitored by pH-paper) was added until neutral,
then the mixture was filtered and concentrated. The residue was dissolved in
dichloromethane and trifluoroacetic acid was added at 0 eC. The reaction mixture was
then stirred at rt for 55 min (TLC: dichloromethane-methanol 9:1, ninhydrine staining),
then concentrated. Column chromatography (diameter: 2 cm, silica: 15 g, packing

eluent dlchloromethane-methanol 95:5) of the residue (dissolved in dichloromethane-
methanol 95:5) using methanol in dichloromethane 5:95 (150 ml), 7:93 (100 ml) and 1:9
(200 ml) gave a hard syrup which crystallized upon standing (0.39 g, 1.50 mmol, 88%).
NMRdata (400 MHz, DMSO-d6): 1H, 3.34, 3.44 (2 dd, 1H, H-6a), 3.60-3.70 (m, 2H, H-
1a and H-6b), 3.89 (dd, 1H, H-1b), 4.15 (d, 1H, H-3), 4.51 (brs, 1H, H-2),4.76 (dd, 1H,
H-4), 5.26 (dd, 2JH,F = 48.3 Hz, H-5).

To a stirred solution of compound 64 (0.34 g, 1.30 mmol), N-ethyl-N'-(3-
dlmethylamInopropyl)carbodiimide hydrochloride (0.28 g, 1.43 mmol), 1-
hydroxybenzotriazole hydrate (0.22 g) and A/-(terf-Butoxycarbonyl)-L-leucine
monohydrate (0.34 g, 1.37 mmol) in DMF (10 ml) was added triethylamine (0.54 ml, 3.9
mmol), then stirred at rt for 24 h. The reaction mixture was the partitioned between 10%
aq. citric acid (30 ml) and ethyl acetate (10 ml). The water layer was extracted with ethyl
acetate (3x10 ml), then the organic layers were combined, and washed successively
with water (1 x 20 ml) and 1M aq. sodium hydrogen carbonate (3 x 20 ml), then dried
(sodium sulfate), filtered and concentrated onto silica. Flash chromatography with ethyl
acetate in petroleum ether (40-60 %, stepwise gradient elution) of the residue gave 15
(0.35 g, 0.98 mmol, 75%) as a colourless amorphous solid.
LR-MS: Calcd for C13H22FN2O5:305.1. Found: 305.1 [M+2H-f-Butyl].
Example 3
Elongation with a tvoical P3


To a solution of compound 65 (0.11 g, 0.31 mmol) in dichloromethane (2 ml) at 0 °C
was added trifluoroacetic acid (2 ml), then stirred at rt for 45 min. The reaction mixture
was then concentrated and co-concentrated with toluene. To a suspension of the
residue, N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.064 g, 0.34
mmol), 1-Hydroxybenzotriazole hydrate (0.051 g) and benzo[b]furan-2-carboxylic acid
(0.052 g, 0.32 mmol) in DMF (3 ml) was added triethylamine (0.13 ml, 0.9 mmol), then
stirred at rt for 24 h. The reaction mixture was then concentrated. The residue was then
partitioned between 10% aq. citric acid (30 ml) and ethyl acetate (10 ml). The water
layer was extracted with ethyl acetate (2x10 ml), then the organic layers were
combined, and washed successively with water (1x10 ml) and 1M aq. sodium
hydrogen carbonate (3x10 ml), then dried (sodium sulfate), filtered and concentrated
onto silica. Rash chromatography with ethyl acetate in petroleum ether (50-60 %,
stepwise gradient elution) of the residue gave 66 (0.11 g, 0.27 mmol, 89 %) as a
colourless glassy solid.


To a stirred solution of compound 66 (0.10 g, 0.25 mmol) in dichloromethane (4 ml) at rt
was added Dess-Martin periodinane (0.12 g, 0.28 mmol). After stirring for 90 minutes
the reaction mixture was diluted with dichloromethane (10 ml), washed with 1:11M aq.
sodium hydrogen carbonate-10 % aq. sodiumthiosulfate (4x10 mi), then dried (sodium
sulfate), filtered and concentrated onto silica. Flash chromatography with ethyl acetate
in petroleum ether (50-60 %, stepwise gradient elution) of the residue gave 67 (0.072 g,
0.18 mmol, 71 %) as a colourless foam. Compound 67 is obtained as a mixture of
geometrical isomers (rotamers) and their hydrates.
LR-MS: Calcd for C2iH24FN205: 403.2. Found: 403.0 [M+H]. A NMR sample of the
ketoforms of 67 was obtained as follows; 5 mg of compound 67 (mixture of geometrical
isomers and hydrate forms with the ratio: hydrate/keto 6:4) was dissolved in DMSO-d6,
then heated up to 100 °C in the NMR apparatus and then allowed to reach 50 °C upon
which NMR indicated only trace amounts of the hydrate forms and the ratio of the
rotamers were 2:1.
NMR data (500 MHz, DMSO-d6, 50 °C): 1H, 0.90-1.04 (m, 4 x CH3, major and minor
forms), 1.39-1.82 (m, 2 x CH2CH(CH3)2 and 2 x CH2CH(CH3)2, major and minor forms),
3.56 (m, H-6a, minor), 3.82 (m, H-6A, major), 3.97-4.25 (m, 4 x H-1, major and minor
forms and H-6b, minor), 4.37 (dd, H-6b, major), 4.62 (d, H-3, minor), 4.79 (m, H, major),
4.84 (d, H-3, major), 4.94 (m, H-4, major), 5.12 (m, H-4, minor), 5.15-5.34 (m, H-5 major
and H-5 minor, H minor, JH.F major — 49.1 Hz, JH,F minor = 49.4 Hz), 7.35 (t, 1H, Ar-H), 7.47
(t, 1H, Ar-H), 7.57-7.70 (m, 2H, Ar-H), 7.78 (d, 1H, Ar-H), 8.18 (d, -NH, minor), 8.70 (d, -
NH, major).


To a solution of compound 55 (0.11 g, 0.32 mmol) in dichloromethane (2 ml) at 0 °C
was added trifluoroacetic acid (2 ml). After stirring for 45 mln at rt (TLC: petroleum
ether-ethyl acetate 1:1 and ethyl acetate-methanol-acetic acid-water 40:3:3:2), Ihe
reaction mixture was concentrated and co-concentrated from toluene (3x5 ml). To a
suspension of the residue, 4-(dimethylamino)benzoic acid (0.055 g, 0.33 mmol), N-
ethyl-NX3-dimethylaminopropyl)carbodiimide hydrochloride (0.067 g, 0.35 mmol) and
1-hydroxybenzotriazole hydrate (0.053 g) in DMF (3 ml) was added triethylamine (0.13
ml, 0.95 mmol), then stirred at rt overnight (TLC: petroleum ether-ethyl acetate 2:3 and
ethyl acetate-methanol-acetic acid-water 40:3:3:2). The reaction mixture was then
concentrated, partitioned between 8% aq. KH2PO4 (30 ml) and ethyl acetate (10 ml).
The water layer was extracted with ethyl acetate (3x10 ml), and the combined organic
layers were washed with water (1x10 ml) and 1M aq. sodium hydrogen carbonate (3 x
10 ml), then dried (sodium sulphate), filtered and concentrated. The residue was
redissofved in dichloromethane and concentrated onto silica. Flash chromatography
(diameter 2 cm, Silica: 8 g, packing eluent: petroleum ether-ethyl acetate 1:1) of the
residue (stepwise gradient elution, ethyl acetate in petroleum ether 50-100%) gave a
colourless foam (0.10 g, 0.25 mmol, 80%).

To a stirred solution of the mono-ol 68 (0.096 g, 0.24 mol) in dichloromethane at rt was
added Dess-Martin periodinane (0.11 g, 0.26 mmol). The reaction mixture turned red
and after stirring for approximately 35 min (TLC: petroleum ether-ethyl acetate 2:3), the
reaction mixture was diluted with dichloromethane (10 ml), washed with 1:1 1M aq.
sodium hydrogen carbonate-10 % aq. sodiumthiosulfate (4x10 ml), then dried (sodium
sulfate), filtered and concentrated onto silica. Flash chromatography (diameter: 2 cm,
Silica: 7 g, Packing eluent: petroleum ether-ethyl acetate 1:1) of the residue (stepwise

gradient elution, ethyl acetate in petroleum ether 50-100%) gave a colourless foam
(0.039 g, 0.10 mmol, 41%).

To a solution of compound 55 (0.12 g, 0.32 mmol) in dichloromethane (2 ml) at 0 °C
was added trifluoroacetic acid (2 ml), then stirred at rt for 45 min. The reaction mixture
was then concentrated and co-concentrated with toluene (3x5 ml). To a suspension of
i:theresidue, N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride (0.068g,
0.36 mmol), 1-hydroxybenzotriazole hydrate (0.055 g) and 4-phenoxybenzoic acid
(0.073 g, 0.34 mmol) in DMF (3 ml) was added triethylamine (0.14 ml, 0.97 mmol), then
stirred at rt for 24 h. The reaction mixture was then concentrated. The residue was then
partitioned between 10% aq. citric acid (30 ml) and ethyl acetate (10 ml). The water
layer was extracted with ethyl acetate (2x10 ml), then the organic layers were
combined, and washed successively with water (1x10 ml) and 1M aq. sodium
hydrogen carbonate (3x10 ml), then dried (sodium sulfate), filtered and concentrated
onto silica. Flash chromatography of the residue with 1:1 ethyl acetate in petroleum
ether gave colourless hard syrup (0.14 g, 0.30 mmol, 91%).
LR-MS: Calcd for C25H30FN2O5:457.2. Found: 457.2 [M+H].


To a stirred solution of the mono-ol (0.128 g, 0.28 mmol) in dichloromethane (4 ml) at rt
was added Dess-Martin periodlnane (0.12 g, 0.28 mmol). After stirring for 90 minutes
the reaction mixture was diluted with dichloromethane (10 ml), washed with 1:11M aq.
sodium hydrogen carbonate-10 % aq. sodiumthiosulfate (4x10 ml), then dried (sodium
sulfate), filtered and concentrated onto silica. Flash chromatography with ethyl acetate
in petroleum ether (50-60 %, stepwise gradient elution) of the residue gave 71 (0.072 g,
0.18 mmol, 71 %) as a colourless foam.

To a stirred solution of compound (60) (1.58g, 4.19mmol) In methanol (20mL) was
added a solution of 0.5 M sodium methoxide in methanol (5mL) at room temperature,

then stirred for 40 min. The reaction mixture was then neutralized with Dowex 50 WX 8
(H+-form), filtered, added triethylamine until slight alkaline, then concentrated and
concentrated from toluene (2 x 20mL). To a stirred solution of the residue and imidazole
(0.43g, 6.28mmol) in DMF (10ml_) at 0 °C was added te/f-Butyldimethylchlorosilane
(0.76g, 5.02mmol), then stirred at room temperature overnight The reaction mixture
was then diluted with ethyl acetate (lOOinL), washed successively with 10% aq. citric
acid (3 x 50ml_) and 1M aq. sodium hydrogen carbonate (3 x 50mL), dried (sodium
sulphate), filtered and concentrated onto silica. Column chromatography (stepwise
gradient elution, ethyl acetate in toluene, 5-20%) of the residue afforded the fully
protected intermediate as a syrup (1.86g). A mixture of palladium on charcoal (Aldrich
10%, 0.28g) and the intermediate obtained above (1.80g, 4.00mmol) in ethyl acetate
(40mL) was hydrogenated at slight overpressure for 1 h, then filtered through celtte and
concentrated. The material crystallized upon drying in vacuum to afford 72 as needles
(1.34g,90%).
NMR data (400 MHz, CDCI3): 1H, delta 0.14 (m, 6H, Si(CH3)2), 0.90 (m, 9H, SiCfOfcfe),
1.48 (m, 9H, C(CH3)3), 2.53 (m, 1H, OH), 2.78 (dd, 1H, - H-6A), 3.67-4.05 (m, 3H, H-1A,
H-1B and H-6B), 4.05-4.21 (m, 2H, H-3 and H-5), 4.35-4.50 (2 brs, 1H, H-2), 4.57 (m,
1H, H-4).

To a stirred solution of (72) (1.068g, 2.97mmol), benzoic acid (0.50g, 4.46mmol) and
triphenylphosphine (1.17g, 4.46mmol) in THF (15 mL) at 0 °C was added dropwise a
solution of diisopropyl azodicarboxylate (0.88mL, 4.46mmol) in THF (5mL) during 20
minutes. The reaction mixture was then stirred at room temperature overnight, then

concentrated onto silica. Flash chromatography of the residue using petroleum ether-
ethyl acetate 9:1 as eluent, gave a colorless syrup (1.34g, 97%).
NMR data (400 MHz, CDCI3): 1H, delta 0.08-0.21 (m, 6H, Si(CH3)2), 0.90 (s, 9H,
SiC(CH3)3), 1.42-1.56 (m, 9H, C(CH3)3), 3.48 (m, 1H, H-6A), 3.70-4.01 (m, 3H, H-1 A, H-
1B, H-6B minor and major), 4.21, 4.30 (2d, 1H, H-3), 4.44,4.56 (2 brs, 1H, H-2), 4.72
(m, 1H, H-4), 5.34 (d, 1H, H-5), 7.45 (t, 2H, Ar-H), 7.58 (t, 1H, Ar-H), 8.00 (d, 2H, Ar-H).

To a stirred solution of (73) (1,34g, 2.89mmol) in methanol (6mL) was added a solution
of 0.5 M sodium methoxide in methanol (6mL) at room temperature, then stirred for 15
min. The reaction mixture was then neutralized with Dowex 50 WX 8 (HVorm) and
filtered. The obtained solution was added a solution obtained similarly as above starting
from (II) (0.187g, 0.40mmol), then concentrated. Flash chromatography of the residue
using toluene-ethyl acetate 3:2 as eluent gave 74 as a colorless syrup which crystallized
upon drying in vacuum (1.091 g, 92%).
NMR data (400 MHz, CDCI3): 1H, delta 0.06-0.20 (m, 6H, Si(CW3)2), 0.89 (s, 9H,
SIC(CW3)3), 1-42-1.54 (m, 9H, C(CH3)3), 2.03 (brs, 1H, OH), 3.28 (dd, 1H, H-6A), 3.53-
3.79 (m, 3H, H-1A, H-1B, H-6B), 4.19 and 4.34-4.56 (2 m, 4H, H-2, H-3, H-4 and H-5).


To a stirred solution of (74) (0.428g, 1.19mmol) in dichloromethane (10mL) in a Teflon
coated flask was added Deoxofluor (50% in THF, 0.53mL) at room temperature
resulting in a slight temperature increase. The reaction mixture was stirred at room
temperature for 72 h, then diluted with dichloromethane (20ml_), washed with 1M aq.
sodium hydrogen carbonate (2 x 20mL), dried (sodium sulphate), filtered and
concentrated onto silica. Flash chromatography of the residue using petroleum ether-
ethyl acetate 9:1 as eluent gave (IV) as a colorless oil (0.118g, 27%).
NMR data (400 MHz, CDCI3): 1H, delta 0.08-0.20 (m, 6H, Si(Ctf3)2), 0.89 (s, 9H,
SiC(CW3)3), 1.42-1.53 (m, 9H, C(CWb)3), 3.26 and 3.36 (2 dd, 1H, H-6A), 3.64 (m, 1H, H-
1A), 3.73-4.04 (m, 3H, H-1B, H-6B), 4.20 (dd, 1H, H-3*), 4.40, 4.51 (2 s, 1H, H-2), 4.69
(m, 1H, H-4*) 4.86,4.98 (2 brs, 1H, H-5). * Could be interchanged.

To a stirred solution of (75) (0.229g, 0.63mmol) in THF (8mL) was added 1M
tetrabutylammonium fluoride in THF (0.70mL), then stirred at room temperature for 40
min. The reaction mixture was then concentrated onto silica. Column chromatography of
the residue using toluene-ethyl acetate 1:1 as eluent gave 75 as a colorless hard syrup
(0.150g, 96%).
NMR data (400 MHz, CDCI3): 1H, delta 1.461.53 (m, 9H, C(CH3)3), 2.70 (d, 0.3H, OH-
minor), 3.26-3.46 (m, 1.7H, H-6A and OH-major), 3.75-4.04 (m, 3H, H-1A, H-1B and H-
6B), 4.29,4.34 (2d, 1H, H-3* minor and major), 4.43,4.50 (2 brs, 1H, H-2 minor and
major), 4.74 (m, 1H, H^*), 4.89, 5.02 (2 brs, 1H, H-5).


To a solution of (75) (0.099g, 0.40mmol) in dichloromethane (2mL) at 0 °C, was added
trifluoroacetic acid (2mL), then stirred at room temperature for 35 min, then
concentrated and concentrated from toluene (3 x 5mL). To a suspension of the residue,
1-hydroxybenzotrazole hydrate (0.067g, 0.44mmol), N-ethyl-N'-(3-
dimethylaminopropyl)carbodiimide x HCI (0.084g, 0.44mmol) and IM-(ferf-
Butoxycarbonyl)-L-leucine monohydrate (0.105g, 0.42mmo!) in DMF (4mL) was added
triethylamine (0.17mL, 1.2mmol), then stirred at room temperature overnight The
reaction was then concentrated into half the volume, diluted with ethyl acetate (25mL),
washed successively with 10% aq. citric acid (3 x 15mL), and 1M aq. sodium hydrogen
carbonate (3 x 15mL), dried (sodium sulphate), filtered and concentrated. Column
chromatography of the residue using ethyl acetate-toluene 3:2 afforded (76) as a
colorless hard syrup (0.137g, 95%).
NMR data (400 MHz, CDCI3, selected signals): 1H, delta 0.891.01 (m, 6H, C(CH)z),
4.98,5.07 (2 dd, 1H, H-5major and H-5 minor).
LR-MS: Calcd for C17H3QFN205: 361.2. Found: 361.1 [M+H].

To a solution of (76) (0.137g, 0.38mmol) in dichloromethane at 0 °C was added TFA,
then stirred at room temperatre for 30 min, then concentrated and concentrated from
toluene (3x5 mL). To a suspension of the residue, 1-hydroxybenzotrazole hydrate

(0.064g, 0.42mmol), N-ethyl-N*-(3-dimethylaminopropyl)carbodiimide x HCI (0.080g,
0.42mmol) and benzo[b]furan-2-carboxylic acid (0.065g, 0.40mmol) in DMF (3mL) was
added triethylamine (0.16mL, 1.2mmol), then stirred at room temperature overnight The
The reaction was then concentrated into half the volume, diluted with ethyl acetate
(25mL), washed successively with 10% aq. citric acid (3 x 15mL), and 1M aq. sodium
hydrogen carbonate (3 x 15mL), dried (sodium sulphate), filtered and concentrated.
Column chromatography of the residue using ethyl acetate-toluene 3:2 afforded (77) as
a colorless hard syrup (0.148g, 96%).

To a stirred solution of (77) (0.148g, 0.37mmol) in dichloromethane at room temperature
was added Dess-Martin periodinane (0.171g, 0.40mmol). After stirring for 2 h, the
reaction mixture was diluted with dichloromethane (20mL), then washed with 1:1 1M aq.
sodium hydrogen carbonate/10% aq. sodium thiosulphate (3 x 12mL), dried (sodium
sulphate), filtered and concentrated. Column chromatography of the residue (stepwise
gradient elution, ethyl acetate in toluene, 40-50%) afforded (VIII) as a colorless foam
(0.105g, 71%).
IMMR data (100 MHz, CDCI3, selected signals): 13C, delta 206.7 and 206.5 (C=0 major
and minor).
LR-MS: Calcd for Cjn^FNzOs: 403.2. Found: 403.1 [M+H].
Example 8
Additional cathepsin K inhibitors

Compounds 8.1 - 8.13 & 8.15 - 8.20 depicted in the table below were synthesised by
successively coupling the N-protected P2 and P3 acids itemised in the table, to the P1
building block of Example 1 using the solid phase methodology outlined below.
Compound 8.14 was synthesised in solution phase as outlined below. The construction
of P2 and P3 building blocks not readily accessible from commercial sources appears
below.























Solid phase synthesis of 8.1 - 8.13 & 8.15 - 8.67 was generally carried out using
Murphy's linker methodology using known chemistries as described in WO02/88106.
The ketone function of the FmocNH bicycle was derivatised as an acid labile
semicarbazone which provided a carboxylic acid for attachment to the aminomethyl
functionalised polymer support resin using HBTU, HOBt and NMM. After Fmoc removal
the corresponding P2 Fmoc acid was coupled on where the symmetric anhydride was
preformed. Coupling was first carried out for 8 h, and then repeated with fresh reagents
overnight. After Fmoc removal the P3 acids were introduced using standard coupling
conditions. Washing, drying and cleavage from the resin provided the crude desired
material which was purified either by column chromatography or preparative hplc.
Compounds which required modified procedures are described below.

1 H-indote-2-carboxvlic add n-(6-fluoro-3-oxo-hexahydrofuror3.2-b7pyrrote-4-cart)onv(>-
3-methyi-butyfl-amide (Example 8.6)
To the resin bound H2N-L-Leu-P1 (150 mg, 0.03 mmol) was added a solution of indole-
2-carboxylic acid (24.2 mg, 0.15 mmol) in DMF (1.0 ml_). A solution of 1,3-
diisopropylcarbodiimide (19 mg, 0.15 mmol) and 1-hydroxybenzotriazole hydrate (23
mg, 0.15 mmol) in DMF (1 mL) was then added. The reaction was agitated overnight
and then washed with DMF (7x10 mL), MeOH (5 x 10 mL) and TBME (5 x 10 mL).
After drying under vacuum for 17 h, the product was cleaved from the resin by
suspension in 10 mL of 95: 5 TFA: water for 45 mins. The filtrate was then concentrated
under N2 stream, purified by semi preparative HPLC and then freeze dried to give the
title compound as a white solid. Compounds were characterised by HPLC, 1H NMR and
MS which showed both the ketone and hydrate forms to be present.
4-Piperidin-4-vl benzoic acid (Example 8.9)
4-Phenylpiperidine (10.0 g, 62 mmol) and pyridine (5.74 mL, 71 mmol) were dissolved
in DCM (80 mL) and cooled to 0 "C. A solution of acetyl chloride (4.00 mL, 71 mmol) in
DCM (20 mL) was added drop wise to the above solution. The mixture was then stirred
for 2 h at RT and when deemed to be complete by hplc, extracted with water, dried and
concentrated In vacuo to afford a light yellow oil (10.6 g, 84%) which solidified on
standing and was used without further purification. The yellow oil (10.6 g, 52.2 mmol)
was dissolved in DCM and cooled to - 78 °C and treated with oxalyl chloride (18.3 mL,
209 mmol) drop wise followed by the addition of aluminium chloride (20.9 g, 157 mmol)
in portions. When the addition was complete, the flask was placed in an ice-salt bath,
and the mixture stirred at - 20 *C for 3h and then at RT overnight. The mixture was then
poured onto ice-water and extracted with DCM (100 mL x 3), dried and concentrated In
vacuo. The residue was dissolved in aq. NaOH (2N) and HCI (6N) was added at 0 "C to
acidify the solution to pH 5. The precipitate (7.9 g) was filtered off and washed with
water (200 mL). The residue was then suspended in 6N HCI and heated at reflux for
18h. The solvent was evaporated and the residue was recrystallised from ethanol.
Crystals were filtered off and provided the title compound (5.05 g, 63%).
1

4-f5-PiDeridin-1-vlmethvl-thioDhen-2-vl^benzoic acid (Example 8.15)
5-Bromo-2-thiophenecarboxaldehyde (10 mmol) and piperidine (10 mmol)were mixed
in THF (10 mL) and dibutyltin dichloride (0.2 mmol) was added. After stirring at RT for 5
minutes, phenylsilane (11 mmoi) was added and the reaction allowed to stir at room
temperature for a further 17 h. The reaction was then concentrated in vacuo and the
residue purified by flash chromatography (silica gel, DCM) to give 1-(5-bromo-thiophen-
2-ylmethyl)-piperidine: m/z = 260,262 in MS ES+ as a golden oil which was used
directly in the subsequent step. A reaction tube containing a magnetic stirrer bar was
charged with 4-carboxyphenylboronic acid (0.05 mmol), the thiophene bromide (0.06
mmol), Pd(PPh3)4 (0.025 mmol), acetcnitrile (2 mL) and 1M Na2C03 (aq) (2 mL). The
reaction tube was then sealed and heated by microwave irradiation (100W, 4 mins) to
150 *C and held at that temperature for 10 mins. After being allowed to cool to room
temperature the reaction were acidified to pH 1 with 1M HCI and the resulting
precipitate filtered off. This crude product was then passed through a silica plug to
remove any inorganic species and concentrated to give a the titel compound as a brown
powder m/z = 304 in MS ES+, which was characterised by hplc and MS and used in the
next step without any further purification.
4-(5-Morpholin-4-ylmethyl-thiophen-2-vl)benzoicacid (Example 8.16)
To synthesise 4-(5-morpholin-4-ylmethyl-thiophen-2-yl)benzoic acid, the piperidine was
substituted by morpholine in the previous experimental.
5-r2-f4.4-Difluoro-piperidin-1-vn-ethoxv1-benzofuran-2-carboxvlic acid (Example 8.17)
To a solution of 4,4-difluoropiperidine hydrochloride (1 g, 6.3 mmol) in THF (20 mL) was
added methylbromoacetate (0.63 mL, 6.6 mmol) and triethylamine (2.65 mL, 19.0
mmol). The reaction was heated at reflux for 4 h. The reaction was diluted with water
(50 mL) and the product extracted with ethyl acetate (3 * 20 mL). The combined organic
fractions were washed with brine, dried over magnesium sulphate and concentrated In
vacuo to yield (4,4-difIuoropiperidin-1-yl)acetic acid methyl ester as a brown oil (1.17g,
96%). MS 194 (M + H)+. To a solution of (4,4-difluoropiperidin-1-yl)acetic acid methyl
ester (1.17 g, 6.1 mmol) in THF (15 mL) at 0 *C was added potion wise lithium
aluminium hydride (0.46 g, 12.2 mmol). Once the effervescence had ceased the
reaction was heated at 60 'C for 1.5 h. The reaction was quenched with water (10 mL)
followed by sodium hydroxide solution (2N, 10 mL)then water (10 mL). The reaction

was filtered and the filtrate extracted with ethyl acetate (3 * 20 mL). The combined
organic fractions were washed with brine, dried over magnesium sulphate and
concentrated in vacuo to yield 2-(4,4-difluoropiperidin-1-yl)-ethanol as a brown oil (0.99
g, 99%). MS 166 (M + H)+. To a solution of diisopropylazodicarboxylate (0.36 mL, 1.82
mmol) in DCM (20 mL) was added polymer supported triphenylphosphine (728 mg, 2.18
mmol). The reaction was stirred at RT for 10 min. 5-Hydroxybenzofuran-2-carboxylic
acid ethyl ester (0.25 g, 1.21 mmol) and 2-(4,4-difluoropiperidin-1-yl)-ethanol (210 mg,
1.27 mmol) were added and the reaction stirred at RT for 16 h. The reaction was
filtered and the filtrate concentrated in vacuo. The product was purified on silica eluting
with 50 % tert-butyl methyl ether in n-heptane to yield 5-[2-(4,4-difluoropiperidin-1-
yl)ethoxy]benzofuran-2-carboxylic acid ethyl ester as a yellow solid (375 mg, 88%). MS
354 (M + H)+. To a solution of 5-[2-(4,4-difIuoropiperidin-1 -yl)ethoxy]benzofuran-2-
carboxylic acid ethyl ester (375 mg, 1.06 mmol) in THF (5 mL) and water (1 mL) was
added lithium hydroxide (34 mg, 2.12 mmol). The reaction was stirred at RT for 16 h.
The THF was removed in vacuo and the remaining aqueous solution dried overnight in
a freeze dryer to yield the crude title compound as a brown solid. MS 326 (M + H, 5.3
min) and used for coupling onto H2N-Leu-P1 without any further purification.
i /
5-f2-(4-Trifluoromethyl-piperidin-1-yl)-ethoxyI-benzofuran-2-carboxylicacid (Example {
8.18)
To a solution of 4-trifluoromethylpiperidine hydrochloride (1 g, 5.3 mmol) in THF (20 mL)
was added methylbromoacetate (0.52 mL, 5.5 mmol) and triethylamine (2.2 mL, 15.8
mmol). The reaction was heated at reflux for 4 h and then diluted with water (50 mL)
and the product extracted with ethyl acetate (3 * 20 mL). The combined organic
fractions were washed with brine, dried over magnesium sulphate and concentrated in
vacuo to yield (4-trifluoromethylpiperidin-1-yl)aceticacid methyl ester as a brown oil
(1.19 g, 98%). MS 226 (M + H)+. To a solution of (4-trifluoromethylpiperidin-1-yl)acetic
acid methyl ester (1.19 g, 5.3 mmol) in THF (15 mL) at 0 °C was added portion wise
lithium aluminium hydride (0.4 g, 10.6 mmol). Once the effervescence had ceased the
reaction was heated at 60 'C for 1.5 h. The reaction was quenched with wafer (10 mL)
followed by sodium hydroxide solution (2N, 10 mL) then water (10 mL). The reaction
was filtered and the filtrate extracted with ethyl acetate (3 x 20 mL). The combined
organic fractions were washed with brine, dried over magnesium sulphate and
concentrated in vacuo to yield 2-(4-trifluoromethylpiperidin-1-yl)-ethanol as a brown oil

(1.0 g, 99%). MS 198 (M + H)+. To a solution of diisopropylazodicarboxylate (0.58 mL,
2.28 mmol) in DCM (20 mL) was added polymer supported triphenylphosphine (1.14 g,
3.4 mmol). The reaction was stirred at RT for 10 mins. 5-Hydroxybenzofuran-2-
carboxylic acid ethyl ester (0.47 g, 2.3 mmol) and 2-(4-trifluoromethylpiperidin-1-yl)-
ethanol (0.45 g, 2.28 mmol) were added and the reaction stirred at RT for 16 h. The
reaction was filtered and the filtrate concentrated in vacuo. The product was purified on
silica eluting with 50 % tert-butyl methyl ether in n-heptaneto yield 5-[2-(4-
trifluoromethylpiperidin-1 -yl)ethoxy]benzofuran-2-carboxylic acid ethyl ester as a yellow
solid (548 mg, 62%). MS 386 (M + H)+. To a solution of 5-[2-(4-trifluoromethylpiperidin-
1-yl)ethoxy]benzofuran-2-carboxylic acid ethyl ester (548 mg, 1.42 mmol) in THF (5 mL)
and water (1 mL) was added lithium hydroxide (45 mg, 2.84 mmol). The reaction was
stirred at RT for 16 h. The THF was removed in vacuo and the remaining aqueous
solution dried overnight in a freeze dryer to yield the crude title compound as a brown
solid. MS 358 (M + H)+ which was used directly for coupling with HaN-Leu-P1.
4-[2-(4-Methvl-piDerazin-1-vn-thiazol-4-vn-benzoic acid (Examples 8.19 & 8.20)
To thtocarbonyldiimidazole (2 g, 11.5 mmol) in THF (30 mL) at RT was added N-
methylpiperazine (1.00 g, 10 mmol) drop wise. The reaction was stirred at RTfor2 h
and then at 55 'C for 1 h. The reaction was cooled to RT and 20 mL of THF was
removed in vacuo. 2M NH3 (10 mL) in MeOH was added and the reaction stirred for 15
h. A further 2M NH3 (10 mL) in MeOH was added and the reaction maintained at 55 *C
for 8 h. A pale yellow precipitate (1.00 g) was observed and filterered off, dried and
used directly in the next step. The thiourea (0.84 g, 5.2 mmol) was dissolved in EtOH
(30 mL) and 4-(2-bromo-acetyl)-benzoic acid (1.28 g, 5.2 mmol) was added. The
reaction was heated at reflux for 3 h. The reaction was cooled to RT and the solid
filtered off. The solid was washed with Et20 and dried thoroughly. This procedure
provided the title compound as a pale yellow solid (1.23 g, 77 %).
ri-f6-Fluoro-3-oxo-hexahydro-furor3,2-b1pyrrole-4-carbonyl)-cyclohexyl1-carbamlcacld
9H-fluoren-9-vlmethvl ester (Example 8.20)
Fmoc-1-amino-1-cyclohexane carboxylic acid (0.300 mg, 0.82 mmol) was dissolved in
DCM (8 mL) and DAST (1 mL, 8.2 mmol) was added. After 1.5 h the starting material
was consumed and H2O (5 mL) was added drop wise with care. The organic layer was
removed, dried (Na2S04) and concentrated in vacuo to afford a pale brown solid (0.287

g, 96 %). This material was used crude in the next step. (1 -Fluorocarbonyl-cyclohexyl)-
carbamicacid 9H-fluoren-9-ylmethyl ester (0.050 g, 0.135 mmol) was dissolved in DMF
(1 ml_) and added to H2N-P1 in DMF (1 mL). NMM (0.027 g, 0.27 mmol) was added and
the reaction left overnight. The resin was filtered off to remove spent reagents and fresh
reagents were added and the reaction repeated for a further 24 h. After washing with
DMF (10 mL x 10) and DCM (10 mL x 10) the title compound (loading equivalent to 50%
yield) was obtained bound to resin.
ri-(6-Fluoro-3-oxo-hexahydro-furor3,2-blpyrrole-4-carbonyl)-3-methyl-butyn-carbamic
acid benzyl ester (Example 8.14)

Example 8.14 was prepared in solution rather than on solid phase.
6-Fluoro-3-hydroxy-hexahydro-furo[3,2-b]pyrrole-4-carboxylic acid tert-butyl ester (0.200
g, 0.81 mmol) was dissolved in DCM (4 mL) at 0 °C and TFA (4 mL) added. After
stirring at 0 -4 °C for 1 h, the solvent was evaporated in vacuo and the residue left
under high vacuum for 4 h to afford a brown oil. The residue was dissolved in DMF (5
mL) and WSC.HCI (171 mg, 0.89 mmol), HOBt (137 mg, 1.01 mmol), Cbz-Leu-OH (226
mg, 0.85 mmol) and Et3lM (337 ul, 2.43 mmol) added. After stirring at room temperature
overnight, the reaction mixture was concentrated in vacuo, dissolved in EtOAc (10 mL),
washed with H20 (5 mL) and saturated NaHC03 solution (5 mL), dried (Na2S04) and
evaporated in vacuo to afford a colourless oil (242 mg; [M+H]+ 395). [1-(6-Fluoro-3-
hydroxy-hexahydro-furo[3,2-b]pyrrole-4-carbonyl)-3-methyl-butyl]-carbamic acid benzyl
ester (242 mg, 0.62 mmol) was dissolved in dry DCM (8 mL) and Dess-Martin
periodinane (261 mg, 0.62 mmol) added. The reaction immediately turned light brown.
After stirring at room temperature for 2.5 h, the yellow solution was diluted with DCM (8
mL) and washed with saturated NaHC03 solution (5 mL), dried (NaaSCM) and
evaporated in vacuo to afford a yellow residue. Purification by column chromatography
(EtOAc: heptane; 1:2) yielded the title compound as a colourless oil, 147 mg; [H+HJ*
393.

4-Thioraroamoyl-plperazfne-1-carboxylic acid tert-butyl ester (Example 8.21)
To a solution of piperazine-1-carboxylic acid tert-butyl ester (32.2 mmol) in
tetrahydrofuran (60 ml) was added thiocarbonyldiimidazole (37.0 mmol). The reaction
waa stirred at RT for 2 h then heated at 55°C for 1 h. The reaction was concentrated in
vacuo to approximately half the volume and methanolic ammonia added (7N, 1074
mmol). The reaction was heated at 55°C for 16 h. The reaction was concentrated in
vacuo to approximately half the volume and cooled to 0°C at which point the product
precipitated from solution. The product was collected by filtration ID yield the title
compound as a white solid (3.3 g). 1H NMR (400MHz,.d6-DMSO) 1.40 (9H, s), 3.32
(4H,s), 3.71 (4H,s), 7.42 (1H,s).
4-f4-(4-Carboxy-phenvl)-thiazol-2-vn-piperazine-1 -carboxylic acid_tert-butyl ester
To a suspension of 44hiocart)amoyl-piperazine-1-carboxylic acid tert-butyl ester (13.3
mmol) in ethanol (60 ml) was added 4-(2-bromoacetyl)-benzoic acid (13.3 mmol) and 4-
methylmorpholine (13.9 mmol). The reaction was heated at reflux for 2.5 h. The reaction
was concentrated in vacuo and the solid washed with water (200 ml) to yield the title
compound as a white solid (3.9 g). 1H NMR (400MHz, CDCI3) 1.45 (9H, s), 3.58 (8H,
m), 4.86 (1H, s), 6.95 (1 H,s), 7.97 (2H, d, J 8 Hz), 8.1 (2H, d, J 8Hz).
4^2-f4-(2-Methoxv-ethvl)-piperazin-1 -vn-thiazol-4-yl}-benzoic acid
4-[4-(4-Carboxy-phenyl)-thiazol-2-yl]-piperazine-1-carboxylic acid tert-butyl ester (5.0
mmol) was dissolved in hydrochloric acid in dioxane (4N, 25 ml) and the reaction stirred
at RT for 2 h. The reaction was concentrated in vacuo to yield 4-(2-piperazin-1 -yl-
thiazol-4-yl)-benzoic acid. Trimethoxyethane (6.5 mmol) was dissolved in aqueous
hydrochloric acid (1N, 10 ml) and the reaction heated at 50°C for 1.5 h. The reaction
was allowed to cool to RT and was then added to a suspension of 4-(2-piperazin-1-yl-
thiazol-4-yl)-benzoic acid (5.0 mmol) in acetonitrile (25 ml) and sodium acetate buffer
(IN, pH 5.5,10ml). The reaction was stirred at RT for 1.5 h. Sodium cyanoborohydrlde
(6.5 mol) was added and the reaction stirred at RT for 16 h. The reaction was
concentrated in vacuo and the product purified by flash chromatography (silica gel, 10%
methanol in dichloromethane) to give the title product as a colourless oil (0.9 g). mfe =
348 (100% M+H) in MS ES+.
4-f1-f5-Bromo-thiophen-2-vl)-ethvn-morpholine (Example 8.22}
To a solution of morpholine (1.20 mmol) in titanium (IV) isopropoxide (1.95 mmol) was
added 2-acetyl-5-bromothiophene (1.20 mmol). The reaction was heated in a

microwave at 150°C for 5 minutes. Sodium borohydride (1.95 mmol) was added and the •
reaction stirred at RT for 16 h. The reaction was diluted with sodium hydroxide solution j
(2N, 10 ml) and the solids formed removed by filtration. The filtrate was extracted with
ethylacetate (3 x 20 ml), the combined organics were washed with brine and dried over
magnesium sulphate. The product was purified by flash chromatography (silica gel, 10- }
20% ethylacetate in /so-hexane) to give the title product as a brown oil: m/z = 276
(100%, M+H), 278 (100%, M+H) in MS ES+.
4-f5-(1-Morpholin-4-yl-ethyl)-thiophen-2-vf|-benzoicacid
441-(5-Bromo-thiophen-2-yl)-ethyl]-morpholine (0.36 mmol), 4-
methoxycarbonylphenyiboronic acid (0.43 mmol) and sodium carbonate (1.09 mmol)
were suspended in dioxane:water (2 ml, 2:1). Nitrogen gas was bubbled through the
reaction for 5 minutes then tetrakis(triphenylphosphine)palladium(0) (0.04 mmol) added.
The reaction was heated in a microwave at 150 °C for 10 min. The reaction was
concentrated in vacuo and the product was purified by flash chromatography (silica gel,
10% methanol in dichloromethane) to give the title product as a brown oil: m/z = 318
(50% M+H), 231 (100%, M+H-morpholine) in MS ES+.
4-ff(1-Methylimidazol-2-yl)methyl]amino}benzoic acid (Example 8.23)
1-Methyl-2-imidazolecarboxaldehyde (5.0 mmol) and methyl-4-aminobenzoate (5.0
mmol) were mixed in MeOH (7 mL). Acetic acid (0.3 mL) was added and the mixture
stirred for 30 minutes at room temperature. The reaction mixture was cooled, sodium
cyanoborohydride (5.0 mmol) was added and the reaction allowed to stir at room
temperature for a further 17 h. The reaction mixture was then concentrated under
vacuum and partitioned between H2O and EtOAc. The aqueous layer was extracted
with EtOAc, and the combined organic layers were washed with H20, brine, dried over
MgS04 and the solvent removed under vacuum. The residue was purified by flash
chromatography (silica gel, 5% MeOH in DCM) to give methyl 4-{[(1-methylfmidazol-2-
yl)methyfJamino}benzoate: m/z = 246 in MS ES+ as a pale yellow solid which was used
directly in the subsequent step.
To a solution of methyl ester (2.5 mmol) in 1,4-dioxane (5 mL) was added 1M aqueous
KOH solution (5.5 mmol) and the reaction mixture stirred for 18 h. The reaction mixture
was neutralised to pH 7 with 1M HCI and concentrated by N2 stream. The product was
resuspended in water and lyophilised to give 4-{[(1-methylimidazol-2-yl)methyl]arnino}

benzoic acid: m/z 232 in MS ES+ as a white solid which was used directly in the
subsequent step.
4^5^1^orDholin^vi-etnviVfuran--2-vn-benzoicacid (Example 8.25)
2-Acetyifuran (20 mmol) and morpholine (20 mmol) were added to neat titanium
isopropoxide (32 mmol) and the reaction stirred under N2 at room temperature for 3h.
Methanol (90 ml) was then added followed by the careful portionwise addition of NaBH4
(32 mmol). After stirring at room temperature for 10 mins, the reaction was quenched by
addition of 0.1 M NaOH and the resultant mixture filtered through a celite pad. The
filtrate was extracted twice with DCM, dried over Na2S04 and concentrated h-vacuo.
Flash chromatography of the residue (silica, 5 to 20% EtOAc in Heptane) yielded pure
4-(1-furan-2-yl-ethyl)-morpholine as a golden oil: m/z in MS ES+ = 182 [M+Hf,
2.76mmol, 14% yield.
4-(1-furan-2-yl-ethyl)-morpholine (1.1 mmol) was taken up in DCM (5 ml) and stirred at
0°C. Nitrogen was passed through the reaction vessel and bubbled out through a
Dreschel bottle containing a saturated aqueous solution of sodium thiosulphate, whilst
bromine (1.54mmol in 2ml DCM) was added dropwise. After addition the reaction was
stirred at room temperature for 2h, then diluted with more DCM, washed twice with 2M
Na2C03 solution, dried over Na2S04 and concentrated h-vacuo. After purification by
flash chromatography (silica, 5 to 10% EtOAc in hexane), 4-[1-(5-bromo-furan-2-yl)-
ethyi]-morphoiine was obtained as a goiden oil: m/z in MS ES+ = 260,262 [M+Kf,
0.46mmol, 42% yield.
4-[1-(5-bromo-furan-2-yl)-ethyl]-morpholine (0.54mmol) was taken up in 7ml toluene and
4-carboxymethyiphenylboronlc acid (0.54mmol) was added as a solution in 0.7ml of
EtOH. 12ml of 2M aqueous Na2C03 solution was added, followed by Pd(PPh3)4
(0.054mmol). Reaction was stirred at 70°C for 17h under a nitrogen atmosphere and
then cooled to room temperature and extracted with DCM (x2). Combined organic
layers were washed with brine, concentrated in vacuo and the residue purified by flash
chromatography (silica, 20-50% EtOAc in hexane). This furnished the pure 4-[5-(1-
Morpholin-4-yl-ethyl)-furan-2-yfI-benzoic acid methyl ester as a powdery grey solid: m/z
in MS ES+ = 316 [M+Hf, 0.08mmol, 15% yield.
This ester (0.08mmol) was heated to 70°C in 18% Ha for 2h at which point HPLC
showed all the starting material to have been hydrolysed. The reaction was cooled and

the product that precipitated out of solution was collected by filtration as a white solid
and used directly in the next step.
4-f2-Methvl-Dvridin-3-vloxvVbenzoic acid (Example 8.26)
A reaction tube containing a magnetic stirrer bar was charged with ethyl-4-
fluorobenzoate (1 mmol), 2-methyl-3-pyridol (1.0 mmol), potassium carbonate (1.08
mmol) and DMF (2 ml). The reaction tube was then sealed and heated by microwave
Irradiation (100W, 4 mins) to 150 'C and held at that temperature for 80 mins. The
solution was filtered to remove the insoluble potassium carbonate and then
concentrated in vacuo. The residue was purified by preparative HPLC and freeze dried
to give 4-{2-Methyl-pyridin-3-yloxy)-benzoic acid ethyl ester as a white solid which was
hydrolysed by 6N aqueous HCI solution heated by microwave irradiation (200W) for 3
mins at 150 *C. The solution was freeze dried to give to 63 mg of hydrochloride salt of
the title compound as a white powder m/z = 229 in MS ES+, which was characterised
by HPLC and MS.
4-f2-n -Dlmethylamlno-ethvlHhlazol-4-vfl-benzoic acid (Example 8.27)
4-J2-H -(tert-Butoxycarbonyl-methyl-amino)-ethyl>-thiazol-4-vl)-benzoic acid methyl ester
Boc-L-NMe-Alanine-OH (1.0g, 4.92mmols) was dissolved in dioxan (10mls) and to this
was added pyridine (0.25mls), di-tert-butyl dicarbonate (1.4g, 6.4mmols) and
ammonium hydrogen carbonate (0.49g, 6.2mmols). After stirring for 18 hours the crude
reaction mixture was concentrated in vacuo and re-suspended in ethyl acetate. This
was washed with 1M KHSO4 and the organic layer dried over magnesium sulphate.
After concentration, a clear oil was obtained (0.79g). This was dissolved in ethylene
glycol dimethyl ether (10mls) and to this was added Lawesson's reagent (4.31mmols,
1.74g). After stirring at room temperature for 3 hours the reaction mixture was
concentrated in vacuo and the residue re-suspended in ethyl acetate. This was washed
with 1M Na2CC-3and the organic layer dried over magnesium sulphate. After
concentration a yellow oil was obtained. This was purified by flash chromatography
(heptane/ethyl acetate) to give a white solid (0.73g). This was dissolved in ethanol
(10mls) and 4-(2-Bromo-acetyl>benzoic acid methyl ester (3.34mmo!s, 0.86g) was
added. The reaction was heated to 50°C for one hour. The crude product was purified
by flash chromatography
(heptane/ethyl acetate) to give a white solid (0.39g). ESMS (M + H = 377.23).

4-T2-(1-Dimethvlamino-ethvlV-thiazol-4-vIH)enzoicacid
4^2^1-(tert-ButoxycarbonylHmethyl-amino)-GthyO-thiazol-4-yl}-benzoic acid methyl ester
was deprotected with a solution of 4N HCI in dioxan for 1h. The solvent was removed in
vacuo and the residue freeze dried to get a white solid which was methylated as
followed. 4-[2-(1 -Methylamino-ethyl)-thiazol-
4-yfJ-benzoic acid methyl ester (0.44 mmol) was stirred for one hour with formaldehyde
(1.1 equivalent) in methanol (2ml) and sodium acetate buffer (1N, pH 5.5,1 ml). Sodium
cyanoborohydride (0.49 mmol) was added and the reaction stirred at RT for 2 h. The
reaction was concentrated in vacuo and the residue was extracted in EtOAc and
washed with a 1 M aqueous solution of sodium carbonate. The organic layer was
concentrated in vacuo and the residue was hydrolysed by 6N aqueous HCI solution
heated by microwave irradiation (200W) for 3 mins at 150 'C. The solution was freeze
dried to give to 134 mg of hydrochloride salt of the title compound as a white powder
m/z = 277 in MS ES+, which was characterised by HPLC and MS.
E-4-f2-f1H-lmidazol-4-vn-vinvn-benzoicacid (Example 8.28)
{4- Methyl-4-bromomethyl benzoate (26 mmol) were added to a suspension of 4.4 g of PS-
Triphelphosphine resin (Fiuka, 3 mmolg-1) in 40 ml of DMF. The solution was gently
stirred at 65°C for 48 hours. The phosphonium resin was washed with DMF (4x40 ml),
DCM (4x40 ml) and TBME (2x40 ml) and dried in vacuo for 18 h.
E-4-[2-(1 H-lmldazol-4-yl)-vinyl]-benzoic acid
A reaction tube containing a magnetic stirrer bar was charged with 1-Methyl-1 H-
imidazole-2-carbaldehyde(1.5mmol), potassium carbonate (2.1 mmol), {4-
(methoxycarbonyl) benzyl(triphenyl)} phosphonium bromide on polymer support (1.5
mmol) and methanol (4 ml). The reaction tube was then sealed and heated by
microwave irradiation (100W, 3 mins) to 150 *C and held at that temperature for 5 mins.
The solution was filtered to remove the insoluble potassium carbonate and then
concentrated in vacuo. The residue was purified by preparative HPLC and freeze dried
to give E-4-[2-(1-Methyl-1 H-imidazol-2-yl)-vinyl]-benzoic acid methyl ester as a white
solid which was hydrolysed by 6N aqueous HCI solution heated by microwave

irradiation (200W) for 3 mins at 150 "C. The solution was freeze dried to give to 90 mg
of hydrochloride salt of the title compound as a white powder m/z = 215 in MS ES+,
which was characterised by HPLC and MS.
E-4-f2-(1-Methvl-1H-imidazol-2-vn-vinvn-benzoic add (Example 8.291
Same as example 8.28.1-Methyl-1 H-imidazole-2-carbaldehyde was used as the
aldehyde. The title compound was obtained as a white powder m/z = 229 in MS ES+,
which was characterised by HPLC and MS.
E-4-r2-f3-Methvl-3H-lmidazol-4-vlVvinvn-benzoic acid (Example 8.30)
Same as example 8.28. 3-Methyl-3H-imidazole-4-carbaldehyde was used as the
aldehyde. The title compound was obtained as a white powder m/z = 229 in MS ES+,
which was characterised by HPLC and MS
4-12-41 H-lmidazol-4-vn-ethvn-benzoic acid (Example 8.31)
E-4-[2-(1-Methyl-1H-imidazol-2-yl)-vinyl]-benzoic acid methyl ester was hydrogenated
using Pd/C (10% of substrate weight), ammonium formate (5 equivalents) in
isopropanol heated by microwave irradiation (200W) for 5 mins at 150 'C. The solution
was filtered through celite to remove the insoluble catalyst diluted with water and
freeze-dried to remove the excess of ammonium formate. The obtained solid was
hydrolysed by 6N aqueous HCI solution heated by microwave irradiation (200W) for 3
mins at 150 *C. The solution was freeze dried to give to the hydrochloride salt of the title
compound as a white powder m/z = 217 in MS ES+, which was characterised by HPLC
and MS.
4-r2-(1-Methvl-1H-imidazol-2-vl)-ethvl1-benzoicacid (Example 8.321
Same as example 8.31.4-[2-(1 -Methyl-1 H-imidazol-2-yl)-vlnyl]-benzoic acid methyl
ester (Example 8.29) was used as the methyl ester. The title compound was obtained
as a white powder m/z = 231 In MS ES+, which was characterised by HPLC and MS.
Potassium 4-methvl(pvridin-2-vl)aminomethvlbenzoate (Example 8.33)
2-Methylaminopyridine (1.0 mmol) and methyl-4-formylbenzoate (1.0 mmol) were mixed
in THF (2 mL) and dibutyltin dichloride (0.1 mmol) was added. After stirring at RT for 10
minutes, phenylsilane (1.1 mmol) was added and the reaction mixture allowed to stir at

room temperature for a further 17 h. The reaction mixture was then concentrated by N2
stream and the residue purified by flash chromatography (silica gel, heptane:EtOAc) to
give methyl 4-[methyl(pyridin-2-yl)amino]methylbenzoate: m/z = 257 in MS ES+ as a
yellow oil which was used directly in the subsequent step.
To a solution of methyl ester (0.27 mmol) in 1,4-dioxane (0.6 mL) was added 1M
aqueous KOH solution (0.59 mmol) and the reaction mixture stirred for 18 h. The
reaction mixture was concentrated by N2 stream, resuspended in water and the product
lyophilised to give potassium 4-methyl(pyridin-2-yl)aminomethyl benzoate: m/z 243 in
MS ES+ as an off-white solid which was used directly in the subsequent step.
Sodium 4-f2-f1 fS Vr(tBrt-butoxvcarbonvl)( methvflamlno1ethvll-5-methvl-1.3-th?azol-4-
vl)benzoate (Example 8.34)
Ethyl 4-propionylbenzoate (2.0 mmol), pyrrolidinone hydrotribromide (2.1 mmol) and 2-
pyrrolidinone (2.2 mmol) were heated in THF (20 mL) at 50C for 2.5 h. The mixture was
cooled, filtered, concentrated under vacuum and the residual oil partitioned between
H2O and MTBE. The aqueous layer was extracted with MTBE, and the combined
organic layers were washed with saturated aqueous sodium metabisulfite solution, H20,
brine, dried over MgSd* and the solvent removed under vacuum. The residue was
purified by flash column chromatography (9:1 'Hexane: MTBE) to afford ethyl-4(2'-
bromopropionyl)benzoate as a clear oil.
Ethyl 4(2,-bromopropionyl)benzoate (0.5 mmoi), BOC(Me)AIa thioamide (0.5 mmol) and
NMM (0.5 mmol) were heated in EtOH (2 mL) at 80C for 3 h. The mixture was cooled,
concentrated by N2 stream and the crude product partitioned between H20 and MTBE.
The aqueous layer was extracted with MTBE, and the combined organic layers were
washed twice with 1M KHSO4, brine, dried over MgS04 and the solvent removed under
vacuum to give a yellow oil. The residue was purified by flash column chromatography
(9:1 'Hexane: EtOAc) to afford an intense yellow fraction. On standing for several
hours, this fraction decolorises and ethyl 4-(2-{1(S)-[(tert-
butoxycarbonyl)(methyl)amino]ethyl}-5-methyl-1,3-thiazol-4-yl)benzoate
was isolated by flash column chromatography (9:1 'Hexane: EtOAc) as a clear oil: m/z
= 405 (MH+) and 349 (M-BOC+) in MS ES+.
To a solution of ethyl ester (0.24 mmol) in 1,4-dioxane (5 mL) and water (1 mL) was
added 1M NaOH (0.53 mL) and the reaction mixture stirred for 18 h. The reaction
mixture was concentrated under vacuum, the product resuspended in water and

lyophilised to give sodium 4-(2-{1(SH(tert-butoxycarbonyl)(methyl)amino]elhyI}-5-
methyl-1,3-thiazol-4-yl)benzoate
: m/z 377 (MH+) and 321 (M-BOC+) in MS ES+ as a white solid which was used directly
in the subsequent step.
Lithium 4-12-M rS)-fdimethvlamino)ethvll-5-methyl-1.3-thiazol-4-vllbenzoate (Example
8.35)
Ethyl 4-(2-{1 (S)-[(tert-butoxycarbonyl)(methyl)amino]ethyl}-5-methyl-1,3-thiazol-4-
yl)benzoate was prepared as described previously.
To a solution of BOC-protected amine (0.25 mmol) in 1,4-dioxane (3 mL) was added
4M HCI in dioxane (4 mL) and the reaction mixture stirred for 2 h. The reaction mixture
was concentrated under vacuum to afford a viscous pale yellow oil. The oil was
dissolved in 1:1 H20:MeCN and lyophilised to afford ethyl 4-{2-[1(S)-
(methylamino)ethyl]-5-methyl-1,3-thiazol-4-yl}benzoate hydrochloride salt
A pH 5.5 buffer was prepared by adding AcOH to 1M NaOAc until pH 5.5 was reached.
The amine hydrochloride (0.28 mmol) was dissolved in 1:1 buffenMeOH (4 mL) and
formaldehyde (37 weight % in water; 0.31 mmol) was added. The mixture was stirred
for 1 h and then sodium cyanoborohydride (0.31 mmol) was added portionwise. After 1
h, the reaction mixture was concentrated by N2 stream. The residue was partitioned
between saturated aqueous NaHC03 and EtOAc. The aqueous layer was extracted with
EtOAc and the combined organic layers were washed with H20, brine, dried over
Na2S04 and the solvent removed under vacuum. The residue was purified by
preparative HPLC (0.1 % TFA in H20 : MeCN). The combined HPLC fractions were
partitioned between saturated aqueous NaHC03 and EtOAc. The aqueous layer was
extracted with EtOAc and the combined organic layers were washed with brine, dried
over Na2S04 and the solvent removed under vacuum. The absence of EtOAc was
confirmed by 1H-NMR.
The ethyl ester (0.19 mmol) was dissolved in 1:1 H20:1,4-dioxane (8 mL) and 1M LiOH
(0.42 mL) was added and the reaction mixture stirred for 17 h. The reaction mixture was
adjusted to pH 8 by addition of 1M HCI. The mixture was concentrated under vacuum,
resuspended in 1:1 H20:MeCN and lyophilised to give lithium 4-(2-{1(S)-
[(dimethylJaminolethylJ-S-methyl-I.S-thiazol-^yObenzoate: m/z 291 (MH+) in MS ES+
as a white solid and was used directly in the subsequent step.

5-(4-Methv(-morohoiin-2-vlmethoxvVbenzoftiran-2-cafboxv)ic acid (Example 8.36)
Ethyl 5-methoxybenzofuran carboxylate (22.7 mmol) was dissolved in dichloromethane
(20 ml) and a 1.0 M solution of boron tribromide methyl sulphide complex in
dichloromethane (68.1 mmol) was added. The mixture was heated at reflux over night
The solvent was evaporated under vacuo and the residue purified by flash
chromatography to obtain ethyl 5-hydroxybenzofuran carboxylate as a white solid.
Triphenyl phosphine polymer bound (8.96 mmol) was suspended in anhydrous
dichloromethane (20 ml) then diisopropyl azodicarboxylate (7.76 mmol) was added and
the mixture was stirred for 15 minutes at room temperature. Then ethyl 5-
hydroxybenzofuran carboxylate (5.97 mmol) was added over 5 minutes followed by the
addition of 4-N-boc-3-morpholinecarboxylic acid (5.97 mmol) over 5 minutes too. The
mixture was stirred at room temperature over night. The solvent was evaporated under
vacuo and the residue purified by flash chromatography to obtain ethyl 5-(4-Boc-
morpholin-2-ylmethoxy)-benzofuran-2-carboxylate: m/z = 406 in MS ES+, as dear oil.
Ethyl 5-(4-Boc-morpholin-2-ylmethoxy)-benzofuran-2-carboxyIate (2.47 mmol) was
dissolved in 30 ml of a 4.0 M solution of hydrochloric acid in dioxan and stirred at room
temperature for 1 hour. After removing the solvent under vacuo the resulting amine was
dissolved In anhydrous dichloromethane and N-methylmorpholine (5.67 mmol) was
added and stirred at room temperature for 5 minutes. Then allylchloroformate (2.71
mmol) was added and the mixture was stirred at room temperature over night under an
Inert atmosphere. The mixture was washed with a 1.0 M solution of hydrochloric acid,
water, dried over Na2S04 and the solvent was evaporated in vacuo. The residue was
purified by flash chromatography to yield ethyl 5-(4«alloc-morpholin-2-ylmethoxy)-
benzofuran-2-carboxylate: m/z = 390 in MS ES+, as a white solid.
Ethyl 5-(4-alloc-morpholin-2-ylmethoxy)-benzofuran-2-carboxylate (2.09 mmol) was
dissolved in 3 ml of tetrahydrofuran. Then 3 ml of a 1.0 M solution of lithium hydroxide
were added and the mixture stirred at room temperature over night. After removing the
tetrahydrofuran under vacuo the mixture was acidified with a 1 M solution of
hydrochloric acid to Congo red, extracted with dichloromethane, washed with water,
dried over Na2SC>4 and the solvent was removed under vacuo to yield 5-(4-alloc-
morpholin-2-ylmethoxy)-benzofuran-2-carboxyiic acid as a white solid.
5-(4-Alloc-morpholin-2-ylmethoxy)-benzofuran-2-carboxylic acid (3 equiv) was then
incorporated on the peptide as described previously (600 mg; 0.32 mmol/g) with HBTU
(3 equiv), HOBt (3 equiv) and NMM (6 equiv) in DMF over night at room temperature.

The Alloc group was removed with (1) DCM (4x1 min); (2) borane dimethylamine
complex (40 equlv), tetrakis (triphenylphosphine) palladium (0) (0.1 equiv) In anhydrous
DCM (2x15 min); (3) DCM (3 x 1 min); (4) DMF (3 x 1 min); (5) dioxan-water (9:1) (3 x
1 min); (6) DMF (3 x 1 min); (7) MeOH (3 x 1 min); (6) DCM (3 x 1 min) and the peptide
resin was treated with dibutyltin dichloride (5 equiv), phenylsilane (5 equiv) and a 37%
solution of formaldehyde in water (5 equiv) in THF for 2 hours at room temperature. The
reminder of the procedure was carried out as described in the general protocol.
3-Methyl-5-(4-methyl-morpholin-2-ylmethoxy)-benzofuran-2-carboxylicacid
(Example 8.37)
4-Methoxyphenol (0.119 mol) was dissolved in dry toluene and treated with sodium
hydride (0.120 mol) at room temperature for 60 h. The sodium phenolate solution was
heated to +100 °C and cc-chloroacetoacetate (0.09 mol) was added. After stirring at
+110 °C for a further 4 hours, the mixture was cool to room temperature, washed with
water and brine, dried over Na2S04 and the solvent was evaporated in vacuo to yield a
crude o(4-methoxyphenoxy)acetoacetate as a dark brown oil. Phosphoric acid (0.22
mol) was mixed with P205 (0.35 mol) at room temperature and stirred at +130 °C for 4
hours. The mixture was allowed to cool to +100 °C, the acetoacetate was slowly added,
and held at that temperature for 2 hours. After cooling to room temperature, ice was
carefully added to the stirred solution, extracted with toluene, concentrated in vacuo and
purified by flash chromatography on silica to yield ethyl 3-methyl-5-methoxybenzofuran
carboxylate: m/z ~ 235 In MS ES+, as a white solid.
Ethyl 3-methyl-5-methoxybenzofuran carboxylate (8.53 mmol) was dissolved in
dtahloromethane (10 ml) and a 1.0 M solution of boron tribromide methyl sulphide
complex In dlchloromethane (25.59 mmol) was added. The mixture was heated at reflux
over night. The solvent was evaporated under vacuo and the residue purified by flash
chromatography to obtain ethyl 3-methyl-5-hydroxybenzofuran carboxylate as a white
solid.
Triphenyl phosphine polymer bound (1.37 mmol) was suspended in anhydrous
dichloromethane (10 ml) then diisopropyl azodicarboxylate (1.18 mmol) was added and
the mixture was stirred for 15 minutes at room temperature. Then ethyl 3-methyl-5-
hydrtixybenzofuran carboxylate (0.91 mmol) was added over 5 minutes followed by the
addition of 4-N-boc-3-morpholinecarboxylic acid (0.91 mmol) over 5 minutes too. The

mixture was stirred at room temperature over night. The solvent was evaporated under
vacuo and the residue purified by flash chromatography to obtain ethyl 3-methyl-5-(4-
Boc-morpholin-2-ylmethoxy)-benzofuran-2-carboxylate: m/z = 419 in MS ES+, as dear
oil.
Ethyl 3-methyl-5-(4-Boc-morpholin-2-ylmethoxy)-benzofuran-2-carboxylate (1.05 mmol)
was dissolved in 30 ml of a 4.0 M solution of hydrochloric acid in dioxan and stirred at
room temperature for 1 hour. After removing the solvent under vacuo the resulting
amine was dissolved in 20 ml of a mixture 2 to 1 of methanol and a buffered solution of
acetic acid and sodium acetate at pH=5.3. A 37% solution of formaldehyde in water
(1.16 mmol) was added and the mixture was stirred at room temperature for 1 hour.
Then sodium cyanoborohydride (1.16 mmol) was added and the mixture was stirred
over night at room temperature. The methanol was removed under vacuo and the water
was eliminated by liophylisation. The solid obtained was purified by flash
chromatography to yield ethyl 3-methyl-5-(4-methyl-morpholin-2-ylmethoxy)-
benzofuran-2-carboxylate: m/z = 334 in MS ES+, as a white solid.
Ethyl 3-methyl-5-(4-methyl-morpholin-2-ylmethoxy)-benzofuran-2-carboxylate (0.12
mmol) was dissolved in 300 jtil of tetrahydrofuran. Then 300 u.l of a 1.0 M solution of
lithium hydroxide were added and the mixture stirred at room temperature for 3 hours.
The tetrahydrofuran was removed under vacuo and the water eliminated by
lyophiilsation to yield the tilted compound as a white solid: m/z = 304 in MS ES-.
4-f2-f1-Dimethyiamino-ethyl)-thiazol-5-vll-benzoic acid lithium salt (Example 8.38)
4-(2-Azido-acetyl)-benzoic acid methyl ester.
4-(2-Bromo-acetyl)-benzoic acid methyl ester (15.5mmol) was dissolved in ethanol
(120ml) and acetic acid (4.8ml). Sodium azide (31 mmol) was added and the reaction
stirred at 4°C overnight. The ethanol was removed and the mixture diluted with ethyl
acetate (100ml). The organic layer was washed with saturated sodium hydrogen
carbonate (2x50ml) and dried (MgS04). The solvent was removed in vacuo to give a
yellow solid, which was re-crystallized from ethanol to give the title compound as a pale
yellow solid (2.6g). IR 2117cm"1
4-(2-Amino-acetyl)-benzoic acid methyl ester hydrochloride .
4-(2-Azido-acetyl)-benzoic acid methyl ester_(6.53 mmol) was suspended in methanol
(30ml) and aqueous hydrochloric acid (6.53 mmol, 1M) was added. A catalytic amount
of palladium on carbon (10% wt) was added and the reaction stirred over an

atmosphere of hydrogen for 3h. The reaction was filtered and the solvent removed in
vacuo to give the title compound (1.3g) as a yellow solid m/z = 194 in MS ES+, which
was used in the next step without purification.
4-^-[2-(S)-(tert-Butoxycarbonyl-methyl-amino)-propionylamino]-acetyl}-benzoic acid
methyl ester.
4-(2iAmJno-aoetyl)-benzoic acid methyl ester hydrochloride (2.22mmol), WSC.HCI
(2.44mmol), Boc-N-methyl-(L.)-alanine (2.44mmol) and HOBt (2.77mmol) were
suspended in dichloromethane (10ml). NMM (2.44mmol) was added and the reaction
stirred for 2h. The reaction was diluted with ethyl acetate (50ml) and washed with 10%
citric acid (2x25ml) and saturated sodium hydrogen carbonate (2x25ml). The organic
layer was dried (MgSCX») and the solvent removed in vacuo to give a brown oil residue.
Purification by silica chromatography eluting with 10-50% ethyl acetate/ iso-hexane
gave the title compound (620mg) as a pale yellow oil m/z = 379 in MS ES+.
4-{2-(SH1 -(tert-Butoxycarbonyl-methyl-am ino)-ethyl]-thiazol-5-yl}-benzoic acid methyl
ester.
4-{2-[2-(S)-(tert-Butoxycarbonyl-methyl-amino)-propionylamino]-acetyl}-benzoicacid
methyl ester (1.65mmol) was dissolved in dry THF and Lawesson's reagent (2.5mmol)
was added. The reaction was heated at reflux for 5h and cooled to room temperature.
The solvent was removed in vacuo and the residue was dissolved in ethyl acetate
(100ml). The organic layer was washed with 10% citric acid (2x50ml) and saturated
sodium hydrogen carbonate (2x50ml) and dried (MgS04). The solvent was removed in
vacuo to give a yellow oil residue, which was purified by silica chromatography to give
the title compound (570mg) as a pale yellow solid. 1H NMR (CDCI3l 400MHz) 1.5(s, 9H),
1.6(d, 7Hz), 2.8 (brs, 3H), 3.9(s, 3H), 5.6(brm, 1H), 7.6(m, 2H), 7.9(s, 1H), 8.0(m, 2H).
4-{2-(SH1 -(tert-Butoxycarbonyl-methyl-am ino)-ethyl]-thiazol-5-yl}-benzoic acid .
4-{2-(S)-[1-(tert-Butoxycarbonyl-methyl-amino)-ethyl]-thiazol-5-yl}-benzoic acid methyl
ester (0.75mmol) was dissolved in methanol (10ml) and lithium hydroxide (10ml, 1M)
was added. The reaction was stirred at room temperature overnight and the methanol
removed in vacuo. The aqueous solution was taken to pH=3 with 1M hydrochloric acid
and extracted with ethyl acetate (2x50ml). The organic layer was dried (MgS04) and the
solvent removed in vacuo to give an off-white powder, which was purified by silica

chromatography eluting with 50-80% ethyl acetate/ iso-hexane. The title compound was
obtained as a white powder (252mg) m/z = 363 in MS ES+.
4-|2- 4-{2-(SH1-(tert-Butoxycarbonyl-methyl-amino)-ethyl]-thiazol-5-yl}-ben2oicacid
(0.75mmo!) was dissolved in 50% TFA/ DCM (2ml) and stirred for 1h. The solvent was
removed in vacuo and the residue placed under high vacuum for 3h to give a light
brown oil residue. The residue was dissolved in methanol (2ml) and buffer (1ml, 1M
sodium acetate/ acetic acid, pH=5.5) was added. Formaldehyde (0.83mmol, 37wt% in
water) was added and the reaction stirred for 30 minutes. Sodium cyanoborohydride
(0.83mmol) was added and the reaction stirred overnight at room temperature. The
methanol was removed in vacuo and the aqueous diluted with saturated sodium
hydrogen carbonate (25ml). The aqueous layer was extracted with ethyl acetate
(2x25ml) and the organic layer dried (MgSO-i). The solvent was removed in vacuo and
the residue purified by silica chromatography to give the title compound (158mg) as an
off-white solid 1H NMR (CD3OD, 400MHz) 1.5(d, J 7Hz), 2.3(s, 6H), 3.9(s, 3H), 3.95(q,
J 7Hz), 7.7(m, 2H), 8.0(m, 3H).
4-(2-(1-Dimethylamino-ethyl)-thiazol-5-yl]-benzoic acid lithium salt.
4-{2-(S)-[1-(tsrt-Butoxycarbonyl-methyl-amino)-ethyl]-thiazol-5-yl}-benzoicacid
(0.54mmol) was dissolved in methanol (2ml) and lithium hydroxide (0.54mmol, 1M) was
added. The reaction was stirred overnight and the methanol removed in vacuo. The
residue was diluted with water (5ml) and the aqueous layer extracted with ethyl acetate.
The aqueous layer was freeze-dried to give the title compound as an off-white solid
(143mg) which was used in the next step without further purification m/z = 277 in MS
ES+.
5-N.N-Dimethylamino-1H-indole-2-carboxylicacid (Example 8.39)
5-Amino-1H-indole-2-carboxyIic acid ethyl ester.
5-Nitro-1 H-indole-2-carboxylic acid ethyl ester (14.9 mmol) was suspended in acetone
(50 ml) and added to a mixture of titanium(lll) chloride (91ml, >10% in 2M hydrochloric
acid) and ammonium acetate (265ml, 4M). The reaction was stirred for 2h and
neutralized with saturated sodium hydrogen carbonate. The mixture was extracted with
ethyl acetate (100ml) and the organic layer dried (MgS04). The solvent was removed in

vacuo to give a light brown solid which was purified by silica chromatography to give the
title compound as an off-white solid (1.57g) m/z = 205 in MS ES+.
5-N,N-Dimethylamino-1 H-indole-2-carboxylic acid ethyl ester.
5-Amino-1H-indole-2-carboxylic acid ethyl ester (7.7mmol) was dissolved in acetonitrile
(30ml) and formaldehyde (19.2mmol, 37%wt in water) was added. Sodium
cyanoborohydride (7.7m mol) was added and the reaction stirred at room temperature
overnight. The acetonitrile was removed in vacuo and the residue was purified by silica
chromatography to give the title compound was a pale yellow solid (244mg). m/z = 233
in MS ES+.
&-N,N-Dimethylamino-1 H-indole-2-carboxylic acid
5-N,N-Dimethylamino-1 H-indole-2-carboxylic acid ethyl ester (1.05mmol) was
suspended in ethanol (1ml) and lithium hydroxide (1.2ml, 1M in water) was added. The
reaction was stirred at room temperature overnight. The solution was taken to pH=7
with 1M hydrochloric acid and the ethanol removed in vacuo. The aqueous layer was
extracted with ethyl acetate and the organic layer dried (MgS04). The ethyl acetate was
removed in vacuo to give the title compound as a yellow powder (75mg), which was
used in the next step without purification, m/z = 205 in MS ES+.
442-ri-ftert-Butoxvcarbonyl-methvl-aminoVethvll-thiazol-4-yl}-benzoicacid (Example
840)
Boc-L-NMe-Alanine-OH (1.0g, 4.92mmols) was dissolved in dioxan (1 Qmls) and to this
was added pyridine (0.25mls), di-tert-butyl dicarbonate (1.4g, 6.4mmols) and
ammonium hydrogen carbonate (0.49g, 6.2mmo!s). After stirring for 18 hours the crude
reaction mixture was concentrated in vacuo and re-suspended in ethyl acetate. This
was washed with 1M KHS04 and the organic layer dried over magnesium sulphate.
After concentration a clear oil was obtained (0.79g). This was dissolved in ethylene
glycol dimethyl ether (10mls) and to this was added Lawesson's reagent (4.31mmols,
1.74g). After stirring at room temperature for 3 hours the reaction mixture was
concentrated in vacuo and the residue re-suspended in ethyl acetate. This was washed
with 1M Na2C(>3 and the organic layer dried over magnesium sulphate. After
concentration a yellow oil was obtained. This was purified by flash chromatography
(heptane/ethyl acetate) to give a white solid (0.73g). This was dissolved in ethanol

(10mls) and 4-(2-Bromo-acetyl)-benzoic acid methyl ester (3.34mmols, 0.86g) was
added. The reaction was heated to 50°C for one hour. The crude product was purified
by flash chromatography
(heptane/ethyl acetate) to give a white solid (0.39g). ESMS (M + H = 377.23) which was
subsequently hydrolysed to the corresponding acid.
442-n-Dimethvlamino-2-methoxv-ethvlV-thia2ol-4-yn-benzolc acid (Example 8.41)
Boc-L-Serine(OMe)-OH (2.4g, 6.0mmols) was dissolved In dioxan (20mls) and to this
was added pyridine (0.31 mis), di-tert-butyl dicarbonate (1.7g, 7.8mmols) and
ammonium hydrogen carbonate (0.62g, 7.2mmols). After stirring for 18 hours the crude
reaction mixture was concentrated In vacuo and re-suspended in ethyl acetate. This
was washed with 1M KHSO4 and the organic layer dried over magnesium sulphate.
After concentration the crude product was purified by flash chromatography to yield
0.55g of a clear oil. This was dissolved in ethylene glycol dimethyl ether (20mls) and to
this was added Lawesson's reagent (2.78mmols). After stirring at room temperature for
3 hours the reaction mixture was concentrated in vacuo and the residue purified by flash
chromatography (silica gel, DCM) to give a yellow oil (2-Methoxy-1-thiocarbamoyl-
ethyl)-carbamic acid tert-butyl ester (0.49g).
The ester (0.25g, 1.07mmols) was dissolved in ethanol (10mls) and 4-(2-Bromo-acetyl>-
benzoic acid methyl ester (1.18mmols, 0.30g) was added. The reaction was heated to
50°C for one hour. The crude product was purified by preparative HPLC (MeCN/H20) to
yield 0.138g of a yellow solid. The Boc group was removed via treatment with 4M
HCI/dioxan for one hour after which time the reaction mixture was concentrated in
vacuo. The free amine (0.093g, 0.265mmols) was then dimethylated. The crude HCI
salt was dissolved in 5mls of methanol and buffered with 2.5mls pH 5.5 Sodium
acetate/acetic acid. Formaldehyde was added (0.58mmols) and the reaction stirred for
one hour. Sodium cyanoborohydride was then added (0.58mmols, 0.036g) and the
reaction stirred for a further thirty minutes. The reaction mixture was concentrated in
vacuo and purified by preparative HPLC to yield 60mg of a yellow solid. Finally, the
methyl ester was hydrolysed with 1M LiOH (5mls) and dioxan (5mls) at room
temperature for two hours. The reaction mixture was concentrated in vacuo and
lyophilised from water to yield 62mg of the desired acid as the lithium salt ESMS (M +
H = 307.04)

4-f2-f4-Fluoro-1^tiethvl 4-Fluoro-2-[4-(4-*riethoxycarbon^ acid tert-
butyi ester (0.1g) was treated with 4M HCI/dioxan (10mls) for 2 hours. The reaction
mixture was then concentrated in vacuo and lyophilised from water to yield a yellow
solid (0.8g). The crude HCI salt was dissolved in 5mls of methanol and buffered with
2.5mls pH 5.5 Sodium acetate/acetic acid. Formaldehyde was added (O.SSmmols,
0.0032mls) and the reaction stirred for one hour. Sodium cyanoborohydride was then
added (0.38mmols, 0.024g) and the reaction stirred for a further thirty minutes. The
reaction mixture was concentrated in vacuo and the residue re-suspended in ethyl
acetate. This was washed with 1M NaaCOaand the organic layer dried over magnesium
sulphate. After concentration a yellow solid was obtained (0.075g). Finally, the methyl
ester was hydrolysed with 1M LiOH (5mls) and dioxan (5mls) at room temperature for
two hours. The reaction mixture was concentrated in vacuo and lyophilised from water
to yield 62mg of the desired acid as the lithium salt ESMS (M + H = 306.88)
2-f4-f4-Carboxv-ohenyl)-thia2ol-2-vn-4-fluoro-Dyrrolidine-1 -carboxylic acid (Example
8.43)
2-i4-(4-Carboxv ester The amide (0.32g, 1.37mmols) was dissolved in ethylene glycol dimethyl ether
(10mls) and to this was added Lawesson's reagent (0.61g, 1.5mmols). After stirring at
room temperature for 3 hours the reaction mixture was concentrated in vacuo and the
residue re-suspended in ethyl acetate. This was washed with 1M Na2C03and the
organic layer dried over magnesium sulphate. After concentration a yellow oil was
obtained. This was purified by flash chromatography (heptane/ethyl acetate) to give a
white solid (0.36g). This was dissolved in ethanol (10mls) and 4-(2-Bromo-acetyl)-
benzoic acid methyl ester (0.41 g ,1.59mmols) was added. The reaction was heated to
50°C for one hour. The crude product was purified by flash chromatography
(heptane/ethyl acetate) to give a white solid (0.34g). The methyl ester was then treated
with 1M LiOH (10mls) and dioxan (10mls) for 3 hours. After quantitative hydrolysis, the
crude product was concentrated in vacuo and the residue re-suspended in ethyl
acetate. This was washed with 1M KHS04 and the organic layer dried over magnesium
sulphate. After concentration In vacuo the product was lyophilised from
acetonitrile/water to yield the title compound as a white solid (0.32g). ESMS (M + H =
393.03).

Lithium 5-f4-N-methylmorDhollno-2S-methvloxv)benzofuran carboxvlate (Example 8.36
as single enantiomers)
Ethyl 5-(4-boo-morpholino-2R-methyloxy)benzofuran carboxylate.
4-Boc»2R-hydroxymethylmorpholine was prepared according to method described in
Heterocycles, 1993 35,105-109. To a mixture of polymer supported triphenylphosphine
(2.4mmol) and thehydroxymethylmorpholine (1.2mmol)in dry dichloromethane (5
ml) ethyl-5-hydroxybenzofuran-2-carboxylate (1.2 mmol) and DIAD (1.2 mmol) was
added at room temperature. The mixture was stirred further 16 hours, titrated, diluted in
dry dichloromethane (5 ml) and stirred at room temperature another 16 hours.
Filtrated, concentrated In vacuo and purified by y flash chromatography on silica
(ethylacetate, hexane)to yield ethyl-5-(4-bcc-morpholino-2R-methyloxy)benzofuran
carboxylate (0.3 mmol), m/z = 406 in MS ES+, as an oil.
Lithium 5-(4-N-methylmorpholino-2R-methyloxy)benzofuran carboxylate.
Ethyl-5-(4-boc-morpholino-2R-methyloxy)benzofuran carboxylate (0.3 mmol) was
dissolved in HCI in dioxane (4M, 15 ml), stirred at room temperature for 4 hours,
concentrated in vacuo to a pale yellow oil. The crude benzofuran hydrochloride (0.3
mmol) and formaldehyde (0.35 mmol) were mixed in THF (5 mL) and dibutyltin
dichloride (0.05 mmol) was added. After stirring at RT for 5 minutes, phenylsilane (0.6
mmol) was added and the reaction allowed to stir at room temperature for a further 17
h. The reaction was then concentrated in vacuo and the residue purified by flash
chromatography (silica gel, ethyl acetate, isopropanol, triethylamine) to give ethyl-5-(4-
N-methylmorpholino-2R-methyloxy)benzofuran carboxylate: m/z = 320 in MS ES+ as a
clear oil.
To ethyl-5-(4-N-nr>ethylmorpholino-2R-methyloxy)benzofuran carboxylate (0.6 mmol) In
5 ml dioxane was added LiOH (0.6 mmol) in 1 ml of water. The mixture was refluxed for
16 hours, concentrated In vacuo to give lithium 5-(4-N-methylmorpholino-2R-
methy!oxy)benzofuran carboxylate: m/z 292 in MS ES+ as a white solid.
Lithium 5-(4-N-methylmorpholino-2S-methyloxy)benzofuran carboxylate.
S-isomen m/z 292 in MS ES+ was prepared as a white solid white solid according to
method used to prepare R-isomer but substituting 4-Boc-2R-hydroxymethylmorpholine
by 4-Boc-2S-JTydroxymethylmorpholine.

4^3-Methvl-5-morpholino^vlmethyl-thiophen-2-vlVbenzoic add (Example 8.44)
5-Bromo-4-methyl-thiophene-2-carboxylic acid methyl ester (8.51 mmol) was dissolved
in EtOH (100ml) and NaOH (42.5 mmol) added, as a 1M solution in water. Reaction
was heated to 80°C for 2h, after which time all starting material had been consumed.
Reaction was then concentrated in vacuo and the residue taken up in DCM and shaken
with 1M HCI. The resulting triphasic mixture was then filtered and the filtrant washed
with hexane and dried under vacuum. This gave 5-Bromo-4-methyl-thiophene-2-
carboxylic acid as off white solid: m/z in MS AP- = 219,221 [M-H]', 6.79 mmol, 80%.
5-Bromo-4-methyl-thiophene-2-carboxylic acid (6.79 mmol) was taken up in 30ml DMF
and morpholine (747 mmol), WSC.HCI (7.47 mmol) and HOBt (7.47 mmol) were
added. Reaction was stirred at room temperature for17h and then diluted with EtOAc,
washed with 1M HCI and brine, dried over Na2S04 and concentrated In vacuo. Rash
chromatography of the residue (silica, 33-50% EtOAc in hexane) gave (5-Bromo-4-
methyi-thiophen-2-yl)-morpholln-4-yl-methanone as a pale golden oil: m/z in MS ES+ =
290, 292 [M+HT, 4.67mmol, 69%.
(5^Bromo-4-methyl-thiophen-2-yl)-morpholin-4-yi-methanone (1.78 mmol) was added to
a flask containing 1M THF.BH3 complex in THF (4.45mmol). Reaction was stirred at
reflux for 2.5h under N2. Methanol was then added until gas evolution ceased, followed
by 10ml of 1M NaOH and the reaction stirred at reflux for a further 7h. The mixture was
cooled to room temperature and extracted with EtOAc. This extract was concentrated in
vacuo and the residue taken up in 1M HCI and washed with EtOAc. The acid layer was
then basified with 1M NaOH and extracted back into EtOAc. Removal of solvent gave 4-
(5-Brorno-4-methyl-thiophen-2-ylmethyl)-morpholine as a colourless oil: m/z in MS ES+
= 276,278 IM+Hf, 0.98 mmol, 55%.
4-(5-Bromo-4-methyl-thiophen-2-ylmethyl)-morpholine (0.98mmol) was taken up in 10ml
toluene and 4-carboxymethyiphenylboronic acid (0.98mmol) was added as a solution in
1ml of EtOH. 6ml of 2M aqueous Na2003 solution was added, followed by Pd(PPh3)4
(0.098mmol). Reaction was stirred at 70°C for 17h under a nitrogen atmosphere and
then cooled to room temperature and extracted with DCM (x2). Combined organic
layers were washed with brine, concentrated in vacuo and the residue purified by flash
chromatography (silica, 33-99% EtOAc in hexane). This furnished the pure 4-(3-Methyl-

5-morpholi-4-ylmethyl-thiophen-2-yl)-benzoic acid methyl ester as a waxy white solid :
mfe in MS ES+ = 332 [M+H]+, 0.090mmol, 9%.
This ester (0.09mmol) was heated to 70°C in 18% Ha for 2h at which point HPLC
showed all the starting material to have been hydrolysed. The reaction was cooled and
the product that precipitated out of solution was collected by filtration as a white solid.
With no further purification this was coupled using the standard procedure.
3-Methvl-4-(5-morpholin~4-vlmethvl-furan-2-vl)-benzoic acid (Example 8.45)
A three necked flask was charged with methyl 4-bromo-3-methylbenzoate (2.18mmol),
bis(pinacolato)diboron (2.29mmol), palladium acetate (0.065mmol), potassium acetate
(6.54mmol) and DMF (10ml). The solution was degassed by bubbling through N2 gas
for 30mins and was then heated to 80% under isfe for 3h. Reaction was then cooled to
room temperature and 4-(5-Bromo-furan-2-ylmethyl)-morphollne (2.18mmol), cesium
carbonate (3.27mmol) and Pd(PPh3)4 (0.065mmol) added. The reaction was heated to
80°C and stirred for a further 17h. Mixture was then diluted with EtOAc and water and
filtered through a celite pad to remove black particulates. The organic layer was
separated, washed with brine and dried over Na2S04 and concentrated in vacuo. Rash
chromatography of the residue (silica, 10-99% EtOAc in hexane) gave 3-Methyl-4-(5-
morpholin-4-ylmethyl-furan-2-yl)-benzoic acid methyl ester as a grey powdery solid: m/z
in MS ES+ a 316 [M+Hf, 0.51 mmol, 23%.
This ester (0.51mmol) was heated to 70°C in 18% HCI for 2h at which point HPLC
showed all the starting material to have been hydrolysed. The reaction was cooled and
the product that precipitated out of solution was collected by filtration as a white solid.
With no further purification this was coupled using the standard procedure.
4-f5-Methvl-2-(4-rn9tf>yl-piDerazin-1-vl)-thiazol-4-yn-benzoic acid (Example 8.46)
4-propionylbenzoicacid (890 mg, 5 mmol), NaHC03 (1.26 g, 15 mmol), and
iodomethane (935 uL, 15 mmol) in DMF (10 ml_) were stirred at RT overnight. The
mixture was diluted with saturated aqueous NaCI (50 mL) and extracted with ether (3 x
50 mL). The organic phase was washed with water (50 mL), dried, and evaporated.
Flash chromatography (90 g silica, 2/1 petroleum ether- EtOAc) gave white solids of 4-
Propionyl-benzoic acid methyl ester (744 mg, 77%).

1H NMR (CDCI3> 400MHz) 6 1.24 (t, 3H, J = 7 Hz), 3.03 (q, 2H, J = 7 Hz), 3.95 (s, 3H),
8.0 and 8.12 (ABq, 4H)
4-Propionyl-benzoic acid methyl ester (744 mg, 3.87 mmol), pyrrolidone
hydrotribromide (1.98 g), and 2-pynrolidinone (380 mg, 4.5 mmol) in THF (38 mL) were
heated at 50 °C under nitrogen for 3 h. The mixture was cooled, filtered, concentrated,
and then redissolved in ether (50 mL). The ether solution was washed successively with
water (20 mL), saturated aqueous sodium thiosulphate (20 mL), saturated aqueous
NaCI (20 mL), and water (20mL), dried and evaporated to give crude 4-(2-Bromo-
propionyl)-benzoic acid methyl ester as a yellow oil (1.025 g) that was used directly in
the Hanizsch coupling. This material contained 91% of the desired bromoketone, 5%
starting material, and 4% 4-bromo-1-butanol, as determined by 1H NMR.
1H NMR (CDCI3l 400MHz) 51.92 (d, 3H, J = 7 Hz), 3.96 (s, 3H), 5.28 (q, 1H, J = 7 Hz),
8.07 and 8.14 (ABq, 4H)
All of the 4-(2-Bromo-propionyl)-benzoic acid methyl ester above and piperazine-1-
carboxylic acid /erf-butyl ester (J. Med. Chem., 1998,5037-5054, 917 mg, 3.73 mmol)
were refluxed in 36 mL THF at 70 °C for 2 h, under N2. The precipitate was filtered and
the filtrate evaporated to give yellow solids. Flash column chromatography (silica, 5/1
petroleum ether- EtOAc) gave 624 mg of 4-[4-(4-Methoxycarbonyl-phenyl)-5-rnethyl-
thiazol-2-y!]-piperazine-1 -ca
rboxylic acid tert-butyl ester as a light yellow solid. Chromatography of the precipitate
(silica, 2/1 petroleum ether - EtOAc) gave 32 mg more of compound. Total yield is 44%.
1H NMR (CDCI3,400MHz) 81.46 (s, 9H), 2.43 (s, 3H), 3.42, (m, 4H), 3.54 (m, 4H), 3.90
(s, 3H), 7.68 and 8.04 (ABq, 4H).
The above methyl ester (564 mg, 1.35 mmol) was heated with 1.35 mL 2N NaOH, 5 mL
THF, and 3.65 mL water at 60 °C for 4 h. The reaction mixture was evaporated, poured
into 20 mL saturated aqueous NaCI and 20 mL CH2CI2, and then acidified to pH 3 with
5% citric acid, in an ice bath. The layers were separated and the organic phase was

extracted farther with 2 x 10 mL CH2CI2. The organic phases were combined, washed
with water (10 mL), dried, and evaporated to give 4-[4-(4-Carboxy-phenyl)-5-methyl-
thiazol-2-yl]-piperazine-1-carboxylic acid tort-butyl ester as a light yellow solid (537 mg,
98%).
1H NMR (CDQIg, 400MHz) 8 1.48 (s, 9H), 2.47 (s, 3H), 3.47 (m, 4H), 3.57 (m, 4H), 7.74
and 8.12 (ABq,4H).
13C NMR (CDCI3> 100MHz) 5 ppm: 12.6, 28.3, 42.8, 48.1, 80.3, 119.1, 127.8, 128.2,
130.1,140.5,145.6,154.6,167.2,171.4.
LCMS: (M + H)+ 404, (M - HV 402.
4-[4^4- acid tert-butyl ester (0.421 mmoi) was dissolved In 4M HCI In 1,4-dioxane, and stirred
at room temperature for 1 h. The solvent was then removed under vacuum, and the
residue 4-(5-Methyl-2-piperazin-1-yl-thiazol-4-yl)-benzoic acid was suspended in
methanol (10 ml) and treated with AcOH/AcONa buffer (pH ~5.5, 5 ml), and
formaldehyde (0.547 mmol). The reaction mixture was stirred at room temperature for 1
h, then treated with NaCNBH3 (0.547 mmol) and stirred at room temperature overnight
The solvent was then removed under vacuum, and the residue was purified by column
chromatography to afford the title compound (0.403 mmol, 95%). MS(ES) m/z 318
(100%, [M+Hf).
4-f2-Moroholin-4-vl-thiazol-4-vlVben20ic acid (Example 8.471
4-(2-bromoacetyl)benzoicacld (1.23 mmol) and 1-morpholinethlocarboxamide (1.23
mmol, J. Med. Chem 1998,41, 5037-5054) were mixed in THF (10 mL), then refluxed
for 3.5 h. The reaction mixture was then allowed to reach room temperature and the
obtained precipitate was collected by filtration and washed with 4 portions of diethyl
ether. The crude product was crystallized from hot 1:1 EtOH-EtOAc to give a first
harvest of colorless needles (0.16 g, 0.55 mmol). 1H NMR (DMSO-Oe, 400 MHz) 5 7.94
(4H, m), 7.49 (1H, s). 3.72 (4H, m), 3.44 (4H, m).

4-(2-Piperidin-1-vl-thiazol-4-vl^-ben7oic add (Example 8.48)
4-(2-bromoacetyl)benzoic acid (1.23 mmoi) and 1-piperidlnethlocarboxamlde (1.23
mmol) were mixed in THF (10 mL), then refluxed for 3 h. The reaction mixture was then
allowed to reach room temperature and the obtained precipitate was collected by
filtration and washed with 3 portions of diethyl ether. The crude product was crystallized
from hot 1:1 EtOH-EtOAc to give a first harvest of colorless needles (0.28 g, 0.95
mmol). 1H NMR (DMSO-de, 400 MHz,) 87.93 (4H, m), 7.40 (1H, s), 3.48 (4H, m), 1.60
(6H, m).
4-(2-Dlmethylamlno-thlazol-4-Yt)-benzqlc add (Example 8.49)
To a stirred mixture of thiocarbonyldiimidazole (44.9 mmol) in THF (40 mL) at room
temperature was added portionwise 2 M Dimethylamine in THF (44 mmoi) and a
temperature increase was observed. 40 min after final addition the reaction mixture was
heated to 55 °C for 1 h, then allowed to reach room temperature again. The reaction
was then concentrated in vacuo and the residue purified by flash chromatography (silica
gel, Petroleum ether-EtOAc) to give the intermediate lmidazole-1-carbothioic acid
dimethylamide. This material was treated with freshly prepared sat ammonia in
methanol (40 ml) for 60 h, then concentrated in vacuo and the precipitated residue was
suspended in diethyl ether and collected by filtration. The precipitate was washed with
diethyl ether and air-dried to give a slight yellow solid (1.71 g, 16.4 mmol) which was
used in the subsequent step. 4-{2-bromoacetyl)benzoic acid (1.23 mmol) and 1-
piperidinethiocarboxamide (1.23 mmol) were mixed in THF (10 mL), then refluxed for 3
h. The reaction mixture was then allowed to reach room temperature and the obtained
precipitate was collected by filtration and washed with 3 portions of diethyl ether. The
crude product was crystallized from hot 1:1 EtOH-EtOAc to give a first harvest of
colorless needles (0.1 g, 0.40 mmol). 1H NMR (DMSO-d6,400 MHz) 5 7.94 (4H, m),
7.37 (1H,s), 3.11 (6H,m).
4-r2-(lsoDropvl-methvl-amino)-5-metrivl-thiazol-4-vn-benzoic acid (Example 8.50)
To a solution of 4-propionylbenzoic acid (11.2 mmol), benzyl alcohol (1.1 mL,10.7mmol)
and dimethylamlnopyridlne (0.14 g, 1.1 mmol) in dichloromethane (90 ml) at 0 °C was
added N-Ethyi-N'-(3-dimethylaminopropyl)carbodiimide x HCI (2.4 g, 12.3 mmol), then
stirred at room temperature overnight. The obtained solution was then diluted with

DCM, washed successively with aq. 10% citric acid and aq. sat sodium hydrogen
carbonate, then dried (Na2S04), filtered and concentrated In vacuo. Rash
chromatography of the residue (silica gel, Petroleum ether-EtOAc) gave a colorless oil
which crystallized upon standing (2.67 g). A portion of the benzyl ester from above (1 g,
3.73 mmol) was refluxed with 2-pyrrolidlnone (0.37 g, 4.33 mmol) and pyrrolidone
hydrotribromide (1.85 g, 3.73 mmol) in THF for 1.5 h. The resulting reaction mixture was
allowed to reach room temperature, then diluted with EtOAc, washed successively with
water, aq. 10% sodium thiosulphate, aq. sat sodium hydrogen carbonate and brine,
then dried (Na2S04), filtered and concentrated in vacuo. The obtained bromide from
above was directly mixed with isopropyrthiocarboxamide (0.44 g, 3.73 mmol) in THF (20
ml_) and refluxed overnight, then concentrated onto silica. Flash chromatography of the
residue (silica gel, Petroleum ether-EtCAc-EklM) gave a light red oil (1.28g, 3.49 mmol).
To a stirred solution of the thiazole derivative (0.250 g, 0.68 mmol), obtained above, in
acetonitrile (7 ml_), acetic acid (1.3 mL) and aq. 37% formaldehyde (2 ml_) at 0 °C was
added sodium cyanoborohydride (0.09 g), then stirred at room temperature overnight
Additional sodium cyanoborohydride (0.08 g) was added, and after stirring for additional
2 h, the reaction mixture was diluted with water, neutralized using aq. 0.5 M sodium
carbonate, then extracted win dichloromethane. The dichloromethane layers were
collected, dried (Na2S04), filtered and concentrated. Rash chromatography (silica gel,
Petroleum ether-EtOAc) of the residue gave a slight yellow crystalline solid (0.115 g).
1H NMR (CDCI3s 400 MHz,) 8 8.09 (2H, d), 7.72 (2H, d), 7.27-7.32 (5H, m), 5.38 (2H, s),
4.27 (1H, m), 2.92 (3H, s), 2.42 (3H, s), 1.22 (6H, d). The benzyl ester from above (0.25
g, 0.66 mmol) was hydrolysed by treating with aq. 1M LiOH (1.3 mL) in THF (2 mL) at
60 °C overnight. The obtained solution was then made slight acidic with aq. 10% citric
acid and then extracted using dichloromethane. The organic layer was then dried
(Na2S04), filtered and concentrated. Column chromatography of the residue (Silica gel,
dichloromethane-methanol) gave the title compound as a crystalline solid (0.19 g) m/z =
304 in MS ES+, which was characterised by hplc and MS.
4-(2-Methvlamino-thiazol-4-vn-benzoic acid (Example 8.5H
To 25 ml of ethanol were added 4-(2-bromoacetyl)benzoic acid (486 mg, 2 mmole) and
N-methyi thiourea (180 mg, 2 mmole). The reaction mixture was refluxed for 3 hr and
the TLC showed the disappearing of the starting materials and the formation of a
fluorescent product The reaction was cooled on ice. The product was collected on

filtration and washed with ethanol pre-cooled to 0 °C twice (2 x 3ml), followed by
diethyl ether. After drying, 486 mg product was obtained. 1H NMR (DMSO-de,400 MHz)
8 7.97 (2H, d), 7.89 (2H, d), 7.32 (1H, s), 2.96 (3H, s).
442^4.4-Difluoro-oiperidln-1-vl)-thiazol-4-vn-benzoicadd (Example 8.52^
4,4-difluoropiperidine (hydrochloride salt 1-57 g, 10 mmole) and diisopropylethylamine
(1.74 ml, 10 mmole) In acetone (10 ml) was slowly dropped into a mixture of
ethoxycarbonylisothiocyanate (1.02 ml, 10 mmole) in aceton (10 ml) at 0 °C. When
the addition was completed, the reaction was kept under stirring at room temperature
for one hour. 3N hydrochloric acid (15 ml) was added and the reaction mixture was
extracted with ethyl acetate. The organic phase was concentrated In vacuo.
To the residue was added concentrated hydrochloric acid (20 ml) and the reaction was
kept at 80 °C for 5 hours. Water (30 ml) was added to the reaction. After the
neutralization with ammonium carbonate, the reaction mixture was extracted with ethyl
acetate. The organic phase was washed with water and dried in vacuo to obtain the
crude intermediate 4,4-Drfluoro-piperidine-1-carbothioicacid amide (1.21 g). To the
residue from above (360 mg, 2 mmole) and 4-(2-bromoacetyf)benzoic acid (486 mg,
2mmole) in THF (20 ml) were refluxed for 5 hours. TLC showed the disappearing of
the starting materials and the formation of a fluorescent product. The reaction was
cooled on tee. The solid was collected by filtration. The product was recrystallized from
ethanol (380mg). 1H NMR (DMSO-da, 400 MHz) 5 7.96 (4H, m), 7.51 (1H, s), 3.66 (4H,
m) 2.12 (4H, m).
Yields of the following title compounds in examples 8.53-8.61 and 8.63 were in general
between 30 and 90%.
4-(2-isopropvlamino-thiazol-4-ylVbenzolc acid (Example 8.53)
Isopropyl-thiourea (2.47 mmol) and 4-(2-Bromo-acetyl)-benzoic acid (2.47 mmol) were
mixed in THF (12 mL). After stirring at room temperature for 5 minutes the mixture was
heated to 80 °C for 2 hours. The volume was reduced to 5 mL and the mixture was then
cooled to -20 °C and filtered. The solid was washed with a small amount of diethylether

and dried, m/z = 263.1 in MS ES+, which was characterized by hplc and MS and used
in the next step without any further purification.
3-f2-(4-Methvl-Diperazin-1-vlVthia2o^4-vn-bergoic acid (Example 8.54)
4-Methyl-piperazine-1-carbothioic acid amide (2.47 mmol) and 3-(2-Bromo-acetyl)-
benzoic acid (2.47 mmoi) were mixed in THF (12 ml_). After stirring at room temperature
for 5 minutes the mixture was heated to 80 °C for 2 hours. The mixture was then cooled
to room temperature and filtered. The solid was washed with a small amount of
diethylether and dried, m/z = 304.1 in MS ES+, which was characterized by hplc and
MS and used in the next step without any further purification.
3-(2-lsopropylamlno-thiazol^4-vl)-benzoic acid (Example 8.55)
Isopropyl-thiourea (2.47 mmol) and 3-(2-Bromo-acetyl)-benzoic acid (2.47 mmol) were
mixed in THF (12 mL). After stirring at room temperature for 5 minutes the mixture was
heated to 80 °C for 2 hours. The volume was reduced to 5 mL and the mixture was then
cooled to -20 °C and filtered. The solid was washed with a small amount of diethylether
and dried, m/z - 263.1 in MS ES+, which was characterized by hplc and MS and used
in the next step without any further purification.
4-(2-Piperidin-4-vl-th?azol-4-vl>-benzo?c acid (Example 8.56)
4-Thiocarbamoyi-piperidine-1-carboxylic acid tert-butyl ester (2.47 mmol) and 4-(2-
Bromo-acetyl)-benzoic acid (2.47 mmol) were mixed in THF (12 mL). After stirring at
room temperature for 5 minutes the mixture was heated to 80 °C for 2 hours. The
volume was reduced to 5 mL and diethylether (5 mL) was added. The mixture was then
cooled to -20 °C and filtered. The solid was washed with a small amount of diethylether
and dried, m/z = 289.1 in MS ES+, which was characterized by hplc and MS and used
in the next step without any further purification.
4-r2-f1-Methvl-piperidin-4-vn-thiazol^-vn-benzolcacid (Example 8.571
To a solution of 4-(2-PIperidin-4-yl-thiazol-4-yl)-benzoic acid (1 mmol) in acetic acid (0.5
mL), methanol (3 mL) and tetrahydrofurane (4.5 mL) was added formaldehyde (aq.
37%, 300 mL) and polystyrene bound cyanoborohydride (2.36 mmol/g, 900 mg). The
slurry was then agitated for 16 hours at room temperature. The slurry was then filtered
and the resin washed with methanol (2 mL). The solution was concentrated to dryness

in vacuo, mfz = 303.1 in MS ES+, which was characterized by hplc and MS and used in
the next step without any further purification.
4"i2-(Pyridin-3-vlaminoVthia2ol-4-vn-benzoicacid (Example 8.58)
Pyridin-3-yl-thiourea (2.06 mmol) and 4-{2-Bnomo-acetyl)-benzolc acid (2.06 mmol)
were mixed in THF (12 mL). After stirring at room temperature for 5 minutes the mixture
was heated to 80 °C for 2 hours. The mixture was then cooled to room temperature and
filtered. The solid was washed with a small amount of diethylether and dried, m/z =
298.0 In MS ES+, which was characterized by hplc and MS and used in the next step
without any further purification.
4-f2-fF>yrldln-2-vlamlno)-thiazol-4-vl1-benzoicacid (Example 8.59)
Pyridin-2-yl-thiourea (2.06 mmol) and 4- were mixed in THF (12 mL). After stirring at room temperature for 5 minutes the mixture
was heated to 80 °C for 2 hours. The mixture was then cooled to room temperature and
filtered. The solid was washed with a small amount of diethylether and dried, m/z =
298.0 in MS ES+, which was characterized by hplc and MS and used in the next step
without any further purification.
4-(2-Cvclopentvlamino-fhiazol-4»vlV-benzoic acid (Example 8.60)
Isothiocyanato-cyclopentane (4 g) in ammonia (37% in water, 8 mL) and methanol (32
mL) was stirred for 16 hours, filtered of and dried. The Cyclopentyl-thiourea (2.06 mmol)
and 4-(2-Bromo-acetyl)-benzoic acid (2.06 mmol) were mixed in THF (12 mL). After
stirring at room temperature for 5 minutes the mixture was heated to 80 °C for 2 hours.
The mixture was then cooled to room temperature and filtered. The solid was washed
with a small amount of diethylether and dried, m/z = 289.05 in MS ES+, which was
characterized by hplc and MS and used in the next step without any further purification.
4-(2-Cvclopropvlamlno-thiazol-4-vh-benzoic acid (Example 8.61)
Isothiocyanato-cyclopropane (4 g) was mixed with ammonia (37% in water, 8 mL) and
methanol (32 mL) at 0 °C and then stirred for 16 hours at room temperature. The
mixture was then cooled to 0 °C, filtered, washed with a little water and dried. The
Cyclopropyl-thiourea (2.06 mmol) and 4-(2-Bromo-acetyi)-benzoic acid (2.06 mmol)
were mixed in THF (12 mL). After stirring at room temperature for 5 minutes the mixture

was heated to 80 °C for 2 hours. The mixture was then cooled to room temperature and
filtered. The solid was washed with a small amount of diethylether and dried, m/z =
261.0 in MS ES+, which was characterized by hplc and MS and used in the next step
without any further purification.
4-f2-(CyctoDroovl-methyl-amino)-thiazol-4-Yl]-benzoic acid (Example 8.62)
4-(2-Cyclopropylamino-thiazol-4-yl)-bQnzoic acid (1.98 mmol), methyliodide (4.36 mmol)
and potassium carbonate were mixed in DMF (20 mL) and stirred for 72 hours at room
temperature. The mixture was concentrated to dryness and partitioned between
dichloromethane and water. The organic layer was dried (MgS04) and concentrated to
dryness. This solid was mixed with THF (4 mL), methanol (2 mL) and 1N LiOH (3 mmol)
and heated to 50 °C for 1 hour. The mixture was then cooled to room temperature and
1N HCI was added until pH 4. The mixture was concentrated in vacuo, then the
obtained residue was redissotved In dichloromethane-methanol and concentrated onto
silica. Flash chromatography of the residue (silica gel, dichloromethane-methanol) gave
the title compound as an off-white solid (0.1g), m/z = 275.0 in MS ES+, which was
characterised by hplc and MS.
4-f2-f1-Methvl-pvrrolidin-3-vl)-thiazol-5-vn-benzoic acid (Example 8.63)
3-Thiocarbamoyl-pyrrolidine-1-carboxylicacid tert-butyl ester (2.47 mmol) 4-(2-Bromo-
acetyl)-benzoic acid (2.47 mmol) were mixed in THF (12 mL). After stirring at room
temperature for 5 minutes the mixture was heated to 80 °C for 1 hour. The mixture was
then cooled to room temperature and filtered. The solid was washed with a small
amount of diethylether and dried, m/z = 304.1 in MS ES+. This solid was then mixed in
dichloromethane-trifluoroacetic acid (2:1) and kept at room temperature for 20 minutes.
The mixture was concentrated to near dryness and the concentrated once from
dichloromethane and once from 1 N HCI in diethylether. The remaining solid was mixed
with acetic acid (0.5 mL), methanol (3 mL) and tetrahydrofurane (4.5 mL) and
formaldehyde (aq. 37%, 300 mL) and polystyrene bound cyanoborohydride (2.36
mmol/g, 900 mg) was added. The slurry was then agitated for 16 hours at room
temperature. The slurry was then filtered and the resin washed with methanol (2 mL).
The solution was concentrated to dryness In vacuo, m/z = 289.0 in MS ES+, which was
characterized by hplc and MS and used in the next step without any further purification.

2-(1^TiethylDipei^ne-^Yl)-6-(4-carfaoxYphen-1-v mi
2.6-dibromopyridine (11.8g, 50mmol) was dissolved in dimethyHbrmamide (50ml) and 1-
methyipiperazine (5.0g, 50mmol) and sodium iodide (0.6g) was added. The solution
was heated to 80 °C for 30 minutes, then allowed to reach room temperature and
diluted with ethyl acetate and water. The water layer was carefully extracted and the
organic layers were collected, dried (Na2S04) and concentrated. Flash chromatography
of the residue on silica-gel (packed with ethyl acetate) using ethyl acetate-methanoi-
triethylamine 20:2:1 as the eluant. Pure fractions were collected and concentrated.
1.54g of the residue was dissolved in dimethoxymethane(48ml) and
tetrakistriphenylphosphine palladium(O) (5.0g) was added. The solution was degassed
and stirred for 15minutes under N2.4-ethoxycarbonyiphenylboronic acid (1.16g) was
added followed by 36ml aq. 1M sodium hydrogen carbonate solution. The solution was
degassed one more time and heated to reflux and stirred for 12hours. The solution was
filtered and the ffltercake was carefully extracted with ethyl acetate and
dimethoxymethane. The extracts were evaporated and purified by
flashchromathography on silica-gel (packed with ethyl acetate) using ethyl acetate-
methanol-triethylamine 20:2:1 as the eluant. Pure fractions were collected and
concentrated. The residue was dissolved in 30ml concentrated hydrochloric acid and
was refluxed for 12hours. The solution was evaporaded to yield the title compound as a
solid. 1H-NMR (400MHz, DMSO-d8) 8 2.7 (3H, m) 3.3 (4H, m) 4.5 (4H, m) 7.0 (1H, m)
7.4 (1H, m) 7.8 (1H, m) 8.0 (1H, m) 8.15 (1H, m) 11.5 (1H, bs).
4-(6-Moroholin-4-vl-Pvridin-2-vO-benzoic acid hydrochloride (Example 8.65)
2.6-dibromopyridine (2.0g) was dissolved in dimethoxymethane and morpholine (4.0ml)
and sodium iodide (0.3g) was added. The solution was heated to reflux for 1 hour, then
allowed to reach room temperature. The obtained solution was diluted with ethyl
acetate, washed with water, then dried (NaaSO^), filtered and concentrated. Flash
chromatography of the residue using stepwise gradient elutlon (ethyl acetate in hexane
20-33%). Pure fractions were concentrated and then subjected to Suzuki coupling as
described in example 8.64, then purified by flash chromatography as described above.
Pure fractions where collected and concentrated, then the residue was dissolved in
30ml concentrated hydrochloric acid and was refluxed for 1 hour. The obtained solution
was evaporated io yield the title compound as a solid. 1H-NMR (400MHz, DMSO-d6) 5

3.6 (4H, m) 3.7 (4H, m) 6.9 (1H, m) 7.3 (1H, m) 7.7 (1H,M) 7.9 (2H, m) 8.1 (2H, m).
Example 9
Fluid phase synthesis of l^ffSVW(3aS.^.6aS)-6-nuoro-3-oxo-hexahvdro-fijror3.2-
b1pvrrole^K»rbomri)-3-methv^
vri-bertzamide

a) [(^S)-1-((3f?l3aR,6S,6aS)^FIuoit>^4iydroxy+exahydro-furo[3l2-b]pyrrole-4-
carbonyl)-3-methyl-butyri-carbamic add benzyl ester (2')
(3/^,3a/?,6S,6aS)^-Fluoro-34iydroxy-hexahydro-furo[3)2-b]pyrrol0-4-carix)xylicacid
ferf-butyl ester (1') (20.24 mmol) was dissolved in 4M HCI in 1,4-dioxane, and stirred at
room temperature for 1 h. The solvent was then removed under vacuum, and the
residue was suspended in dichloromethane (50 ml), and treated with Cbz-Leu-OH
(20.24mmol), WSCHCI (22.26 mmol), HOBt(22.26 mmol)andNMM (40.48 mmol).
The reaction mixture was stirred at room temperature overnight, then washed with
saturated aqueous NaHC03, dried and concentrated. The residue was purified by
column chromatography (ethyl acetate-hexanes 1:1, Rf 0.23) to afford compound 2
(15.62 mmol, 77%). MS(ES) mfe 395 (100%, [M+Hf).

b) [(^^^((SaS.eS.eaS^FIuoro-S^x^hexahydro-furoP^-blpyiTole-^-carbonyl)-
3-methyl-butyl]-carbamic add benzyl ester (3')
A solution of compound 2' (15.61 mmol) in dichloromethane (70 ml) was treated with
Doss-Martin periodinone (15.61 mmol), and the reaction mixture was stirred at room
temperature overnight. The solution was then washed with saturated aqueous NaHC03,
dried and concentrated, and the residue was purified by column chromatography (ethyl
acetate-hexanes 1:1, Rf 0.37) to afford compound 3 (9.38 mmol, 60%). MS(ES) m/z 393
(15%, [M+HF), 411 (100%, [MH+HzOD.
c) [(^S)-1-((3aS,6S,6aS)^FIuoro^,3Klimethoxy4iexahydro-furo[3,2-b]pyrT0le^4-
carbonyl>3-methyl-butyl]-carbamic acid benzyl ester (4')
A solution of compound 3' (8.49 mmol) in anhydrous methanol (50 ml) was treated with
1,1,2-trimethylorthoformate (24 ml) and p-TsOH (catalytic amount), and stirred at 60 °C
for 3 h. The reaction mixture was then cooled down to room temperature, the solvent
was removed under vacuum and the residue was purified by column chromatography to
afford compound 4' (7.42 mmol, 87%). MS(ES) m/z 439 (100%, [M+Hf).
d) N^(fS)-1-((3aS,eS,6aS)^FIuoro-3,3 carbonyl)-3-methyl-butyn^[5-methyl-2-(4-methyli)iperazin-1-yO-thiazol^yQ-
benzamide (5')
Compound 4" (0.625 mmol) was dissolved in ethanol (15 ml), and treated with a
catalytic amount of Pd (10% wt Pd in carbon). The reaction was stirred under hydrogen
atmosphere for 3-4 h. The reaction mixture was then filtered through a celite cake, and
the cake was washed with ethanol, the organic extracts were combined and
concentrated under vaccum. The residue was then dissolved in dichloromethane (15
ml) and treated with 4-[5-Methyl-2-(4-methyl-plperazln-1-yl)-thiazol-4-yl]-benzoic acid
(0.60 mmol), WSCHCI (0.625 mmol) and HOBt (0.625 mmol). The reaction mixture was
monitored by HPLC. When the reaction had finished (4 h) the organic solution was
washed with saturated aqueous NaHC03, dried and concentrated, and the residue was
purified by preparative HPLC to afford compound 5 (0.30 mmol, 50%). MS(ES) m/z 604
(100% [M+H]*).

e) N-[(fS)-1-((3aS,0S,6aS)^Ruon> carlx)nyl)-3-methyl^utyl]^[5-methyl-2^4-n^
benzamide (6')
Compound 5' (0.21 mmol) was dissolved in neat trifluoroacetic acid (2 ml) and stirred at
room temperature. The reaction was monitored closely by HPLC, to avoid cleaveage of
the tertiary amide. As soon as the starting material disappeared (3 h 45 min) the TFA
was removed under a stream of nitrogen, and the residue was partitioned between ethyl
acetate and saturated aqueous NaHCOa, the organic extracts were dried and
concentrated under vacuum, and the residue was dissolved in acetonitrile-water 1:1 (2
ml) and freeze-dried overnight to afford compound 6' as a white solid (0.20 mmol, 94%).
MS(ES) m/z 558 (10%, [M+H]*), 576 (100%, [MH+HaOft.
Biological Examples
Determination of cathepsin K proteolytic catalytic activity
Convenient assays for cathepsin K are carried out using human recombinant enzyme,
such as that described in PDB.
ID BC016058 standard; mRNA; HUM; 1699 BP.
DE Homo sapiens cathepsin K (pycnodysostosis), mRNA (cDNIA clone MGC:23107
RX MEDLINE;. RX PUBMED: 12477932.
DR RZPD; IRALo962G1234.
DR SWISS-PROT; P43235;
The recombinant cathepsin K can be expressed in a variety of commercially available
expression systems including E coli, Plchia and Baculovirus systems. The purified
enzyme is activated by removal of the prosequence by conventional methods.
Standard assay conditions for the determination of kinetic constants used a fluorogenic
peptide substrate, typically H-D-Ala-Leu-Lys-AMC, and were determined in either
100 mM Mes/Tns, pH 7.0 containing 1 mM EDTA and 10 mM 2-mercaptoethanol
orlOOmMNa phosphate, imM EDTA, 0.1%PEG4000 pH 6.5 or 100 mM Na acetate, pH
5.5 containing 5 mM EDTA and 20 mM cysteine, in each case optionally with 1M DTT
as stabiliser. The enzyme concentration used was 5 nM. The stock substrate solution

was prepared at 10 mM in DMSO. Screens were carried out at a fixed substrate
concentration of 60 uM and detailed kinetic studies with doubling dilutions of substrate
from 250 uM. The total DMSO concentration in the assay was kept below 3%. All
assays were conducted at ambient temperature. Product fluorescence (excitation at
390 nm, emission at 460 nm) was monitored with a Labsystems Fluoroskan Ascent
fluorescent plate reader. Product progress curves were generated over 15 minutes
following generation of AMC product.
Inhibition Studies
Potential inhibitors are screened using the above assay with variable concentrations of
test compound. Reactions were initiated by addition of enzyme to buffered solutions of
substrate and inhibitor. K( values were calculated according to equation 1

where v0 is the velocity of the reaction, V is the maximal velocity, S Is the concentration
of substrate with Michaelis constant of KM, and / is the concentration of inhibitor.
Compounds of the invention bearing the distinctive halogen substltuent in the P1 group
were assayed against the closest individualised compound of the abovementioned WO



It will be apparent that introduction of at least one halogen atom to P1, according to the
invention has surprisingly resulted In a 10 fold Increase in potency.
* Note that the Ki indicated in WO 02057270 for the prior art compound is the less
potent 0.1 micromolar, whereas the above trials reflect accurate side by side trials in the
same assay system.

Throughout the specification and the claims which follow, unless the context requires
otherwise, the word 'comprise', and variations such as 'comprises' and 'comprising', will
be understood to imply the inclusion of a stated integer, step, group of integers or group
of steps but not to the exclusion of any other integer, step, group of integers or group of
steps.


We Claim:
1. A compound of the formula II

wherein
one of R1 and R2 is halo and the other is H or halo;
R3 is C1-C5 straight or branched chain, optionally fluorinated, alkyl;
R4 is H;
R6 phenyl which is substituted with thiazolyl, 5-methylthiazolyl or thienyl,
wherein the thiazolyl, 5-methylthiazolyl or thienyl is substituted with a substituent
selected from the group: morpholinyl, morpholinylmethyl-, piperidinyl, piperidinylmethyl-,
piperazinyl, piperazinylmethyl-, any of which in turn is optionally substituted with C1-C3
alkyl, fluoro, difluoro or C1-C3alkyl-O-C1-C3alkyl;
or a pharmaceutically acceptable salt or solvate thereof.
2. A compound as claimed in claim 1, wherein R2 is halo and R1 is H.
3. A compound as claimed in claim 2, wherein R2 is fluoro.
4. A compound as claimed in claim 1, wherein R3 is C1-C4 branched chain alkyl.
5. A compound as claimed in claim 4, wherein R3 is iso-butyl.
6. A compound as claimed in claim 1, wherein the phenyl is substituted with thiazol-
4-yl, 5-methylthiazol-4-yl or thien-2-yl, wherein the thiazolyl, 5-methylthiazolyl or theinyl
is substituted with a substituent selected from the group: morpholinyl,
morpholinylmethyl-, piperidinyl, piperidinylmethyl-, piperazinyl, piperazinylmethyl-, any
of which in turn is optionally substituted with C1-C3 alkyl, fluoro, difluoro or C1-C3alkyl-O-
C1-C3alkyl-.

7. A compound as claimed in claim 1, wherein the substituent to the thiazolyl, 5-
methylthiazolyl or thienyl is piperid-4-yl which is substituted with methyl, piperazinyl
which is N-substituted with C1-C3 alkyl or methyloxyethyl-, or piperid-1-ylmethyl- which is
unsubstituted or 4-substituted with fluoro or di-fluoro.
8. A compound as claimed in claim 1 selected from:



or a pharmaceutically acceptable salt of any one thereof.
9. A compound as claimed in claim 8 with the formula

or a pharmaceutically acceptable salt thereof.
10. A pharmaceutical composition comprising a compound as defined in any one of
claims 1 to 9 and a pharmaceutically acceptable carrier or diluent therefor.
11. The composition as claimed in claim 10, wherein said composition is useful for
the treatment of a disorder is selected from:
osteoporosis,
gingival diseases such as gingivitis and periodontitis,
Paget's disease,
hypercalcaemia of malignancy
metabolic bone disease
diseases characterised by excessive cartilege or matrix degradation, such as
osteoarthritis and rheumatoid arthritis.
bone cancers including neoplasia,
pain.


The invention discloses a compound of the formula II

wherein R1 ,R2 ,R3 ,R4 and R6 are as defined in the specification.
The invention is also for a pharmaceutical composition comprising said compound.

Documents:

01960-kolnp-2006 abstract.pdf

01960-kolnp-2006 claims.pdf

01960-kolnp-2006 correspondence others.pdf

01960-kolnp-2006 description(complete).pdf

01960-kolnp-2006 form-1.pdf

01960-kolnp-2006 form-3.pdf

01960-kolnp-2006 form-5.pdf

01960-kolnp-2006 international publication.pdf

01960-kolnp-2006 international search authority report.pdf

01960-kolnp-2006 pct form.pdf

01960-kolnp-2006 priority document.pdf

01960-kolnp-2006-correspondence others-1.1.pdf

01960-kolnp-2006-correspondence-1.2.pdf

01960-kolnp-2006-form-18.pdf

01960-kolnp-2006-form-3-1.1.pdf

01960-kolnp-2006-g.p.a.pdf

1960-KOLNP-2006-ABSTRACT 1.1.pdf

1960-KOLNP-2006-ABSTRACT 1.2.pdf

1960-KOLNP-2006-AMANDED CLAIMS.pdf

1960-KOLNP-2006-ASSIGNMENT.pdf

1960-kolnp-2006-assignment1.1.pdf

1960-KOLNP-2006-CANCELLED PAGES.pdf

1960-KOLNP-2006-CLAIMS 1.1.pdf

1960-KOLNP-2006-CORRESPONDENCE 1.1.pdf

1960-kolnp-2006-correspondence.pdf

1960-KOLNP-2006-DESCRIPTION (COMPLETE) 1.1.pdf

1960-KOLNP-2006-DESCRIPTION (COMPLETE) 1.2.pdf

1960-kolnp-2006-examination report.pdf

1960-KOLNP-2006-FORM 1 1.2.pdf

1960-KOLNP-2006-FORM 1.1.1.pdf

1960-KOLNP-2006-FORM 13.1.1.pdf

1960-kolnp-2006-form 13.2.pdf

1960-KOLNP-2006-FORM 13.pdf

1960-kolnp-2006-form 18.pdf

1960-KOLNP-2006-FORM 2 1.2.pdf

1960-KOLNP-2006-FORM 2.pdf

1960-KOLNP-2006-FORM 3 1.2.pdf

1960-KOLNP-2006-FORM 3.1.1.pdf

1960-kolnp-2006-form 3.pdf

1960-kolnp-2006-form 5.pdf

1960-kolnp-2006-gpa.pdf

1960-kolnp-2006-granted-abstract.pdf

1960-kolnp-2006-granted-claims.pdf

1960-kolnp-2006-granted-description (complete).pdf

1960-kolnp-2006-granted-form 1.pdf

1960-kolnp-2006-granted-form 2.pdf

1960-kolnp-2006-granted-specification.pdf

1960-KOLNP-2006-OTHERS 1.2.pdf

1960-KOLNP-2006-OTHERS.pdf

1960-kolnp-2006-others1.1.pdf

1960-KOLNP-2006-PETITION UNDER RULE 137.pdf

1960-KOLNP-2006-REPLY TO EXAMINATION REPORT 1.2.pdf

1960-KOLNP-2006-REPLY TO EXAMINATION REPORT.pdf

1960-kolnp-2006-reply to examination report1.1.pdf

abstract-01960-kolnp-2006.jpg


Patent Number 249758
Indian Patent Application Number 1960/KOLNP/2006
PG Journal Number 45/2011
Publication Date 11-Nov-2011
Grant Date 08-Nov-2011
Date of Filing 12-Jul-2006
Name of Patentee MEDIVIR AB
Applicant Address LUNASTIGEN 7, S-141 44 HUDDINGE
Inventors:
# Inventor's Name Inventor's Address
1 NILSSON MAGNUS MEDIVIR AB, LUNASTIGEN 7, S-141 44 HUDDINGE
2 ODEN LOURDES MEDIVIR AB, LUNASTIGEN 7, S- 141 44 HUDDINGE
3 CLASSON BJORN MEDIVIR AB, LUNASTIGEN 7, S- 141 44 HUDDINGE
4 NOREN ROLF MEDIVIR AB, LUNASTIGEN 7, S- 141 44 HUDDINGE
5 GRABOWSKA URSZULA MEDIVIR UK LTD. CHESTERFORD RESEARCH PARK, LITTLE CHESTERFORD, ESSEX CM 10 1XL,
6 JACKSON PHILIP MEDIVIR UK LTD. CHESTERFORD RESEARCH PARK, LITTLE CHESTERFORD, ESSAX CM10 1XL,
7 FALLON PHILIP MEDIVIR UK LTD. CHESTERFORD RESEARCH PARK, LITTLE CHESTERFORD, ESSAX CM10 1XL,
8 CARR ANDREW MEDIVIR UK LTD. CHESTERFORD RESEARCH PARK, LITTLE CHESTERFORD, ESSAX CM10 1XL,
9 LILEY MARK MEDIVIR UK LTD. CHESTERFORD RESEARCH PARK, LITTLE CHESTERFORD, ESSAX CM10 1XL,
10 TOZER MATT MEDIVIR UK LTD. CHESTERFORD RESEARCH PARK, LITTLE CHESTERFORD, ESSAX CM10 1XL,
11 JOHNSON TONY MEDIVIR UK LTD. CHESTERFORD RESEARCH PARK, LITTLE CHESTERFORD, ESSAX CM10 1XL,
12 DIAZ VICTOR MEDIVIR UK LTD. CHESTERFORD RESEARCH PARK, LITTLE CHESTERFORD, ESSAX CM10 1XL,
13 CRESPO LAIA MEDIVIR UK LTD. CHESTERFORD RESEARCH PARK, LITTLE CHESTERFORD, ESSAX CM10 1XL,
14 KANGASMETSA JUSSI MEDIVIR UK LTD. CHESTERFORD RESEARCH PARK, LITTLE CHESTERFORD, ESSAX CM10 1XL,
15 BONNAUD THIERRY MEDIVIR UK LTD. CHESTERFORD RESEARCH PARK, LITTLE CHESTERFORD, ESSAX CM10 1XL,
16 ZHOU XIAO-XIONG MEDIVIR AB, LUNASTIGEN 7, S- 141 44 HUDDINGE
PCT International Classification Number C07D 491/04
PCT International Application Number PCT/GB2005/050003
PCT International Filing date 2005-01-06
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 0400022-0 2004-01-08 Sweden
2 0401332-2 2004-05-26 Sweden