(12) United States Patent (10) Patent No.: US 8,946,224 B2 Craighead Et Al

USOO8946224B2

(12) United States Patent (10) Patent No.: US 8,946,224 B2 Craighead et al. (45) Date of Patent: Feb. 3, 2015

(54) SUBSTITUTED (2013.01); C07C 257/14 (2013.01); C07C 1.2.4TRIAZOLO4,3-APYRAZINES FOR 251/48 (2013.01); C07C 57/00 (2013.01); MEDCAMENTS AND PHARMACEUTICAL C07C233/36 (2013.01); C07H 19/173 COMPOSITIONS (2013.01); C07D411/10 (2013.01); C07C 229/18 (2013.01); C07D 295/02 (2013.01); (75) Inventors: Mark Craighead, Manchester (GB); C07H 19/06 (2013.01); Ronald Palin, Manchester (GB); Neil (Continued) Murray, Manchester (GB); Derek Lindsay, Manchester (GB) (58) Field of Classification Search CPC ...... A61K 31/4985; C07D 487/04 (73) Assignee: Redx Pharma Limited, Liverpool (GB) USPC ...... 514/249; 544/350 See application file for complete search history. (*) Notice: Subject to any disclaimer, the term of this patent is extended or adjusted under 35 (56) References Cited U.S.C. 154(b) by 0 days. U.S. PATENT DOCUMENTS (21) Appl. No.: 13/883,713 7,326,708 B2 * 2/2008 Cypes et al...... 514,249 (22) PCT Filed: Nov. 11, 2011 FOREIGN PATENT DOCUMENTS (86). PCT No.: PCT/GB2O11AOS2211 CN 101429115 A 5, 2009 S371 (c)(1), EP 2397141 A1 12/2011 (2), (4) Date: May 6, 2013 (Continued) (87) PCT Pub. No.: WO2012/063085 OTHER PUBLICATIONS PCT Pub. Date: May 18, 2012 International Search Report mailed Jun. 21, 2012 for International Application No. PCT/GB2011/052211 (20 pages). (65) Prior Publication Data (Continued) US 2013/022.5594 A1 Aug. 29, 2013 (30) Foreign Application Priority Data Primary Examiner — Douglas MWillis (74) Attorney, Agent, or Firm — Morgan, Lewis & Bockius Nov. 11, 2010 (GB) ...... 1O19078.3 LLP Nov. 18, 2010 (GB) ...... 1019527.9 (57) ABSTRACT (51) Int. Cl. The present invention relates to derivatives of known active A6 IK3I/4985 (2006.01) pharmaceutical compounds. These derivatives are differenti CO7D 487/04 (2006.01) ated from the parent active compound by virtue of being (Continued) redox derivatives of the active compound. This means that (52) U.S. Cl. one or more of the functional groups in the active compound CPC ...... C07D 487/04 (2013.01); C07D498/06 has been converted to another group in one or more reactions (2013.01); C07D 413/12 (2013.01); C07C which may be considered to represent a change of oxidation 63/64 (2013.01); C07D 213/60 (2013.01); state. We refer to these compounds generally as redox deriva C07C2 17/58 (2013.01); C07D 403/06 tives. The derivatives of the invention may be related to the (2013.01); C07C 69/12 (2013.01); C07C original parent active pharmaceutical compound by only a 235/16 (2013.01); C07D 257/04 (2013.01); single step transformation, or may be related via several Syn C07D 313/04 (2013.01); C07D407/06 thetic steps including one or more changes of oxidation state. (2013.01); C07C 2102/10 (2013.01); C07C Exemplary derivatives have the formula 43/205 (2013.01); C07C 215/46 (2013.01); C07C 255/25 (2013.01); C07D 205/08 (2013.01); C07D 281/06 (2013.01); C07D 417/06 (2013.01); C07D 413/04 (2013.01); C07D401/04 (2013.01); C07C 63/66 (2013.01); C07D499/88 (2013.01); C07D 401/12 (2013.01); C07C 2101/16 (2013.01); C07D 519/00 (2013.01); C07D 209/18 (2013.01); C07D 307/00 (2013.01); C07D 501/00 (2013.01); C07C233/22 (2013.01); C07D473/00 (2013.01); C07H 19/052 (2013.01); C07C 251/40 (2013.01); C07D 403/12 (2013.01); C07D 513/06 (2013.01); C07C 2103/74 (2013.01); C07C 251/54 10 Claims, 1 Drawing Sheet US 8,946,224 B2 Page 2

(51) Int. Cl. C07C 235/28 (2006.01) CO7D 49.8/06 (2006.01) C07D 233/54 (2006.01) CO7D 413/2 (2006.01) CO7I5/00 (2006.01) CD7C 63/64 (2006.01) C07D 47L/04 (2006.01) CO7D 213/60 (2006.01) C07D 24I/04 (2006.01) C07C 21 7/58 (2006.01) C 25.1% 30.8 CO7D 403/06 (2006.01) C07D 417/12 (2006.01) CD7C 69/2 (2006.01) C07D 309/32 (2006.01) CD7C 235/76 (2006.01) C07C 237/20 (2006.01) C07D 257/04 (2006.01) C07C 251/58 (2006.01) CO7D 313/04 (2006.01) C07D40 L/4 (2006.01) CO7D 407/06 (2006.01) C07D 49/04 (2006.01) CD7C 43/205 (2006.01) (52) U.S. Cl. CD7C 25/246 (2006.01) CPC ...... C07C 235/28 (2013.01); C07D 233/54 C07C 255/25 (2006.01) (2013.01); C07J 5/00 (2013.01); C07D 471/04 CO7D 205/08 (2006.01) (2013.01); C07D 24I/04 (2013.01); C07D CO7D 28/06 (2006.01) 235/16 (2013.01); C07D 277/40 (2013.01); CO7D 417/06 (2006.01) C07D 417/12 (2013.01); C07D309/32 CO7D 413/04 (2006.01) (2013.01); C07C 237/20 (2013.01); C07C CO7D40 L/04 (2006.01) 251/58 (2013.01); C07D401/14 (2013.01); CD7C 63/66 (2006.01) C07D491/04 (2013.01) C07D 499/88 (2006.01) USPC ...... 514/249; 544/350 CO7D 40/12 (2006.01) C07D 519/00 (2006.01) (56) References Cited C07D 209/18 (2006.01) WTO 3:08: FOREIGN PATENT DOCUMENTS C07C 233/22 (2006.01) WO WO-2004087650 A2 10, 2004 CO7D 473/00 (2006.01) WO WO-2009084024 A2 7, 2009 C07H 19/052 (2006.01) WO WO-2010O78440 A1 T 2010 CD7C 25/240 (2006.01) WO WO 2010.122578 * 10/2010 CO7D 403/2 (2006.01) OTHER PUBLICATIONS CO7D 53/06 (2006.01) C07C 251/54 (2006.01) International Written Opinion mailed Jun. 21, 2012 for International C07C 257/14 (2006.01) Application No. PCT/GB2011/052211 (29 pages). CD7C 2.5L/48 (2006.01) English translation of Chinese Office Action mailed May 29, 2014 for C07C 57/00 (2006.01) sponding Chinese Patent Application No. 20118.0053804.2. (5 CD7C233/36 (2006.01) finitional Search Reportmailed Mav 9, 2014 for Singapore Appli C07H 19/173 (2006.01) styleiga's My gapore App CO7D 4II/O (2006.01) International Written Opinion mailed Sep. 9, 2014 for Singapore C07C 229/18 (2006.01) Application No. 2013,035365 (13 pages). C07D 295/02 (2006.01) C7H 9/06 (2006.01) * cited by examiner U.S. Patent Feb. 3, 2015 US 8,946,224 B2

1.60 1.40 1.20

1.OO O.80 O. 60 0.40 O.20 O.OO US 8,946,224 B2 1. 2 SUBSTITUTED In many cases, the compounds of the invention have inher 1.2.4ITRIAZOLO4,3-APYRAZINES FOR ent therapeutic activity on their own account. In some cases, this activity relative to the same target or targets of the parent MEDCAMENTS AND PHARMACEUTICAL compound is as good as or better than the activity which the COMPOSITIONS parent compound has against the target or targets. However, the present invention also concerns such redox derivatives of CROSS-REFERENCE TO RELATED active compounds which have only a low level activity rela APPLICATIONS tive to that of the parent compound but which are easily capable of metabolising in Vivo to active pharmaceutical This application is the U.S. national stage application of compounds, including the parent active compound itself. These compounds perform a useful function as prodrugs of International (PCT) Patent Application Serial No. PCT/ 10 the active compound. GB2011/052211, filed Nov. 11, 2011, which claims the ben Generally, the present invention thus relates to redox efit of GB Application No. 1019078.3, filed Nov. 11, 2010, derivatives which have the same type of activity i.e. against and GBApplication No. 1019527.9, filed Nov. 18, 2010. The the same targets as the parent known active pharmaceutical entire disclosure of each of these applications is hereby incor compound itself does. In some instances, the compounds may porated by reference. 15 have new activity against a different target also in addition to that of the parent, or may have activity against a different SUMMARY target in preference to that of the parent. It is generally intended however that the activity of the compounds of the The present invention relates to derivatives of known active invention is the same in terms of its type as that of its respec pharmaceutical compounds. These derivatives are differenti tive ultimate parent compound i.e. the known pharmaceuti ated from the parent active compound by virtue of being cally active compound upon which the redox compound of redox derivatives of the active compound. This means that the invention is ultimately based. one or more of the functional groups in the active compound This invention provides compounds that achieve one or has been converted to another group in one or more reactions more of the above aims. The compounds may be active in which may be considered to represent a change of oxidation their own right or may metabolise or react in aqueous media state. We refer to these compounds generally as redox deriva 25 to yield a parent active compound. Ultimately, the overall tives. skeleton i.e. gross structure of the parent active molecule is Many known drugs are less stable than the ideal. For retained but the various functional groups have been modified example, drug molecules containing carboxylic acids may and we have identified “islands of activity” in these new undergo decarboxylation of the terminal acid. This represents compounds. The activity of these compounds of the present a significant problem during manufacture of an active princi 30 invention cannot be predicted empirically based on knowl pal or during extended storage of the same in a pharmacy. edge of the respective parent compounds because the change Similarly, amides can be subject to hydrolysis to the carboxy of potency of an inhibitor depends on the binding of the lic acid derivatives. The resulting decomposition products inhibitor to the protein. Generally, only molecules having the may have reduced activity and potentially increased toxicity correct shape and electronic properties will be suitable for when compared with the parent active. binding at the relevant site on the protein. However, we have It is therefore an aim of the present invention to provide 35 identified a small group of compounds related to each parent reduced or oxidised derivatives of active compounds which for which we have evidence of activity. This evidence shows are able to demonstrate similar to or better longevity than the that in the case of each of our “islands of compounds’ i.e. for parent active compound. It is also an aim of the present each of the individual genera represented by formulae 1 to invention to provide compounds which have an IC50 value 159 there is activity across the group of compounds. This is so comparable to or better than that of the parent active. Ideally, 40 despite each of these genera having a different shape, due to these reduced or oxidised derivatives will have good stability changes in Substitution, and having a different electron dis and/or bioavailability relative to the parent active compound. tribution, due to different electronic properties in the new It is thus an aim to provide reduced or oxidised derivatives substituents, relative to the relevant parent compound. This having improved stability. Another aim of the present inven activity across the Small but diverse range of compounds tion is to provide compounds having improved bioavailabil 45 within each formula is quite Surprising but can be seen from ity. Ideally, the reduced or oxidised derivatives will have an the various examples provided later below which all show extended shelf-life. activity. In addition, conventional wisdom in the pharmaceu The derivatives of the invention may be related to the tical field specifically aims to avoid having Substituent groups original parent active pharmaceutical compound by only a Such as those utilised in the present invention, for example single step transformation, or may be related via several Syn Such as aldehydes and Oximes etc., present in active molecules thetic steps including one or more changes of oxidation state. 50 on account of expected instability or unwanted reactivity. The In certain cases, the functional group obtained after two or compounds of the invention have Surprisingly been found to more transformations may be in the same oxidation state as be active and stable. the parent active compound (and we include these com According to a first aspect, the present invention provides pounds in our definition of redox derivatives). In other cases, a compound or compounds according to any one of the for the oxidation state of the derivative of the invention may be 55 mulae below taken alone or any combination of more than one regarded as being different from that of the parent compound. of the formulae 1-161 taken together:

Formula Name of Parent number Active Compound 1. Metronidazole US 8,946,224 B2

-continued

Formula Name of Parent number Active Compound Formula

PDO332991

Sitagliptin F F F F r N \ N N S. M N

V Y F

Cefadroxi Z O

Cefazolin

I N N N H Na

Cefacetrile Z Z2 O Y OCS1 2. H --

Cefaloglycin Z2 Z1 O Y t(S1 2. US 8,946,224 B2 6 -continued

Formula Name of Parent number Active Compound Formula

Cefalonium Z O NN a NN Y

2 S 2. S \ G 11 N N

Cefaloridine Z O NN a NN Y S 2 S N \ H N

10. Cefalotin Z2 Z O 21 N Y S S1 2. Y. \ H N

11. Cefapirin Z2 Z O 21 N Y s1% H su | S. leN

12. Cefatrizine 'N Z N O N Q a NN Y S OH 11 N

13. Cefazedone US 8,946,224 B2 8 -continued

Formula Name of Parent number Active Compound Formula

14. Cefazafur ^N NN Z

NullsN Q a NN O / Y s12

F F

1S. Cefraidine Z O a NN Y s12

16. Cefroxadine Z O O 1. 21 NN Y s1%

17. Ceftezole

O ClauS Q a NN Y e NN S % N F \ H NaN

18. Cefaclor Z O C 21 NN Y s12

19. Cefamandole ŽSN Z NN ls Q a NN O / Y S e OH 1 N US 8,946,224 B2 10 -continued

Formula Name of Parent number Active Compound Formula

20. Cefninox

21. Cefonicid

22. Ceforanide

23. Cefotiam

24. CefbuperaZone US 8,946,224 B2 11 12 -continued

Formula Name of Parent number Active Compound Formula

25. Cefuroxime O Z

HN lsO 21 NN O Y

H N O V

26. Cefuzonam N M Z

N H H N S

27. Cefoxitin O Z

HN lsO a NN O Y S S1S \ H V N N

28. Cefotetan NNN 1 Z1

29. Cefnmetazole

30. Flomoxef US 8,946,224 B2 13 14 -continued

Formula Name of Parent number Active Compound Formula

31. Loracarbef Z O C 21 NN Y

S 2. YH N

32. Cefixime

21 NH2 Y N N S N O

33. Ceftazidime Z -- O SN 21 N NH2 Y 2 e N S e N H H | N S N >O

34. Ceftriaxone Os nN.1 Z O O Nals Q 21 NN NH2 Y N S 2.Y N H H S S

35. Cefcapene O Z

US 8,946,224 B2 15 16 -continued

Formula Name of Parent number Active Compound Formula

36. Cefaloxime Z No Y N=(NH2 N S

37. Cefetamet Z O 21 NN NH2 Y S1 % it N NN=( V

38. Cefnmenoxime Z

yNulls Q a NN O Y NH2

S1 2. in N NN=( V

39. Cefodizime Z1

7 alsS N Q Y N=(NH2 S S

40. CefoperaZone ? N Z Nulls O so-NYS Y S OH 11 N

O y N

O N US 8,946,224 B2 17 18 -continued

Formula Name of Parent number Active Compound Formula

41. Cefotaxime

42. Cefpimizole

43. Cefpiramide N N --, a NN O / Y1 s1% OH

N NH

44. Cefpodoxime

45. Cefsulodin US 8,946,224 B2

-continued

Formula Name of Parent number Active Compound Formula

46. Cefiteram Z

N O / NN 21 NN NH2 e N N N s1 2 Y Y =( H H S

47. Ceftibuten Z1 O 21 NN S1 2. H

48. Ceftiolene

Y N

49. Ceftizoxime Z O 21 NN NH2 Y NS S e Y N H H N S

SO. MoxalactOX88C8 MN NN Z N NullsN Q 21 N O / Y O S OH H 1. N Z2

S1. Cefepime Z US 8,946,224 B2

-continued

Formula Name of Parent number Active Compound Formula

52. CefoZopran O N NH2 Y s N 1 NH N 1 S N V

53. Cefpirome Z

-- O n N 21 N NH2 2 s1 - 2 Y Y NS={ H H N S

54. Cefaulinome Z O -- n N 21 N NH2 Y N 2 s S e N H H S S

55. Ceftobiprole Z Y HN 21 . I I IN

56. Ceftaroline Z O

Q S 21 NN H voH Y NE NNP?W, OH S Y N ( H H N / S N V

57. Neratinib N-1 O NN

HN 21-w

1. T -N-N Y HN C

O N US 8,946,224 B2 23 2 4 -continued

Formula Name of Parent number Active Compound Formula

S8. Sutinib

59. Imatinib

60. Faropenem

61. Biapenem

62. Doripenem VS / w w N1 n H

63. Ertapenem

N US 8,946,224 B2 25 26 -continued

Formula Name of Parent number Active Compound Formula

64. Imipenem

65. Meropenem

66. Panipenem

67. Cefdinir

68. Cefprozil

69. Cefalexin

70. Enoxacin US 8,946,224 B2 27 28 -continued

Formula Name of Parent number Active Compound Formula 71. Feroxacin F

SirN- Nr

F Z

72. Lomefloxacin RN F r N- N

F Z

73. Nadifloxacin

74. Norfloxacin

75. Rufloxacin

76. Balofloxacin US 8,946,224 B2 29 30 -continued

Formula Name of Parent number Active Compound Formula

77. Grepafloxacin RNC

78. PaZufloxacin

79. Sparfloxacin

80. Temafloxacin

81. Tosufloxacin US 8,946,224 B2 31 32 -continued

Formula Name of Parent number Active Compound Formula

82. Besifloxacin C

83. Clinafloxacin C V

84. Garenoxacin F

85. Gemifloxacin W

86. Gatifloxacin

87. Sitafloxacin V F

C US 8,946,224 B2 33 34 -continued

Formula Name of Parent number Active Compound Formula

88. Trovafloxacin

89. Prulifloxacin

y- N N O F Z

90. Ciprofloxacin RN ~ \ y N- N F Cl Z G

91. Clindamycin C G3 Y

N H H O G QS s

92. Mupirocin G3 G2 w 21 O

O Y

93. Verapamil US 8,946,224 B2 35 36 -continued

Formula Name of Parent number Active Compound Formula 94. Alitretinoin 2n-4N4

95. Aliskiren

96. Eprosartan

97. Doxorubicin

98. Etoposide

OH US 8,946,224 B2 37 38 -continued

Formula Name of Parent number Active Compound Formula

99. Gemcitabine NH2

100. Chlorambucil C

N N-1N

101. Megestrol G1

102. Bexaroteine

103. BIBF-1120 H Y2 N NN-4

104. Eprotirome Br

N Br OH US 8,946,224 B2

-continued

Formula Name of Parent number Active Compound Formula 105. Remikiren

106. Acadesine

107. Aleglitazar

108. Nifedipine

J Z1 Z2

N H

109. Alvocidib OH G1

110. Amrubicin O OH G1 COO 'NH, H O OH O, G3

O US 8,946,224 B2 41 42 -continued

Formula Name of Parent number Active Compound Formula

111. Apaziquone O / / Z2 / N

V O Z

112. AZilsartan O - NH V OY NF '- Y O N X- O\— 113. Bendamustine C /

114. Canagliflozin Z G1 O

S F G G3 O /

115. Cladribine NH (1.N Z N als O N C

H H G H

116. Dabigatran etexilate Y US 8,946,224 B2 43 44 -continued

Formula Name of Parent number Active Compound Formula

117. Fluocinolone Acetonide

118. Forodesine

119. Nabumetone

12O. Laninamivir

121. Lixivaptain US 8,946,224 B2 45 46 -continued

Formula Name of Parent number Active Compound Formula 122. Mirabegron G H

HN Or - C

Y N)- N HN

123. Motesanib

N N NH 2 T NH

N 2T2

124. Otamixaban W 2NT N H zy w N E Y

125. Pemetrexed Z Y 2

H Y\ Z2

in-QN f \ N H

126. Rivaroxaban O O H N C N- )- N S Y Y2 US 8,946,224 B2 47 48 -continued

Formula Name of Parent number Active Compound Formula

127. Safinamide

128. Sapacitabine

129. Saredutant

130. SemagaceStat

131. Teriflunomide

132. Trabected in US 8,946,224 B2 49 -continued

Formula Name of Parent number Active Compound Formula 133. Ramelteon N--Y H

134. Ombrabulin Z (AVE8062) H N o O V

Y O O

1.O

135. Adapalene Z

136. Bimatoprost

137. Candesartan Cilexetil US 8,946,224 B2 51 52 -continued

Formula Name of Parent number Active Compound Formula

138. Ezetimibe G

F 139. Fenofibrate C x

140. Latanoprost Z 21 G1 W

G2 G3

141. Losartan HN-N Z ( \ /N N C le N

142. Olopatadine O

143. Quetiapine US 8,946,224 B2 53 54 -continued

Formula Name of Parent number Active Compound Formula

144. Telmisartan

N -

145. Valaciclovir

146. Valsartan

147. Amlodipine H Besylate N 1nw

Z C

148. Omacetaxine Mepesuccinate K O

OH ON Y US 8,946,224 B2 55 -continued

Formula Name of Parent number Active Compound Formula

149. Voreloxin

1SO. ABT-263

S-NH

151. Clopidogrel

152. Dilitazem

153. Etodolac

154. Felodipine C

N US 8,946,224 B2 57 58 -continued

Formula Name of Parent number Active Compound Formula

155. Fexofenadine

156. Gemfibrozil

157. Hydroxyzine

158. Indometacin C

159. Acyclovir

16O. Aztreonam US 8,946,224 B2

-continued

Formula Name of Parent number Active Compound Formula 161. Apixaban N O Y1

N Y2

N N N M N

W wherein: 2O -continued Z, Z and Z are independently, at each occurrence, R3 R4 selected from the group comprising: l l V

ORI O OR2 25

is independently, at each occurrence, selected from the group OR2 R3 comprising: N1 x1 Y 30 ; and R: w R7 s w X - * * s N1 OR2 RHN H N1 R2 Y Y and vy W is independently, at each occurrence, selected from the 40 group comprising are independently, at each occurrence, selected from the group comprising: RHN O NH O H. H. RO H. H X 45 H; NR2R2. NR2R2: and -OR2 ORI N aN H 50 R7 ; and H;

and J is independently, at each occurrence, selected from the 55 group comprising: - NO; and NHR'. Q, Q and Q are independently at each occurrence selected from the group comprising: are independently, at each occurrence, selected from the group comprising: 60 O Rio H. 1" 2 S vy| WyW

; and 65 U is independently at each occurrence selected from the group comprising: US 8,946,224 B2 61 62 Otamixaban, Pemetrexed, Rivaroxaban, Safinamide, Sapac O O itabine, Saredutant, Semagacestat, Teriflunomide, Trabect \/ SH edin, Ramelteon, Ombrabulin (AVE8062), PD 0332991, YOH, and Sunitinib, Adapalene, Aripiprazole, Bimatoprost, Cande sartan, Cilexetil, Ezetimibe, Fenofibrate, Latanoprost, Losa rtan, Clopidogrel, Olopatadine, Quetiapine, Sitagliptin, T. T. and T is independently at each occurrence selected Telmisartan, Valaciclovir, Valsartan, Acyclovir, Amlodipine, from the group comprising: N and NO; Besylate, Omacetaxine Mepesuccinate, Voreloxin, ABT-263, L is independently at each occurrence selected from the Diltiazem, Etodolac, Felodipine, Fexofenadine, Gemfibrozil, group comprising: 10 Hydroxy Zine, aztreonam, apixaban and Indometacin. The compound may be selected from the group of com pounds defined by all of the formulae 161, or it may be H selected from a smaller group Such as that defined by a single N 15 formula from within the formulae 1 to 161, or from a group of compounds defined by a combination of from two to twenty -N- ". N1 of any of the above formulae. ; and In an embodiment, W is independently at each occurrence selected from the group comprising R is Hor Ac: R" is independently at each occurrence H or Ac; R’ is independently at each occurrence H, C, alkyl, Calkyl, RHN O NH C. alkyl or C alkyl; H; R and R are independently, at each occurrence, selected H NR2R2 NR2R2 from the group comprising: Hand C alkyl, or alternatively 25 R and R', together with the X atoms to which they are OR: and attached and the carbon atom bearing the X atoms, form a 5-, N1 6- or 7-membered ring which is saturated or unsaturated: N R is independently at each occurrence selected from the H group comprising: H, Ac, and C alkyl, C2 alkyl, C alkyl or 30 Calkyl; > x1 R is independently at each occurrence selected from the group comprising: H, C, alkyl, C, alkyl, Chaloalkyl and C wherein R' and Rare as described above: haloalkyl: In an embodiment R is H. R’ is independently at each occurrence selected from the 35 In an embodiment, R is H. group comprising: H, C, alkyl, C, alkyl, C, haloalkyl, C In an embodiment R, is H. haloalkyl and NR'R'': and The compounds of the invention are based on the parent X is independently, at each occurrence, —O— or —S—; approved pharmaceutically active compounds disclosed provided that the compound is not selected from the group below. The synthetic routes to each of the compounds are comprising: 40 available in the literature and in the relevant EMA and FDA Cafedroxil, Cefazolin, Cefacetrile, Cefaloglycin, Cefalo regulatory files and accordingly are not reproduced here. nium, Cefaloridine, Cefalotin, Cefapirin, Cefatrizine, These disclosures insofar as the synthetic procedures are Cefazedone, Cefazaflur, Cefradine, Cefroxadine, Ceftezole, concerned form part of the disclosure of the present invention. Cefaclor, Cefamandole, Cefninox, Cefonicid, Ceforanide, In the interests of brevity, the details of these synthetic pro Cefotiam, CefbuperaZone, Cefuroxime, CefuZonam, Cefox 45 itin, Cefotetan, Cefnmetazole, Flomoxef, Loracarbef, cedures are not reproduced here but it is intended that this Cefixime, Ceftazidime, Ceftriaxone, Cefcapene, Cefdal Subject matter is specifically incorporated into the disclosure oxime, Cefetamet, Cefnmenoxime, Cefodizime, Cefopera of these documents by reference. Zone, Cefotaxime, Ce?pimizole, Ce?piramide, Cefpodoxime, Equally, the compounds can be prepared by total or partial Cefsulodin, Cefteram, Ceftibuten, Ceftiolene, Ceftizoxime, 50 synthesis. Thus, conveniently, the derivatives of each parent Moxalactam, Cefepime, Cefozopran, Ce?pirome, Cefaui active may be prepared directly from the respective parent nome, Ceftobiprole, Ceftaroline, Faropenem, Biapenem, active itself by reactions known to the skilled person. How Doripenem, Ertapenem, Imipenem, Meropenem, Panipenem, ever, in practice the skilled person will design a Suitable Cefdinir, Cefprozil, Cefalexin, Enoxacin, Fleroxacin, Lom synthetic procedure, including convergent synthesis, to pre efloxacin, Nadifloxacin, Norfloxacin, Rufloxacin, Balofloxa 55 pare a given derivative depending on its particular function cin, Grepafloxacin, PaZufloxacin, Sparfloxacin, Temafloxa ality and oxidation state. The skilled person is familiar with cin, ToSufloxacin, Besifloxacin, Clinafloxacin, Garenoxacin, Such procedures and these represent common general knowl Gemifloxacin, Gatifloxacin, Sitafloxacin,Trovafloxacin, Pru edge as set out in textbooks such as Warren “Organic Syn lifloxacin, Ciprofloxacin, Clindamycin, Metronidazole, thesis: The disconnection’ Approach; Mackie and Smith Mupirocin, Verapamil, Alitretinoin, Aliskiren, Eprosartan, 60 “Guidebook to Organic Chemistry’; and Clayden, Greeves, Doxorubicin, Etoposide, Raloxifene, Fulvestrant, Gemcitab Warren and Wothers “Organic Chemistry”. ine, Imatinib, Chlorambucil, Megestrol, Bexarotene, BIBF For convenience only, the derivatives of the invention may 1120, Eprotirome, Remikiren, Acadesine, Aleglitazar, Nife be obtained by effecting oxidation or reduction of the target dipine, Alvocidib, Amrubicin, Apaziquone, AZilsartan, functional group at an intermediate stage in the synthesis Bendamustine, Canagliflozin, Cladribine, Dabigatran etexi 65 rather than as a final stage in the synthesis of the derivatives of late, Fluocinolone Acetonide, Forodesine, Nabumetone, the present invention. Where necessary, the skilled person Laninamivir, Lixivaptan, Mirabegron, Motesanib, Neratinib, will be aware of the need to use Suitable protecting groups to US 8,946,224 B2 63 64 protect other functionalities in the molecule from unwanted acute pyelonephritis, pharyngitis, tonsillitis, eColi, prophy oxidation or reduction during transformation of the target laxis before dental Surgery, cellulitis, acnes, cystitis, infec functional group. tious diarrhoea, typhoid fever, infections caused by anaerobic The skilled man will appreciate that adaptation of methods bacteria, peritonitis, malaria, babesiosis bacterial vaginosis, known in the art could be applied in the manufacture of the pelvic inflammatory disease, pseudomembranous colitis, compounds of the present invention. helicobacter pylori, amoebiasis, giardasis, acute gingivitis, For example, the skilled person will be immediately famil Crohn's Disease, rosacea, fungating Tumours, MRSA, impe iar with standard textbooks such as “Comprehensive Organic tigo. Transformations—A Guide to Functional Group Transfor The compounds and formulations of the present invention mations”, RC Larock, Wiley-VCH (1999 or later editions), 10 can be used to treat viral infections including HIV, influenza “March's Advanced Organic Chemistry Reactions, Mecha virus A & B, hepatitis B, herpes simplex and herpes Zoster. nisms and Structure', M B Smith, J. March, Wiley, (5th The compounds and formulations of the present invention edition or later) Advanced Organic Chemistry, Part B. Reac can be used to treat cancers such as colon cancer, breast tions and Synthesis’, FA Carey, RJ Sundberg, Kluwer Aca cancer (hormone-receptor positive, postmenopausal, meta demic/Plenum Publications, (2001 or later editions), 15 static breast cancer), prostate cancer, chronic myelogenous “Organic Synthesis The Disconnection Approach, S War leukaemia, GI stromal tumours (including imatinib resistant ren (Wiley), (1982 or later editions), “Designing Organic GI stromal tumours), endometrium cancer, cutaneous T cell Syntheses' S Warren (Wiley) (1983 or later editions), lymphoma, ovarian cancer (including platinum resistant ova “Guidebook To Organic Synthesis' R K Mackie and D M rian cancer), acute lymphoblastic leukaemia, chronic lym Smith (Longman) (1982 or later editions), etc., and the refer phocytic leukaemia, lung cancer (including both Small cell ences therein as a guide. and non Small cell lung cancers), Superficial non-muscle inva The skilled chemist will exercise hisjudgement and skill as sive bladder cancer, hairy cell leukaemia, relapsed B-cell to the most efficient sequence of reactions for synthesis of a chronic lymphocytic leukaemia, pleural mesothelioma, Solid given target compound and will employ protecting groups as & haematological tumours, acute myeloid leukaemia, necessary. This will depend inter alia on factors such as the 25 advanced soft tissue sarcoma, refractory advance Soft tissue nature of other functional groups present in a particular Sub sarcoma, ovarian & peritoneal neoplasms, head & neck can strate. Clearly, the type of chemistry involved will influence cers, glioma, multiple myeloma, renal cell carcinoma, non the choice of reagent that is used in the said synthetic steps, Hodgkins lymphoma, stage III or IV melanoma, HER2 nega the need, and type, of protecting groups that are employed, tive metastatic breast cancer, neoplastic disorders and B-Cell and the sequence for accomplishing the protection/deprotec 30 malignancies. tion steps. These and other reaction parameters will be evi The compounds of the present invention can be used to dent to the skilled person by reference to standard textbooks treat incontinence and overactive bladder disorder. and to the examples provided herein. The compounds and formulations of the present invention Sensitive functional groups may need to be protected and can be used to treat cutaneous lesions in patients with AIDS deprotected during synthesis of a compound of the invention. 35 related Kaposi's sarcoma, chronic hand eczema, asthma, This may be achieved by conventional methods, for example nasal polyposis, allergic rhinitis, Crohn's disease, prevention as described in “Protective Groups in Organic Synthesis” by of rejection in organ transplants, lupus, acne, keratosis, TW Greene and P G M Wuts, John Wiley & Sons Inc (1999), pilaris, allergies, hay fever, angioedema, chronic obstructive and references therein. pulmonary disease, idiopathic thrombocytopenic purpura, Each of the compounds of the present invention may be 40 allergic conjunctivities & other eye allergies (eg. from contact used as a medicament. lenses), bronchospasms, idiopathic urticaria, itching, hyper The compounds of the present invention can be used in the algesia. treatment of the human body. They may be used in the treat The compounds and formulations of the present invention ment of the animal body. In particular, the compounds of the can be used to treat diabetic macular edema, open angle present invention can be used to treat commercial animals 45 glaucoma and ocular hypertension. Such as livestock. Alternatively, the compounds of the present The compounds and formulations of the present invention invention can be used to treat companion animals such as cats, can be used to treat stomach ulcers, Zollinger Ellison Syn dogs, etc. drome, gastroesophageal reflux disease, erosive oesophagitis, The compounds and formulations of the present invention H Pylori, functional dyspepsia, ulcerative colitis and Crohn's may be used in the treatment of diabetes, bacterial infections 50 disease. and viral infections. They may be used in the fields of oncol The compounds and formulations of the present invention ogy, urology, immunology and ophthalmology. They may be can be used to treat bipolar depression, Schizophrenia includ used to treat diseases and disorders of the gastrointestinal ing acute relapsed Schizophrenia), narcolepsy, Parkinson's system, the central nervous system, the bones and joints, and disease (both early stage and advanced Parkinson's disease), the cardiovascular system. 55 Alzheimer's Disease, restless leg syndrome, epilepsy, relaps The compounds and formulations of the present invention ing/remitting multiple Sclerosis, insomnia, delayed sleep can be used to treat type II diabetes including non-insulin phase disorder, bipolar I and II disorders, clinical depression, dependent diabetes mellitus (adult onset) diabetes, or as an ADHD, postural orthostatis, tachycardia syndrome, nausea, adjunct therapy to hyperglycaemia. Vomiting (in chemotherapy regimens), gastric emptying in The compounds and formulations of the present invention 60 patients with gastroparesis, gastroesophageal reflux disease, can be used to treat both Gram positive and Gram negative migraine, mania, major depressive disorder, generalised bacterial infections such as infections of the urinary tract, the anxiety disorder, obsessive compulsive disorder, Social anxi respiratory tract, the ear, the skin, the throat, soft tissue, bone ety disorder, panic disorder, menopausal hot flushes, acute and joints (including infections caused by Staph Aureus). The psychosis, parasomnia, rapid eye movement disorder, spinal compounds can be used to treat pneumonia, sinusitis, acute 65 chord injury, spastic diplegia, amyotrophic lateral Sclerosis, bacterial sinusitis, bronchitis, acute bacterial exacerbation of peripheral neuropathy, trigeminal and glossopharyngeal neu chronic bronchitis, anthrax, chronic bacterial prostatitis, ralgias, alcohol withdrawal, Smoking cessation, sexual dys US 8,946,224 B2 65 66 function, obesity, seasonal affected disorder, prolactinomas, rhosis, benign prostatic hyperplasia (BPH), cardiac arryth hyperprolactinaemia and psychoneurosis, neuropathic pain mia, congestive heart failure, coronary artery disease, acute from diabetic neuropathy, post herpetic neuralgia, partial Sei coronary syndrome chest pain, statin-treated dyslipidaemia, Zures, fibromyalgia. hyponatremia (with liver cirrhosis or congestive heart failure, The compounds and formulations of the present invention venous thrombo embolism, phytosterolemiaypercholes can be used to treat osteoporosis in menopause, rheumatoid terolaemia, hypertriglyceridaemia, combined dyslipi arthritis, osteoarthritis, arthritic gout, reactive arthritis, Pagets daemias, diabetic nephropathy, essential hypertension, Ven disease of bone, Barter syndrome, and pseudogout and ten tricular fibrillation, ventricular tachycardia, atrial fibrillation, donitis. peripheral vascular disease, cerebrovascular disease, preven The compounds and formulations of the present invention 10 tion of ischaemic events in patients with atherosclerosis, can be used to treat orthostatic hypotension, hypertension, Graves disease, pre-eclampsia, oesophageal spasm, mild congestive heart failure, MI, renal and retinal complications achalasia, oedema associated with heart failure, hepatic cir of diabetes, tachycardia, Angina, heart failure, migraine pro rhosis, renal impairment and hyperlipidaemia. phylaxis, Vasovagal Syncope, adjunctive treatment of hyper In an embodiment, the parent of the derivative of the inven thyroidism, long QT syndrome (in patients with Asthma), 15 tion is selected from one of the compounds identified in the hypertension of pheochromocytoma, Supraventricular tach table below. In each case, the therapeutic class and target yarrhythmias, cluster headaches, migraine, non-Surgical indication is identified for the derivatives of the invention. treatment of gall Stones, hypercholesterolaemia, biliary cir This can be seen in the second and third columns respectively. TABLE Name of parent active compound Therapeutic class Target indication Cefadroxi Antibacteria Gram Positive & Gram Negative infections (Skin, UTI, ENT), Pharyngitis, Tonsilitis, e Coli, Prophylaxis before dental Surgery azolin Antibacteria Bacterial infections acetrile Antibacteria Bacterial infections aloglycin Antibacteria Bacterial infections alonium Antibacteria Bacterial infections aloridine Antibacteria Bacterial infections alotin Antibacteria Bacterial infections apirin Antibacteria Bacterial infections atrizine Antibacteria Bacterial infections aZedone Antibacteria Bacterial infections azafur Antibacteria Bacterial infections radine Antibacteria Bacterial infections roxadine Antibacteria Bacterial infections Ceftezole Antibacteria Bacterial infections aclor Antibacteria Bacterial infections amandole Antibacteria Bacterial infections minox Antibacteria Bacterial infections onicid Antibacteria Bacterial infections oranide Antibacteria Bacterial infections otiam Antibacteria Bacterial infections buperaZone Antibacteria Bacterial infections Cefuroxime Antibacteria Bacterial infections Cefuzonam Antibacteria Bacterial infections oxitin Antibacteria Bacterial infections otetan Antibacteria Bacterial infections metazole Antibacteria Bacterial infections Flomoxef Antibacteria Bacterial infections Loracarbef Antibacteria Bacterial infections Cefixime Antibacteria Bacterial infections Ceftazidime Antibacteria Bacterial infections Ceftriaxone Antibacteria Bacterial infections capene Antibacteria Bacterial infections daloxime Antibacteria Bacterial infections etallet Antibacteria Bacterial infections menoxime Antibacteria Bacterial infections odizime Antibacteria Bacterial infections operaZone Antibacteria Bacterial infections otaxime Antibacteria Bacterial infections bimizole Antibacteria Bacterial infections biramide Antibacteria Bacterial infections bodoxime Antibacteria Bacterial infections Sulodin Antibacteria Bacterial infections Cefiteram Antibacteria Bacterial infections Ceftibuten Antibacteria Bacterial infections Ceftiolene Antibacteria Bacterial infections Ceftizoxime Antibacteria Bacterial infections Moxalactam Antibacteria Bacterial infections epime Antibacteria Bacterial infections oZopran Antibacteria Bacterial infections pirome Antibacteria Bacterial infections guinome Antibacteria Bacterial infections US 8,946,224 B2 67 68 TABLE-continued Name of parent active compound Therapeutic class Target indication Ceftobiprole Antibacteria Bacterial infections Ceftaroline Antibacteria Bacterial infections Faropenem Antibacteria Bacterial infections Biapenem Antibacteria Bacterial infections Doripenem Antibacteria Bacterial infections Ertapenem Antibacteria Bacterial infections Imipenem Antibacteria Bacterial infections Meropenem Antibacteria Bacterial infections Panipenem Antibacteria Bacterial infections Cefinir Antibacteria Bacterial infections of ear, sinus, throat & skin, CAP, Bronchitis Cefprozil Antibacteria Bronchitis, ear & skin infections Cefalexin Antibacteria UTIs, respiratory tract infections, skin & Soft tissue infections, cellulitis, acne Enoxacin Antibacteria Bacterial infections Feroxacin Antibacteria Bacterial infections Lomefloxacin Antibacteria Bacterial infections Nadifloxacin Antibacteria Bacterial infections Norfloxacin Antibacteria Bacterial infections Rufloxacin Antibacteria Bacterial infections Balofloxacin Antibacteria Bacterial infections Grepafloxacin Antibacteria Bacterial infections Pazufloxacin Antibacteria Bacterial infections Sparfloxacin Antibacteria Bacterial infections Temafloxacin Antibacteria Bacterial infections ToSufloxacin Antibacteria Bacterial infections Besifloxacin Antibacteria Bacterial infections Clinafloxacin Antibacteria Bacterial infections Garenoxacin Antibacteria Bacterial infections Gemifloxacin Antibacteria Bacterial infections Gatifloxacin Antibacteria Bacterial infections Sitafloxacin Antibacteria Bacterial infections Trovafloxacin Antibacteria Bacterial infections Prulifloxacin Antibacteria Bacterial infections Aztreonam Antibacteria Bacterial infections Ciprofloxacin Antibacteria UTIs, Cystitis, Chronic Bacterial Prostatits, Lower Respiratory Tract infections, Sinusitis, skin infections, bone & joint infections, Infectious Diarrhoea, Typhoid Fever Clindamycin Antibacterial Combination therapy in Acne, infections caused by anaerobic bacteria: respiratory tract, skin and soft tissue infections, Peritonitis, Bone & Joint infections caused by Staph Ali retis, Combination treatment for Malaria & Babesiosis Metronidazole Antibacterial Bacterial Vaginosis, Pelvic Inflammatory Disease, Anaerobic Bacterial Infections, Pseudomembranous Colitis, Helicobacter Pylori, Amoebiasis, Giardasis, Acute Gingivitis, Crohn's Disease, Rosacea, Fungating Tumours Mupirocin Antibacterial Gram-Positive Bacteria, including MRSA, Skin infections, Impetigo, Staph Aureus infections which are resistent to other anibiotics Verapamil Cardiovascular Angina, Hypertension, Supraventricular Tachyarrhythmias, Cluster Headaches, Migraine prevention, Potential combined use in treatment of Malaria Alitretinoin Immunology Cutaneous Lesions in patients with AIDS-related Kaposi's Sarcoma, Chronic Hand Eczema Aliskiren Cardiovascular Hypertension Eprosartan Cardiovascular Hypertension Doxorubicin Oncology Oncology (Chemotherapy) Etoposide Oncology Oncology (Chemotherapy) Gemcitabine Oncology Oncology (Chemotherapy) Imatinib Oncology Chronic Myelogenous Leukaemia & GI Stromal Tumours Chlorambucil Oncology Oncology (Chemotherapy) Megestrol Oncology Breast & Endometrium Cancer Bexaroteine Oncology Cutaneous T Cell Lympoma BIBF-1120 Oncology Ovarian Cancer Eprotirome Cardiovascular Statin-Treated Dyslipidaemia Remikiren Cardiovascular Hypertension US 8,946,224 B2 69 70 TABLE-continued Name of parent active compound Therapeutic class Target indication Acadesine Oncology Acute Lymphoblastic Leukaemia Aleglitazar Diabetes Type II Diabetes Nifedipine Cardiovascular Anti-anginal (Prinzmetal's Angina) & Hypertension, Raynaud's, Premature Labor, Oesophageal Spasm (in cancer and tetanus patients) Alvocidib Oncology Chronic Lymphocytic Leukaemia Amrubicin Oncology Lung Cancer Apaziquone Oncology Superficial non-muscle invasive Bladder Cancer AZilsartan Cardiovascular Hypertension Bendamustine Oncology Chronic Lymphocytic Leukaemia Canagliflozin Diabetes Type II Diabetes Cladribine CNS/Oncology Hairy Cell Leukaemia & Multiple Sclerosis Dabigatran Etexilate Fluocinolone Ophthalmology Diabetic Macular Edema & Acetonide Dermatitis Eczema Psoriasis Forodesine Oncology Cutaneous T-Cell Lymphoma & Relapsed B-Cell Chronic Lymphocytic Leukaemia Nabumetone Bones and joints Rheumatoid Arthritis, Osteoarthritis Laninamivir Antiviral influenza virus A & B Lixivaptain Cardiovascular Hyponatremia (with Liver Cirrhosis or Congestive Heart Failure) Mirabegron Urology incontinence - Overactive Bladder Motesanib Oncology Non Small Cell Lung Cancer Neratinib Oncology Breast Cancer Otamixaban Cardiovascular Acute Coronary Syndrome Apixaban Cardiovascular Acute Coronary Syndrome Pemetrexed Oncology Non Small Cell Lung Cancer & Pleural Mesothelioma Rivaroxaban Cardiovascular Venous Thrombo Embolism ACS following hip or knee replacements Safinamide CNS Alzheimer's Disease, Restless Leg Syndrome, Epilepsy Sapacitabine Oncology Solid & Haematological Tumours & Cutaneous T-Cell Lymphoma & Acute Myeloid Leukaemia in elderly) Saredutant CNS Anti-depressant & Anxiolytic Semagacestat CNS Alzheimer's Disease Teriflunomide CNS Rheumatoid Arthritis & relapsing/remitting Multiple Sclerosis Trabectedlin Oncology Advanced Soft Tissue Sarcoma & Ovarian Cancer Ramelteon CNS nsomnia & Delayed Sleep Phase Disorder Ombrabulin Oncology Refractory Advance Soft Tissue (AVE8062) Sarcoma & Non Small Cell Lung Cancer PDO332991 Oncology Multiple Myeloma Sunitinib Oncology Renal Cell Carcinoma & Imatinib resistant GI Stromal Tumour Adapalene Immunology Acne & Keratosis Pilaris Aripiprazole CNS Acute Relapsed Schizophrenia, Bipolar Disorder & Clinical Depression Bimatoprost Ophthalmology Open Angle Glaucoma & Ocular Hypertension Candesartan Cardiovascular Hypertension & in heart failure where Cilexetil ACE Inhibitors not tolerated Ezetimibe Cardiovascular Hypercholesterolaemia, Phytosterolemia Fenofibrate Cardiovascular Hypercholesterolaemia & Hypertriglyceridaemia or Combined Dyslipidaemias Latanoprost Ophthalmology Glaucoma & Ocular Hypertension Losartan Cardiovascular Hypertension, Diabetic Nephropathy Olopatadine Immunology Allergic Conjunctivities & other eye allergies (eg. from contact lenses) Quetiapine CNS Schizophrenia, Bipolar I & II mania & depression. insomnia & anxiety disorders Sitagliptin Diabetes Type II Diabetes Telmisartan Cardiovascular Essential Hypertension Valaciclovir Antiviral Herpes Simplex & Herpes Zoster Valsartan Cardiovascular Hypertension, Congestive Heart Failure, post-MMI US 8,946,224 B2 71 72 TABLE-continued Name of parent active compound Therapeutic class Target indication Acyclovir Antiviral Herpes Simplex & Herpes Zoster Amlodipine Cardiovascular Hypertension, Angina Besylate Omacetaxine Cardiovascular Acute coronary syndrome Mepesuccinate Voreloxin Oncology Acute Myeloid Leukaemia & Platinum Resistant Ovarian Cancer ABT-263 Oncology Small Cell Lung Cancer & B-Cell Malignancies Clopidogrel Cardiovascular Coronary Artery Disease, Peripheral Vascular Disease, Cerebrovascular Disease, prevention of ischaemic events in patients with atherosclerosis, Acute Coronary syndrome without NSTEMI, Diltiazem Cardiovascular Hypertension, Angina, Arrhythmia, Prevention of Migraine Etodolac Bones and joints Osteoarthritis & Rheumatoid Arthritis Felodipine Cardiovascular Hypertension, Pre-Eclampsia, Angina, Oesophageal Spasm, Mild Achalasia Fexofenadine Immunology Hay Fever, Allergies, Allergic Rhinitis, Chronic Idiopathic Urticaria Gemfibrozil Cardiovascular Hyperlipidaemia, Hypertriglyceridaemia Hydroxyzine Immunology/CNS tching, Allergies, Hyperalgesia, Motion Sickness-induced Nausea, Insomnia, Mild Anxiety, Psychoneurosis Indometacin CNS/bones and joints Patent Ductus Arteriosus, Retinopathy of Prematurity, Ankylating Spondylitis, Rheumatoid Arthritis, Arthritic Gout, Osteoarthritis, Reactive Arthritis (ReA), Pagets Disease of Bone, Barter Syndrome, Pseudogout, Dysmenorrhoea, Pericarditis, Bursitis, Tendonitis, Nephrogenic Diabetes insipidus, Renal Colic, Migraine Perfloxacin Antibacterial Pneumonia, UTIs, anthrax, acutebacterial sinusitis, chronic bacterial prostitis, acute pyelonephritis, skin infections Moxifloxacin Antibacterial Pneumonia, UTIs, anthrax, acutebacterial sinusitis, chronic bacterial prostitis, acute pyelonephritis, skin infections Ofloxacin Antibacterial Pneumonia, UTIs, anthrax, acutebacterial sinusitis, chronic bacterial prostitis, acute pyelonephritis, skin infections Oseltamivir Antiviral influenza virus Pregabalin CNS Neuropathic pain from Diabetic Neuropathy & Post Herpetic Neuralgia, Partial Seizures, Fibromyalgia, Generalised Anxiety Disorder Darifenacin Urology Urinary Incontinence, Over Active Bladder Peramivir Antiviral influenza virus Zanamivir Antiviral influenza virus

The compounds of the present invention may also be used state one or more oxidation states lower than the phar in treating other conditions treatable by modulating the maceutically active compound; appropriate receptor. (ii) isolating the oxidised or reduced derivative; and In a second aspect of the present invention, there is pro 55 (iii) mixing the oxidised or reduced derivative with one or vided a method of preparing a formulation of an oxidised or more pharmaceutically acceptable excipients to produce reduced derivative of a pharmaceutically active compound, the pharmaceutical formulation. the method comprising: In an embodiment, step (i) of the method comprises oxid (i) either synthesising a derivative of a pharmaceutically ising the pharmaceutically active compound to provide an active compound as defined in the first aspect of the 60 oxidised derivative. invention; or In an embodiment, step (i) of the method comprises reduc oxidising the pharmaceutically active compound to pro ing the pharmaceutically active compound to provide a vide an oxidised derivative which is in an oxidation reduced derivative. state one or more oxidation states higher than the Compounds of the invention intended for pharmaceutical pharmaceutically active compound; or 65 use may be administered as crystalline or amorphous prod reducing the pharmaceutically active compound to pro ucts. They may be obtained, for example, as solid plugs, vide an reduced derivative which is in an oxidation powders, or films by methods such as precipitation, crystal US 8,946,224 B2 73 lization, freeze drying, or spray drying, or evaporative drying. -continued Microwave or radio frequency drying may be used for this purpose. Name of Parent The above in silico methods have been demonstrated in Formula Active predicting activity against target receptors. The more prom 5 number Compound Formula ising candidates are then taken forwards into in vitro assays. 167. pefloxacin N In another aspect the present invention provides a pharma N ceutical formulation comprising a compound selected from N- N N the compounds of formulae 1-161 and a pharmaceutically 10 acceptable excipient. In another aspect the present invention provides a pharma F Z ceutical formulation comprising a compound selected from the compounds of formula 162-169 and a pharmaceutically G 15 acceptable excipient: 168. moxifloxacin

Name of Parent Formula Active number Compound Formula F Z

G 162. Oseltamivir 25 169. ofloxacin N N ~ r O., Z N- N N

30 F Z L G V

163. pregabalin W 35 wherein R. Z. L. G. W. and V are as defined above: H provided that the compound is not selected from the group comprising: pefloxacin, moxifloxacin, ofloxacin, oseltamivir, pregabalin, darifenacin, peramivir and Zanamivir. Z Compounds of the invention containing one or more asym 40 metric carbon atoms can exist as two or more stereoisomers. 164. darifenacin Where a compound of the invention contains a double bond N Such as a C=C or C–N group, geometric cis/trans (or Z/E) W. O isomers are possible. Where structural isomers are intercon Vertible via a low energy barrier, tautomeric isomerism (tau Phi Ph 45 tomerism) can occur. This can take the form of proton tau tomerism in compounds of the invention containing, for 165. peramivir NH. L. example, an imino, keto, or oxime group, or so-called Valence HN tautomerism in compounds which contain an aromatic moi H ety. It follows that a single compound may exhibit more than HN 50 one type of isomerism. Included within the scope of the present invention are all Stereoisomers, geometric isomers and tautomeric forms of the compounds of the invention, including compounds exhib iting more than one type of isomerism, and mixtures of one or 55 more thereof. Also included are acid addition or base salts wherein the counterion is optically active, for example, d-lac 166. Zanamivir tate or 1-lysine, or racemic, for example, d1-tartrate or d1 arginine. Cis/trans isomers may be separated by conventional tech 60 niques well known to those skilled in the art, for example, chromatography and fractional crystallisation. Conventional techniques for the preparation/isolation of individual enantiomers when necessary include chiral Syn thesis from a suitable optically pure precursor or resolution of 65 the racemate (or the racemate of a salt or derivative) using, for example, chiral high pressure liquid chromatography (HPLC). US 8,946,224 B2 75 76 Alternatively, the racemate (or a racemic precursor) may be The activity of the compounds of the present invention may reacted with a suitable optically active compound, for be predicted using one or more of the in silico techniques example, an alcohol, or, in the case where the compound of mentioned below as a precursor to in vitro testing. Structure-based drug design works by positioning com the invention contains an acidic or basic moiety, a base or acid pounds or fragments of compounds from a database into a Such as 1-phenylethylamine or tartaric acid. The resulting selected region of a target structure. These compounds or diastereomeric mixture may be separated by chromatography fragments of compounds are scored and ranked based on their and/or fractional crystallization and one or both of the steric and electrostatic interactions with the target site. The diastereoisomers converted to the corresponding pure enan best scoring and ranking compounds are then tested with tiomer(s) by means well known to a skilled person. biochemical assays (Anderson, A. C., Chemistry & Biology, Chiral compounds of the invention (and chiral precursors 10 Vol. 10,787-797). thereof) may be obtained in enantiomerically-enriched form The target structure is first chosen on the basis of biological using chromatography, typically HPLC, on an asymmetric and biochemical properties. Ideally, a target structure is one resin with a mobile phase consisting of a hydrocarbon, typi that is (i) linked to a human disease, (ii) binds a small in order to carry out a function and (iii) has a well-defined binding cally heptane or hexane, containing from 0 to 50% by volume pocket. Once a target structure has been identified, it is nec of isopropanol, typically from 2% to 20%, and from 0 to 5% 15 essary to obtain accurate structural information. This can be by volume of an alkylamine, typically 0.1% diethylamine. achieved using X-ray crystallography, NMR and/or homology Concentration of the eluate affords the enriched mixture. modelling. Once the structural information has been obtained When any racemate crystallises, crystals of two different through these techniques, the structure of target can then be types are possible. The first type is the racemic compound prepared for the drug design computer program by e.g. add (true racemate) referred to above wherein one homogeneous ing hydrogenatoms which may be absent and correctly defin form of crystal is produced containing both enantiomers in ing tautomeric structures. Alternatively, structural informa equimolar amounts. The second type is the racemic mixture tion of target structures may also be available commercially. After the structural information of the target structure has or conglomerate whereintwo forms of crystal are produced in been obtained, a potential ligand binding site on the target equimolar amounts each comprising a single enantiomer. structure must then be identified. The target site is ideally a While both of the crystal forms present in a racemic mix 25 pocket or a protrusion having a number of possible hydrogen ture have identical physical properties, they may have differ bond donors and acceptors and particular hydrophobic/hy ent physical properties compared to the true racemate. Race drophilic characteristics. Again, information relating to mic mixtures may be separated by conventional techniques ligand binding sites on target structures may be readily avail known to those skilled in the art—see, for example, “Stere able commercially. ochemistry of Organic Compounds” by E. L. Eliel and S. H. 30 After identification of the target structure binding site, Wilen (Wiley, 1994). databases of small molecules can be virtually screened for The activity of the compounds of the present invention can docking into the target site of interest in silico. Each small be assessed by a variety of in silico, invitro and in vivo assays. molecule of the database can be scored based on the predicted In silico analysis of a variety of compounds has been demon interaction with the target site. strated to be predictive of ultimate in vitro and even in vivo Examples of algorithms for docking Small molecules and/ activity, which is illustrated in the Examples below. or fragments against the target binding site include:

Name Description Reference DOCK Docks either small molecules Kuntz, I., Blaney, J., Oatley, S., or fragments and can include Langridge, R., and Ferrin, T. (1982). A solvent effects: geometric approach to macromolecular igand interactions. J. Mol. Biol. 161, 269-288. Lorber, D., and Shoichet, B. (1998). Flexible ligand docking using conformational ensembles. Protein Sci. 7,938-950. Ewing, T., Makino, S., Skillman, G., and Kuntz, I. (2001). DOCK 4.0: search strategies for automated molecular docking of flexible molecule databases. . Comput. Aided Mol. Des. 15, 411 428. Shoichet, B., Leach, A., and Kuntz, I. (1999). Ligand salvation in molecular docking. Proteins 34, 4-16. FlexX Utilises incremental Kramer, B., Metz, G., Rarey, M., and construction; Lengauer, T. (1999). Ligand docking and screening with FlexX. Med. Chem. Res. 9,463-478. FlexE Utilises incremental Claussen, H., Buning, C., Rarey, M., construction and can sample and Lengauer, T. (2001). FlexE. ensembles of receptor Efficientmolecular docking considering structures; protein structure variations. J. Mol. Biol. 308,377–395. SLIDE Operates by firstly anchoring Schnecke, V., Swanson, C., Getzoff, fragments and then E., Tainer, J., and Kuhn, L. (1998). Subsequently adding the Screening a peptidyl database for remainder of the ligand; potential ligands to proteins with side chain flexibility. Proteins 33, 74-87. US 8,946,224 B2 77 78 -continued

Name Description Reference Fo98 Rapidly dock a large number of McMartin, C., and Bohacek, R. (1997). ligand molecules and enables QXP: Powerful, rapid computer the user to graphically view algorithms for structure-based drug results; design. J. Comput. Aided Mol. Des. 11, 333-344. ADAM Aligns fragments based on Mitzutani, M., Tomioka, N., and Itali, A. hydrogen bonding: (1994). Rational automatic search method for stable docking models of protein and ligand. J. Mol. Biol. 243, 31O-326. AUTODOCK Uses averaged interaction Goodsell, D., Morris, G., and Olson, A. energy grid to account for (1996). Automated docking of flexible receptor conformations and igands: applications of AutoDock. J. simulated annealing for ligand Mol. Recognit. 9, 1-5. conformations; MCDOCK Uses Monte Carlo to sample Liu, M., and Wang, S. (1999). ligand placement; MCDOCK: A Monte Carlo simulation approach to the molecular docking problem. J. Comput. Aided Mol. Des. 13,435-451. ProDOCK Uses Monte Carlo minimization Trosset, J., and Scheraga, H. (1999). for flexible ligands; Prodock: software package for protein modeling and docking. J. Comput. Chem. 20, 412–427. ICM Uses Monte Carlo minimization Abagyan, R., Totrov, M., and for protein-ligand docking; and Kuznetsov, D. (1994). ICM-a new method for protein modeling and design-applications to docking and structure prediction from the distorted native conformation. J. Comput. Chem. 15, 488-506. DockVision Uses Monte Carlo Hart, T., and Read, R. (1992). Proteins minimization. 13, 206-222.

Once a small molecule has been identified as potentially An alternative to the QSAR methods includes molecular binding to the target molecule, it must be evaluated before field-based similarity analysis. These methods rely on the fact proceeding to further stages. Usually, several molecules 35 that similar field patterns will bind at the same target site which scored well during the docking run are evaluated in regardless of their underlying structure. In fact, it has been further tests e.g. visually with computer graphics or their reported that there may be a linear correlation between ligand likelihood to be orally bioavailable using the so-called “rule similarity and biological activity. of 5” which states that good leads generally have less than five 40 Molecules interact via their electronic properties: electro hydrogenbond donors and less than 10 hydrogen bond accep static and Vander Waals forces. If two molecules with diverse tors, a molecular weight less than 500 and the calculated log structures interact with an enzyme or receptor in a similar of the partition coefficient less than 5. way, their bound conformations will have similar properties, In many cases, the docked and experimental confirmations although this might not be immediately apparent from a con are within 2 A root mean standard deviation (rmsd) using 45 sideration of their structures alone. The idea of a field pattern structure-based drug design methods. around a ligand is intuitively appealing as the main criterion Alternative methods to structure-based design methods for binding recognition and has been acknowledged for many include three-dimensional quantitative structure-activity years. There exist in silico methods for defining molecular relationship (3D-QSAR) methods for deriving ligand-based fields in a form that enables similarity comparisons across models to estimate the activities of new compounds. Some 50 molecules in three dimensions and defining how molecular methods also provide a graphical output indicating regions fields can be used as non-structural templates for defining where increases in affinity might be expected from modifying similar biological behaviour. physical properties such as steric book, partial charge, hydro Field Templating and Field Screening rely on the assump phobicity, or hydrogen-bond donor/exceptability. Compara tion that those molecules whose field patterns are most simi 55 lar to those of an active search molecule will be the ones most tive molecular field analysis (CoMFA) and comparative likely to show the same patterns of biological activity and molecular similarity indices analysis (CoMSIA) are well should be chosen for further investigation. known examples of these techniques. These methods com It is reported in C. M. R.J.Med. Chem., 2008,51,565-573 pare molecules in terms of grid-based field energies or simi that the field patterns of three potent and selective CCK2 larity indices and use partial least-squares statistics to 60 antagonists can be amalgamated to give a ligand based view generate models that have been widely applied to medicinal of the active site of the receptor in field point terms. A test set chemistry problems. However, specific receptor antagonists of compounds can then be selected from a very diverse col may encompass a wide range of structures. For example, lection of CCK2 receptor-ligands and each compared to the cholecystokinin 2 receptor antagonists include molecules of “receptor template'. The field overlay scores for the model varying structure (C. M. R., J. Med. Chem., 2008, 51, 565 65 system can then be compared to experimentally determined 573). This can make certain receptor antagonists unsuitable affinity estimates (pKB values) for the compounds in a func candidates for 3D-QSAR. tional in vitro CCK2 bioassay. US 8,946,224 B2 79 80 The above in silico methods have been demonstrated in predicting activity against target receptors. The more prom ising candidates are then taken forwards into in vitro assays. The following embodiments apply independently to com pounds according to any one, or any combination of more than one, of formulae 1-169. In an embodiment, when Z is COH, G is not —O. In an embodiment, when G is —O, Z is not CO.H. Preferably, Z, Z and Z are independently at each occurrence In an embodiment, Z, Z or Z are independently at each 10 OCCUCC OR2. N O

15 x In these embodiments, R may be H. Alternatively, R may be Thus, Z, Z or Z may independently at each occurrence be selected from C alkyl, C. alkyl, C alkyl or C alkyl. For example, R may be methyl ethyl, propyl, isopropyl, butyl or tert-butyl. In particular embodiments, R is methyl. OH In a further alternative embodiment, Z, Z or Z are inde pendently at each occurrence

vs. 25 or, alternatively, Z, Z or Z may independently at each occur rence be

OAc 30 H. In a preferred embodiment, Z, Z or Z are independently at v'sH each occurrence In an alternative embodiment, Z, Z or Z are indepen 35 dently at each occurrence

40 R6. Preferably X is O. In these embodiment, RandR may both v. be C alkyl, Calkyl, C alkyl or C alkyl. R and R may be Preferably, Z, Z or Z are independently at each occur the same or different. For example, R and R* may both be CC 45 methyl or may both be ethyl. Alternatively, R and R. together with the X atoms to which they are attached and the carbon atom bearing the X atoms, form a 5 membered ring. For example, Z, Z and Z may independently at each occur rence be CH-ethylene glycol acetal, i.e. Z, Z or Z are inde 50 pendently

In a further alternative embodiment, Z, Z or Z are inde pendently O) 55 O

OR2

x, 60 Y Y Y In this embodiment, R may be H. Alternatively, R may be selected from C alkyl, C, alkyl, C, alkyl or C alkyl. For example, R may be methyl ethyl, propyl, isopropyl, butyl or tert-butyl. In particular embodiments, R is methyl. 65 In a further alternative embodiment, Z, Z or Z are inde are independently, wherever they occur, selected from the pendently at each occurrence group comprising: US 8,946,224 B2 8 1. 82 -continued O H. H.

xy . Thus, 5

Y Y are independently at each occurrence

s vy O 10 HO H may independently at each occurrence be xy 15 In an alternate embodiment, Alternatively, OR2 G3 G4 N1 25 vy O vy O vy 8t vy In this embodiment, R may be H. Alternatively, R may be may independently at each occurrence be 30 selected from C alkyl, C. alkyl, C alkyl or C alkyl. For example, R may be methyl, ethyl, propyl, isopropyl, butylor tert-butyl. In particular embodiments, R’ is methyl. In a further alternate embodiment 35

In an embodiment, 40 R3 R4 G3 G4 l l

45 vy O vy 8t xy Preferably X is O. In an embodiment, RandR may both be O r Calkyl, Calkyl, Calkyl or C alkyl. R and R may be the 50 same ordifferent. For example, RandR may both be methyl are independently or may both be ethyl. Alternatively, RandR, together with the X atoms to which they are attached and the carbon atom bearing the X atoms, form a 5 membered ring. For example, G, G, G, G and G' may independently at each occurrence xy 55 be ethylene glycol acetal, i.e. Alternatively, 60 x/ v/

65 xy vy r vy US 8,946,224 B2 83 84 may independently at each occurrence be 'V/ . ( ), vyS vy In an embodiment, W is

In an embodiment, 10 HN

15 Alternatively, W may be is independently at each occurrence 2O 2

N -OR. x In an alternative embodiment, W is 25 In this embodiment, R may be H. Alternatively, R may be selected from C alkyl, C. alkyl, C alkyl or C4 alkyl. For example, R may be methyl ethyl, propyl, isopropyl, butyl or 30 NR2R2. tert-butyl. In particular embodiments, R is methyl. In an alternative embodiment, In this embodiment, W may be selected from

V 35 O O x's x and may be 40 In a further alternative embodiment, W is HN H

45 NH

NR2R2. In an embodiment, Q, Q or Q may independently at each occurrence be 50 In this embodiment, W may be selected from Wy NH NH NH

In an alternative embodiment, Q, Q or Q may independently 55 x's x and at each occurrence be In a further alternative embodiment, W is 60 Wy N1 OR2. 65 R7 In a further alternative embodiment, Q, Q or Q may inde pendently at each occurrence be US 8,946,224 B2 85 86 In a preferred alternative embodiment, W is Isotopically-labelled compounds can generally be pre pared by conventional techniques known to those skilled in the art or by processes analogous to those described using an OR2. appropriate isotopically-labelled reagent in place of the non N1 labelled reagent previously employed. Throughout the description and claims of this specifica tion, the words “comprise and “contain” and variations of the words, for example “comprising and "comprises'. means “including but not limited to’, and is not intended to In these embodiments, R may be H. Alternatively, R may be 10 (and does not) exclude other moieties, additives, components, selected from C alkyl, C. alkyl, C. alkyl or C alkyl. For integers or steps. example, R may be methyl, ethyl, propyl, isopropyl, butylor Throughout the description and claims of this specifica tert-butyl. In particular embodiments, R is methyl. tion, the singular encompasses the plural unless the context In an embodiment, T, T or T. may independently at each 15 otherwise requires. In particular, where the indefinite article occurrence beN. Alternatively.T.T. or T. may independently is used, the specification is to be understood as contemplating at each occurrence be NO. plurality as well as singularity, unless the context requires In an embodiment, L is otherwise. Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment -N- or example described herein unless incompatible therewith. O 25 BRIEF DESCRIPTION OF THE DRAWING FIG. 1 illustrates the efficacy of particular rosuvastatin Alternatively, L is compounds in Vivo as described in example 1.

30 DETAILED DESCRIPTION wn Example 1 This example serves to illustrate that the activity of the In an embodiment, two adjacent G. V or Y groups when 35 compounds of the present invention derived by in silico meth present in a vicinal arrangement may form a 5- or 6-mem ods can be predictive of ultimate in vitro and even in vivo bered ring, optionally Substituted with an oxo group. In a activity. preferred embodiment, two adjacent G. V or Ygroups when In Silico present in a vicinal arrangement may form a 5-membered The structures of a number of rosuvastatin analogues were ring, optionally Substituted with an oxo group. 40 screened in silico to determine whether or not these com The present invention also includes the synthesis of all pounds are active against the enzyme 3-hydroxy-3-methyl pharmaceutically acceptable isotopically-labelled com glutaryl-coenzyme A reductase (HMG-CoA). The results are pounds of formulae (I) to (VI) wherein one or more atoms are given as the binding free energy (kcal/mol) when each com replaced by atoms having the same atomic number, but an pound is docked with the 1HWL structure (i.e. the complex of atomic mass or mass number different from the atomic mass 45 the catalytic portion of human HMG CoA reductase with or mass number usually found in nature. rosuvastatin) in silico. Two different conformations of the Examples of isotopes suitable for inclusion in the com binding site were also modelled for comparison. It can be pounds of the invention include isotopes of hydrogen, Such as deduced that all compounds listed in the table below have a *H and H. carbon, such as 'C and ''C, chlorine, such as binding energy comparable to rosuvastatin and therefore can Cl, fluorine, such as 'F, iodine, such as 'I and 'I, 50 be expected to have an activity comparable to rosuvastatin. nitrogen, such as 'N and 'N, oxygen, such as "O, O and In Vitro 'O, phosphorus, such as P. and sulphur, such as S. The following procedure was followed using a HMG-CoA Certain isotopically-labelled compounds, for example, Reductase assay kit obtained from Sigma-Aldrich (catalogue those incorporating a radioactive isotope, are useful in drug number CS1090). The assay is based on the spectrophotomet and/or substrate tissue distribution studies. The radioactive 55 ric measurement of the decrease in absorbance at 340 nm of isotopes tritium, i.e. H. and carbon-14, i.e. ''C, are particu NADPH in solution. A decrease in absorbance is caused by larly useful for this purpose in view of their ease of incorpo the oxidation of NADPH by the catalytic subunit of HMGR in ration and ready means of detection. the presence of the substrate HMG-CoA. Effective inhibition Substitution with heavier isotopes such as deuterium, i.e. of the HMG-CoA leads to a reduction in oxidation of NADPH fH, may afford certain therapeutic advantages resulting from 60 which in turn leads to a smaller reduction in the absorbance at greater metabolic stability, for example, increased in vivo 340 nm over time. This is illustrated in the following reaction half-life or reduced dosage requirements, and hence may be scheme: preferred in some circumstances. Substitution with positron emitting isotopes, such as 'C, HMG-CoA-2NADPH-2H-emevalomate+2NADP+ CoA-SH 'F, 'O and 'N, can be useful in Positron Emission Topog 65 raphy (PET) studies for examining substrate receptor occu Compounds showing the best inhibitory action are those pancy. which reduce the absorbance least. US 8,946,224 B2 87 88 Preparation of the Assay Solution TV=total volume of the reaction in ml (1 ml for cuvettes) Ultrapure water (17 MS.2-cm or equivalent was used for the V-volume of enzyme used in the assay (ml) preparation of reagents and throughout the procedure. 0.6-enzyme concentration in mg-protein (mgPO/ml (0.55 First, an assay buffer Solution was prepared using the fol 0.65 mgP/ml) lowing method: 0.2 ml of assay buffer, 5x (catalogue number LP-light path in cm (1 for cuvettes). A5981) was diluted with 0.8 ml of ultrapure water. The result The ICso values for particular rosuvastatin analogues are ing buffer solution was kept on ice or stored at -20°C. for provided in the table below. It can be seen that the rosuvastatin further use. analogues have a comparable ICs value to rosuvastatin itself. Next, 25 mg of NADPH (catalogue number N6505) was 10 This confirms the conclusion derived from the in silico data. reconstituted with 1.5 ml of the buffer solution. The recon In Vivo stituted NADPH was stored in working aliquots at -20°C. The efficacy of particular rosuvastatin compounds was The HMG-CoA substrate solution (catalogue number then determined in vivo. The Example demonstrates the S7447), HMG-CoA reductase (catalogue number H8789) effect of 3 or 5 days BID treatment with rosuvastatin ana and inhibitor Solution (e.g. pravastatin, catalogue number 15 logues and rosuvastatin (all at 25 mg/kg po) on rat plasma 15909) were kept on ice throughout the procedure. triglyceride levels 16 hours after the last treatment dose. The 1. Before beginning, the spectrophotometer was set at 37°C. measurement of the change in rat plasma triglyceride levels is and 340 nm, with a kinetic programme: 1 ml sample, read considered to be a fair test for determining HMG CoA reduc every 20 seconds for up to 10 minutes. tase activity. 2. The appropriate volumes of the reaction solutions were 112 male SD rats (Harlan) were housed in groups of 6 added according to Table 1 (1 ml assay). under a 12 h light dark cycle (lights on 07.00 h) with free access to food (normal laboratory chow) and water. Animals TABLE 1. between 148-183 g were allocated to treatment groups of 8 25 Reaction volumes for 1 ml samples balanced by body weight and treatments were balanced acroSS cages. 1x Test Assay compound The rosuvastatin analogues were made up in 10% PEG300/ Sample buffer Pravastatin NADPH HMG-CoA HGMG 10% cremophor/80% methyl cellulose (0.5%) (vehicle 1) to Blank 920 ul 20 ul 60 ul 30 make a 5 mg/mL Solution. The rosuvastatin compounds used Activity 915 ul 20 ul 60 ul Were Inhibition 910 ul 20 ul 60 ul Rosuvastatin lactol iso-propyl acetal benzyl ether; and Rosuvastatin lactol methyl acetal nicotinoyl ester (diaste The reagents were added to the reaction in the following reomeric ratio 2/1). order: 35 Rosuvastatin was formulated in 0.5% Tween in 0.5% a. Add a buffer to all samples. methyl cellulose (vehicle 2) at 5 mg/kg as a suspension. b. Add the inhibitor (test compound/Pravastatin) to the Rats were orally dosed with vehicle 1, one of the rosuvas inhibition sample. tatin analogues in vehicle 1 (25 mg/kg), vehicle 2 or rosuv c. Add the reconstituted NADPH to all samples. d. Add Substrate Solution (HMG-CoA) to all samples. 40 astatin in vehicle 2 (25 mg/kg po), BID for 3 or 5 days. e. Add HMG-CoA Reductase (HMGR) to the Activity and Sixteen hours after the last treatment, terminal plasma Inhibition samples. samples were taken, Stored at -20°C., and transported on dry f. Mix the samples thoroughly. ice for analysis of triglyceride levels. 3. The kinetics programme was started immediately. The Data for each time-point were analysed by 1-way ANOVA activity of the product was calculated according to the fol 45 and post-hoc Dunnett's test. lowing equation: The results are provided in FIG. 1 from which it can be deduced that administration of rosuvastatin (25 mg/kg po) BID for 3 or 5 days causes a marked reduction in plasma Units/mgP= 12.44x WX0.6.x LP 50 triglycerides. All rosuvastatin analogues also significantly reduced plasma triglycerides after both 3 and 5 days BID where: treatment. All animals tolerated the rosuvastatin treatments 12.44=e, the extinction coefficient for NADPH at 340 mm well and there was no evidence of any adverse events. is 6.22 mM'cm. 12.44 represents the 2 NADPH consumed The magnitude of the effect of the rosuvastatin analogues in the reaction. was equivalent to that of rosuvastatin.

Binding free energy (kcal/mol) ICso

Structure 1HWL (configuration 2) 1HWL (configuration 3) (nm) In vivo

Rosuvastatin -937 -8.78 -8.83 4 Reduction in plasma triglycerides US 8,946,224 B2 89 90 -continued

Binding free energy (kcal/mol) ICso

Structure 1HWL (configuration 2) 1HWL (configuration 3) (nm) In vivo

-8.56 -8.98 NA NA

-8.20 -9.08 NA 22 NA

-8.00 -8.70 -8.81 Reduction in plasma triglycerides

NA -852 -8.63 1 Reduction in plasma triglycerides US 8,946,224 B2 91 92 -continued Binding free energy (kcal/mol ICso Structure 1HWL 1HWL (configuration 2) 1HWL (configuration 3) (nm) In vivo NA NA -8.75 8 NA

Example 2 plex of the catalytic portion of human HMG CoA reductase

25 with atorvastatin) in silico. It can be deduced that all com This example serves to illustrate that the activity of the pounds listed in the table below have a binding energy com compounds of the present invention derived by in silico meth parable to either rosuvastatin oratorvastatin and therefore can ods can be predictive of ultimate in vitro and even in vivo be expected to have an activity comparable to rosuvastatin or activity. 30 atorvastatin. In Silico In Vitro The structures of a number of rosuvastatinandatorvastatin analogues were screened in silico to determine whether or not The above assay procedure described in example 1 was followed. these compounds are active against the enzyme 3-hydroxy 35 3-methylglutaryl-coenzyme A reductase (HMG-CoA). The The ICso values for particular rosuvastatinandatorvastatin results are given as the binding free energy (kcal/mol) when analogues are provided in the table below. It can be seen that each compound is docked with the 1HWL structure (i.e. the the analogues have a comparable ICso value to rosuvastatin complex of the catalytic portion of human HMG CoA reduc 40 and atorvastatin themselves. This confirms the conclusion tase with rosuvastatin) or the 1 HWK structure (i.e. the com derived from the in silico data.

Docking energy Structure (kcal/mol) ICso (nm)

Rosuvastatin -9.83 4

-939 3 US 8,946,224 B2 93 94 -continued Docking energy Structure (kcal/mol) ICso (nm)

-9.73 4

Atorvastatin -11.07 7

-1007 3

-10.49 1

Synthetic Examples 50 T (min) B(%) A(%) Materials and Methods O 10 90 O.2 10 90 Equipment: 55 'HNMR Spectra were recorded at 400 MHz using a Bruker 3 10 90 AVANCE 400 MHz spectrometer. LC-MS equipment and 5 10 90 conditions are as follows: LC-MS (Agilent): 2. MS; G6110A, Quadrupole LC/MS, Ion Source: ES-API, 1. LC: Agilent Technologies 1200 series, Binary Pump, TIC: 50-900 m/z. Fragmentor: 60, Drying gas flow: 10 Diode Array Detector. Ultimate AQ-C18, 3 um, 2.1x50 L/min, Nebulizer pressure: 35 psi, Drying gas tempera mm column. Mobile phase: B (MeOH) and A (0.07% ture: 350° C., Vcap: 3500V. HCOOH adueous solution). Flow Rate: 0.4 mL/min at 65 3. Sample preparation: samples were dissolved in metha 25°C. Detector: 214 nm, 254 nm. Gradient stop time, 5 nol at 1-10 ug/mL, then filtered through a 0.22 um filter min. Timetable: membrane. Injection Volume: 1-10 uL. US 8,946,224 B2 95 96 LC-MS (Waters): THF (tetrahydrofuran); TLC (thin layer chromatography): 1. LC: Waters 2695, Quaterary Pump, Waters 2996 Photo Tol (toluene); Ts-OH (p-toluene sulfonic acid); V/v (volume/ diode Array Detector Xbridge-C18, 3.5um, 2.1x50mm Volume). column. Mobile Phase: B (MeOH) and A (0.07% Example 3 HCOOH acqueous solution). Flow Rate: 0.3 mL/min at 30°C. Detector: 214 nm, 254 nm. Gradient stop time, 10 Formula 1—Compounds 3a & 3b min. Timetable:

10 T (min) B(%) A(%) OH ON O 10 90 oxalyl choride, DMSO 2.5 75 25 Her S.O 95 5 W N Et3N, CHCl2, -78°C. 7.5 95 5 15 7.6 10 90 N > 10 10 90 A 2. MS: Micromass QZ, TIC: 100-900 m/z, Ion Source: ES, / / Nir O Capillary: 3 kV. Cone: 3V. Extractor: 3V. Drying gas ON / / ON / / flow: 600 L/hr, cone: 50 L/hr, Desolvation temperature: N NH2OCH, N 300° C., Source temperature: 100° C. W HCI W 3. Sample preparation: samples were dissolved in metha 2 HeMeOH, RT 2n nol at 1-10 ug/mL, then filtered through a 0.22 um filter N N membrane. Injection Volume: 1-10 uL. 25 B 3b Compound Synthesis: CH(OCH3)3MeOH The compounds of the invention may be prepared by meth ToS-OH reflux ods well known to those skilled in the art, and as described in the synthetic experimental procedures shown below. 30 O DEFINITIONS Ac2O (acetic anhydride); AcOK (potassium acetate); Boc ON N -- (tert-butoxycarbonyl); Boc-O (di-tert-butyl dicarbonate); cat 35 (catalytic); Cbz-OSu (N-(benzyloxycarbonyloxy)succinim O)-N ide); CDC1 (deuterated chloroform): CDOD (deuterated 3a methanol); conc (concentrated); DIBAI-H (diisobutylalu minium hydride); DIPEA (N,N-diisopropylethylamine); DMAP (4-dimethylaminopyridine); DMF (N,N-dimethyl 40 2-(2-Methyl-5-nitro-1H-imidazol-1-yl)acetaldehyde formamide); DMP (Dess-Martin Periodinane); DMSO (dim ethylsulfoxide); DMSO-d (deuterated dimethylsulfoxide); To a solution of anhydrous DMSO (10 mL) in CHCl (120 EDC1 (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide); mL) at -78°C. was added a 2 M solution of oxalyl chloride in eq (equivalent); ES-API (electrospray atmospheric pressure 45 CHCl (10 mL. 20 mmol) slowly dropwise. The reaction ionization); EtN (triethylamine); EtO (diethyl ether); mixture was allowed to stir for 20 min and a solution of EtOAc (ethyl acetate); EtOH (ethanol); g (gram); h (hour); compound A (2.00 g, 11.7 mmol) in DMSO (15 mL) and HATU (2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetram CHCl (25 mL) was added at -78° C. The mixture was ethyluronium hexafluorophosphate); HBTU (2-(1H-benzot stirred at -78°C. for 1 h then triethylamine (14.2 g 140.3 riazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophos 50 mmol) was added and stirring was continued at -78°C. for phate); H NMR (proton nuclear magnetic resonance); HOBt another 1 h. The mixture was allowed to warm to room (hydroxybenzotriazole); HPLC (high-performance liquid temperature then poured into water (70 mL) and extracted chromatography); HZ (hertz): IBX (2-iodoxybenzoic acid); with CHCl (50 mLX3). The combined organic layers were washed with brine then dried (MgSO) and concentrated i-PrCH (isopropanol); L (liter); LAH (lithium aluminium 55 hydride); LC-MS (liquid chromatography-mass spectrom under reduced pressure. The residue was purified by flash chromatography (CH.Cl/MeOH, 100-40/1, V/v) to give etry); M (molar): m-CPBA (meta-chloroperoxybenzoic 2-(2-methyl-5-nitro-1H-imidazol-1-yl)acetaldehyde (1.30 g, acid); MeCN (acetonitrile); MeOH (methanol); mg (milli grams); MHZ (megahertz), min (minutes); mL (milliliters), 66%) as a yellow oil. mmol (millimoles); MTBE (methyl tert-butyl ether); NaOMe 60 LC-MS (Agilent): R, 2.61 min; m/z calculated for (sodium methoxide); PCC (pyridinium chlorochromate); Pet. CHNOM+MeOH+H" 202.2. found 202.1. ether (petroleum ether); ppm (parts per million); PPTS (pyri Compound 3a: 1-(2,2-Dimethoxyethyl)-2-methyl-5- dinium p-toluenesulfonate); psi (pounds per square inch); R, nitro-1H-imidazole (retention time); RT (room temperature); TBAF (tetra-n-bu 65 tylammonium fluoride); TBS-C1 (tert-butyldimethylsilyl A solution of intermediate B (200 mg, 1.18 mmol. 1.0 eq), chloride); t-BuOH (tert-butanol); TFA (trifluoroacetic acid); CH(OCH) (376 mg, 3.55 mmol. 3 eq) and Tos-OH (10 mg) US 8,946,224 B2 97 98 in MeCH (4 mL) was heated at reflux overnight. The reaction mmol. 2.0 eq) in MeOH (3 mL) was stirred at room tempera mixture was allowed to cool to room temperature and con ture for 16 h. The mixture was concentrated under reduced centrated under reduced pressure. The residue was diluted pressure and the residue was diluted with water (5 mL) and with EtOAc and washed with water, brine then dried over brine (5 mL) and extracted with EtOAc (10 mLX3). The NaSO and concentrated under reduced pressure. The resi due was purified by flash chromatography (Pet. ether/ combined organic layers were washed with brine, dried over CHCl2, 1/2 to CHCl, V/v) to give 1-(2,2-dimethoxyethyl)- Na2SO and concentrated under reduce pressure to give 2-(2- 2-methyl-5-nitro-1H-imidazole (120 mg, 47%) as a light methyl-5-nitro-1H-imidazol-1-yl)acetaldehyde O-methyl brown oil. oxime (120 mg, 53%) as a light brown oil, 'H-NMR spec LC-MS (Agilent): R, 2.86 min; m/z calculated for 10 troscopy revealed a 2:3 mixture of isomers. CHNO IM+H 216.2. found 216.1. LC-MS (Agilent): R, 2.87 min; m/z calculated for 'HNMR: (400 MHz, CDC1,) & (ppm): 7.97 (s, 1H), 4.57 (t, CHNO, M+H" 1992. found 199.1. J=5.2 Hz, 1H), 4.39 (d. J=5.2 Hz, 2H), 3.45 (s, 6H), 2.53 (s, H NMR: (400 MHz, CDC1) & (ppm): 7.98 (s, 0.4H), 7.97 3H). 15 (s, 0.6H), 7.52 (t, J=4.8 Hz, 0.6H), 6.75 (t, J–4.4 Hz, 0.4H), 5.16 (d. J–4.4 Hz, 0.8H), 5.05 (d. J–4.8 Hz, 1.2H), 4.0 (s, Compound 3b: 2-(2-Methyl-5-nitro-1H-imidazol-1- 1.2H), 3.85 (s, 1.8H), 2.53 (s, 1.8H), 2.50 (s, 1.2H). yl)acetaldehyde O-methyl oxime Example 4 A solution of intermediate B (200 mg, 1.18 mmol, 1.0 eq) and O-methylhydroxylamine hydrochloride (197 mg, 2.36 Formula 141—Compounds 4a & 4b

OH Method 1 N-->N MnO2, H2O/THF reflux, 24h NaN Method 2 MnO, t-BuOH cat MeSO3H reflux, 24h

NH2OHHCl, KHCO -e- EtOH, H2O, 60° C., 16 h

4a NHOMe-HCI EtOH, HO KHCO 60° C., 16 h

US 8,946,224 B2 99 100 Intermediate B: 1-((2-(2H-Tetrazol-5-yl)-1,1'-bi mg, 3.27 mmol. 6.0 eq). The resulting mixture was heated at phenyl-4-yl)methyl)-2-butyl-4-chloro-1H-imida Zole-5-carbaldehyde 60° C. for 16 h then poured into water (20 mL) and extracted with EtOAc (20 mLX3). The combined organic layers were Method 1: To a solution of compound A (922 mg, 2.0 5 washed with brine, dried over NaSO and concentrated mmol) in water (15 mL) and THF (10 mL) was added MnO, under reduced pressure and the residue was purified by silica (522 mg, 6.0 mmol. 3.0 eq) and the resulting mixture was gel column chromatography (MeOH/CHCl2, 0-1/20, V/v) to heated at reflux for 24 h. The MnO, was removed by suction give 1-((2-(2H-tetrazol-5-yl)-1,1'-biphenyl-4-yl)methyl)- filtration and the filtrate was concentrated under reduced 10 2-butyl-4-chloro-1H-imidazole-5-carbaldehyde oxime (100 pressure. The residue was dissolved in EtOH (30 mL) and the mg, 39%) as a light yellow solid. Solvent was removed by rotary evaporation to remove LC-MS (Agilent): R, 3.20 min; m/z calculated for residual water before purification by silica gel column chro CHCINO IM+H" 435.9. found 436. matography (MeOH/CHC1, 0-1/50, V/v) to give 1-((2-(2H 15 "H NMR: (400 MHz, DMSO-d) & (ppm): 11.39 (s, 1H), tetrazol-5-yl)-1,1'-biphenyl-4-yl)methyl)-2-butyl-4- 8.01 (s, 1H), 7.66-7.50 (m, 2H), 7.09 (d. J=8.0 Hz, 2H), 6.97 chloro-1H-imidazole-5-carbaldehyde (280 mg, 33%) as a (d. J=8.4 Hz, 2H), 5.56 (s. 2H), 2.50 (t, J–7.6 Hz, 2H), 1.48 brown solid. (quint, 2H), 1.28-1.19 (m, 2H), 0.80 (t, J=7.4 Hz, 3H). LC-MS (Agilent): R, 3.23 min; m/z calculated for CHCINO IM+H" 420.9, M+Na" 442.9, found 421.1, 443.1. Compound 4b. 1-((2-(2H-Tetrazol-5-yl)-1,1'-biphe "H NMR: (400 MHz, CDOD) 8 (ppm): 9.77 (s, 1H), nyl-4-yl)methyl)-2-butyl-4-chloro-1H-imidazole-5- 7.71-7.67 (m, 2H), 7.60-7.55 (m, 2H), 5.65 (s. 2H), 2.53 (s, carbaldehyde O-methyl oxime 3H), 2.69 (t, J=7.8 Hz, 2H), 1.64-1.56 (m, 2H), 1.31-1.39 (m, 25 2H), 0.90 (t, J=7.4 Hz, 3H). To a solution of intermediate B (300 mg. 0.713 mmol) in Method 2: To a solution of compound A (2.31 g, 5.0 mmol) EtOH (10 mL) and water (15 mL) was added O-methylhy int-BuOH (20 mL) was added MnO, (2.17 mg, 25.0 mmol. droxylamine hydrochloride (298 mg, 3.57 mmol. 5.0 eq) and 5.0 eq) and MeSOH (238 mg, 2.5 mmol. 0.5 eq) and the 30 KHCO (428 mg, 4.28 mmol. 6.0 eq). The resulting mixture resulting mixture was heated at reflux for 16 hours. The was heated at 60°C. for 16 h then poured into water (15 mL) mixture was allowed to cool to room temperature and MeOH and extracted with EtOAc (20 mLX3). The combined organic (50 mL) was added. The MnO, was removed by suction layers were washed with brine, dried over NaSO and con filtration and the filtrate was concentrated under reduced 35 centrated under reduced pressure and the residue was purified pressure. The residue was purified by silica gel column chro by silica gel column chromatography (MeOH/CHC1, 0-1/ matography (MeOH/CHC1, 0-1/50, V/v) to give 1-((2-(2H 50, V/v) to give 1-((2-(2H-tetrazol-5-yl)-1,1'-biphenyl-4- tetrazol-5-yl)-1,1'-biphenyl-4-yl)methyl)-2-butyl-4- yl)methyl)-2-butyl-4-chloro-1H-imidazole-5-carbaldehyde chloro-1H-imidazole-5-carbaldehyde (1.27 g. 60%) as a 40 O-methyl oxime (100 mg, 31%) as a white solid, 'H-NMR brown solid. spectroscopy revealed a 8:92 mixture of isomers. LC-MS (Agilent): R, 3.23 min; m/z calculated for LC-MS (Agilent): R, 3.34 min; m/z calculated for CHCINO IM+H" 420.9, M+Na" 442.9, found 421.1, CHCINO IM+H" 449.9. found 450.1. 443.1. 45 "H NMR: (400 MHz, DMSO-d) & (ppm):8.02 (s, 0.92H), 7.70-7.52 (m, 4H), 7.46 (s, 0.08H), 7.09 (d. J=8.4 Hz, 2H), 6.97 (d. J=8.0 Hz, 2H), 5.51 (s, 1.84H), 5.23 (s, 0.16H), 3.80 Compound 4a: 1-((2-(2H-Tetrazol-5-yl)-1,1'-biphe (s, 0.24H), 3.76 (s, 2.76H), 2.60 (t, J–7.6 Hz, 2H), 1.52(quint, nyl-4-yl)methyl)-2-butyl-4-chloro-1H-imidazole-5- 2H), 1.33-1.24 (m, 2H), 0.83 (t, J=7.2 Hz, 3H). carbaldehyde oxime 50

To a solution of intermediate B (250 mg. 0.545 mmol) in Example 5 EtOH (5 mL) and water (10 mL) was added hydroxylamine hydrochloride (189 mg, 2.72 mmol. 5.0 eq) and KHCOs (327 Formula 135 Examples 5a & 5b

US 8,946,224 B2 101 102

-continued

NH2OHHCI MeOH, RT

Intermediate B: (6-(3-(Adamantan-1-yl)-4-methox overnight. The reaction mixture was concentrated under yphenyl)naphthalen-2-yl)methanol 25 reduced pressure and the residue was purified by Silica gel column chromatography (CH2Cl2/Pet. ether, 1/10-1/2, V/v) A solution of compound A (2.00 g, 4.85 mmol. 1 eq) in to give 6-(3-(adamantan-1-yl)-4-methoxyphenyl)-2-naph THF (180 mL) was cooled to 0°C. before adding BH.THF thaldehyde oxime (180 mg. 80%) as an off-white solid. (1M solution in THF, 14.6 mL, 14.6 mmol. 3 eq) dropwise. 'HNMR: (400 MHz, CDC1)ö (ppm): 8.32 (s, 1H), 8.00 (s, The reaction mixture was warmed to room temperature and 30 1H), 7.92-7.84 (m, 4H), 7.77 (dd, J=6.8, 2.0 Hz, 1H), 7.67 (s, stirred for 3 h then diluted with water and extracted with 1H), 7.61 (d. J=2.4 Hz, 1H), 7.55 (dd, J=6.4, 2.0 Hz, 1H), 7.01 CHCl (30 mLX3). The organic layers were combined, (d. J=8.4 Hz, 1H), 3.93 (s.3H), 2.21 (d. J=2.8 Hz, 6H), 2.13 washed with brine and dried over anhydrous MgSO. The (brs, 3H), 1.82 (s, 6H). Solvent was removed under reduced pressure and the residue was purified by silica gel column chromatography (CH2Cl2/ 35 5b: 6-(3-(Adamantan-1-yl)-4-methoxyphenyl)-2- Pet. ether, 1/2, V/v) to give (6-(3-(adamantan-1-yl)-4-meth naphthaldehyde O-methyl oxime oxyphenyl)naphthalen-2-yl)methanol (1.90 g, 98%) as a white solid. A solution of intermediate C (100 mg, 0.25 mmol) and LC-MS (Agilent): R. 4.11 min; m/z calculated for O-methylhydroxylamine hydrochloride (42 mg, 0.5 mmol) in CHOM--Na 421.5. found M--Na' 421.2 40 CHCl (5 mL) and MeOH (5 mL) was stirred at room tem "H NMR: (400 MHz, CDC1) & (ppm): 8.00 (s, 1H), 7.92 perature overnight. The reaction mixture was concentrated 7.75 (m, 4H), 7.62 (d. J=2.4 Hz, 1H), 7.57-7.51 (m, 2H), 7.01 under reduced pressure and the residue was purified by silica (d. J=8.4 Hz, 1H), 4.90 (s.2H), 3.93 (s.3H), 2.21 (s, 6H), 2.13 gel column chromatography (CH.Cl/Pet. ether, 1/10-1/2, (s, 3H), 1.83 (m, 6H). V/v) to give 6-(3-(adamantan-1-yl)-4-methoxyphenyl)-2- 45 naphthaldehyde O-methyl oxime (67 mg, 62%) as an off Intermediate C. 6-(3-(Adamantan-1-yl)-4-methox white solid. yphenyl)-2-naphthaldehyde H NMR: (400 MHz, CDC1) & (ppm): 8.24 (s, 1H), 7.99 (d. J=1.6 Hz, 1H), 7.89 (m, 4H), 7.77 (dd, J=6.8, 2.0 Hz, 1H), A solution of intermediate B (1.00g, 2.51 mmol) and PCC 7.61 (d. J=2.4 Hz, 1H), 7.55 (dd, J–6.0, 2.4 Hz, 1H), 7.01 (d. (1.62 g, 7.53 mmol) in dry CHCl (80 mL) was stirred at 50 room temperature for 3 h. The solids were removed by filtra J=8.4 Hz, 1H), 4.05 (s.3H), 3.92 (s.3H), 2.21 (d. J=2.8 Hz, tion and rinsed with CHC1. The filtrate was concentrated 6H), 2.12 (s.3H), 1.82 (s, 6H). under reduced pressure and the residue was purified by silica gel column chromatography (CH2Cl2/Pet. ether, 1/10-1/2, Example 6 V/v) to give 6-(3-(adamantan-1-yl)-4-methoxyphenyl)-2- naphthaldehyde (800 mg, 80%) as a light red solid. 55 "H NMR: (400 MHz, CDC1,) & (ppm): 10.18 (s, 1H), 8.37 Formula 113—Compounds 6a & 6b (s, 1H), 8.07 (m, 2H), 7.99 (s, 1H), 7.87 (dd, J=7.2, 1.6 Hz, 1H), 7.63 (d, J-2.0 Hz, 1H), 7.58(dd, J=6.0, 2.0 Hz, 1H), 7.03 (d. J=8.8 Hz, 1H), 3.94 (s.3H), 2.21 (d. J=2.8 Hz, 6H), 2.13 60 V (s, 3H), 1.83 (s, 6H). N 5a: 6-(3-(Adamantan-1-yl)-4-methoxyphenyl)-2- M Ol C BH THF naphthaldehyde oxime N N1)a-1 oHC Oo C. HO C A solution of intermediate C (200 mg, 0.25 mmol) and 65 O N hydroxylamine hydrochloride (42 mg, 0.5 mmol) in CHCl, (5 mL) and MeOH (5 mL) was stirred at room temperature US 8,946,224 B2 103 104 -continued Example 6a

N 4-(5-(Bis(2-chloroethyl)amino)-1-methyl-1H-benzo DMP dimidazol-2-yl)butanal M Her N CH2Cl2. 5 O RT To a stirred solution of intermediate B (1.03 g, 3.0 mmol) in HO dry CHCl (50 mL) was added Dess-Martin Periodinane N (1.90 g, 4.5 mmol) at room temperature and the mixture was allowed to stir overnight. The reaction was quenched with B water and extracted with CHCl (30 mLX3). The combined V 10 N NHOHHCl, organic layers were washed with brine, dried over NaSO NaHCO and the solvent was removed under reduced pressure. The -e- residue was purified by flash chromatography (CH2Cl2/ EtOH, MeOH, 100/1 to 25/1, v/v) to give 4-(5-(bis(2-chloroethyl) CO reflux amino)-1-methyl-1H-benzodimidazol-2-yl)butanal (590 mg, 59%) as a white solid. N 15 LC-MS (Agilent): R, 2.74 min; m/z calculated for CHCN39 M+H 342.26. found M+H 342.1 H NMR: (400 MHz, CDC1) & (ppm): 8.46 (brs, 1H), 7.94 (brs, 1H), 7.35 (d. J=1.6 Hz, 1H), 6.93 (dd, J=7.6, 1.6 Hz, 1H), 3.98 (m, 4H), 3.89 (s.3H), 3.82 (t, J=6.0 Hz, 4H), 3.33 (m. 1H), 2.89 (m, 1H), 1.96-2.10 (m, 4H). Example 6b 4-(5-(Bis(2-chloroethyl)amino)-1-methyl-1H-benzo dimidazol-2-yl)butanal oxime 25 A stirred solution of example 4a (171 mg, 0.5 mmol), hydroxylamine hydrochloride (208 mg 3 mmol) and Intermediate B: 4-(5-(Bis(2-chloroethyl)amino)-1- NaHCO, (252 mg, 3 mmol) in EtOH (20 mL) was heated at methyl-1H-benzodimidazol-2-yl)butan-1-ol reflux overnight. The reaction mixture was allowed to cool to 30 room temperature and concentrated under reduced pressure. The residue was purified by flash chromatography (CH2Cl2/ To a stirred solution of compound A (393 mg, 1 mmol) in MeOH, 50/1 to 25/1, v/v) to give 4-(5-(bis(2-chloroethyl) dry THF (20 mL) was added borane in THF (1M, 3.0 mL, 3 amino)-1-methyl-1H-benzodimidazol-2-yl)butanal oxime mmol) dropwise at 0°C. under nitrogen. The resulting mix (130 mg, 73%) as a white solid, 'H-NMR spectroscopy ture was allowed to warm to room temperature and stirred revealed a ~2:1 mixture of isomers. overnight. The reaction was quenched with water at 0°C., 35 LC-MS (Agilent): R, 2.79 min; m/z calculated for extracted with CHCl (20 mLX3) and the combined organic ClHC1.N. M+H" 357.28. found M+H 357.1 layers were washed with brine then dried over anhydrous H NMR: (400 MHz, CDC1) & (ppm): 7.48 (t, J=6.0 Hz, NaSO. The solvent was removed under reduced pressure 0.65H), 7.20 (dd, J=8.8, 1.6 Hz, 1H), 7.11 (t, J=1.6 Hz, 1H), and the residue was purified by flash chromatography 6.79 (m. 1.35H), 3.76-3.64 (m, 11H), 2.89 (t, J=7.6 Hz, 2H), (CH.Cl/MeOH, 50/1 to 25/1, v/v) to give 4-(5-(bis(2-chlo 40 roethyl)amino)-1-methyl-1H-benzodimidazol-2-yl)butan 2.55 (m. 1H), 2.40 (m, 1H), 2.11 (m, 2H). 1-ol (300 mg, 86%) as a white solid. Example 7 LC-MS (Agilent): R, 2.84 min; m/z calculated for CHCINo M+H" 344.28. found M+H" 344.1 Formula 99-7a & 7b

O BH-THF PCC -e- -e- HO THF CH2Cl2, RT O° C. -RT HO O

O A B

CH(OCH3)3 TOS-OH O O MeOH, reflux O

- O C 7a.

NHOHHCI MeOH, RT US 8,946,224 B2 105 106 -continued HO -X- 7b

Intermediate B: 10 mmol. 3 eq) and Tos-OH (5 mg). The mixture was heated at 5-(2,5-Dimethylphenoxy)-2,2-dimethylpentan-1-ol reflux for 5h then allowed to cool tort and concentrated under vacuum. The residue was diluted with a Saturated aqueous To a stirred solution of compound A (250 mg, 1.0 mmol. solution of NaHCO and extracted with EtOAc (20 mLX3). 1.0 eq) in dry THF (25 mL) was added BH THF (1M solu The combined organic layers were washed with brine, dried tion in THF, 3 mL, 3 mmol. 3 eq) dropwise at 0°C. and the 15 over NaSO and the solvent was removed under reduced mixture was stirred at 0°C. for 1 h. The mixture was allowed pressure. The residue was purified by chromatography (Pet. ether/EtOAc, 100/1 to 50/1, v/v) to give 2-((5,5-dimethoxy to warm to room temperature and stirred for 16h then diluted 4,4-dimethylpentyl)oxy)-1,4-dimethylbenzene (150 mg. with water and extracted with EtOAc (20 mLX3). The com 63%) as a colourless oil. bined organic layers were washed with a saturated aqueous LC-MS (Agilent): R, 3.70 min; m/z calculated for solution of NaHCO, then brine and dried over NaSO. The C7H9. M+Na' 303.4. found 303.2. solvent was removed under reduced pressure to give 5-(2,5- H NMR: (400 MHz, CDC1) & (ppm): 7.02 (d. J=7.6 Hz, dimethylphenoxy)-2,2-dimethylpentan-1-ol (229 mg, 97%) 1H), 6.67 (d. J=7.6 Hz, 1H), 6.64 (s, 1H), 3.94 (t, J–6.4 Hz, as a colourless oil. 2H), 3.89 (s, 1H), 3.54 (s, 6H), 2.33 (s, 3H), 2.22 (s, 3H), LC-MS (Agilent): R, 3.51 min; m/z calculated for 1.81-1.76 (m, 2H), 1.50-1.46 (m, 2H), 0.94 (s, 6H). CHO, M+H 237.35. found 237.2. 25 Example 7b Intermediate C: 5-(2,5-Dimethylphenoxy)-2,2-dimethylpentanal 5-(2,5-Dimethylphenoxy)-2,2-dimethylpentanal Oxime To a solution of intermediate B (400 mg, 1.69 mmol. 1 eq) To a solution of intermediate C (90 mg, 0.384 mmol. 1 eq) in CHCl (5 mL) was added PCC (1.09 g, 5.09 mmol. 3 eq) 30 in MeOH (5 mL) was added hydroxylamine hydrochloride and the mixture was stirred at room temperature for 16 h. The (54 mg. 0.768 mmol. 2 eq) and the mixture was stirred at room solids were removed by filtration and washed with CHCl2. temperature for 16h. The solvent was removed under reduced The filtrate was concentrated under reduced pressure and the pressure and the residue was purified by chromatography residue was purified by chromatography (Pet. ether/EtOAc, (Pet. ether/EtOAc, 50/1, V/v) to give 5-(2,5-dimethylphe 50/1, V/v) to give 5-(2,5-dimethylphenoxy)-2,2-dimethyl 35 noxy)-2,2-dimethylpentanal oXime (65 mg, 68%) as a colour pentanal (215 mg, 54%) as a brown oil. less oil. LC-MS (Agilent): R, 3.51 min; m/z calculated for LC-MS (Agilent): R, 3.45 min; m/z calculated for CHO, M+Nat 257.33, M+MeOH+Nat 30 289.33. CHNO, M+H" 250.35, M+Nat 272.35, found found M+Nat 257.2, M+MeOH+Na" 289.2. M+H" 250.2, M+Nat 272.2. Example 7a 40 'HNMR: (400 MHz, CDC1) & (ppm):745 (brs, 1H), 7.36 (s, 1H), 7.02 (d. J=7.6 Hz, 1H), 6.89 (d. J=7.6 Hz, 1H), 6.63 2-((5.5-Dimethoxy-4,4-dimethylpentyl)oxy)-1,4- (s, 1H), 3.94 (t, J=6.4 Hz, 2H), 2.33 (s, 3H), 2.20 (s, 3H), dimethylbenzene 1.81-1.77 (m, 2H), 1.63-1.59 (m, 2H), 1.15 (s, 6H). Example 8 To a solution of intermediate C (200 mg. 0.85 mmol. 1 eq) ' in MeOH (10 mL) was added CH(OCH) (271 mg, 2.57 Formula 138 Compounds 8a & 8b F F O O F F N N MnO2 s He- w w CH2Cl2/Reflux w

OH O

OH OH

A B NHOHHCI

EtOH reflux NH2OCHHCI EtOH reflux US 8,946,224 B2 107 108 -continued F F O O F F N N w w HO1 N r N

OH OH

8a. 8b

Intermediate B: (3S,4R)-1-(4-Fluorophenyl)-3-(3-(4- water and extracted with EtOAc (3x50 mL). The combined fluorophenyl)-3-oxopropyl)-4-(4-hydroxyphenyl) organic layers were concentrated under reduced pressure and aZetidin-2-one the residue was purified by silica gel chromatography (Pet. ether/EtOAc, 5/1, V/v) to give (3S4R)-1-(4-fluorophenyl)-3- To a rapidly stirred solution of compound A (1.0 g, 2.4 (3-(4-fluorophenyl)-3-(methoxyimino)propyl)-4-(4-hydrox mmol) in CHCl (50 mL) was added activated manganese yphenyl)azetidin-2-one (120 mg, 56%) as a white solid. (IV) oxide (1.0 g, 12 mmol) in small portions over 15 min. LC-MS (Agilent): R, 3.33 min; m/z calculated for The mixture was heated at reflux for 18 h then additional CHF.N.O. M+H" 437.16, M+Na" 459.15, found activated manganese (IV) oxide (0.5 g. 6.0 mmol) was added 25 M+H" 437.2, M+Na" 459.1. in portions. The mixture was heated at reflux for another 24h H NMR: (400 MHz, CDC1) & (ppm): 7.63 (m, 2H), 7.25 then cooled to room temperature. The solids were removed by (m, 4H), 7.05 (app t, J=8.8 Hz, 2H), 6.93 (app t, J=8.8 Hz, filtration and washed with CHCl (3x50 mL). The filtrate 2H), 6.86 (d. J=8.4 Hz, 2H), 5.85 (s, 1H), 4.62 (d. J=2.4 Hz, was concentrated under reduced pressure and the residue was 1H), 3.89 (s.3H), 3.14 (m. 1H), 2.93 (m, 2H), 2.13 (m, 2H). purified by silica gel chromatography (Pet. ether/EtOAc, 5/1, 30 V/v) to give (3S,4R)-1-(4-fluorophenyl)-3-(3-(4-fluorophe Example 9 nyl)-3-oxopropyl)-4-(4-hydroxyphenyl)azetidin-2-one (410 mg, 41%) as a white solid. Formula 133—Compound 9a LC-MS (Agilent): R, 3.21 min; m/z calculated for CHFNO, M+H" 408.41, M+Na" 430.41, found 35 M+H 408.0, M+Nat 430.1. O 8a; (3S,4R)-1-(4-Fluorophenyl)-3-(3-(4-fluorophe nyl)-3-(hydroxyimino)propyl)-4-(4-hydroxyphenyl) U- NH o -e-BH THF aZetidin-2-one 40 THF A solution of intermediate B (180 mg, 0.44 mmol) and hydroxylamine hydrochloride (92 mg, 1.33 mmol) in EtOH A (50 mL) was heated at reflux for 5 h. The mixture was cooled to room temperature, poured into water and extracted with 45 EtOAc (3x50 mL). The organic layers were combined, dried -N/N O over NaSO and concentrated under reduced pressure. The residue was purified by silica gel chromatography (Pet. ether/ HCI EtOAc, 5/1, V/v) to afford (3S,4R)-1-(4-fluorophenyl)-3-(3- (4-fluorophenyl)-3-(hydroxyimino)propyl)-4-(4-hydrox 50 9a yphenyl)azetidin-2-one (108 mg, 60%) as a white solid. LC-MS (Agilent): R, 2.70 min; m/z calculated for CHFNO IM+H 423.14, M+Na 445.14, found 9a: (S)- N-(2-(2,6,7,8-Tetrahydro-1H-indeno5,4-b M+H 423.1, M+Na" 445.1. furan-8-yl)ethyl)propan-1-amine hydrochloride H NMR: (400 MHz, DMSO-d) & (ppm): 11.3 (s, 1H), 55 9.55 (s, 1H), 7.69 (dd, J=8.8, 5.6 Hz, 2H), 7.21 (m, 8H), 6.76 To a solution of compound A (200 mg, 0.77 mmol) in (d. J–8.4 Hz, 2H), 4.89 (d. J–2.0 Hz, 1H), 3.15 (m. 1H), 2.87 anhydrous THF (50 ml) was added BH THF (1M solution in (m. 2H), 2.22 (m. 2H). THF, 2.3 mL, 2.3 mmol) and the mixture was stirred at room temperature for 2 h. A 1M aqueous HCl solution was then 8b: (3S,4R)-1-(4-Fluorophenyl)-3-(3-(4-fluorophe 60 added dropwise into the reaction mixture until pH 7. The nyl)-3-(methoxyimino)propyl)-4-(4-hydroxyphenyl) solution was extracted with EtOAc (3x50 mL) and the aZetidin-2-one organic layers were combined, dried over Na2SO and con centrated under reduced pressure. The residue was purified by A solution of intermediate B (200 mg, 0.49 mmol) and silica gel column chromatography (Pet. ether/EtOAc, 5/1, O-methylhydroxylamine hydrochloride (123 mg, 1.47 65 V/v) to give (S)- N-(2-(2,6,7,8-tetrahydro-1H-indeno5,4-b mmol) in EtOH (50 mL) was heated at reflux for 5 h. The furan-8-yl)ethyl)propan-1-amine hydrochloride (105 mg. mixture was cooled to room temperature and poured into 56%) as a white solid. US 8,946,224 B2 109 110 LC-MS (Agilent): R, 2.84 min; m/z calculated for LC-MS (Agilent): R, 3.15 min; m/z calculated for CHNOM+H" 246.36. found 246.2. CHNO, M+H" 427.18, M+Nat 449.18, found HNMR:(400 MHz, CDC1)8(ppm): 9.56(brs, 2H), 6.94 IM+H" 427.2. M+Nal" 449.2. 3.36(d. J=8.0 (m. 1H),Hz, 1H),3.24-3.16 6.62 (d. (m. J=8.0 2H), Hz,3.13-275 1H), 4.62-4.50 (m, 6H), (m,2.48 2H), (m, 5 Intermediate C: 1-(2-(1H-Tetrazol-5-yl)-1,1-biphe 1H), 2.29 (m. 1H), 2.12 (m, 1H), 1.89 (m, 2H), 1.81-1.71 (m, nyl-4-yl)methyl)-2-ethoxy-1H-benzodimidazole 1H), 0.92 (t, J=7.6 Hz, 3H). 7-carbaldehyde E 1e 10 To a stirred solution of intermediate B (1.5 g., 3.5 mmol) in Xample 10 DMSO (50 mL) was added Dess-Martin Periodinane (2.2g, 5.25 mmol). The mixture was stirred at room temperature for Formula 137 Compounds 10a & 10b 6h then poured into a saturated aqueous solution of NaHSO

O HO On HO-Ns AN AN AN OH N LiAlH4. N -( Dess- N -K N -K

O- THF O HerMartin o-v HipNH2OHHCI o-w RT DMSO, AcOK, RT MeOH HN-N HN-N HN-N HN-N W W W W CryNN Orn N CrsNN CrsNN A B C 10a

NHOMe-HCl AcOK, MeOH

Y, -N N N -( O \

HN-N N \

1Ob

Intermediate B: (1-((2-(1H-Tetrazol-5-yl)-1,1'-bi- (300 mL). The precipitate formed was collected by filtration phenyl-4-yl)methyl)-2-ethoxy-1H-benzodimida- and washed with a saturated aqueous solution of NaHCO (50 Zol-7-yl)methanol mLX3) to give 1-((2-(1H-tetrazol-5-yl)-1,1'-biphenyl-4-yl) 55 methyl)-2-ethoxy-1H-benzodimidazole-7-carbaldehyde To a solution of compound A (2.0g, 4.54 mmol) in dry THF (0.8 g. 54%) as a white solid. (50 mL) at room temperature was added LAH (345 mg, 9.1 LC-MS (Agilent): R, 3.26 min; m/z calculated for mmol) in five portions. The mixture was stirred at room CHNO. M+H 425.16, M+Na 447.16, found temperature overnight then cooled to 0°C. and quenched with M+H 425.2, M+Nat 447.1. water (100 mL) and stirred for an additional 30 min. The 60 Example 10a reaction mixture was filtered and the filtrate was acidified slowly with a 1M aqueous HCl solution. The resulting crys- 1-((2-(1H-Tetrazol-5-yl)-1,1'-biphenyl-4-yl)me talline precipitate was collected by suction filtration and thyl)-2-ethoxy-1H-benzodimidazole-7-carbalde washed with a saturated aqueous solution of NaHCOs (3x50 hyde oxime mL) to give (1-((2-(1H-tetrazol-5-yl)-1,1'-biphenyl-4-yl) 65 methyl)-2-ethoxy-1H-benzodimidazol-7-yl)methanol (1.5 A mixture of intermediate C (100 mg 0.24 mmol), AcOK g, 77%) as a white solid. (46 mg, 0.47 mmol) and hydroxylamine hydrochloride (33 US 8,946,224 B2 111 mg, 0.47 mmol) in MeOH (5 mL) was stirred at room tem -continued perature for 20 min. The solvent was removed under reduced pressure at room temperature and the residue was poured into water and the mixture was stirred at room temperature for 10 C min. The solid formed was filtered, washed with water (10 O O mLX3) and dried under vacuum at 50° C. for 3 h to give 1-((2-(1H-tetrazol-5-yl)-1,1'-biphenyl-4-yl)methyl)-2- O NH, 0°C. ethoxy-1H-benzodimidazole-7-carbaldehyde oxime (80 mg, 77%) as a white solid. N LC-MS (Agilent): R, 3.23 min; m/z calculated for 10 H CH, NO, M+H" 440.47, M+Na" 462.47, found M+H 440.2, M+Na" 462.2. s H NMR: (400 MHz, DMSO-d) & (ppm): 11.3 (brs, 1H), O 8.30 (s, 1H), 7.47-7.63 (m, 5H), 7.29 (d. J=7.6 Hz, 1H), 7.12 15 D (t, J=8.0 Hz, 1H), 7.03 (d. J=8.0 Hz, 2H), 6.95 (d. J=8.0 Hz, 2H), 5.48 (s. 2H), 4.58 (q, J–7.2 Hz, 2H), 1.38 (t, J–7.2 Hz, 3H). C O O Ag2O, CH2Cl2, Example 10b Hip Br CN 1-((2% (1 H-Tetrazol-5-yl)-1,1'-biphenyl-4-yl)me thyl)-2-ethoxy-1H-benzodimidazole-7-carbalde N hyde O-methyl oxime H 25 OH A mixture of intermediate C (150 mg, 0.35 mmol), AcOK E (69 mg, 0.71 mmol) and O-methylhydroxylamine hydrochlo ride (59 mg 0.71 mmol) in MeOH (5 mL) was stirred at room temperature for 20 min. The solvent was removed under C reduced pressure at room temperature and the residue was O 30 O NHCl, NaOMe, poured into water and the mixture was stirred at room tem MeOH perature for 10 min. The solid formed was filtered and washed O with water (10 mLX3). The solid was collected and dried under reduced pressure at 50° C. for 3 h to give 1-((2-(1H tetrazol-5-yl)-1,1'-biphenyl-4-yl)methyl)-2-ethoxy-1H N benzodimidazole-7-carbaldehyde O-methyl oxime (95 mg. 35 H 59%) as a white solid. LC-MS (Agilent): R, 3.30 min; m/z calculated for s C.H.N.O. M+H" 454.50, M+Na". 476.50, found CN M+H" 454.2, M+Nat 476.2. "H NMR: (400 MHz, DMSO-d) & (ppm): 8.36 (s, 1H), 40 F 748-7.64 (m, 5H), 7.30 (d. J=8.0 Hz, 1H), 7.15 (t, J=8.0 Hz, 1H), 7.04 (d. J=8.0 Hz, 2H), 6.92 (d. J=8.0 Hz, 2H), 5.49 (s. C 2H), 4.58 (q, J–7.2 Hz, 2H), 3.81 (s, 3H), 1.38 (t, J–7.2 Hz, O O 3H). 45 Example 11 No || || O Formula 147 Compound 11a N 50

O O ulus -e-Br, CH3COOH HN NH 1N 55 11a A CHCOOK, DMF -e- BN-N-N- Intermediate B: Ethyl 4-bromo-3-oxobutanoate B 60 O O To a solution of compound A (10.0g, 76.9 mmol. 1.0 eq) in acetic acid (30 mL) was added bromine (12.3 g, 76.9 mmol. O Sulus i-ProH, 859 C. e 1.0 eq) at 0°C. over 10 min. The mixture was stirred at 0°C. Y 1\ for 1 h, the solvent was removed under reduced pressure and O 65 the residue was diluted with water (50 mL). The aqueous C mixture was extracted with CHCl (50 mLX3). The com bined organic layers were washed with brine (60 mLx2), US 8,946,224 B2 113 114 dried over MgSO and concentrated under reduced pressure mmol. 3.0 eq) at room temperature. The mixture was stirred at to give ethyl 4-bromo-3-oxobutanoate (14.3 g, 85%) as a room temperature for 1 h before addition of AgO (1.1 g, 4.8 yellow oil. mmol. 3.0 eq) and n-Bu-NI (586 mg, 1.6 mmol. 1.0 eq). LC-MS (Agilent): R, 3.06 min; m/z calculated for Stirring was continued at room temperature for an additional CHBrO, M+H 208.97. found 209.1. 16 h in the dark. The solids were removed by filtration through Celite and the filtrate was concentrated under Intermediate C: Ethyl 4-acetoxy-3-oxobutanoate reduced pressure. The residue was purified by flash chroma tography (Pet. ether/EtOAc, 1/10 to 1/2, V/v) to give 3-ethyl To a solution of intermediate B (10.0 g, 47.4 mmol. 1.0 eq) 5-methyl 4-(2-chlorophenyl)-2-((cyanomethoxy)methyl)-6- in dry DMF (60 mL) was added potassium acetate (13.9 g, 10 methyl-1,4-dihydropyridine-3,5-dicarboxylate (0.40 g. 60%) 142.2 mmol. 3.0 eq) at room temperature. The mixture was as a yellow solid. heated at 80° C. for 16 h then allowed to cool to room tem LC-MS (Agilent): R, 3.30 min; m/z calculated for perature, diluted with EtOAc (150 mL) and washed with CHCINOs M+H" 405.1, M+Na" 427.1, found water (120 mLX3). The organic layer was washed with brine M+H" 405.1, M+Na" 427.1. (60 mLx2), dried over NaSO and concentrated under 15 H NMR: (400 MHz, CDC1) & (ppm): 7.38 (m, 1H), 7.25 reduced pressure. The residue was purified by flash chroma (m. 1H), 7.16 (m, 1H), 7.10 (m, 1H), 6.71 (brs, 1H), 5.43 (s, tography (Pet. ether/EtOAc, 1/10 to 1/2, V/v) to give ethyl 1H), 4.95 (d. J=16.0 Hz, 1H), 4.88 (d. J=14.8 Hz, 1H), 4.41 (s, 4-acetoxy-3-oxobutanoate (1.44g, 16%) as a yellow oil. 2H), 4.08 (m,2H), 3.64 (s.3H), 2.36 (s.3H), 1.21 (t, J–7.2 Hz, LC-MS (Agilent): R, 3.12 min; m/z calculated for 3H). CHOs M+H" 189.07. found 189.1 11a: 3-Ethyl 5-methyl 2-((amidinomethoxy)methyl)- Intermediate D: 3-Ethyl 5-methyl 2-(acetoxym 4-(2-chlorophenyl)-1,4-dihydro-6-methylpyridine-3, ethyl)-4-(2-chlorophenyl)-1,4-dihydro-6-methylpyri 5-dicarboxylate dine-3,5-dicarboxylate To a solution of intermediate F (380 mg, 0.940 mmol) and To a solution of intermediate C (1.2g, 6.4 mmol. 1.0 eq) 25 NHCl (127 mg, 2.35 mmol. 2.5 eq) in toluene (35 mL) was and 2-chlorobenzaldehyde (890 mg., 6.4 mmol. 1.0 eq) in added NaOMe (127mg, 2.35 mmol. 2.5 eq) and the resulting isopropanol (30 mL) was added (Z)-methyl 3-aminobut-2- mixture was stirred at 80°C. for 40 min. After cooling to room enoate (736 mg, 6.4 mmol. 1.0 eq) and the mixture was heated temperature, the mixture was treated with a methanolic at reflux for 16 h. The mixture was concentrated under ammonia solution (1.0M, 10 mL, 10 mmol) and stirred for an reduced pressure and the residue was diluted with water (50 30 additional 2 h. The solvent was removed under reduced pres mL). The aqueous mixture was extracted with EtOAc (60 sure and the residue was diluted with water (50 mL) and mLX3) and the combined organic layers were washed with extracted with CHCl (50 mLX3). The combined organic brine, dried over NaSO and concentrated under reduced layers were washed with brine (60 mLX2), dried over MgSO pressure to give 3-ethyl 5-methyl 2-(acetoxymethyl)-4-(2- and concentrated under reduced pressure. The residue was chlorophenyl)-1,4-dihydro-6-methylpyridine-3,5-dicar purified by flash chromatography (Pet. ether/EtOAc, 1:10 to 35 1:2, V/v) to give 3-ethyl 5-methyl 2-((amidinomethoxy)me boxylate (1.2g, 53%) as a light yellow solid. thyl)-4-(2-chlorophenyl)-1,4-dihydro-6-methylpyridine-3, LC-MS (Agilent): R, 3.20 min; m/z calculated for 5-dicarboxylate (210 mg, 53%) as a light yellow solid. CHCINO IM+H" 408.11. found 408.1. LC-MS (Agilent): R, 3.00 min; m/z calculated for Intermediate E: 3-Ethyl 5-methyl 4-(2-chlorophe CHCINOs M+H 422.1. found 422.1. nyl)-2-(hydroxymethyl)-6-methyl-1,4-dihydropyri 40 "H NMR: (400 MHz, CDC1), 8 (ppm): 8.38 (brs, 3H), dine-3,5-dicarboxylate 8.21 (s, 1H), 7.36-7.39 (m, 1H), 7.21 (d. J=8.0 Hz, 1H), 7.11 (t, J=7.6 Hz, 1H), 7.00 (t, J=7.2 Hz, 1H), 5.38 (s, 1H), 4.56 To a solution of intermediate D (1.2g, 2.9 mmol. 1.0 eq) in 4.84 (m, 4H), 3.93-4.06 (m, 2H), 3.58 (s, 3H), 2.37 (s, 3H), methanol (20 mL) was added a methanolic ammonia Solution 1.14 (t, J=6.8 Hz, 3H). (1.0M, 15 mL, 15 mmol). The mixture was stirred at 0°C. for 45 2h then the solvent was removed under reduced pressure and Example 12 the residue was diluted with water (50 mL). The aqueous mixture was extracted with CHCl (50 mLX3) and the com Formula 139 Compounds 12a & 12b bined organic layers were washed with brine (60 mLx2), dried over MgSO and concentrated under reduced pressure. 50 O The residue was purified by flash chromatography (Pet. ether/ LiOH, EtOAc, 1/10 to 1/2, V/v) to give 3-ethyl 5-methyl 4-(2-chlo THF, rophenyl)-2-(hydroxymethyl)-6-methyl-1,4-dihydropyri H2O dine-3,5-dicarboxylate (0.70 g. 65%) as a yellow solid. He LC-MS (Agilent): R, 3.28 min; m/z calculated for 55 )- O C CHCINOs M+H 366.1, M+Na" 388.1, found M+H 366.1, M+Nat 388.1. O "H NMR: (400 MHz, CDC1) & (ppm): 7.39 (m, 1H), 7.25 A (m. 1H), 7.13 (m. 1H), 7.05 (m, 1H), 5.41 (s, 1H), 4.75 (d. O J–4.4 Hz, 2H), 4.06 (m, 2H), 3.63 (s.3H), 2.33 (s.3H), 1.20 (t, J–7.2 Hz, 3H). 60

Intermediate F: 3-Ethyl 5-methyl 4-(2-chlorophe HO nyl)-2-((cyanomethoxy)methyl)-6-methyl-1,4-dihy O C dropyridine-3,5-dicarboxylate 65 O To a solution of intermediate E (0.6 g. 1.6 mmol. 1.0 eq) in B CHCl (20 mL) was added 2-bromoacetonitrile (0.59 g, 4.8 US 8,946,224 B2 116 -continued EtOAc (10 mLX3). The combined organic layers were OH washed with brine, dried over MgSO and the solvent was Dess removed under reduced pressure. The residue was purified by Martin flash chromatography (Pet. ether/EtOAc, 5/1 to 2/1, V/v) to He 5 give 2-(4-((4-chlorophenyl)(hydroxy)methyl)phenoxy)-2- to-X CHCl2 methylpropan-1-ol (452 mg, 94%) as a white solid. O C LC-MS (Agilent): R, 3.00 min; m/z calculated for C CHCIO, M+Na" 329.1. found 329.0. O 12a: 2-(4-(4-Chlorobenzoyl)phenoxy)-2-methylpropanal

Hip O O NHOHHCIPyridine To a stirred solution of intermediate C (453 mg, 1.4 mmol) NN-X O C y in CHCl (10 mL) at room temperature was added Dess 12a 15 Martin Periodinane (1.8 g. 4.3 mmol) and the resulting mix O ture was stirred overnight. The reaction was quenched with water and the mixture was extracted with CHCl (10 mLX3) and the combined organic layers were washed with brine and dried over MgSO. The solvent was removed under reduced pressure and the residue was purified by flash chromatogra HO1 N O C phy (Pet. ether/EtOAc, 5/1 to 2/1, V/v) to give 2-(4-(4-chlo 12b robenzoyl)phenoxy)-2-methylpropanal (284 mg. 66%) as a white solid. LC-MS (Agilent): R, 3.37 min; m/z calculated for Intermediate B: 25 CHCIOM+MeOH+H" 335.1. found 335.1. 2-(4-(4-chlorobenzoyl)phenoxy)-2-methylpropanoic 'H-NMR: (400 MHz, CDC1) & (ppm): 9.82 (s, 1H), 7.74 acid (d. J=9.2 Hz, 2H), 7.71 (d. J–8.4 Hz, 2H), 7.45 (d. J=8.4 Hz, 2H), 6.89 (d. J=8.8 Hz, 2H), 1.52 (s, 6H). To a stirred solution of compound A (1.0g, 2.77 mmol) in THF (10 mL) was added LiOH.HO (0.7g, 16.6 mmol) and 30 12b: (E)-2-(4-(4-Chlorobenzoyl)phenoxy)-2-methyl H2O (10 mL). The resulting mixture was heated at reflux propanal oXime overnight then quenched with a 1 Maqueous HCl solution and extracted with EtOAc (10 mLX3). The combined organic A solution of example 11a (80 mg, 0.26 mmol) and layers were washed with brine, dried over MgSO and con hydroxylamine hydrochloride (18 mg, 0.26 mmol) in pyri centrated under reduced pressure. The residue was purified by 35 dine (2.5 mL) was stirred at 10°C. for 90 min. The solvent flash chromatography (CH.Cl/MeCH, 15/1, V/v) to give was removed under reduced pressure the residue was purified 2-(4-(4-chlorobenzoyl)phenoxy)-2-methylpropanoic acid by flash chromatography (Pet. ether/EtOAc, 10/1 to 5/1, V/v) (130 mg, 15%) as a white solid. to give (E)-2-(4-(4-chlorobenzoyl)phenoxy)-2-methylpropa LC-MS (Agilent): R, 3.00 min; m/z calculated for nal oxime (52 mg, 62%) as a white solid. CHCIO, M+H" 319.07. found 319.1. 40 LC-MS (Agilent): R, 3.32 min; m/z calculated for CHCINOM+H" 318.08, M+Nat 340.1, found Intermediate C: 2-(4-((4-Chlorophenyl)(hydroxy) M+H" 318.1, M+Nat 340.1. methyl)phenoxy)-2-methylpropan-1-ol H-NMR: (400 MHz, DMSO-d) & (ppm): 11.1 (s, 1H), 7.70 (m, 4H), 7.62-7.59 (m, 3H), 7.06 (d. J=8.8 Hz, 2H), 1.53 To a stirred solution of intermediate B (500 mg, 1.57 45 (s, 6H). mmol) in dry THF (10 mL) at 0°C. under nitrogen was added a solution of borane in THF (1M, 4.7 mL, 4.7 mmol) drop Example 13 wise. The resulting mixture was heated at 50° C. for 3 h then cooled to 0°C., quenched with MeOH and extracted with Formula 102 Compounds 13a & 13b OO THF 2-Iodoxyben CICOEt Zoin acid NH2OHHCI O He-2. NaBH4 O He-DMSO O HipMeOH O

Sa n O1 OH OH O OH A B C 13a Norto US 8,946,224 B2 117 118 -continued

13b

Intermediate B: (4-(1-(3.5,5,8,8-Pentamethyl-5.6.7. due was partitioned between EtOAc (300 mL) and water (300 8-tetrahydronaphthalen-2-yl) vinyl)phenyl)methanol mL). The layers were separated and the aqueous phase was extracted with EtOAc (200 mLx2). The combined organic To a stirred solution of compound A (4.0 g, 11.5 mmol) in layers were dried over NaSO and the solvent was removed THF (100 mL) at room temperature was added ethyl chloro under reduced pressure to give 4-(1-(3,5,5,8,8-pentamethyl formate (1.43 mL, 14.3 mmol) and triethylamine (2.26 mL). 25 5,6,7,8-tetrahydronaphthalen-2-yl)Vinyl)benzaldehyde The mixture was stirred at room temperature for 30 min and then filtered. The filtrate was diluted with water and the sol oxime (160 mg, 100%) as a white solid. vent was removed under reduced pressure. To the residue was LC-MS (Agilent): R, 3.93 min; m/z calculated for added ice water (200 mL) and NaBH (15g, 38 mmol). The CHNO IM+H" 348.2, M+Na" 370.5. found M+H" resulting mixture was stirred at 0°C. for 1 h then water (100 30 348.2, M+Nat 370.2. mL) and methyl t-butyl ether (300 mL) was added. The H NMR: (400 MHz, CDC1) & (ppm): 8.12 (s, 1H), 7.51 organic layer was separated, washed with brine, dried over (d. J=8.4 Hz, 2H), 7.31 (d. J=8.4 Hz, 2H), 7.12 (s, 1H), 7.07 Na2SO and concentrated under reduced pressure to give (s, 1H), 5.77 (d. J=1.2 Hz, 1H), 5.25 (d. J=1.2 Hz, 1H), 1.96 (4-(1-(3.5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen (s, 3H), 1.70 (s, 4H), 1.30 (s, 6H), 1.27 (s, 6H). 2-yl) vinyl)phenyl)methanol (3.6 g., 93%) as a white solid. 35 LC-MS (Agilent): R, 3.77 min; m/z calculated for 13b: 4-(1-(3,5,5,8,8-Pentamethyl-5,6,7,8-tetrahy CHOM--Na' 357.2. found 357.2. dronaphthalen-2-yl) vinyl)benzaldehyde O-methyl Oxime Intermediate C: 4-(1-(3.5,5,8,8-Pentamethyl-5,6,7,8- tetrahydronaphthalen-2-yl) vinyl)benzaldehyde 40 To a stirred solution of intermediate C (100 mg, 0.3 mmol) in methanol (5 mL) was added O-methylhydroxylamine To a stirred solution of intermediate B (0.5g, 1.50 mmol) in hydrochloride (75 mg. 0.9 mmol) and the mixture was stirred DMSO (20 mL) was added 2-iodoxybenzoic acid (0.84g, 3.0 at room temperature overnight. The methanol was removed mmol) and the mixture was stirred at room temperature for 2 under reduced pressure and the residue was partitioned h. The reaction was quenched with NaHSO, and the mixture 45 between EtOAc (200 mL) and water (200 mL). The layers was diluted with EtOAc (400 mL) and washed with water were separated and the aqueous phase was extracted with (400 mLx4). The organic layer was dried over NaSO and EtOAc (150 mLx2). The combined organic layers were dried the solvent was removed under reduced pressure to give 4-(1- over NaSO and the solvent was removed under reduced (3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl) pressure to give 4-(1-(3.5,5,8,8-pentamethyl-5,6,7,8-tetrahy vinyl)benzaldehyde (0.48g, 97%) as a white solid. 50 dronaphthalen-2-yl) vinyl)benzaldehyde O-methyl oxime (70 LC-MS (Agilent): R, 3.93 min; m/z calculated for mg, 64%) as a white Solid. CHO M+H 333.5, M+Na" 355.5. found M+H" LC-MS (Agilent): R. 4.42 min; m/z calculated for 333.2, M+Nat 355.2. CHNO IM+H" 362.2, M+Nat 384.5. found M+H" 362.3, M+Nat 384.2. 13a: 4-(1-(3,5,5,8,8-Pentamethyl-5,6,7,8-tetrahy 55 "H NMR: (400 MHz, CDC1,) & (ppm): 8.04 (s, 1H), 7.51 dronaphthalen-2-yl) vinyl)benzaldehyde oxime (d. J=8.4 Hz, 2H), 7.29 (d. J=10.8 Hz, 2H), 7.12 (s, 1H), 7.07 (s, 1H), 5.76 (s, 1H), 2.25 (s, 1H), 3.97 (s.3H), 1.95 (s.3H), To a stirred solution of intermediate C (150 mg, 0.45 1.69 (s, 4H), 1.30 (s, 6H), 1.27 (s, 6H). mmol) in methanol (10 mL) at room temperature was added hydroxylamine hydrochloride (94 mg, 1.35 mmol) and the 60 Example 14 mixture was stirred at room temperature overnight. The methanol was removed under reduced pressure and the resi Formula 151—Compounds 14a & 14b

US 8,946,224 B2 121 122 Intermediate B: Methyl 2-(2-chlorophenyl)-2-(6,7- din-5(4H)-yl)acetaldehyde (80 mg) as a light yellow oil, dihydrothieno 3.2-cpyridin-5(4H)-yl)acetate which was used directly in the next step. 14a: 2-(2-Chlorophenyl)-2-(6,7-dihydrothieno 3.2-c. Compound A (1.25 g, 0.03 mol) was treated with a satu pyridin-5(4H)-yl)ethyl acetate rated aqueous NaCO Solution (10 mL) and the mixture was extracted with CHCl (30 mLx2). The combined organic To a stirred solution of intermediate C (270 mg. 0.9 mmol) layers were dried over NaSO and the solvent was removed in CHCl (20 mL) at room temperature was added acetyl under reduced pressure to give methyl 2-(2-chlorophenyl)-2- 10 chloride (235 mg, 3 mmol. 3.0 eq) and the resulting mixture (6,7-dihydrothieno3.2-cpyridin-5(4H)-yl)acetate (0.96 g. was stirred at this temperature overnight. The mixture was 99%) as a yellow oil. concentrated under reduced pressure and the residue was purified by column chromatography (Pet. ether/EtOAc, 5/1, Intermediate C: 2-(2-Chlorophenyl)-2-(6,7-dihy V/v) to give 2-(2-chlorophenyl)-2-(6,7-dihydrothieno 3.2-c. drothieno 3.2-cpyridin-5(4H)-yl)ethanol 15 pyridin-5(4H)-yl)ethyl acetate (80 mg, 35%) as a light yellow oil. To a stirred solution of intermediate B (960 mg, 3.0 mmol. LC-MS (Agilent): R, 3.15 min; m/z calculated for 1.0 eq) in CHCl (15 mL) at 0°C. was added a 1.0M solution CHCINOS M+H"336.07. found 336.1. of DIBAI-H in hexanes (9 mL, 9.0 mmol. 3.0 eq) dropwise "H NMR: (400 MHz, CDC1) & (ppm): 7.61 (m, 1H), 7.38 and the mixture was stirred at room temperature for 1 h. The (m. 1H), 7.26 (m, 2H), 7.09 (d. J–4.8 Hz, 1H), 6.97 (d. J–4.8 reaction was quenched with water (10 mL) and the mixture HZ, 1H), 4.55 (m, 1H), 4.41 (t, J=5.6 Hz, 1H), 4.37 (m. 1H), 3.83 (d. J–14.4 Hz, 1H), 3.62 (d. J=14.8 Hz, 1H), 2.76-2.93 was extracted with CHCl (25 mLx2). The combined 25 (m, 4H), 2.00 (s.3H). organic layers were washed with brine, dried over NaSO and the solvent was removed under reduced pressure to give 14b: (E)-2-(2-Chlorophenyl)-2-(6,7-dihydrothieno3, 2-(2-chlorophenyl)-2-(6,7-dihydrothieno 3.2-cpyridin-5 2-cpyridin-5(4H)-yl)acetaldehyde O-methyl oxime (4H)-yl)ethanol (850 mg, 95%) as a yellow oil. LC-MS (Agilent): R, 2.88 min; m/z calculated for 30 To a solution of intermediate D (70 mg, 0.24 mmol) in CHCINOS M+H 294.06. found 294.1. methanol (2 mL) at room temperature was added O-methyl H NMR: (400 MHz, CDC1) & (ppm): 749 (m, 2H), 7.28 hydroxylamine hydrochloride (40 mg, 0.48 mmol. 2.0 eq). (m. 2H), 7.10 (d. J=5.2 Hz, 1H), 6.75 (d. J=5.2 Hz, 1H), 4.55 The resulting mixture was heated at 70° C. for 2 h and the (dd, J=4.8, 4.4 Hz, 1H), 4.00 (dd, J=11.2, 7.6 Hz, 1H), 3.84 35 reaction was quenched by addition of a Saturated aqueous (dd, J=11.2, 4.8 Hz, 1H), 3.80 (d. J=14.4 Hz, 1H), 3.68 (d. NaCO solution until pH a' 8. The mixture was extracted J=14.4 Hz, 1H), 3.06 (m. 1H), 2.90 (m, 2H), 2.80 (m, 1H). with EtOAc and the combined organic layers were washed with brine, dried over NaSO and the solvent was removed Intermediate D: 2-(2-Chlorophenyl)-2-(6,7-dihy 40 under reduced pressure to give (E)-2-(2-chlorophenyl)-2-(6. drothieno 3.2-cpyridin-5(4H)-yl)acetaldehyde 7-dihydrothieno 3.2-cpyridin-5(4H)-yl)acetaldehydeO-me thyl oxime (50 mg, 40%) as a light yellow solid. To a stirred solution of intermediate C (440 mg, 1.5 mmol. LC-MS (Agilent): R, 2.88 min; m/z calculated for 1.0 eq) in CHCl (10 mL) at room temperature was added 45 C.H.CINOS M+HO+H" 339.08. found 339.1 PCC (645 mg, 3 mmol) and Celite (-0.5 g). The mixture was H NMR: (400 MHz, CDC1) & (ppm): 8.15 (s, 1H), 7.22 stirred at room temperature for 3 h, additional PCC (645 mg. 7.39 (m, 5H), 7.14 (d. J=5.2 Hz, 1H), 4.35 (dd, J–8.4, 4.0 Hz, 3 mmol) was added and stirring was continued for an addi 1H), 3.95 (s, 3H), 3.79 (dd, J=10.8, 4.0 Hz, 1H), 3.50 (dd, tional 5 h at 40°C. The mixture was filtered and the filtrate 50 J=10.8, 8.4 Hz, 1H), 3.08 (m, 2H), 2.83 (m, 2H). was washed with water, dried over NaSO and the solvent was removed under reduced pressure. The residue was puri Example 15 fied by column chromatography (Pet. ether/EtOAc, 20/1, V/v) to give 2-(2-chlorophenyl)-2-(6,7-dihydrothieno 3.2-cpyri Formula 121—Compounds 15a and 15b

O C

C OHC CC OHC Pol/C, N NO 1N NO -e- -- HN NaH, DMF EtN, CHCl2 le N NO H B C US 8,946,224 B2 123 124 -continued O C O C

N EtOH:HO (2:1) N NO NH2

D E BH THF

CrsN NH2

O F C

Et3N, F CHCl2

O C O C

N N N N H H

F F 15a 15b

Intermediate E: (4-Amino-2-chlorophenyl)(5H LC-MS (Agilent): R, 3.41 min; m/z calculated for benzoelpyrrolo 1,2-a 1,4-diazepin-10(11H)-yl) C.H.CIN, M+H 324.12. found 324.1. methanone 45 15a: N-(4-((5H-Benzoelpyrrolo 1,2-a 1,4-diaz (4-Amino-2-chlorophenyl)(5H-benzoelpyrrolo 1,2-a1, epin-10(11H)-yl)methyl)-3-chlorophenyl)-5-fluoro 4diazepin-10(11H)-yl)methanone was obtained from com 2-methylbenzamide pound A in four steps and 27% overall yield according to the procedures described in J. Med. Chem. 1998, 41,2442-2444 50 and J. Med. Chem. 1980, 23, 462-465. To a solution of intermediate F (100 mg, 0.3 mmol. 1.0 eq) in CHCl (15 mL) was added triethylamine (1.0g, 0.9 mmol. Intermediate F: 4-((5H-Benzoelpyrrolo 1,2-a 1.4 3.0 eq) at room temperature and the resulting mixture was diazepin-10(11H)-yl)methyl)-3-chloroaniline stirred for 30 min. A solution of 5-fluoro-2-methylbenzoyl 55 chloride (50.0 mg, 0.36 mmol. 1.2 eq) in CHCl (5 mL) was To a solution of intermediate E (600 mg, 1.8 mmol. 1.0 eq) then added and stirring was continued for a further 18 h. The Solvent was evaporated under reduced pressure to give a solid, in dry THF (20 mL) was added a 1.0 M solution of BH in which was purified by flash chromatography (Pet. ether/ THF (4.5 mL, 4.5 mmol. 2.5 eq) and the resulting mixture was EtOAc,571, V/v) to give N-(4-((5H-benzoelpyrrolo 1,2-a1, stirred at room temperature for 18 h. Water (20 mL) was 60 added and mixture was stirred for 15 minutes then extracted 4diazepin-10(11H)-yl)methyl)-3-chlorophenyl)-5-fluoro with EtOAc. The combined organic layers were dried over 2-methylbenzamide (72 mg, 51%) as a white solid. NaSO and concentrated under reduced pressure to give a LC-MS (Agilent): R, 3.69 min; m/z calculated for solid which was purified by flash chromatography (Pet. ether/ CHCIFNO IM+H" 460.15. found 460.1. EtOAc, 10/1, V/v) to give 4-((5H-benzoelpyrrolo 1,2-a1, 65 H NMR: (400 MHz, DMSO-d) & (ppm): 10.5 (s, 1H), 4diazepin-10(11H)-yl)methyl)-3-chloroaniline (110 mg. 8.01 (d.J=2.0 Hz, 1H), 7.57 (dd, J=8.4, 2.0 Hz, 1H), 7.37-7.31 19%) as a white solid. (m,3H), 7.25 (m. 1H), 7.15 (dd, J-7.6., 1.6 Hz, 1H), 7.08 (m, US 8,946,224 B2 125 126 1H), 6.82 (m. 1H), 6.70 (m, 1H), 6.63 (d. J=8.4 Hz, 1H), -continued 5.89-5.92 (m, 2H), 5.29 (s. 2H), 4.48 (d. J–3.6 Hz, 4H), 2.35 O (s, 3H). N)-()—a NHOMe-HCI 15b: (5H-Benzoelpyrrolo 1,2-a 1,4diazepin-10 MeOH (11H)-yl)(2-chloro-4-(5-fluoro-2-methylbenzy / lamino)phenyl)methanone Niv OH

A solution of intermediate E (200 mg, 0.6 mmol. 1.0 eq) 10 and 5-fluoro-2-methyl-benzaldehyde (124 mg. 0.9 mmol. 1.5 16a eq) in MeCH (20 mL) was heated at reflux for 18 h then cooled to room temperature. NaBH (45 mg, 1.2 mmol. 2.0 eq) was then added and the mixture was stirred at room temperature for 4 h. The solvent was removed under reduced 15 pressure to give a solid, which was purified by flash chroma tography (Pet. ether/EtOAc, 5/1, V/v) to give (5H-benzoe pyrrolo 1,2-a 1,4-diazepin-10(11H)-yl)(2-chloro-4-(5- fluoro-2-methylbenzylamino)phenyl)methanone (50 mg. 16b 18%) as a white solid. LC-MS (Agilent): R, 3.49 min; m/z calculated for CHCIFNO IM+H" 460.15. found 460.1. Intermediate B: (4-Chlorophenyl)(3-(hydroxym ethyl)-5-methoxy-2-methyl-1H-indol-1-yl)metha "H NMR: (400 MHz, CDC1) & (ppm): 7.25 (m, 1H), 25 7.19-6.79 (m, 7H), 6.65 (m, 1H), 6.43 (m. 1H), 6.24 (m, 1H), O 6.10-5.98 (m, 2H), 5.38-4.77 (m, 4H), 4.17 (m, 2H), 2.26 (s, 3H). To a stirred solution of compound A (200 mg. 0.559 mmol) in THF (2 mL) was added a 2 M solution of BH MeS in Example 16 30 THF (0.31 mL, 0.615 mmol) at -20° C. The mixture was allowed to warm to room temperature and stirred for 18 h. The solvent was removed under reduced pressure and the residue Formula 158—Compounds 16a & 16b was purified by silica gel column chromatography (CH2Cl2/ MeOH, 50/1, V/v) to give (4-chlorophenyl)(3-(hydroxym 35 ethyl)-5-methoxy-2-methyl-1H-indol-1-yl)methanone (150 mg, 83%) as a yellow solid. LC-MS (Agilent): R, 3.45 min; m/z calculated for CHCINO IM+Nat 366.1. found 366.1. BH MeS H NMR: (400 MHz, CDC1) & (ppm): 7.66 (dd, J=6.8, 2.0 -- 40 Hz, 2H), 7.48 (dd, J=6.8, 2.0 Hz, 2H), 6.97 (d. J=2.4 Hz, 1H), / THF 6.89 (d. J=8.8 Hz, 1H), 6.68 (dd, J=8.8 Hz, 2.4 Hz, 1H), 3.88 No (t, J=6.8 Hz, 2H), 3.87 (s.3H), 2.96 (t, J=6.8 Hz, 2H), 2.39 (s. 3H). CO2H 45 A Intermediate C: 2-(1-(4-Chlorobenzoyl)-5-methoxy O 2-methyl-1H-indol-3-yl)acetaldehyde To a solution of intermediate B (150 mg, 0.44 mmol) in N IBX, EtOAc EtOAc (1.5 mL) was added IBX (0.31 g, 1.1 mmol) at room -as 50 temperature and the resulting mixture was heated at 80°C. for / 80° C. 2h. The mixture was filtered and the filtrate was concentrated No under reduced pressure to give 2-(1-(4-chlorobenzoyl)-5- OH methoxy-2-methyl-1H-indol-3-yl)acetaldehyde (100 mg. 67%) as a powder. B 55 LC-MS (Agilent): R, 3.47 min; m/z calculated for CHCINO IM+MeOH+Na" 396.08. found 369.1. O H NMR: (400 MHz, CDC1) & (ppm): 9.73 (s, 1H), 7.70 (d. J=8.4 Hz, 2H), 7.50 (d. J=8.4 Hz, 2H), 7.29 (s, 1H), 6.87 (m. 1H), 6.71 (m. 1H), 3.85 (s, 3H), 3.75 (d. J=1.6 Hz, 2H), N)-( )—c NH2OHHCI 60 2.40 (s.3H). -e- MeOH 16a: 2-(1-(4-Chlorobenzoyl)-5-methoxy-2-methyl N / 1H-indol-3-yl)acetaldehyde oxime O Z O 65 To a solution of intermediate C (200 mg, 0.6 mmol) in C MeOH (2 mL) and pyridine (0.2 mL) was added NH-OH.HCl (48 mg, 0.7 mmol) at room temperature and the resulting

US 8,946,224 B2 130 -continued (Z)-methyl 3-(2,3-dichlorophenyl)-2-(2-methyl-1,3-diox O O olan-2-yl)acrylate (1.0 g, 43%) as a white solid. LC-MS (Waters): R, 7.46 min; m/z calculated for -----> CHOOM+Na"339.03. found 338.9 Intermediate D: (E)-3-(2,3-Dichlorophenyl)-2-(2- Riot methyl-1,3-dioxolan-2-yl)prop-2-en-1-ol NH O O ulus o1\ To a solution of intermediate C (500 mg, 1.6 mmol. 1.0 eq) 10 in CHCl (10 mL) was added a 1.0 M solution of DIBAI-H N-1- NN1 N I in hexanes (6.3 mL, 6.3 mmol. 4.0 eq) dropwise at -78°C. isopropanol The resulting mixture was warmed to room temperature and C reflux stirred overnight. Water (0.24 mL), 15% aqueous NaOH (0.24 mL) and water (0.72 mL) were added to the reaction mixture C 15 in that order and stirring was continued for 15 min at room H temperature. MgSO was then added and stirring was contin C ued for a further 15 min. The mixture was filtered, the filtrate was concentrated under reduced pressure and the residue was purified by column chromatography (Pet. ether/EtOAc, 10/1, O C Pd(PPh3)4 V/v) to give (E)-3-(2,3-dichlorophenyl)-2-(2-methyl-1,3-di He N-O HCOOH.EtN oxolan-2-yl)prop-2-en-1-ol (250 mg, 55%) as a colourless 1\o N1'N-1s EtOH/H2O oil. reflux LC-MS (Waters): R., 6.88 min; m/z calculated for N CHClO, IM+Na" 311.03. found 311.0. H 25

Intermediate E: (E)-3-(2,3-Dichlorophenyl)-2-(2- methyl-1,3-dioxolan-2-yl)acrylaldehyde To a solution of intermediate D (1.3 g, 4.5 mmol. 1.0 eq) in 30 CHCl (20 mL) was added PCC (1.9 g, 9.0 mmol. 2.0 eq). The resulting mixture was stirred at room temperature for 2 h and then filtered through Celite. The filtrate was concentrated under reduced pressure to give (E)-3-(2,3-dichlorophenyl)-2- (2-methyl-1,3-dioxolan-2-yl)acrylaldehyde (1.3 g, 100%) as 35 a brown oil, which was used directly in the next step. 17a Intermediate F: (1E.2E)-3-(2,3-Dichlorophenyl)-2- (2-methyl-1,3-dioxolan-2-yl)acrylaldehyde oxime Intermediate B: (Z)-Methyl To a solution of intermediate E (1.3 g 4.5 mmol. 1.0 eq) in 2-(2,3-dichlorobenzylidene)-3-oxobutanoate 40 pyridine (2.5 mL) and methanol (25 mL) was added hydroxy lamine hydrochloride (310 mg. 4.5 mmol. 1.0 eq) and the To a solution of compound A (10.1 g, 57 mmol. 1.0 eq) and resulting mixture was stirred at room temperature overnight. methyl 3-oxobutanoate (8.60 g, 74 mmol. 1.3 eq) in isopro The mixture was concentrated under reduced pressure and the panol (100 mL) was added piperidine (0.24g, 2.8 mmol, 0.05 residue was treated with a 1M aqueous HCl solution (10 mL) eq) and picolinic acid (0.35 g, 2.8 mmol, 0.05 eq) at room 45 and EtOAc (10 mL). The organic layer was separated, dried temperature. The resulting mixture was heated at 45° C. over over NaSO and the solvent was removed under reduced night then cooled to 0°C. and the crystalline solid was col pressure to give (1E.2E)-3-(2,3-dichlorophenyl)-2-(2-me lected by suction filtration, washed with isopropanol (20 mL) thyl-1,3-dioxolan-2-yl)acrylaldehyde oxime (1.3 g 96%) as and dried under vacuum to give (Z)-methyl 2-(2,3-dichlo a light green Solid. robenzylidene)-3-oxobutanoate (4.00 g, 25%) as a white 50 LC-MS (Waters): R, 8.03 min: m/z calculated for solid. CHCINO, M+H" 302.03, M+Nat 324.03, found LC-MS (Agilent): R, 3.62 min; m/z calculated for M+H"302.0, M+Nat 324.0. CHCIO, M+H" 273.1, M+Nat 295.0, found M+H" Intermediate G: (1E.2E)-3-(2,3-Dichlorophenyl)-2- 273.0, M+Nat 294.9. 55 (2-methyl-1,3-dioxolan-2-yl)acrylaldehyde O-allyl Oxime Intermediate C: (Z)-Methyl 3-(2,3-dichlorophenyl)- 2-(2-methyl-1,3-dioxolan-2-yl)acrylate To a solution of intermediate F (1.3g, 4.3 mmol. 1.0 eq) in acetone (30 mL) was added KCO (1.2g, 8.6 mmol. 2.0 eq) To a solution of intermediate B (2.0 g, 7.3 mmol. 1.0 eq) in and 3-bromoprop-1-ene (1.6 g. 12.9 mmol. 3.0 eq) and the toluene (50 mL) was added ethylene glycol (0.9 g, 14.6 mmol. 60 resulting mixture was heated at reflux overnight. The mixture 2.0 eq) and p-toluenesulfonic acid (126 mg, 0.7 mmol, 0.1 eq) was concentrated under reduced pressure the residue was and the resulting mixture was heated at reflux for 6 h in a partitioned between water (20 mL) and EtOAc (20 mL). The Dean-Stark apparatus. The mixture was cooled to room tem organic layer was separated, dried over NaSO and the Sol perature, water (50 mL) was added and the organic layer was vent was removed under reduced pressure to give (1E.2E)-3- separated and dried over NaSO. The solvent was removed 65 (2,3-dichlorophenyl)-2-(2-methyl-1,3-dioxolan-2-yl)acryla under reduced pressure and the residue was purified by col ldehyde O-allyl oxime (1.3g, 88%) as a brown oil, which was umn chromatography (Pet. ether/EtOAc, 10/1, V/v) to give used directly in the next step. US 8,946,224 B2 131 132 Intermediate H: H NMR: (400 MHz, DMSO-d) & (ppm): 10.3 (s, 1H), (1E.2E)-2-(2,3-Dichlorobenzylidene)-3-oxobutanal 8.74 (s, 1H), 7.84 (s, 1H), 7.35 (dd, J=7.6, 1.6 Hz, 1H), 7.27 O-allyl oxime (dd, J=8.0, 1.6 Hz, 1H), 7.22 (m. 1H), 5.28 (s, 1H), 3.94 (qd, 5 J=7.2, 1.2 Hz, 2H), 2.22 (s.3H), 1.99 (s.3H), 1.09 (t, J=7.2 To a solution of intermediate G (1.3 g 3.8 mmol. 1.0 eq) in Hz, 3H). THF (30 mL) was added 2 Maqueous HCl solution (60 mL) and the resulting mixture was stirred at room temperature Example 18 overnight. EtOAc (50 mL) was added to the mixture and the 10 organic layer was separated, dried over NaSO and the Sol Formula 98 Compounds 18a & 18b vent was removed under reduced pressure to give (1E.2E)-2- (2,3-dichlorobenzylidene)-3-oxobutanal O-allyl oxime (1.0 g, 91%) as a yellow oil. 15 LC-MS (Waters): R, 4.28 min; m/z calculated for CHCINO, M+H" 298.03, M+Nat 320.03, found M+H 297.9, M+Nat 319.9. - O Intermediate I: Ethyl 3-iminobutanoate DIBAL-H, HO III. I. CHCl2 Hoss

A solution of ethyl acetoacetate (50g,385 mmol. 1.0 eq) in O s 25% aqueous ammonia (300 mL) was stirred at room tem \ 4fa perature for 1 h then extracted with EtOAc (2x300 mL). The 25 combined organic layers were dried over NaSO and the Solvent was removed under reduced pressure to give ethyl 3-iminobutanoate (42g, 85%) as a yellow oil, which was used directly in the next step. 30 A

Intermediate J: (E)-Ethyl 5-((allyloxyimino)methyl)- 4-(2,3-dichlorophenyl)-2,6-dimethyl-1,4-dihydropy ridine-3-carboxylate 35

- O To a solution of intermediate H (1.0 g, 3.4 mmol. 1.0 eq) in isopropanol (20 mL) was added intermediate I (432 mg, 3.4 HO III. I. OH mmol. 1.0 eq) and the resulting mixture was stirred at reflux 40 overnight. The solvent was removed under reduced pressure and the residue was purified by column chromatography (Pet. O s OH ether/EtOAc, 5/1, V/v) to give (E)-ethyl 5-((allyloxyimino) \ Ho-1N methyl)-4-(2,3-dichlorophenyl)-2,6-dimethyl-1,4-dihydro O pyridine-3-carboxylate (0.8 g. 58%) as a yellow solid. 45 LC-MS (Waters): R., 6.67 min: m/z calculated for O -( CHC1.N.O. M+H"409.1. found 409.0. 188 17a: (E)-Ethyl 4-(2,3-dichlorophenyl)-5-(hydroxy 50

imino)methyl)-2,6-dimethyl-1,4-dihydropyridine-3- carboxylate

To a solution of intermediate J (400 mg. 0.98 mmol. 1.0 eq) 55 - O in EtOH (20 mL) and HO (5 mL) was added HCOOH.NEts TBSCI, (431 mg, 2.93 mmol. 3 eq) and Pd(PPha (113 mg, 0.10 HO III. I. OH 1H-imidazole mmol, 0.1 eq) and the resulting mixture was heated at reflux He for 3 h. The solvent was removed under reduced pressure and 60 O S OH the residue was purified by column chromatography (Pet. \ 4\ ether/EtOAc, 5/1, V/v) to give (E)-ethyl 4-(2,3-dichlorophe O nyl)-5-((hydroxyimino)methyl)-2,6-dimethyl-1,4-dihydro pyridine-3-carboxylate (100 mg, 28%) as a yellow solid. 65 o-( LC-MS (Agilent): R, 3.35 min; m/z calculated for CH.ClN.O. M+H"369.07. found 369.1.

US 8,946,224 B2 139 140

BOZ

US 8,946,224 B2 141 142 Intermediate B: (R)-2-(1,8-Diethyl-1,3,4,9-tetrahy and the resulting mixture was stirred at room temperature for dropyrano.3,4-bindol-1-yl)ethanol 4h. The solvent was removed under reduced pressure and the residue was purified by column chromatography (Pet. ether/ To a solution of compound A (2.0 g. 6.97 mmol. 1.0 eq) in EtOAc, 50/1 to 30/1, v/v) to give (R)-2-(1,8-diethyl-1,3,49 dry THF (15.5 mL) under nitrogen was added a solution of 5 tetrahydropyrano3,4-blindol-1-yl)acetaldehyde Oxime (40 LiAlH4 (0.4g, 10.5 mmol. 1.5 eq) in dry THF (10.5 mL) mg, 76%) as a yellow oil, "H NMR spectroscopy revealed a dropwise and the resulting mixture was stirred at room tem ~1:1 mixture of isomers. perature overnight. The reaction was slowly quenched with LC-MS (Agilent): R, 3.48 min; m/z calculated for EtOAc (15 mL) and poured into water. The resulting emul CHNOM+H" 287.17. found 287.2. sion was filtered and the filtrate was extracted twice with 10 'H-NMR: (400 MHz, DMSO-d) & (ppm): 10.8 (s, 0.5H), EtOAc (30 mLx2). The combined organic layers were 10.5 (s, 1H), 10.4 (s, 0.5H), 7.25-7.22 (m. 1H), 7.16 (dd. washed with brine, dried over NaSO and concentrated J=6.8, 5.2 Hz, 0.5H), 6.94-6.87(m,2H), 6.58 (appt, J–4.4 Hz, under reduced pressure. Purification by column chromatog 0.5H), 3.92 (m, 2H), 2.96-2.81 (m, 3.5H), 2.70-2.62 (m, raphy (CHC1/MeOH,50/1 to 20/1) gave (R)-2-(1,8-diethyl 2.5H), 2.0 (m. 1H), 1.83 (m, 1H), 1.25 (m,3H), 0.75 (t, J–7.2 1.3.4.9-tetrahydropyrano3,4-bindol-1-yl)ethanol (835 mg, 15 HZ, 1.5H), 0.71 (t, J–7.2 Hz, 1.5H). 44%) as a yellow oil. LC-MS (Waters): R, 5.89 min: m/z calculated for 20b: (R)-2-(1,8-diethyl-1,3,4,9-tetrahydropyrano.3, CHNOM+Na" 296.17. found 296.1. 4-bindol-1-yl)acetaldehyde O-methyl oxime Intermediate C. (R)-2-(1,8-Diethyl-1,3,4,9-tetrahy dropyrano3,4-bindol-1-yl)acetaldehyde To a solution of intermediate C (50 mg, 0.184 mmol. 1.0 eq) in methanol (10 mL) and pyridine (1 mL) was added To a solution of intermediate B (530 mg, 1.94 mmol. 1.0 methylhydroxylamine hydrochloride (18.5 mg, 0.22 mmol. eq) in acetonitrile (2.5 mL), DMSO (2.5 mL) and Et N (2.5 1.2 eq) and the resulting mixture was stirred at room tempera mL) was added SO-pyridine (1.85g, 11.6 mmol. 6.0 eq) and 25 ture for 4 h. The solvent was removed under reduced pressure the resulting mixture was stirred at room temperature for 40 and the residue was purified by column chromatography (Pet. min. The mixture was poured into water and extracted with ether/EtOAc, 50/1 to 30/1, v/v) to give (R)-2-(1,8-diethyl-1, EtOAc (20 mLx2). The combined organic layers were 34.9-tetrahydropyrano3,4-bindol-1-yl)acetaldehyde washed with 3% aqueous HCl solution (20 mL), saturated O-methyl oxime (50 mg, 91%) as a yellow solid, "H NMR aqueous NaHCO, solution (20 mL) and brine (20 mL) then 30 spectroscopy revealed a ~1:1 mixture of isomers. dried over MgSO and concentrated under reduced pressure. LC-MS (Agilent): R, 3.52 min; m/z calculated for The residue was purified by column chromatography (Pet. C.H.N.O.M+H"301.18, M+Na'323.4, found M+H" ether/EtOAc, 50/1 to 30/1, v/v) to give (R)-2-(1,8-diethyl-1, 301.2, M+Nat 323.2. 34.9-tetrahydropyrano3,4-bindol-1-yl)acetaldehyde (292 'H-NMR (400 MHz, DMSO-d) & (ppm): 10.5 (m. 1H), mg, 58%) as a yellow oil. 35 7.25-7.20 (m. 1.5H), 6.94-6.88 (m, 2H), 6.62 (t, J–4.8 Hz, LC-MS (Waters): R., 6.03 min: m/z calculated for 0.5H), 3.91 (m, 2H), 3.77 (s, 1.5H), 3.67 (s, 1.5H), 2.96-2.82 CHNOM+MeOH+Na" 326.3. found 326.1. (m, 3.5H), 2.73-2.63 (m, 2.5H), 1.96-2.05 (m, 1H), 1.90-1.75 (m. 1H), 1.25 (m, 3H), 0.75 (t, J–7.2 Hz, 1.5H), 0.71 (t, J=7.2 20a: (R)-2-(1,8-diethyl-1,3,4,9-tetrahydropyrano.3, HZ, 1.5H). 4-bindol-1-yl)acetaldehyde oxime 40 To a solution of intermediate C (50 mg, 0.184 mmol. 1.0 Example 21 eq) in methanol (10 mL) and pyridine (1 mL) was added hydroxylamine hydrochloride (38.4 mg. 0.552 mmol. 3.0 eq) Formula 123 Compounds 21a & 21b

O O O

N N N N N Y N N N H HCl, H H H N 2 EtOH 2 BH MeS 2 -e- -e- N NH N NH O 70° C. N NH Reflux N n N 2 N 2N 2N

A B 21a

m-CPBA CHCl2 US 8,946,224 B2 143 144 -continued

O O

N N N HCl, N N N

2 H X- EtOH() 2 H N NH O 70° C. N NH

N N N N 2 No. 2 No.

C 21b

Compounds A and B can be synthesised according to the 21b: 4-(((3-((3.3-Dimethylindolin-6-yl)carbamoyl) procedures described in US2003.0125339. pyridin-2-yl)amino)methyl)pyridine 1-oxide Intermediate C: 4-(((3-((1-Acetyl-3,3-dimethylindo A mixture of intermediate C (120 mg, 0.28 mmol), con lin-6-yl)carbamoyl)pyridin-2-yl)amino)methyl)pyri centrated HCl (5 mL) and ethanol (5 mL) was heated at 70° C. dine 1-oxide overnight and then allowed to cool to room temperature. The Solvent was removed under reduced pressure and the residue To a solution of compound A (200 mg, 0.48 mmol) in dry was diluted with water and washed with EtOAc (3x10 mL). CHCl (10 mL) at 0°C. was added m-CPBA (166 mg, 0.96 25 The aqueous phase was basified to pH 7–8 with a 3 Maqueous mmol) in three portions and the mixture was allowed to warm NaOH solution and extracted with EtOAc (3x20 mL). The to room temperature and stirred for 30 min. A 5% aqueous combined organic layers were washed with brine and dried NaSO solution was added and the mixture was extracted over NaSO. The solvent was removed under reduced pres with EtOAc (3x20 mL). The combined organic layers were sure and the residue was washed with ether to give 4-(((3-((3. washed with a saturated aqueous solution of NaHCO, brine 30 3-dimethylindolin-6-yl)carbamoyl)pyridin-2-yl)amino)me and dried over NaSO. The solvent was removed under thyl)pyridine 1-oxide (80 mg, 74%) a pale yellow solid. reduced pressure and the residue was washed with ether to LC-MS (Agilent): R, 2.85 min: m/z calculated for give 4-(((3-((1-acetyl-3,3-dimethylindolin-6-yl)carbamoyl) CHNO, M+H 390.45, found 390.2. pyridin-2-yl)amino)methyl)pyridine 1-oxide (130 mg, 63%) H NMR: (400 MHz, DMSO-d) & (ppm): 9.98 (s, 1H), as a pale yellow solid. 35 8.42 (t, J=6.0 Hz, 1H), 8.14 (m, 3H), 8.03 (d. J=6.8 Hz, 1H), LC-MS (Agilent): R, 3.24 min; m/z calculated for 7.31 (d. J=6.4 Hz, 2H), 6.97-6.87(m,3H), 6.68 (dd.J–4.8, 2.4 CHNO IM+H 432.49. found 432.2.2. HZ, 1H), 5.55 (s, 1H), 4.62 (d. J=6.0 Hz, 2H), 3.19 (s. 2H), H NMR: (400 MHz, DMSO-d) & (ppm): 10.3 (s, 1H), 1.22 (s, 6H). 8.48 (t, J=6.0 Hz, 1H), 8.35 (s1H), 8.16-8.08 (m, 4H), 7.45 (dd, J=8.0, 1.2 Hz, 1H), 7.33 (d. J=6.8 Hz, 2H), 7.20 (d. J=8.0 40 Example 22 HZ, 1H), 6.70 (m, 1H), 4.62 (d. J=6.0 Hz, 2H), 3.87 (s. 2H), 2.17 (s.3H), 1.30 (s, 6H). Formula 152 Compound 22a 21a: 3.3-Dimethyl-N-((2-(pyridin-4-ylmethylamino) O pyridin-3-yl)methyl)indolin-6-amine 45 To a solution of BH.MeS (1M in THF, 10 mL 10 mmol, 12.5 eq) was added intermediate B (300 mg. 0.8 mmol. 1.0 eq) at 0°C. under nitrogen. The mixture was allowed to warm to room temperature, stirred for 1 h then heated at reflux for 48 -- h. After cooling to 0°C., a 2 Maqueous HCl solution (20 mL) 50 OH DMSO, pyridine was added dropwise and the mixture was heated at 70° C. for 3 h then cooled to room temperature and washed with EtOAc (15 mLX3). The aqueous layer was basified to pH 8-9 with a N

3 Maqueous NaOH solution and extracted with EtOAc (20 mLX3). The combined organic layers were washed with 55 brine, dried over NaSO and the solvent was removed under reduced pressure. The residue was purified by column chro matography (EtOAc/Pet. ether, 1/100 to 1/5, V/v) to give a light yellow sticky oil, which was further purified by prepara tive TLC (EtOAc/Pet. ether, 1/2, v/v) to give 3,3-dimethyl N-((2-(pyridin-4-ylmethylamino)pyridin-3-yl)methyl)indo 60 lin-6-amine (22 mg, 8%) as a pale yellow solid. LC-MS (Agilent): R, 3.19 min; m/z calculated for C2H5N M+H" 360.47. found 360.2. H NMR: (400 MHz, DMSO-d) & (ppm): 8.93 (brs, 3H), 8.09 (brs, 2H), 7.84 (m, 2H), 7.14 (d. J=8.0 Hz, 1H), 6.91 (m, 65 1H), 6.69 (d. J=8.0Hz, 1H), 6.61 (s, 1H), 5.18 (s.2H), 4.37 (s, 2H), 3.38 (s, 3H), 3.17 (s, 1H), 1.29 (s, 6H). US 8,946,224 B2 145 146 -continued Intermediate D: 2-(4-Methoxyphenyl)-2H-spiro benzob 14thiazepine-3,2'-1.3dioxolan-4(5H)- O

A mixture of intermediate C (798 mg, 2.7 mmol), ethane 1,2-diol (661 mg, 10.7 mmol) and Ts-OH (184 mg, 1.1 mmol) HO OH in toluene (40 mL) was heated at reflux in a Dean-Stark Tol, Ts-OH apparatus for 3 h. The mixture was poured into water and the aqueous Solution was extracted with EtOAc. The organic layer was washed with brine, dried over NaSO and concen trated under reduced pressure. The residue was purified by flash chromatography (Pet. ether/EtOAc, 4/1, V/v) to give 2-(4-methoxyphenyl)-2H-spirobenzob 14thiazepine-3, O 15 2-1.3dioxolan-4(5H)-one (390 mg, 43%) as a light yellow HCI solid. N C LC-MS (Agilent): R, 2.83 min; m/z calculated for CHNOS M+H" 344.09. found 344.1. S OC K2CO3, DMF 22a: 5-(2-(Dimethylamino)ethyl)-2-(4-methoxyphe nyl)-2H-spirobenzob 14thiazepine-3,2'-1.3 N O H dioxolan-4(5H)-one O D 25 To a mixture of intermediate D (200 mg. 0.6 mmol) and KCO (241 mg, 1.7 mmol) in DMF (5 mL) was added O 2-chloro-N,N-dimethylethanamine hydrochloride (101 mg, 0.7 mmol). The mixture was stirred at 60° C. for 6 h then cooled to room temperature and poured into water. The aque S ous mixture was extracted with EtOAc and the organic layer was washed with brine, dried over NaSO and concentrated under reduced pressure. The residue was purified by flash chromatography (CH2Cl2/MeOH, 20:1, v/v) to give 5-(2- (dimethylamino)ethyl)-2-(4-methoxyphenyl)-2H-spiro benzob 14thiazepine-3,2'-1.3dioxolan-4(5H)-one (120 mg, 50%) as a white solid. LC-MS (Agilent): R, 2.91 min; m/z calculated for C.H.N.O.S M+H" 415.16. found 415.2. 40 "H NMR: (400 MHz, DMSO-d) & (ppm): 7.51 (d. J=8.8 Hz, 2H), 7.44 (d. J=7.2 Hz, 1H), 7.33 (m, 2H), 7.07 (m, 1H), Intermediate C: 2-(4-Methoxyphenyl)benzob 1.4 6.88 (d. J=8.8 Hz, 2H), 5.41 (s, 1H), 3.82-4.13 (m, 6H), 3.70 thiazepine-3,4(2H,5H)-dione (s, 3H), 2.30-2.46 (m, 2H), 2.17 (s, 6H). 45 Compound A was converted to 2-(4-methoxyphenyl)benzo b1,4thiazepine-3,4(2H.5H)-dione in two steps using the Example 23 procedure described in Journal of Organic Chemistry, 1996, 61, 8586. Formula 104 Compounds 23a & 23b

Br Br O O --- O toluene HN Br OH --> N Br OH CC H A 23a l HBTU, DIPEA O O K2CO3, DMF -sul 1No OH B US 8,946,224 B2 147 148 -continued Br

--- Br OH 23b

10 Compound A can be synthesised according to the proce eq) and DIPEA (323 mg, 2.50 mmol. 2.0 eq) and the mixture dure described in WO2003039456. Compound B can be syn was stirred at room temperature for 30 min. Compound B thesised according to the procedure described in J. Med. (500mg, 1.25 mmol. 1.0 eq) and KCO (172 mg, 1.25 mmol. Chem. 2005, 48, 306. 1.0 eq) were then added and stirring was continued at room 23a; 3-(3,5-Dibromo-4-(4-hydroxy-3 15 temperature overnight. Water (30 mL) was added and the isopropylphenoxy)phenylamino) propanoic acid mixture was extracted with EtOAc (20 mLX3). The combined organic layers were washed with water (50 mL), a saturated A solution of compound A (200 mg, 0.5 mmol. 1.0 eq) and aqueous solution of NaCO (50 mL), brine (50 mL) then acrylic acid (54 mg. 0.75 mmol. 1.5 eq) intoluene (2 mL) was dried over NaSO. The solvent was removed under reduced heated at 100°C. in a sealed steel tube overnight. The reaction pressure and the residue was purified by column chromatog mixture was cooled to room temperature and concentrated raphy (Pet. ether/EtOAc, 7/1, V/v) to give N-(4-(4-hydroxy under reduced pressure. The residue was purified by column 3-isopropylphenoxy)-3,5-dibromophenyl)-3,3-diethoxypro chromatography (CH.Cl/MeOH, 20/1, V/v) to give 3-(3.5- dibromo-4-(4-hydroxy-3-isopropylphenoxy)phenylamino) panamide (72 mg, 15%) as a yellow solid. propanoic acid (60 mg, 25%) as a white Solid. LC-MS (Agilent): R, 3.69 min; m/z calculated for LC-MS (Agilent): R, 3.40 min; m/z calculated for 25 CHBr NOs M+Na" 566.0, 568.0. found 566.0, 568.0. CHSBNO, M+H 473.97. found 474.0. H NMR: (400 MHz, DMSO-d) & (ppm): 10.3 (s, 1H), H NMR: (400 MHz, DMSO-d) & (ppm): 8.95 (s, 1H), 9.05 (s, 1H), 7.98 (s. 2H), 6.66 (m, 2H), 6.27 (dd, J=8.8, 3.2 6.88 (s. 2H), 6.65-6.62 (m, 2H), 6.26-6.23 (dd, J=8.4, 2.8 Hz, HZ, 1H), 4.92 (t, J=5.6 Hz, 1H), 3.66-3.59 (m, 2H), 3.54-3.46 1H), 6.16-6.13 (m. 1H), 3.24 (m, 2H), 3.17 (sept, J–7.2 Hz, (m. 2H), 3.15 (pent, J–7.2 Hz, 1H), 2.65-2.64 (d. J=5.6 Hz, 1H), 2.49 (t, J=6.8 Hz, 2H), 1.10 (d. J–7.2 Hz, 6H). 30 2H), 1.13-1.10 (m. 12H). 23b: N-(4-(4-Hydroxy-3-isopropylphenoxy)-3,5- dibromophenyl)-3,3-diethoxypropanamide Example 24 To a solution of compound A (202 mg, 1.25 mmol. 1.0 eq) in DMF (20 mL) was added HBTU (592 mg, 1.56 mmol. 1.25 Formula 3 Compounds 24a & 24b

F F F F F F

BH THF -e-

24a

1. NaNO2, HSO4. 2. K2Cr2O7

F F F F F F

F N1 N NHOMe-HCI N EtOH N S- M N US 8,946,224 B2 149 150 Intermediate B: 1-(3-(Trifluoromethyl)-5,6-dihydro 3.94 (m, 2H), 3.91 (brs, 1H), 3.82 (brs, 1H), 3.78-3.70 (m, 1,2,4-triazolo 4,3-alpyrazin-7(8H)-yl)-4-(2,4,5- 1H), 3.65 (m, 2H), 3.49-3.40 (m. 1H), 3.37-3.31 (m. 1H). trifluorophenyl)butane-1,3-dione Example 25 Intermediate B can be obtained in two steps from com- 5 Formula 2 Compound 25a pound A according to the procedure described in WO2O101 22578.

24a: (R)-4-(3-(Trifluoromethyl)-5,6-dihydro-1,2,4 CeCl3-7H2O triazolo 4,3-alpyrazin-7(8H)-yl)-1-(2,4,5-trifluo N MeOH rophenyl)butan-2-amine Na2 1. NaBH4THF To a stirred solution of compound A (500 mg, 1.25 mmol) in THF (50 mL) at room temperature was added a 1.0 M NuNH solution of BH THF in THF (5.75 mL, 5.75 mmol) and the 1s A resulting mixture was stirred at room temperature overnight. The reaction was slowly quenched by dropwise addition of

methanol (10 mL) followed by addition of a 0.5 M aqueous HCl solution (5 mL). The mixture was extracted with EtOAc (50 mLX3) and the combined organic layers were dried over Na2SO and concentrated under reduced pressure to give a rs solid. The crude product was washed with CH2Cl and THF to Na2 give (R)-4-(3-(trifluoromethyl)-5,6-dihydro-1,2,4-triazolo 4,3-alpyrazin-7(8H)-yl)-1-(2,4,5-trifluorophenyl)butan-2- amine (68 mg, 14%) as a white solid. LC-MS (Agilent): R, 2.98 min; m/z calculated for 25 CHFNM+H 394.14. found 394.1. "H NMR: (400 MHz, CDOD) 8 (ppm): 7.35 (m. 1H), 7.24 25a: 8-cyclopentyl-6-(1-hydroxyethyl)-5-methyl-2- (m. 1H), 4.26 (t, J=5.6 Hz, 2H), 3.94 (AB, J=15.2 Hz, 1H), (5-(piperazin-1-yl)pyridin-2-ylamino)pyrido2.3-d 3.87 (AB, J=15.6 Hz, 1H), 3.68 (m. 1H), 3.12-2.93 (m, 4H), pyrimidin-7(8H)-one 2.82 (m, 2H), 1.89 (m, 2H). 30 To a stirred solution of compound A (100 mg, 0.22 mmol. 24b: 3-(Methoxyimino)-1-(3-(trifluoromethyl)-5,6- 1.0 eq) in MeOH (50 mL) and THF (20 mL) was added dihydro-1,2,4-triazolo 4,3-alpyrazin-7(8H)-yl)-4-(2, CeC1.7H2O (164 mg. 0.44 mmol. 2.0 eq) then NaBH (16.3 4,5-trifluorophenyl)butan-1-one 35 mg, 0.44 mmol. 2.0 eq). The resulting mixture was stirred at room temperature for 48 hand then quenched with a saturated To a stirred solution of intermediate B (93 mg, 0.23 mmol) aqueous solution of NHCl (10 mL). The aqueous layer was in ethanol (5 mL) and pyridine (5 mL) was added O-methyl extracted with CHCl (10 mLx2) and the combined organic hydroxylamine hydrochloride (30 mg. 0.35 mmol) and the layers were washed with brine and dried over MgSO. The resulting mixture was stirred at room temperature for 4 h. The solvents were removed under reduced pressure and the resi Solvent was removed under reduced pressure and the residue 40 due was purified by flash chromatography (CH2Cl2/MeOH, was dissolved in THF (5 mL) and CHCl (5 mL) thenwashed 10/1, V/v) to give 8-cyclopentyl-6-(1-hydroxyethyl)-5-me with a 2 Maqueous HCl solution and dried over NaSO. The thyl-2-(5-(piperazin-1-yl)pyridin-2-ylamino)pyrido2,3-d Solvent was removed under reduced pressure and the residue pyrimidin-7(8H)-one (31 mg, 30%) as a yellow solid. was purified by column chromatography (CH.Cl/MeOH, LC-MS (Agilent): R, 3.02 min; m/z calculated for 50/1 to 25/1, V/v) to give 3-(methoxyimino)-1-(3-(trifluorom C.H. N.O. M+H" 450.25. found 450.3. ethyl)-5,6-dihydro-1,2,4-triazolo 4,3-alpyrazin-7(8H)-yl)- 45 H NMR: (400 MHz, DMSO-d) & (ppm): 9.86 (s, 1H), 4-(2,4,5-trifluorophenyl)butan-1-one (20 mg, 20%) as a 8.91 (s, 1H), 8.02 (d. J–2.8 Hz, 1H), 7.87 (d. J=9.2 Hz, 1H), white solid, HPLC analysis revealed a ~1:1 mixture of iso 7.44 (dd, J=8.8, 2.8 Hz, 1H), 5.86 (m, 1H), 5.23 (m, 1H), 5.15 CS. (d. J=5.6 Hz, 1H), 3.06 (m, 4H), 2.86 (m, 4H), 2.55 (s.3H), LC-MS (Agilent): R, 3.36 min; m/z calculated for 2.25 (m, 2H), 1.91 (m, 2H), 1.75 (m, 2H), 1.59 (m, 2H), 1.35 CHFNO, M+H" 436.11, M+Na" 458.1, found 50 (d. J=6.4 Hz, 3H). M+H" 436.1, M+Na" 458.1 Example 26 "H NMR: (400 MHz, CDC1) & (ppm): 7.14-7.07 (m, 1H), 6.94-6.90 (m, 1H), 5.04-4.90 (m, 2H), 4.18 (m, 2H), 4.12 Formula 142 Compounds 26a and 26b

EDCL, HOBt, CH2Cl2 DIBAL-H -e- -e- EtN, NHMeOMe-HCI -78° C. US 8,946,224 B2 151

-continued

NH2OHHCI

NHOMe-HCl -es

Intermediate B: (Z)-2-(11-(3-(Dimethylamino)pro ibenzob.eloxepin-2-yl)acetaldehyde oxime (95 mg, 13%) as pylidene)-6, 1'-dihydrodibenzob.eoxepin-2-yl)-N- a white solid, 'H-NMR spectroscopy revealed a ~1:1 mixture methoxy-N-methylacetamide of isomers. 30 LC-MS (Agilent): R, 3.04 min; m/z calculated for To a stirred solution of compound A (2.0 g, 5.9 mmol. 1.0 C.H.N.O. M+H" 337.18. found 337.2. eq), EDC1 (1.7g, 8.9 mmol. 1.5 eq), HOBt (1.2g, 8.9 mmol, "H NMR: (400 MHz, DMSO-d) & (ppm): 11.0 (s, 0.5H), 1.5 eq) and Et-N (1.7g, 17.7 mmol. 3.0 eq) in dry CHCl, 10.6 (s, 0.5H), 7.40-7.25 (m, 4.5H), 7.04 (m, 2H), 6.78 (m, (100 mL) was added O, N-dimethylhydroxylamine hydro 1.5H), 5.68 (t, J=6.8 Hz, 1H), 5.20(m, 2H), 3.53 (d. J=5.2 Hz, chloride (1.1 g, 11.8 mmol. 2.0 eq). The resulting mixture was 35 1H), 3.18 (d. J–4.4 Hz, 0.5H), 2.48-2.39 (m, 4H), 2.11 (s, 6H). stirred at room temperature for 16 h, diluted with CH-Cl (100 mL), washed with water (100 mLx2) and dried over 26b; (Z)-2-(11-(3-(Dimethylamino)propylidene)-6, MgSO. The solvent was removed under reduced pressure 1'-dihydrodibenzob.eloxepin-2-yl)acetaldehyde and the residue was purified by flash chromatography 40 O-methyl oxime (CHC1/MeoH, 100/1 to 10/1, v/v) to give (Z)-2-(11-(3- (dimethylamino)propylidene)-6,11-dihydrodibenzob.eox To a stirred solution of intermediate B (800 mg, 2.1 mmol. epin-2-yl)-N-methoxy-N-methylacetamide (800 mg, 37%) 1.0 eq) in dry CHCl (50 mL) was added a 1.0 M solution of as a light yellow solid. DIBAI-H in hexanes (4.2 mL, 4.2 mmol. 2.0 eq) dropwise at LC-MS (Waters): R, 4.57 min; m/z calculated for 45 -78°C. and the mixture was stirred at this temperature for 1 CHNOM+H 381.21. found 381.1. h. The reaction was quenched with MeOH, methoxylamine hydrochloride (359 mg, 4.2 mmol. 2.0 eq) and EtN (636 mg, 26a. (Z)-2-(11-(3-(Dimethylamino)propylidene)-6, 6.3 mmol. 3.0 eq) were added and stirring was continued at 1'-dihydrodibenzob.eloxepin-2-yl)acetaldehyde room temperature for a further 5 h. The solvent was removed Oxime 50 under reduced pressure and the residue was dissolved in CHCl (100 mL), washed with water (60 mLX2), brine (50 To a stirred solution of intermediate B (800 mg, 2.1 mmol. mLx2) and dried over NaSO. The solvent was removed 1.0 eq) in dry CHCl (50 mL) was added a 1.0 M solution of under reduced pressure and the residue was purified by flash DIBAI-H in hexanes (4.2 mL, 4.2 mmol. 2.0 eq) dropwise at 55 chromatography (CH2Cl2/MeOH, 100/1 to 10/1, V/v) to give -78°C. and the mixture was stirred at this temperature for 1 (Z)-2-(11-(3-(dimethylamino)propylidene)-6,11-dihydrod h. The reaction was quenched with MeOH, hydroxylamine ibenzob.eloxepin-2-yl)acetaldehyde O-methyl oxime (68 hydrochloride (292 mg, 4.2 mmol. 2.0 eq) and EtN (636 mg, mg, 9%) as a white solid, 'H-NMR spectroscopy revealed a 6.3 mmol. 3.0 eq) were added and stirring was continued at ~1:1 mixture of isomers. room temperature for a further 5 h. The solvent was removed 60 LC-MS (Agilent): R, 3.22 min; m/z calculated for under reduced pressure and the residue was dissolved in CHNO, M+H 351.2. found 351.2. CHCl (100 mL), washed with water (60 mLx2), brine (50 H NMR: (400 MHz, DMSO-d) & (ppm): 749 (t, J=6.4 mLx2) and dried over NaSO. The solvent was removed HZ, 0.5H), 7.38-7.25 (m, 4H), 7.02 (m, 2H), 6.87 (t, J=5.6 Hz, under reduced pressure and the residue was purified by flash 65 0.5H), 6.80 (dd, J=8.0, 2.4 Hz, 1H), 5.67 (t, J=6.4 Hz, 1H), chromatography (CH2Cl2/MeOH, 100/1 to 10/1, V/v) to give 5.15 (brs, 2H), 3.83 (s, 1.3H), 3.73 (s, 1.7H), 3.55 (d. J=5.6 (Z)-2-(11-(3-(dimethylamino)propylidene)-6,11-dihydrod HZ, 1H), 3.41 (d. J=6.4 Hz, 1H), 2.54 (m, 4H), 2.23 (s, 6H). US 8,946,224 B2 153 154 Example 27 solvent was removed under reduced pressure. The residue was purified by flash chromatography (CH.Cl/MeOH, 50/1, Formula 29- Compound 27a V/v) to give 2-(cyanomethylthio)-N-((6R,7S)-2-(hydroxym ethyl)-7-methoxy-3-((1-methyl-1H-tetrazol-5-ylthio)me 5 thyl)-8-oxo-5-thia-1-aza-bicyclo4.2.0oct-2-en-7-yl)aceta

mide (2.10g, 22%) as a yellow solid. LC-MS (Agilent): R, 0.91 min; m/z calculated for aN (COCl) - CHN.O.S. M+Na" 480.07. found 479.9. CH2Cl2/ H NMR: (400 MHz, DMSO-d) & (ppm): 9.52 (s, 1H), DMF 10 5.15 (t, J–5.6 Hz, 1H), 5.09 (s, 1H), 4.30 (m, 2H), 4.25 (d. J=13.6 Hz, 1H), 4.04 (d. J=13.6 Hz, 1H), 3.93 (s.3H), 3.76 (m,2H), 3.63 (d. J=17.6 Hz, 1H), 3.48 (brs, 2H), 3.42 (s.3H), 3.31 (d. J=17.6 Hz, 1H). 15 Example 28

Formula 125 Compound 28a

BocO 25 HN -- Dioxane/H2O

Intermediate B: (6R,7S)-7-(2-(Cyanomethylthio) 1. CICOEt acetamido)-7-methoxy-3-((1-methyl-1H-tetrazol-5- ylthio)methyl)-8-oxo-5-thia-1-aza-bicyclo4.2.0 oct 35 2. NaBH4 2-ene-2-carbonyl chloride To a stirred Suspension of compound A (10.0g, 21.2 mmol. 1.0 eq) and DMF (0.5 mL) in dry CHC1 (120 mL) at 0°C. under nitrogen was added a solution of oxalyl chloride (5.2 40 OH mL, 42.5 mmol) in CHCl (20 mL) over 20 min. The result 1 ing mixture was stirred at 0°C. for 1 h to give a clear solution and stirring was continued for a further 3 h. The solvent was removed under reduced pressure keeping the temperature below 10° C. to give crude (6R,7S)-7-(2-(cyanomethylthio) 45 acetamido)-7-methoxy-3-((1-methyl-1H-tetrazol-5-ylthio) methyl)-8-oxo-5-thia-1-aza-bicyclo4.2.0 oct-2-ene-2-car bonyl chloride (12.4 g) as a yellow solid, which was used directly in the next step without purification. LC-MS (Agilent): R, 1.25 min; m/z calculated for 50 CHBr NO IM-Cl-HOCHI'' 486.06. found 485.9. Example 27a 2-(Cyanomethylthio)-N-((6R,7S)-2-(hydroxym ethyl)-7-methoxy-3-((1-methyl-1H-tetrazol-5-ylthio) 55 methyl)-8-oxo-5-thia-1-aza-bicyclo4.2.0 oct-2-en 7-yl)acetamide To a solution of intermediate B (12.4g, 21.2 mmol. 1.0 eq) in THF (160 mL) at 0°C. under nitrogen was added a solution 60 of LiAl(O-tBu)H (10.3 g, 42.5 mmol. 2.0 eq) in THF (50 HN mL) over 30 min. The resulting mixture was stirred at 0°C. for 4 hand then poured into a cold 0.1Maqueous HCl solution (300 mL). The pH of the solution was adjusted to 2 with a saturated aqueous NaHCO Solution and the mixture was 65 extracted with EtOAc (50 mLX3). The combined organic layers were washed with brine, dried over NaSO and the US 8,946,224 B2 156 -continued (2.0 g, 20 mmol) and ethyl chloroformate (2.3 g 20 mmol) O CONa and the mixture was stirred at -10° C. for 25 min. Sodium borohydride (2.2g, 60 mmol) was then added to the mixture N H followed by a slow addition of MeCH (60 mL) over a period O of 1 h at 0° C. The mixture was stirred at 0° C. for an CONa 4MHC HN He additional 10 min and then quenched with a 1 Maqueous HCl solution (20 mL). The organic solvents were removed under H.N-(N-4 / reduced pressure and the aqueous mixture was extracted with N EtOAc. The combined organic extracts were washed with a H 10 1Maqueous HCl solution, water and a 5% aqueous NaHCO F solution, dried over NaSO and the solvent was removed O under reduced pressure. The residue was purified by column chromatography (Pet. ether/EtOAc, 5/1, 2/1, 1/1, V/v) to give H ZO OH E (S)-benzyl 4-(tert-butoxycarbonyl)-5-hydroxypentanoate HN N N-methylmorpholine 15 2 Sk -e-2-chloro-4,6-dimethoxy (3.7 g. 60%) as a yellow oil. N-M 1,3,5-triazine LC-MS (Waters): R, 5.54 min; m/z calculated for N CHNO IM+Nat 346.17. found 346.0. H Intermediate D: (S)-Benzyl G 5-acetoxy-4-(tert-butoxycarbonyl)pentanoate O -OAe To a stirred solution of intermediate C (3.6 g., 11 mmol) and N H DMAP (2.0 g, 14 mmol) in CHCl (15 mL) at room tem O On-Ph H2, perature was added acetic anhydride (1.7g, 16 mmol) and the HN Po?C 25 mixture was stirred for 1 h. The mixture was diluted with H.N-( O CHCl (20 mL), washed with a 2 Maqueous HCl solution 2N VN 4 and a 5% aqueous NaHCO solution then dried over NaSO. N The solvent was removed under reduced pressure to give H (S)-benzyl 5-acetoxy-4-(tert-butoxycarbonyl)pentanoate H 30 OAc (4.0 g, 98%) as a yellow oil, which was used without further O 1 purification. LC-MS (Waters): R, 5.72 min: m/z calculated for N CHNO IM+Na" 388.17. found 388.0. H H NMR: (400 MHz, CDC1) & (ppm): 7.38 (m, 5H), 5.15 35 O OH (s. 2H), 4.61 (d. J=8.4 Hz, 1H), 4.09 (m, 2H), 3.92 (m. 1H), 2.49 (t, J=7.6 Hz, 2H), 2.09 (s.3H), 1.94 (m. 1H), 1.73 (m, HN O 1H), 1.45 (s, 9H). HN \ / Intermediate E: (S)-Benzyl N 40 5-acetoxy-4-aminopentanoate N H To a stirred solution of intermediate D (950 mg, 2.60 28a. mmol) in CHCl (14 mL) at 0°C. was added TFA (14 mL) and the resulting mixture was stirred at 0°C. for 15 min, then 45 at room temperature for a further 2 h. The solvent was Intermediate B: (S)-5-(Benzyloxy)-2-(tert-butoxy removed under reduced pressure and the residue was co carbonyl)-5-oxopentanoic acid evaporated with toluene to remove residual TFA to afford (S)-benzyl 5-acetoxy-4-aminopentanoate, which was used To a solution of compound A (5.0g, 21.1 mmol) in dixoane directly in the next step. and water (1:1, 40 mL) at 0°C. was added BocO(5.06g, 23.1 50 mmol) and the mixture was stirred overnight. The solvent was Intermediate G: 4-(2-(2-Amino-4-oxo-4,7-dihydro removed under reduced pressure and the residue was diluted 3H-pyrrolo2,3-dipyrimidin-5-yl)ethyl)benzoic acid with water (30 mL), basified with NaCO (0.7g) and washed with EtOAc (3x20 mL). The aqueous layer was adjusted to pH 2-3 with a 5 Maqueous HCl solution and extracted with Compound F (1.40g, 2.97 mmol) was suspended in a 4M EtOAc (4x50 mL). The combined organic extracts were 55 aqueous HCl solution (18 mL) and the mixture was heated at washed with brine, dried over NaSO and the solvent was 100° C. for 5 days and then allowed to cool to room tempera removed under reduced pressure to afford (S)-5-(benzyloxy)- ture. The precipitate was filtered and washed with hot water 2-(tert-butoxycarbonyl)-5-oxopentanoic acid (7.1 g, 100%) (30 mL) and EtOH (30 mL), dried in vacuo, then slurried with as a viscous colourless oil. hot EtOH/HO (10:1, 30 mLx2). The solid was collected by LC-MS (Agilent): R, 3.40 min; m/z calculated for 60 filtration and dried in vacuo to afford 4-(2-(2-amino-4-OXo CHNO IM+Nat 360.15. found 360.1. 4,7-dihydro-3H-pyrrolo2,3-dipyrimidin-5-yl)ethyl)benzoic acid (0.326g, 37%) as a green solid. Intermediate C: (S)-Benzyl LC-MS (Waters): R, 5.10 min: m/z calculated for 4-(tert-butoxycarbonyl)-5-hydroxypentanoate CHNO, M+H" 299.11. found 299.1. 65 H-NMR: (400 MHz, DMSO-d) & (ppm): 11.6 (brs, 1H), To a solution of intermediate B (6.5g, 20 mmol) in THF (20 11.5 (s, 1H), 7.84 (d. J=8.0 Hz, 2H), 7.30 (d. J=8.0 Hz, 2H), mL) under nitrogenat -10°C. was added N-methylmorphline 6.49 (s, 1H), 2.85-2.97 (m, 4H). US 8,946,224 B2 157 158

Intermediate H: (S)-Benzyl 5-acetoxy-4-(4-(2-(2- -continued amino-4-oxo-4,7-dihydro-3H-pyrrolo2,3-dipyrimi din-5-yl)ethyl)benzamido)pentanoate To a suspension of intermediate G (0.50 g, 1.68 mmol) in dry DMF (10 mL) was added 2-chloro-4,6-dimethoxy-1,3,5- triazine (0.35g, 2.01 mmol) and N-methylmorpholine (0.37 mL, 3.4 mmol) and the resulting mixture was stirred at room temperature for 3 h. A solution of intermediate E (assumed 2.5 mmol) and N-methylmorpholine (0.37 mL, 3.4 mmol) in 10 DMF (5 mL) was added and stirring was continued at room temperature overnight. The solvent was removed under 29b. reduced pressure and the residue was purified by Silica gel column chromatography (CH.Cl/MeOH, 15/1 to 5/1) to 29a: (3S,10R,13S, 17R)-17-((R)-1-hydroxyethyl)-6, afford (S)-benzyl 5-acetoxy-4-(4-(2-(2-amino-4-oxo-4,7-di 15 10, 13-trimethyl-2,3,8,9,10,11,12,13,14,15,16,17 hydro-3H-pyrrolo2,3-dipyrimidin-5-yl)ethyl)benzamido) dodecahydro-1H-cyclopentaalphenanthrene-3,17 pentanoate (0.70 g, 77%). diol 29b: (3S,10R,13S, 17R)-17-(S)-1- LC-MS (Waters): R., 6.14 min; m/z calculated for hydroxyethyl)-6,10,13-trimethyl-2,3,8,9,10,11,12,13. CHNO IM+H 546.23. found 546.0. 14,15,16,17-dodecahydro-1H-cyclopentaa phenanthrene-3,17-diol Example 28a (S)-5-Acetoxy-4-(4-(2-(2-amino-4- oxo-4,7-dihydro-3H-pyrrolo2,3-dipyrimidin-5-yl) To a solution of compound A (200 mg, 0.58 mmol. 1.0 eq) ethyl)benzamido)pentanoic acid and cerium(w) chloride heptahydrate (653 mg, 1.75 mmol. 3.0 eq) in methanol (20 mL) at 0°C. was added sodium A mixture of intermediate H (100 mg, 0.183 mmol) and borohydride (66 mg, 1.75 mmol. 3.0 eq). The mixture was 10% Pd/C (10 mg) in DMF and THF (1:1, 6 mL) was stirred 25 stirred for 5 min then diluted with water (50 mL) and under a hydrogen atmosphere (1 atm) overnight. The mixture extracted with CHCl (2x50 mL). The combined organic was filtered through Celite and the filtrate was concentrated layers were dried over NaSO and the solvent was removed under reduced pressure. The residue was purified by prepara under reduced pressure. The residue was purified by prepara tive HPLC to give (S)-5-acetoxy-4-(4-(2-(2-amino-4-oxo-4, tive HPLC to give two isomeric products. One isomer (40 mg. 7-dihydro-3H-pyrrolo2,3-dipyrimidin-5-yl)ethyl)benza 30 20%) was obtained as a white solid and assigned as (3S,10R, mido)pentanoic acid as a light green Solid (4.9 mg, 6%). 13S, 17R)-17-((R)-1-hydroxyethyl)-6,10,13-trimethyl-2,3,8. LC-MS (Waters): R. 4.13 min: m/z calculated for 9,10,11,12,13,14,15,16,17-dodecahydro-1H-cyclopentala CHNO IM+H" 456.18. found 456.0. phenanthrene-3,17-diol. H NMR: (400 MHz, DMSO-d) & (ppm): 10.9 (s, 1H), LC-MS (Agilent): R, 3.69 min; m/z calculated for 10.6 (brs, 1H), 8.17 (d.J=8.4 Hz, 1H), 7.74 (d. J=8.0Hz, 2H), 35 CHO, M+Na" 369.25. found 369.2. 7.29 (d. J=8.0Hz, 2H), 6.57 (brs, 2H), 6.40 (s, 1H), 4.24-3.90 H NMR: (400 MHz, DMSO-d) & (ppm): 5.47 (s, 1H), (m,3H), 2.97 (m, 2H), 2.86 (m,2H), 2.28 (m, 2H), 2.0 (s.3H), 5.43 (s, 1H), 4.72 (d. J=5.6 Hz, 1H), 4.12 (d. J=6.4 Hz, 1H), 1.91-1.65 (m, 2H). 4.07 (m, 1H), 3.61 (m. 1H), 3.54 (s, 1H), 1.96 (m, 2H), 1.90-1.65 (m, 6H), 1.65-1.35 (m, 6H), 1.20 (m, 3H), 1.02 (d. Example 29 J=6.4 Hz, 3H), 0.89 (s.3H), 0.85 (m, 1H), 0.69 (s.3H). 40 The other isomer (40 mg, 20%) was obtained as a white Formula 101—Compounds 29a & 29b solid and assigned as (3S,10R,13S,17R)-17-((S)-1-hydroxy ethyl)-6,10,13-trimethyl-2,3,8,9,10,11,12,13,14, 15,16,17 dodecahydro-1H-cyclopentaaphenanthrene-3,17-diol. LC-MS (Agilent): R, 3.66 min; m/z calculated for O 45 C2H M+Nat 369.25. found 369.2. H NMR: (400 MHz, DMSO-d) & (ppm): 5.46 (s, 1H), OH 5.43 (s, 1H), 4.72 (d. J=5.6 Hz, 1H), 4.07 (m, 1H), 4.01 (d. NaBH4 J=6.8 Hz, 1H), 3.75 (quint, J=6.8 Hz, 1H), 3.43 (s, 1H), 2.01 CeCl3-7H2O (m. 1H), 1.85 (m. 1H), 1.75-1.65 (m, 6H), 1.60- 1.40 (m, 5H), MeOH 50 1.4.0-1.10 (m, 4H), 1.01 (d. J=6.0 Hz, 3H), 0.90 (s.3H), 0.86 (m. 1H), 0.78 (s.3H). Example 30

55 Formula 93 Compound 30a OH -O OH Y, 60 \ O LiAlH4 THF

HO o1 CN 65 29a US 8,946,224 B2 159 160 -continued KOH solution (6 mL). The mixture was stirred for 30 minand - O extracted with EtOAc (3x20 mL). The combined organic O extracts were dried over NaSO and the solvent was removed Y, V lsC under reduced pressure to give N1-(3,4-dimethoxyphen N O CHCl2 ethyl)-4-(3,4-dimethoxyphenyl)-4-isopropyl-N1-methyl pentane-1,5-diamine (284 mg, 100%), which was used with o1 out further purification. LC-MS (Agilent): R, 3.24 min; m/z calculated for CHNO IM+H" 459.31. found 459.3. NH2 10 B 30a: N-(5-((3,4-Dimethoxyphenethyl)(methyl) - O amino)-2-(3,4-dimethoxyphenyl)-2-isopropylpentyl) acetamide

Y, \ 15 N O To a solution of intermediate B (284 mg. 0.62 mmol) and EtN (68.7 mg, 0.68 mmol) in anhydrous CHCl (20 mL) at 0° C. was added acetyl chloride (53.5 mg, 0.68 mmol). The o1 mixture was stirred at room temperature for 1 h, washed with water and the organic layer was dried over NaSO. The solvent was removed under reduced pressure and the reside was purified by flash chromatography (Pet. ether/EtOAc, 1/1. "N- V/v) to give N-(5-((3,4-dimethoxyphenethyl)(methyl) O amino)-2-(3,4-dimethoxyphenyl)-2-isopropylpentyl)aceta 25 mide (21 mg, 7%) as a colourless oil. 30a LC-MS (Agilent): R, 3.24 min; m/z calculated for CHNOs M+H" 501.33. found 501.3. "H NMR: (400 MHz, CDC1) & (ppm): 6.84-6.73 (m, 6H), Intermediate B: N1-(3,4-Dimethoxyphenethyl)-4-(3. 6.07 (m. 1H), 3.88 (s.3H), 3.87 (s.3H), 3.86 (s.3H), 3.85 (s, 4-dimethoxyphenyl)-4-isopropyl-N1-methylpentane 30 3H), 3.60 (dd, J=13.6, 4.4 Hz, 1H), 2.75 (m, 2H), 2.63 (m, 1,5-diamine 2H), 2.43 (m, 2H), 2.31 (s.3H), 1.91 (s, 3H), 1.83 (m, 2H), 1.45-1.28 (m, 4H), 0.80 (d. J=6.8 Hz, 3H), 0.76 (d. J=6.8 Hz, To a solution of compound A (300 mg, 0.66 mmol) in THF 3H). (30 mL) at room temperature was added LiAlH (606 mg, 16 mmol) and the resulting mixture was heated at reflux for 10h. 35 Example 31 The mixture was cooled to 0°C., diluted with EtO (150 mL) and the excess LiAlH4 was quenched with a 2 M aqueous Formula 127 Compounds 31a & 31b

US 8,946,224 B2 163 164 Compound A can be synthesised according to the proce EtOAc, 10/1, V/v) to give (S)-tert-butyl 4-(3-fluorobenzy dure described in WO200907.4478. loxy)benzyl (1-(hydroxyimino)propan-2-yl)carbamate (421 mg, 74%) as an oil. Intermediate B: (S)-2-(4-(3-Fluorobenzyloxy)benzy LC-MS (Agilent): R, 3.85 min; m/z calculated for lamino)propan-1-ol CHFNOM+Na" 425.2. found 425.2. 31a: To a solution of compound A (3.36 g. 15 mmol) in metha (S)-2-(4-(3-Fluorobenzyloxy)benzylamino)propanal nol (30 mL) was added (S)-2-aminopropan-1-ol (1.29 mL, Oxime 16.5 mmol) and the resulting mixture was stirred at room temperature overnight. To the mixture was added NaCNBH 10 Intermediate E (380 mg. 0.94 mmol) was dissolved in a 1M (3.78 g. 60 mmol) and stirring was continued at room tem solution of TFA in CHCl (8.5 mL, 8.5 mmol) and the mix perature for 3 h. The solvent was removed under reduced ture was stirred at room temperature for 2 h. The solvent was pressure and the residue was dissolved with EtOAc (300 mL) removed under reduced pressure and the residue was purified and washed with water (3x200 mL) then dried over NaSO. by preparartive silica gel TLC (Pet. Ether/EtOAc, 3/2, V/v) to 15 give (S)-2-(4-(3-fluorobenzyloxy)benzylamino)propanal The solvent was removed under reduced pressure and the oxime (27 mg, 10%) as a light yellow solid. residue was purified by flash chromatography (CH.Cl/ LC-MS (Agilent): R, 3.24 min; m/z calculated for MeOH, 25/1, V/v) to give (S)-2-(4-(3-fluorobenzyloxy)ben CHFN.O. M+H"303.14. found 303.1. Zylamino)propan-1-ol (3.13 g, 72%) as an oil. H NMR: (400 MHz, CDC1) & (ppm): 7.33 (m, 2H), 7.24 LC-MS (Agilent): R, 3.04 min; m/z calculated for (m. 2H), 7.18-7.12 (m, 2H), 7.03 (m, 1H), 6.91 (m, 2H), 5.05 CHFNO, M+H 290.15. found 290.1. (s. 2H), 3.82 (dd, J=12.8, 4.8 Hz, 1H), 3.75 (m, 1H), 3.53 (quint, J=6.4 Hz, 1H), 1.27 (d. J=6.8 Hz, 3H). Intermediate C: (S)-tert-Butyl 4-(3-fluorobenzyloxy) benzyl(1-hydroxypropan-2-yl)carbamate Intermediate F: (S)-tert-Butyl 4-(3-fluorobenzyloxy) 25 benzyl(1-(methoxyimino)propan-2-yl)carbamate To a solution of intermediate B (3.13 g, 10.8 mmol) in To a solution of intermediate D (550 mg, 1.42 mmol) in anhydrous THF (30 mL) was added BocO (3.46 mL, 16.2 methanol (28 mL) at room temperature was added methylhy mmol) and EtN (2.34 mL, 16.2 mmol) and the resulting droxylamine hydrochloride (197 mg, 2.36 mmol) and EtN mixture was stirred at room temperature for 4 h. The solvent (0.41 mL, 2.94 mmol) and the resulting mixture was stirred was removed under reduced pressure and the residue was 30 for 2 h. The solvent was removed under reduced pressure and purified by flash chromatography (Pet. ether/EtOAc, 6/1, V/v) the residue was purified by flash chromatography (Pet. ether/ to give (S)-tert-butyl 4-(3-fluorobenzyloxy)benzyl(1-hy EtOAc, 10/1, V/v) to give (S)-tert-butyl 4-(3-fluorobenzy droxypropan-2-yl)carbamate (3.7g, 80%) as an oil. loxy)benzyl (1-(methoxyimino)propan-2-yl)carbamate (421 LC-MS (Agilent): R, 3.74 min; m/z calculated for mg, 74%) as an oil. CHFNO IM+Na". 412.2. found 412.2. 35 LC-MS (Agilent): R, 3.97 min; m/z calculated for CHFNOM+Na" 439.21. found 439.2. Intermediate D: (S)-tert-Butyl 4-(3-fluorobenzyloxy) benzyl(1-oxopropan-2-yl)carbamate 31b: (S)-2-(4-(3-Fluorobenzyloxy)benzylamino) propanal O-methyl oxime To a solution of intermediate C (3.2 g, 8.22 mmol) in 40 CHCl (50 mL) at room temperature was added Dess-Martin Intermediate F (450 mg, 1.08 mmol) was dissolved in a 1M Periodinane (13.9 g, 32.9 mmol) and the resulting mixture solution of TFA in CHCl (9.72 mL, 9.72 mmol) and the was stirred for 2 h. The solvent was removed under reduced mixture was stirred at room temperature for 2 h. The solvent pressure and the residue was purified by flash chromatogra was removed under reduced pressure and the residue was phy (Pet. ether/EtOAc, 10/1, V/v) to give (S)-tert-butyl 4-(3- 45 purified by preparative silica gel TLC (Pet. Ether/EtOAc, 4/1, fluorobenzyloxy)benzyl(1-oxopropan-2-yl)carbamate (1.2 V/v) to give (S)-2-(4-(3-fluorobenzyloxy)benzylamino)pro g, 38%) as a yellow solid. panal O-methyl oxime (8 mg, 2%) as a light yellow solid. LC-MS (Agilent): R, 3.21 min; m/z calculated for Intermediate E: (S)-tert-Butyl 4-(3-fluorobenzyloxy) CHFNO, M+Nat 317.16. found 317.2. benzyl(1-(hydroxyimino)propan-2-yl)carbamate 50 H NMR: (400 MHz, CDC1) & (ppm): 7.40-728 (m, 4H), 7.21-7.10 (m, 2H), 7.03 (m, 1H), 6.93 (m, 2H), 5.06 (s. 2H), To a solution of intermediate D (550 mg, 1.42 mmol) in 3.88 (s.3H), 3.83-3.71 (m, 2H), 3.51 (quint, J=6.4 Hz, 1H), methanol (28 mL) at room temperature was added hydroxy 1.31 (d. J=6.8 Hz, 3H). lamine hydrochloride (197 mg, 2.84 mmol) and EtN (0.41 mL, 2.94 mmol) and the resulting mixture was stirred for 2 h. 55 Example 32 The solvent was removed under reduced pressure and the residue was purified by flash chromatography (Pet. ether/ Formula 122 Compound 32a

S O BH THF THF rol N lu NX-NH. H A US 8,946,224 B2 165 166 -continued OH

rol N - NX-NH. H 32a

32a: (R)-2-(4-(2-(2-Aminothiazol-4-yl)ethylamino) phenethylamino)-1-phenylethanol -continued

To a solution of compound A (300 mg. 0.76 mmol) in dry 15 ONa O re-()-sco THF (15 mL) was added a 1M solution of BH THF in THF -N-- THF (2.27 mL, 2.27 mmol) dropwise at 0°C. The mixture was stirred at 50° C. for 2 h and then allowed to cool to room temperature and stirring was continued overnight. The reac ONa tion was quenched with a 1M aqueous HCl solution (5 mL) and diluted with water (20 mL). Most of the THF was S. removed under reduced pressure and the aqueous mixture N was adjusted to pH10 with a 1Maqueous NaOH solution and O O extracted with CHC1. The combined organic extracts were FC washed with brine, dried over NaSO and the solvent was 25 removed under reduced pressure. The residue was purified by 33a column chromatography (CH.Cl/MeOH/conc.NHOH, 10/1/0.05, v/v) followed by preparative HPLC to afford the product as a TFA salt (62 mg). An aliquot of the salt (25 mg) 33a: Sodium 3-(dimethylcarbamoyl)-4-oxo-4-(4- was free-based by dissolving in a Saturated aqueous NaCOs 30 (trifluoromethyl)phenylamino)but-2-en-2-olate Solution (5 mL) and extracting with CH2Cl2. The organic layer was dried over NaSO and concentrated under reduced pressure to give (R)-2-(4-(2-(2-aminothiazol-4-yl)ethy To a stirred Suspension of sodium metal (0.25 g, 11 mmol. lamino)phenethylamino)-1-phenylethanol (10 mg. 9%) as a 1.1 eq) in dry THF (50 mL) was added N,N-dimethyl-3- oXobutanamide (1.3g, 10 mmol. 1.0 eq) and the mixture was white foam. 35 stirred overnight. To the resulting white Suspension was LC-MS (Agilent): R, 3.07 min; m/z calculated for added 4-(trifluoromethyl)phenyl isocyanate (1.8 g. 10 mmol. CHNOS M+H" 383.18. found 383.2. 1.0 eq) dropwise at room temperature. The mixture was then 'HNMR: (400 MHz, CDC1/CDOD, -20: 1) & (ppm): 7.29 heated at reflux for 4 h, cooled to room temperature and (m, 4H), 7.22 (m, 1H), 6.95 (d. J=8.4 Hz, 2H), 6.53 (d. J–8.4 diluted with MTBE (80 mL). The solid in the mixture was Hz, 2H), 6.10 (s, 1H), 4.66 (dd, J=9.2, 4.0 Hz, 1H), 3.32 (t, 40 collected by filtration, washed with EtOAc (20 mL) and J=6.8 Hz, 2H), 2.80-2.61 (m, 8H). CHCl (20 mL) and dried under vacuum to give sodium Example 33 3-(dimethylcarbamoyl)-4-oxo-4-(4-(trifluoromethyl)pheny lamino)but-2-en-2-olate (40 mg, 1%) as a yellow solid. Formula 131—Compound 33a LC-MS (Agilent): R, 3.40 min; m/z calculated for 45 CHFNNaO. M+H" 339.09. found 339.1. H NMR: (400 MHz, DMSO-d) & (ppm): 13.5 (s, 1H), 7.65 (d. J=8.8 Hz, 2H), 7.45 (d. J=8.4 Hz, 2H), 2.90 (s, 3H), O O 2.85 (s.3H), 1.69 (s.3H). Na 50 --- THF Semagacestat Example 34 Formula 130 Compounds 34a, 34b and 34c 1.O -e- -e- BocN u N CHCl2TFA H NullsO N HATUDIPEAO N N an N N DMF E H E O E O A B US 8,946,224 B2 167 168

-continued

HO -N O NNulls O E

-as 34b N CHOH N pyridine E O HO 34a -N O

N Nulls N N O E N

34c

Compound A can be synthesised according to the proce LC-MS (Agilent): R, 3.40 min; m/z calculated for dure described in U.S. Pat. No. 7,468,365. It can be obtained 25 CHNO IM+H" 360.18. found 360.2. as a ~1.5:1 mixture of diastereoisomers, determined by inte 'H-NMR: (400 MHz, DMSO-d) & (ppm): 8.88 (d. J=8.0 gration of the NMR spectrum. HZ, 0.66H), 8.82 (d. J=8.0 Hz, 0.33H), 8.44 (d. J=7.6 Hz, 0.33H), 8.36 (d. J=7.6 Hz, 0.66H), 7.26-7.13 (m, 4H), 6.26 Intermediate B: (S)-2-Amino-N-(3-methyl-2-oxo-2, 6.21 (m. 1H), 4.60 (m. 1H), 4.25 (m. 1H), 3.39 (m. 1H), 3,4,5-tetrahydro-1H-benzodiazepin-1-yl)propana 30 3.22-3.15 (m, 2H), 2.93 (m, 1H), 2.92 (m, 3H), 1.40 (m,3H), mide 1.06 (d. J=6.8 Hz, 6H). To a 1M solution of TFA in CHCl (30 mL, 30 mmol) at 34b: 2-(Hydroxyimino)-3-methyl-N-((S)-1-((R)-3- room temperature was added compound A (600 mg, 1.66 methyl-2-oxo-2,3,4,5-tetrahydro-1H-benzod mmol) and the resulting mixture was stirred overnight. A 35 aZepin-1-ylamino)-1-oxopropan-2-yl)butanamide saturated aqueous solution of Na2CO was slowly added to and 34c: 2-(Hydroxyimino)-3-methyl-N-((S)-1- adjust the pH to 8-9. The organic layer was separated and the ((S)-3-methyl-2-oxo-2,3,4,5-tetrahydro-1H-benzod aqueous layer was extracted with CHCl (2x30 mL). The aZepin-1-ylamino)-1-oxopropan-2-yl)butanamide combined organic extracts were washed with brine, dried over NaSO and the solvent was removed under reduced 40 pressure to give (S)-2-amino-N-(3-methyl-2-oxo-2,3,4,5-tet To a solution of example 33a (100 mg, 0.28 mmol. 1.0 eq) rahydro-1H-benzodiazepin-1-yl)propanamide (278 mg, in methanol (20 mL) and pyridine (2 mL) was added hydroxy 64%) as a yellow solid. lamine hydrochloride (23 mg 0.33 mmol. 1.2 eq) and the LC-MS (Agilent): R, 3.90 min; m/z calculated for resulting mixture was stirred at room temperature for 4 h. The CHNO, M+H 262.15. found 262.1. 45 Solvent was removed under reduced pressure and the residue was purified by column chromatography (CH2Cl2/MeOH, 34a: (S)-3-Methyl-N-(1-(3-methyl-2-oxo-2,3,4,5- 50/1 to 30/1, V/v) to separate the diastereoisomers. The minor tetrahydro-1H-benzodiazepin-1-ylamino)-1-oxopro diastereoisomer (25 mg, 24%) was obtained as a colourless pan-2-yl)-2-oxobutanamide oil and was assigned as 2-(hydroxyimino)-3-methyl-N- 50 ((S)-1-((R)-3-methyl-2-oxo-2,3,4,5-tetrahydro-1H-benzod To a solution of 3-methyl-2-oxobutanoic acid (100 mg. azepin-1-ylamino)-1-oxopropan-2-yl)butanamide, 'H-NMR 0.87 mmol. 1.0 eq) in dry DMF (25 mL) at room temperature spectroscopy revealed a ~1:1 mixture of oxime isomers. was added HATU (413 mg 0.87 mmol. 1.0 eq) and DIPEA LC-MS (Agilent): R, 3.43 min; m/z calculated for (561 mg, 1.09 mmol. 1.25 eq) and the resulting mixture was CHNOM+H 375.2. found 375.2. stirred at room temperature for 30 min. Intermediate B (227 55 H-NMR: (400 MHz, DMSO-d) & (ppm): 11.6 (s, 0.5H), mg, 0.87 mmol. 1.0 eq) was then added and the mixture was 11.5 (s, 0.5H), 8.30 (m, 1H), 8.22 (m, 1H), 7.25-7.13 (m, 4H), stirred at room temperature overnight. The mixture was 6.21 (m. 1H), 4.55 (m, 1H), 4.24 (m. 1H), 3.42-3.38 (m, 1H), diluted with water (30 mL) and extracted with EtOAc (3x20 3.32-3.28 (m. 1H), 3.22-3.16 (m, 2H), 2.91 (s, 1.5H), 2.90 (s, mL). The combined organic extracts were washed with water 1.5H), 1.37 (m, 3H), 1.15 (m, 6H). (50 mL), a saturated aqueous NaCO solution (50 mL) and 60 The major diastereoisomer (45 mg, 43%) was obtained as brine (50 mL) then dried over NaSO. The solvent was a colourless oil and was assigned as 2-(hydroxyimino)-3- removed under reduced pressure and the residue was purified methyl-N-((S)-1-((S)-3-methyl-2-oxo-2,3,4,5-tetrahydro by column chromatography (CH.Cl/MeOH, 50/1 to 15/1, 1H-benzodiazepin-1-ylamino)-1-oxopropan-2-yl)butana V/v) to give (S)-3-Methyl-N-(1-(3-methyl-2-oxo-2,3,4,5-tet mide, 'H-NMR spectroscopy revealed a ~1:1 mixture of rahydro-1H-benzodiazepin-1-ylamino)-1-oxopropan-2-yl)- 65 Oxime isomers. 2-oxobutanamide (150 mg, 48%) as a white solid, 'H-NMR LC-MS (Agilent): R, 3.41 min; m/z calculated for spectroscopy revealed the diastereoisomeric ratio to be -2:1. CHNO IM+H" 375.2. found 375.2. US 8,946,224 B2 169 170

H-NMR: (400 MHz, DMSO-d) & (ppm): 11.1 (s, 0.5H), -continued 11.06 (s, 0.5H), 8.80 (d. J=6.8 Hz, 0.5H), 8.68 (d. J=7.6 Hz, 0.5H), 8.27 (d. J=7.2 Hz, 0.5H), 8.23 (d. J=7.6 Hz, 0.5H), 7.31-7.11 (m, 4H), 6.23 (m, 1H), 4.62 (m, 0.5H), 4.50 (m, 0.5H), 4.24 (m. 1H),341-3.36 (m, 1H), 3.18 (m, 2H), 2.92 (s, 3H), 2.68 (m. 1H), 1.33 (m, 3H), 1.15 (m, 6H). Example 35 10 Formula 95—Comparative Compound 35a

35a 15

orO Intermediate B: Benzyl (3S,5S,6S,8S)-3-(4-meth oxy-3-(3-methoxypropoxy)benzyl)-8-((3-amino-2,2- dimethyl-3-oxopropyl)carbamoyl)-6-hydroxy-2.9- dimethyldecan-5-ylcarbamate To a solution of compound A (0.99 g, 1.8 mmol) in CHCl (15 mL) was added Et-N (364 mg., 3.6 mmol) and Cbz-OSu 25 (673 mg, 2.7 mmol) and the resulting mixture was stirred at room temperature for 1 h. The mixture was diluted with EtOAc, washed with water and brine, dried over MgSO and the solvent was removed under reduced pressure to give ben Zyl (3S,5S,6S,8S)-3-(4-methoxy-3-(3-methoxypropoxy) benzyl)-8-((3-amino-2,2-dimethyl-3-oxopropyl)carbam 30 oyl)-6-hydroxy-2,9-dimethyldecan-5-ylcarbamate (1.1 g, 100%) as a colourless oil. Ac2Of EtN Intermediate C: (3S,5S,6S,8S)-8-(4-methoxy-3-(3- methoxypropoxy)benzyl)-3-((3-amino-2,2-dimethyl 35 3-oxopropyl)carbamoyl)-6-(benzyloxycarbonyl)-2.9- dimethyldecan-5-yl acetate To a solution of intermediate B (1.05 g, 1.8 mmol) in CHCl (15 mL) was added EtN (364 mg., 3.6 mmol) and 40 acetic anhydride (364 mg, 2.7 mmol) and the resulting mix ture was stirred at room temperature for 1 h. The mixture was diluted with EtOAc, washed with water and brine, dried over MgSO and the solvent was removed under reduced pressure to give (3S,5S,6S,8S)-8-(4-methoxy-3-(3-methoxypropoxy) 45 benzyl)-3-((3-amino-2,2-dimethyl-3-oxopropyl)carbam oyl)-6-(benzyloxycarbonyl)-2,9-dimethyldecan-5-yl acetate POCl. EN (1.3 g, 99%) as a colourless oil. Intermediate D: (3S,5S,6S,8S)-8-(4-methoxy-3-(3- 50 methoxypropoxy)benzyl)-6-(benzyloxycarbonyl)-3- ((2-cyano-2-methylpropyl)carbamoyl)-2,9-dimethyl decan-5-yl acetate To a solution of intermediate C (1.3g, 1.8 mmol) and EtN 55 (546 mg, 5.4 mmol) in MeCN (10 mL) was added POCl (364 mg, 2.7 mmol) at 0°C. The resulting mixture was stirred at room temperature for 30 min and poured onto ice. The mix ture was extracted with EtOAc and the combined organic extracts were washed with water, brine and dried over NaOHIHO 60 -e- MgSO. The solvent was removed under reduced pressure dioxane and the residue was purified by column chromatography (hexanes/EtOAc, 3/1, V/v) to give (3S,5S,6S,8S)-8-(4-meth oxy-3-(3-methoxypropoxy)benzyl)-6-(benzyloxycarbonyl)- 3-((2-cyano-2-methylpropyl)carbamoyl)-2,9-dimethylde 65 can-5-yl acetate (0.62 g, 4.9%) as a colourless oil. LC-MS (Waters): R., 6.52 min; m/z calculated for CHNOs M+H" 710.43. found 710.5.

US 8,946,224 B2 173 174 quenched with water (5 mL) and the solvent was removed was removed under reduced pressure. The crude product was under reduced pressure. The crude product was purified by collected by filtration and washed with water (5 mL). Purifi preparative TLC (Pet. ether/EtOAc, 1/2, V/v) to give 2-((2S, cation by preparative TLC (Pet. ether/EtOAc. 1/1, V/v) then 6aS,6bR.7S,8aS,8bS,11aR,12aS.12bS)-2,6b-difluoro-7-hy gave 2-((2S,6aS,6bR.7S,8aS,8bS,11aR,12aS.12bS)-2,6b-di droxy-6a,8a,10,10-tetramethyl-4-oxo-2,4,6a,6b,7,8,8a,8b, 5 fluoro-7-hydroxy-6a,8a,10,10-tetramethyl-4-oxo-2,4,6a,6b, 11a, 12.12a, 12b-dodecahydro-1H-naphtho2', 1:4.5indeno 7,8,8a,8b.11a, 12.12a, 12b-dodecahydro-1H-naphtho2", 1":4, 1,2-d1.3dioxol-8b-yl)-2-oxoacetaldehyde oxime (70 mg. 5 indeno1,2-d1.3dioxol-8b-yl)-2-oxoacetaldehyde 15%) as a white powder. O-methyl oxime (70 mg, 15%) as a white powder. LC-MS (Agilent): R, 3.22 min; m/z calculated for LC-MS (Agilent): R, 3.31 min; m/z calculated for CHNOM+H 466.2. found 466.1. 10 "H NMR: (400 MHz, CDOD) 8 (ppm): 8.02 (s, 1H), 7.33 CHNO IM+H" 480.2. found 480.2. (d. J=10.0 Hz, 1H), 6.39 (d. J=10.0 Hz, 1H), 6.32 (s, 1H), 5.51 "H NMR: (400 MHz, CDOD) 8 (ppm): 8.00 (s, 1H), 7.35 (m. 1H), 5.15 (d. J–3.6 Hz, 1H), 4.32 (d. J=8.8 Hz, 1H), 2.71 (d. J=10.0 Hz, 1H), 6.36 (d. J=10.0 Hz, 1H), 6.32 (s, 1H), 5.51 (m. 1H), 2.26 (m,3H), 1.69 (m, 4H), 1.59 (s.3H), 1.45 (s.3H), (m. 1H), 5.13 (d. J=4.8 Hz, 1H), 4.31 (d. J=9.2 Hz, 1H), 4.08 1.14 (s, 3H), 0.95 (s, 3H). 15 (s, 3H), 2.64 (m. 1H), 2.36 (m. 1H), 2.29 (m. 2H), 1.71 (m, 4H), 1.59 (s.3H), 1.45 (s.3H), 1.14 (s.3H), 0.95 (s, 3H). 36b: 2-((2S,6aS,6bR.7S,8aS,8bS,11aR,12aS,12bS)- Methodology Cresset 2.6b-Difluoro-7-hydroxy-6a,8a,10,10-tetramethyl-4- The compounds were analysed for field similarity to the oxo-2,4,6a,6b,7,8,8a,8b.11a, 12.12a, 12b-dodecahy parent. This was determined based on the conformation of the dro-1H-naphtho2.1":4.5indeno1,2-d1,3-dioxol- 2O parent when the parent is in the active site. In some cases the 8b-yl)-2-oxoacetaldehyde O-methyl oxime conformation was determined using crystal structures of the parent in the active site. In some cases the conformation of the A solution of intermediate B (450 mg, 1 mmol. 1.0 eq), parent in the active site was a predicted conformation based O-methylhydroxylamine hydrochloride (92 mg, 1.1 mmol. on what information was available. In some cases the binding 1.1 eq) and triethylamine (110 mg, 1.1 mmol. 1.1 eq) in 25 energies of the compounds were also calculated. The meth MeOH (10 mL) was stirred at room temperature overnight. odology used for these analyses are described in more detail The reaction was quenched with water (5 mL) and the MeOH below:

Step Details Methodology Output(s) 1. Background research: a) Journal papers and c) X-ray crystal literature searching to web-based information structure(s) of identify key information b) Search of PDB database relevant proteins and regarding mechanism of for relevant protein Small molecule action for the parent crystal structure(s) igands molecule and related d) Set of known actives therapeutics. with the same mechanism of action 2. Templating: identification of a) Import the chemical c) Field Template - a key set of active molecules structures of a set of alignment of e.g. 5 from within the therapeutic known actives into known active class, generation model for FieldTemplater, molecules, to give protein ligand binding generate a consensus hypothesis for template containing as interaction with many of the actives as protein possible d) Active conformation b) Output the template or parent molecule - (set of proposed active either from crystal conformations) and the structure or from individual active Field Template conformation of the e) Docking model parent structure. model protein structure which can be used for calculation of interaction energies 3. FieldAlign analysis: a) Load the template files c) Ranked scores for the calculation of Field-based from Step 2 a) into proposed analogues similarity scores between FieldAlign, load based on the Field analogues and analogue structures, similarity score template?parent calculate best d) Aligned best alignment and conformation for each associated similarity analogue score for each analogue. b) Repeat with the single structure of the parent compound from Step 2 b) US 8,946,224 B2 175 176 -continued Step Details Methodology Output(s) 4. Prediction of binding Take aligned analogues d) Aligned energies: docking of poses from Step 3 b) and load conformations from from step 3 into crystal into Accelrys Discovery step 3 b) docked into structure, calculation of Studio. crystal structure with interaction energies. b) Load protein crystal calculated energies structure as found in 1 e) Ranking of analogues c), and prepare for relative to parent docking (apply Structure CHARMm force field, remove ligand from active site, define active site sphere Calculate binding energies for analogues using flexible ligand optimisation under the CHARMm force field Assessment and ranking of a) Calculation of b) Ranked priority list for analogues: generation of a consensus score based the proposed consensus score based on on Sum of ranks for analogues similarities and binding each of the two energies. alignments and the binding energy calculations

The stereoisomeric identity (R vs S or Evs Z) of any group 25 Example 38 described in the following examples is that of the parent active unless otherwise indicated. The compounds analysed in the following examples have A range of structures were tested for their potential as been organised into bands depending on the results obtained analogues of the fluoroquinolone antibitotics, such as cipro in analysis of that compound. In an embodiment, of the inven 30 floxacin. The fluoroquinolone antibitotics are active due to tion the compound is any which falls with band A for a their ability to interact with bacterial DNA gyrase and/or specific analysis for a specific formula. In another embodi topoisomerase II. DNA gyrase (or gyrase for short) is an ment, the compound is any which falls within band A or band important protein involved in DNA replication within bacte B for a specific analysis for a specific formula. In a further ria; mechanistically the gyrase is involved in relaxing Super embodiment, the compound is anywhich falls within band A. 35 coils within the DNA strand which form ahead of the point of band B or band C for a specific analysis for a specific formula. replication (by DNA polymerase). The fluoroquinolones intercalate the DNA and prevent decatenation of the repli Example 37 cated DNA from the gyrase. Based on the 2XCT structure, we can see the complex of A range of structures have been assessed for their potential interactions which are made by the fluoroquinolone ligand: it as analogues of oseltamivir. Oseltamivir is a neuraminidase 40 used to treat flu. It acts by blocking the action of neuramini is intercalating into the DNA strand, fitting in between two of dase in releasing new virus particles from the Surface of an the nucleosides, as well as chelating to a Manganese ion and infected cell. There are many X-ray crystal structures of interacting with the DNA binding site on the gyrase itself. neuraminidase, including several with bound inhibitors. The The complexity of these interactions meant that it was template for analysis was based on the 2HU4 structure of 45 difficult to draw exact conclusions or make quantitative pre oseltamivir bound to viral neuraminidase. dictions of likely binding activity. For field similarity: A is over 80% similarity: B is 60-79% One driving factor for these compounds will be the ability similarity and C is 30-59% similarity. to chelate the Manganese, which is the catalytic metal ion

Field For similarity mula Parent Structure o parent 162 oseltamivir V is H(NH); L is N=CHMe: Z is CH B ethylene glycol acetal 162 oseltamivir V is H(NH); L is NHAc: Z is CH-OH 162 oseltamivir V is H(NH); L is N=CHMe: Z is CH(OEt), 162 oseltamivir V is H(NH); L is N=CHMe: Z is CH=NOH 162 oseltamivir V is H(NH); L is N=CHMe: Z is C(O)H 162 oseltamivir V is H(NH); L is NHAc: Z is C(O)H 162 oseltamivir V is H(NH2); L is NHAc; Z is CH-ethylene glycol acetal 162 oseltamivir V is H(NH); L is NHAc: Z is CH(OEt), 162 oseltamivir V is H(NH); L is NHAc: Z is CH=NOH 162 oseltamivir V is H(NH); L is N=CHMe: Z is COEt 162 oseltamivir V is H(NH); L is N=CHMe: Z is COH Oseltamivir Comparative example: Formula 162 in which V is NH2: L is NHAc: Z is CH=NOBn US 8,946,224 B2 177 178 sitting in the active site of the gyrase. This ability was For field similarity: Field similarity Ameans a similarity of assessed by looking at the intensity of the negative electro 80-85%; and B means a similarity of 70-79%. static field at the Managanese position; a proxy for this was to inspect the magnitude of the negative Field point being gen erated by any given analogue. The negative field point on the 5 Field ring carbonyl for several known fluoroquinolone antibiotics For- similarity are as follows: mula Parent Structure to parent 163 pregabalin W is CH-NH2: Z is CH-OH A. 163 pregabalin W is CH-NH2: Z is C(O)H B Negative field point Antibiotic on ring carbonyl 10 Ciprofloxacin -14.15 Moxifloxacin -16.80 Example 40 Gatifloxacin -16.77 Pefloxacin -16.53 Report 4 15 For the analogues, the values are as follows: A if the nega Penicillin Binding Proteins (named for their propensity for tive field point is between -20 and -15; B if the negative field binding to penicillin and related compounds) are critical pro point is between -10 and -15; and C if the negative field point teins involved in the final stages of the assembly of bacterial is between -5 and -10. cell walls, where they catalyse the cross-linking of pepti doglycan units. Interfering with this process leads to irregu larities in cell wall construction, with concomitant bacteri Negative field point on ring cidal effect. Penicillin Binding Protein 3 (“PBP-3') is a well For carbonyl or characterised member of the group of PBPs and is the target mula Parent Structure equivalent 25 for a variety of antibiotic agents. The B-lactam antibiotics (penicillins, penems, carbapenems, cephalosporins, etc) 90 Ciprofloxacin G is =O; Z is CO-Me 90 Ciprofloxacin G is -O; Z is CH-NOMe inactivate PBPs by covalently bonding to the catalytic serine 90 Ciprofloxacin G is =NOMe: Z is CH-NOMe residue within the PBP active site. 90 Ciprofloxacin G is =O; Z is C(O)H There are several examples in the PDB of compounds 90 Ciprofloxacin G is =O; Z is C(O)Et 30 bound to PBP-3, including Aztreonam (PDB code: 3PBS), meropenem (PDB code: 3PBR), imipenem (3PBQ), ceftazi dime (3PBO) and cefotaxime (2XD1). The analogues of Example 39 meropenem were aligned with a template based on the (open A range of structures have been assessed for their potential ring) configuration of meropenem in 3PBR. The analogues of as analogues of pregabalin. Pregabalin is a primary neuronal 35 faropenem and imipenem were aligned with a template based signalling molecule which mediates a number of processes on the (open ring) configuration of meropenem in 3PBQ. The within neuronal synapses. Its principle activity is as an inhibi analogues of cefimetazole and cefepime were aligned with a tory neurotramsitter and it appears to act through binding to a template based on the (open ring) configuration ofcefotaxime specific Cation channel in the central nervous system. There in 2XD1. is no relevant structural biology information as pregabalin 40 For field similarity: A is over 90% similarity; B is 80-89% binds to an extracellular domain of the ion channel which has similarity; C is 70-79% similarity and D is 60-69% similarity. not been characterised by X-ray studies. Analysis was based For relative binding energy: A means binding energy is on both looking at the quantitative field similarity of the greater than the parent; B means binding energy is within 50 analogues to a set of known active compounds, and also a Kcal of the parent; C means the binding energy is within 100 more qualitative assessment of the field patterns shown by the 45 Kcal and D means the binding energy is within 250 Kcal of molecules. the parent.

Field Field similairt similarity Binding to parent to parent energy For (open (closed relative mula Parent Structure ring) ring) to parent 64 mipenem Q is S; W is CH=NH; Z is A. C B COH: G is =O 64 mipenem Q is S; W is CH=NH; Z is BAB CFC B,C COH: G is —NOH (2 isomers) 64 mipenem Q is S; W is CH=NH; Z is BAB CFC CFC COH: G is =NOMe (2 isomers) 64 mipenem Q is S; W is CH=NH; Z is C D D COH: G is (OMe) 64 mipenem Q is S; W is CH=NH; Z is B C D COH: G is ethylene glycol acetal 64 mipenem Q is S; W is CH=NH; Z is B C D C(O)H: G is H(OH) 64 mipenem Q is S; W is CH=NH; Z is C D D CH-OH: G is H(OH)

US 8,946,224 B2 183 184 assessment was based on aligning the structures to the angio For- Field tensin II molecule extracted from the model in PDB code mula Parent Structure similarity 1ZVO (the alignment being carried out in the presence of the 1 Metronidazole J is NO: Z is C(O)H A. model receptor structure). A second field similarity assess 1 Metronidazole J is NO; Z is CO2H A. 1 Metronidazole J is NO; Z is COEt A. ment is based on a simple field-based alignment of the struc 1 Metronidazole J is NO; Z is CH-ethylene B tures against a template derived from the structures of a series glycol acetal of known Angiotensin Receptor Blockers, including cande 1 Metronidazole J is NO; Z is CH(OMe), A. 1 Metronidazole J is NO: Z is CH=NOH A. Sartan. A binding energy for docking to the angiotensin recep 1 Metronidazole J is NO: Z is CH=NOMe A. 10 tor was also calculated. For candesartan analogues field similarity: A is over 80% similarity; B is 60-79% similarity and C is 30-59% similarity. Example 42 For losartan analogues field similarity: A is over 75% simi larity: B is 60-74% similarity and C is 30-59% similarity. A range of structures have been assessed for their activity 15 For relative binding energy: A means binding energy is at the angiotensin receptor. Angiotensin is a peptidic hormone greater than the parent; B means binding energy is within 50 which is critical in controlling vascular dilation/contraction. Kcal of the parent; C means the binding energy is within 100 Angiotensin receptor blockers lower the blood pressure by Kcal and D means the binding energy is within 250 Kcal of blockading the angiotensin 1 receptor. A first field similarity the parent.

Field Field Binding Similarity Similarity Energy For to Angio to compared mula Parent Structure tensin II Template o parent. 137 Candesartan Z is C(O)H 137 Candesartan Z is CH-NOH 137 Candesartan Z is CH=NOMe 137 Candesartan Z is CH(OMe), 137 Candesartan Z is CH-ethylene glycol acetal 137 Candesartan Z is CH-OH 141 LOSartan Z is C(O)H 141 Losartan Z is CH-NOH 141 Losartan Z is CH=NOMe 141 Losartan Z is CH(OMe), 141 Losartan Z is CH-ethylene glycol acetal 141 Losartan Z is COH C A. A.

40 Example 43 A range of structures have been assessed for their activity as Calcium Channel blockers. Calcium channel blockers are a therapy of choice for various applications in which vasodi 45 lation plays a key role. Such as angina pectoris, migraine, hypertension and cardiac arrhythmia. There are three classes of Calcium channel blockers which bind to different binding sites on the L-type calcium channels: phenylalkylamines Such as Verapimil; benzothiazepines such as dilthiazem and 1,4-dihydropyridines such as amlodipine, felopidine and 50 nifedipine. The assessment has been conducted by aligning the structures to the relevant parent molecule in a likely active confirmation. The likely active confirmation has been derived by comparison and analysis of the parent compound along with other known Cachannel actives from the same class. In 55 the case of Verapamil the active conformation has been derived from prior knowledge of the binding modes, guided by the use of a homology model of the Cachannel. For field similarity: A is over 90% similarity; B is 80-89% similarity; C is 60-79% similarity and D is 40-59% similarity.

For Similarity mula Parent Structure to parent

147 Amlodipine Z is CO-Me: Z, is C(O)H; W is CH-NH2, A. 147 Amlodipine Z is COMe, Z is CH=NOH; W is CH-NH2 A. US 8,946,224 B2 185 186 -continued

For Similarity mula Parent Structure to parent 47 Amlodipine Z is CO-Me: Z2 is CH=NOMe: W is CH-NH2 A. 47 Amlodipine Z is CO-Me: Z2 is CH(OMe); W is CH-NH2 B 47 Amlodipine Z is CO-Me: Z2 is CH-ethylene glycol acetal; B W is CH-NH, 47 Amlodipine Z is CO-Me: Z2 is CH-OH; W is CH-NH2 B 47 Amlodipine Z is CO-Me: Z2 is COEt; W is CN A. 47 Amlodipine Z is CO-Me: Z2 is COEt; W is C(O)NH2 A. 47 Amlodipine Z is CO-Me: Z, is CO-Et; W is C(NH)NH2. A. 47 Amlodipine Z is CO-Me: Z2 is COEt; W is CH-NH2 A. 47 Amlodipine Z is C(O)H: Z2 is COEt; W is CH-NH2 B 47 Amlodipine Z is CH=NOH: Z2 is COEt; W is CH-NH2 B 47 Amlodipine Z is CH=NOMe: Z, is COEt; W is CH-NH2 B 47 Amlodipine Z is CH(OMe); Z is COEt; W is CH-NH2 B 47 Amlodipine Z is CH-ethylene glycol acetal; Z2 is COEt:W B is CH-NH2 54. Felodipine Z is COEt: Z, is C(O)H A. 54. Felodipine Z is COEt; Z is CH=NOMe C 54. Felodipine Z is COEt: Z, is CH=NOH A. 54. Felodipine Z is COEt; Z is CH(OMe), B 54. Felodipine Z is CO-Et; Z is CH-ethylene glycol acetal B 54. Felodipine Z is COEt; Z is CH-OH A. 54. Felodipine Z is C(O)Ht: Z is CO-Me A. 54. Felodipine Z is CH=NOH: Z2 is CO-Me B 54. Felodipine Z is CH=NOMe: Z2 is CO-Me A. 54. Felodipine Z is CH(OMe); Z is CO-Me C 54. Felodipine Z is CH-ethylene glycol acetal; Z2 is CO-Me B 54. Felodipine Z is CH-OH: Z, is CO-Me B 52 Diltiazem Y is =O; G is H(OH) B 52 Diltiazem Y is -O; G is -O B 52 Diltiazem Y is —O; G is —NHOH B 52 Diltiazem Y is -O; G is -NHOMe B 52 Diltiazem Y is =O; G is (OMe), B 52 Diltiazem Y is —O; G is ethylene glycol acetal B 93 Verapamil W is C(O)N.Me, C 93 Verapamil W is C(O)NH, C 93 Verapamil W is CH-NH D 93 Verapamil W is C(O)NHMe C 93 Verapamil W is CH-NHAc C 93 Verapamil W is CH-NOH C 93 Verapamil W is CH-NOMe C 93 Verapamil W is C(NH)NH, D 93 Verapamil W is C(NH)NHMe D 93 Verapamil W is C(NH)N.Me, D 108 Nifedipine J is NO; Z is CO-Me: Z2 is CO-Me A. 108 Nifedipine J is NH2: Z is CO-Me: Z2 is CO-Me B 108 Nifedipine J is NHAc; Z is CO-Me: Z is CO-Me A. 108 Nifedipine J is NO; Z is C(O)H: Z, is CO-Me A. 108 Nifedipine J is NO: Z is CH=NOH: Z, is CO-Me A. 108 Nifedipine J is NO: Z is CH=NOMe: Z, is CO-Me A. 108 Nifedipine J is NO; Z is CH(OMe); Z is CO-Me A. 108 Nifedipine J is NO; Z is CH-cyclic acetal; Z is CO-Me A. 108 Nifedipine J is NO; Z is CH-OH: Z is CO-Me A. 108 Nifedipine J is NO; Z is CHOAc: Z2 is CO-Me B

Example 43 50 Field For- similarity A range of structures were tested for their potential as mula Parent Structure to parent analogues of eZetimibe, which is believed to operate by 138 Ezetimibe G is CH(OAc) C blocking cholesterol absorption in the lower intestines. The 55 138 Ezetimibe Gis =O A. 138 Ezetimibe G is NOH A. mechanism of action is believed to be binding to the 138 Ezetimibe G is (OMe), A. Niemann-Pick C1-Like (NPC1L1) protein which is 138 Ezetimibe Gis =NOMe B expressed in the brush border cells lining the epithelium of the 138 Ezetimibe G is ethylene glycol acetal B lower intestine. There are short-sequence X-ray structures 60 available for the close analogue, NPC1 and for NPC1L1 itself Example 44 but these were insufficiently accurate. Instead, a ligand based approach was adopted to generate a template of the active A range of structures were tested for their potential as conformation of eZetimibe. analogues of otamixaban and apixaban. Otamixaban and 65 apixaban are Factor Xa inhibitors used as anticoagulants. For field similarity: A is over 85% similarity: B is 80-84% Field analysis was performed on the otamixaban structures similarity; and C is 75-80% similarity. by aligning them to the active conformation extracted from US 8,946,224 B2 187 188 the PDB structure of factor Xa, which has otamixaban in the alignment to both the active metabolite of clopidogrel and the active site (PDB code: 1KSN). Field analysis was performed anion of the active metabolite. on the apixaban structures by aligning them to the active conformation extracted from the PDB structure (PDB code: 2P16) of factor Xa, which has apixaban in the active site. O OMe Binding energies have also been calculated. For otamixaban field similarity: A is over 80% similarity; ah and B is 70-80% similarity. N 21 NooH For otamixaban field similarity: A is over 90% similarity; 10 and B is 85-89% similarity. C 'SH For relative binding energy: A means binding energy is clopidogrel active metabolite greater than the parent; B means binding energy is within 50 Kcal of the parent; C means the binding energy is within 100 Kcal and D means the binding energy is within 750 Kcal of For field similarity: A is over 90% similarity; B is 85-89% the parent. similarity and C is 80-84% similarity.

Binding Field energy For- similarity relative mula Parent Structure to parent to parent 24 Otamixaban T is NO;Y is —O; W is A. B C(O)NH: Z is COMe 24 Otamixaban T is NO;Y is —O; W is B B CH=NOH: Z is CO-Me 24 Otamixaban T is NO;Y is —O; W is B D CH=NOMe: Z is COMe 24 Otamixaban T is NO;Y is —O; W is A. A. CH-NH2: Z is CO-Me 24 Otamixaban T is NO;Y is —O; W is CN: A. C Z is CO-Me 24 Otamixaban T is NO;Y is H.; W is A. A. C(NH)NH: Z is CO-Me 24 Otamixaban T is NO;Y is —O; W is A. B C(NH)NH: Z is CH-OH 24 Otamixaban T is NO;Y is —O; W is A. B C(NH)NH2: Z is C(O)H 24 Otamixaban T is NO;Y is —O; W is B B C(NH)NH2: Z is CHOAc 24 Otamixaban T is N;Y is —O; W is A. B C(NH)NH2: Z is CO-Me 24 Otamixaban T is NO;Y is —O; W is B C C(NH)NH2: Z is CH(OMe), 24 Otamixaban T is NO;Y is —O; W is B B C(NH)NH2: Z is CH ethylene glycol acetal 61 Apixaban Y is H.;Y is =O; W is B B C(O)NH2. 61 Apixaban Y is =O;Y is H.; W is B A. C(O)NH, 61 Apixaban Y is =O;Y is =O; W is B A. CH-NH2, 61 Apixaban Y is =O;Y is =O; W is CN A. B 61 Apixaban Y is =O;Y, is =O; W is AAEAZ AA EZ CH-NOH 61 Apixaban Y is =O;Y is =O; W is B.B. E.Z. B.A. E.Z. CH-NOMe

Example 45 55 Field Field similarity A range of structures were tested for their potential as Field similarity to anion analogues of clopidogrel, an ADP-induced platelet aggrega For- similarity to active of active mula Parent Structure to parent metabolite metabolite tion inhibitor. The mechanism of action of clopidogrel 60 requires oxidative activation resulting in opening of the 151 Clopidogrel Z is C(O)H B C C 151 Clopidogrel Z is CH(OMe), B B B thiophene ring to generate the active antithrombotic reagent, 151 Clopidogrel Z is CH- C A. B which is a reversible antagonist of the ADP receptor P2Y. ethylene glycol acetal There is no known crystal structure of the P2Y receptor 65 151 Clopidogrel Z is =NOH ABEZ, CfC EZ. AfC EZ although homology modeals have been constructed. Align 151 Clopidogrel Z is =NOMe A/C E/Z A/C EZ B/C EZ ment of the structures to clopidogrel was performed, as was

US 8,946,224 B2

-continued

Field For- similarity mula Parent Structure to parent 105 Remikiren Q is S(O)2;Y is —O;Y is —O; G is D H(OH); G is ethylene glycol acetal

Example 47 10 enzymes. The three main protein targets which are implicated in the action of these drugs are dihydrofolate reductase A range of structures were tested for their potential as (DHFR), thymidylate synthase (TS) and glycinamide ribo pemetrexed analogues. Folate derivatives have a host of enzymes which process and transport them for use inbiosym nucleotide formyl transferase (GARFT). The pharmacologi thetic pathways leading to DNA/RNA production and one 15 cal activity of the proposed analogues of PMT will depend on carbon transfers. The mode of action of antifolates is compli the overall balance of interactions with these 4 targets and the cated by their molecular similarity to folate such that they are various transporters. X-ray structures of human forms of consequently able to access the same active transport mecha DHFR, TS and GARFT are available from the PDB as com nisms and binding sites of the multiple folate related plexes either of PMT itself or of close analogues

PDB Template Protein Type code Ligandfused used

DHFR Human 2W3M Folic acid Y Y DHFR Human 2W3A DHFRTS Bacterial 3K2H DHFRTS Bacterial 3KJR DHFRTS Bacterial 3NRR TS Human 1HVY Tomludex,Y Y GARFT Human 1MEO GARFT Human 1MEN GARFT Human 1ZLX GARFT Human 1ZLY 10-formyl-5,8,dideaza folate Y GARFT Human 1RBY 10-(trifluoroacetyl)- Y 5,10-dideazaacyclic-5,6,7,8- tetrahydrofolic acid Y GARFT Bacterial 1C2T 10-formyl-5,8,10 tridiazafolic acid Y FPGS Bacterial 1JBW FPGS Bacterial 1JBV FPGS Bacterial 3QCZ Y (from 1RBY) Y

FPGS is not available as the human form, but bacterial examples are available. Analysis of the different forms of FPGS by alignment of the bacterial primary amino acid 45 sequence with the human sequence shows that both the pro tein architecture and the key residues likely to contact PMT are conserved. It was thus deemed that the use of an appro priate bacterial form as a protein template for FPGS is a fair approximation. Thus, field similarity assessments were car 50 ried out for all four targets. Due to the conformational flex ibility of the analogues they were assessed as the benzyl glutamate core only. For field similarity: A is over 70% similarity; B is 65-69% similarity; C is 60-64% similarity and D is 50-59% similarity.

Field Similarity to Parent

Formula Parent Structure DHFR, TS GARFT FGPS

125 Pemetrexed Y is —O; Z is CH(OMe); Z is C B D C COH 125 Pemetrexed Y is =O; Z is CH-ethylene D B C C glycol acetal; Z is CO2H 125 Pemetrexed Y is =O; Z is CH=NOH: Z, is C B C C COH 125 Pemetrexed Y is =O; Z is CH=NOMe: Z, C B C C is CO2H US 8,946,224 B2 193 194 -continued Field Similarity to Parent Formula Paren Structure DHFR, TS GARFT FGPS 25 Pemetrexe Y is —O; Z is CH-OH: Z is C A. C B COH 25 Pemetrexed Y is —O; Z is CHOAc; Z is D B D C COH 25 Pemetrexe Y is —O; Z is COH: Z2 is C B C C CH(OMe), 25 Pemetrexed Y is =O; Z is COH: Z, is CH- C B C C ethylene glycol acetal 25 Pemetrexe Y is —O; Z is COH: Z2 is C A. C B CH-NOH 25 Pemetrexed Y is —O; Z is COH: Z, is C A. C C CH-NOMe 25 Pemetrexe Y is —O; Z is COH: Z2 is D C D C CHOAc 25 Pemetrexed Y is —O; Z is COH: Z, is C A. C B C(O)H 25 Pemetrexe Y is —O; Z is COH: Z2 is B A. C B CHOH 25 Pemetrexed Y is =O; Z is C(O)H: Z, is C A. C B COH

Example 48 25 -continued

A range of structures were tested- for their potential as Fieldsimilarity similarityField analogues of bendamustine, which is a nitrogen mustard anti- For- to parent to parent cancer agent with clinical activity against a variety of cancers mula Parent Structure (neutral) (protonated) including non-Hodgkin's lymphoma, chronic lymphocytic 30 past z is CHOH B B leukemia, multiple myeloma and some solid tumours. It is 113 Bendamustine Z is CHOAc C C presumed that as a nitrogen mustard bendamustine acts by alkylating DNA. In the absence of relevant structural infor mation a low energy extended conformation was chosen for Example 49 the butanoic acid side chain. The analysis was carried out on 35 both the protonated and non-protonated forms of the benz- A range of structures were tested for their potential aS imidazolep group. p analogues1 o f fluocinolone acetonide,s a low- to medium-porum-p 11 group tency corticosteroid used for topical treatment of skin disor For field similarity: A is over 90% similarity: B is 85-89% ders and inflammatory conditions of the eye, ear and nose. similarity and C is 80-84% similarity. 40 The mechanism of action is complex but involves initial bins ing to the cytostolic glucocorticoid receptor. The fused ring system of fluocinolone acetonide provides a Field Field rigid skeleton with a side chain providing the only site of similarity similarity conformational flexibility. The side chain conformation of For- to parent to parent 45 dexamethasone, a related corticosteroid, has been published mula Parent Structure (neutral) (protonated) in a number of crystal structures (3MNE, 3MNO, 3MNP. 113 Bendamustine Z is C(O)H A. A. 3GN8, 1M2Z, 1P93). This conformation is very similar to the 113 Bendamustine Z is CH(OMe), C B lowest energy fluocinolone acetonide side chain conforma 113 Bendamustine Z is CH-ethylene B B tion found by molecular mechanics optimisation. This low glycol acetal energy structure was used as the template for field similarity 113 Bendamustine Z is CH=NOH BiBEZ. BAEZ 50 analysis. 113 Bendamustine Z is CH=NOMe BBEZ. B.A. E.Z. For field similarity: A is over 90% similarity; B is 87-88% similarity and C is 80-86% similarity.

Field For- similarity mula Parent Structure to parent 117 Fluocinolone G is H(OH); G is H(OH); G is —O; Z AfB RAS Acetonide is CH-OH 117 Fluocinolone G is H(OAc); G is H(OH); G is —O; Z C.C RS Acetonide is CH-OH 117 Fluocinolone G is —O; G is H(OH); G is —O; Z is C Acetonide CO2H (anion) 117 Fluocinolone G is —O; G is H(OH); G is —O; Z is B Acetonide COMe 117 Fluocinolone G is —O; G is H(OH); G is —O; Z is A. Acetonide CH(OMe),