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Author(s): Cristina M. Alcantara, and Andres R. Alcantara

Article title: Biocatalyzed synthesis of antidiabetic drugs: A review

Article no: IBAB_A_1323887

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8 a b 61 Complutense University of Madrid, Madrid, Spain; Biotransformations Group, Organic & Pharmaceutical Chemistry Department, 9 Q1 Faculty of Pharmacy, Complutense University of Madrid, Madrid, Spain 62 10 63 11 64 ABSTRACT ARTICLE HISTORY 12 The biocatalyzed production of building blocks for synthesizing drugs is a very attractive Received 25 January 2017 65 13 research field, because of the sustainability introduced in a synthetic schedule when chemical Revised 10 March 2017 66 14 steps are substituted by biocatalyzed protocols. In this article, we will show how different anti- Accepted 29 March 2017 67 15 diabetic drugs, for treating diabetes mellitus Type 1 and Type 2, can be more efficiently and 68 effectively synthetized with the help of different types of biocatalysts. The huge overall drug 16 KEYWORDS 69 market for these drugs, as well as the great number of people suffering from diabetes (the Biocatalysis; diabetes; green 17 prevalence of all types of diabetes is growing), makes this topic attractive enough to focus on chemistry; insulin 70 18 more efficient synthetic protocols for preparing antidiabetic drugs. Examples covering biocata- analogues; drugs 71 lyzed synthesis of insulin analogues, sensitizers (PPAR agonists), secretagogues (GLP-1 analogues, 19 GPR119 agonists) and enzyme inhibitors (a-glucosidase inhibitors, DPP4-inhibitors, SGLT-2 inhibi- 72 20 tors and 11b-HSD1 inhibitors) will be presented. 73 21 74 22 75 23 76 24 Introduction Clearly, the prevalence of all types of diabetes is grow- 77 25 ing, particularly Type 2 DM; this can be seen from 78 Diabetes mellitus (DM) is a disorder of metabolic 26 2014 data (422 million (World Health Organization 79 homeostasis, showing hyperglycaemia and altered lipid 27 2016; Zhou et al. 2016)) or 2015 (415 million, 1 out of 80 metabolism caused by dysfunction of pancreatic islets, 28 11 adults (International Diabetes Federation 2015)), 81 which do not produce enough insulin (a hormone that 29 while the number of people affected by DM is esti- 82 regulates blood sugar or glucose), or rather caused 30 mated to increase up to 439 million people in 2030, 83 when the body cannot effectively use the insulin it 31 reaching 592 million people by 2035 (World Health 84 produces (World Health Organization 2016). According 32 Organization 2016) and 642 in 2040, which means 1 85 to this, there are three main types of DM (American 33 out of 10 adults (International Diabetes Federation 86 34 Diabetes Association 2014; Ramachandran et al. 2017); 2015; Ali et al. 2017). 87 ’ 35 thus, Type 1 DM results from the inherent pancreas s Obviously, DM is a major cause of morbidity and 88 36 failure to produce enough insulin, being this type for- mortality in many countries, although 80% of people 89 “ ” 37 merly known as insulin-dependent diabetes mellitus with diabetes live in low- and middle-income countries 90 “ ” 38 (IDDM) or juvenile diabetes . Conversely, Type 2 DM is (International Diabetes Federation 2015; World Health 91 39 caused by insulin resistance, and it was previously Organization 2016); globally, it caused around 5.0 mil- 92 93 40 referred to as “non-insulin-dependent diabetes lion deaths in 2013 (IDF Diabetes Atlas Group 2015) 41 94 mellitus” (NIDDM) or “adult-onset diabetes”. Finally, and a similar number in 2015 (International Diabetes 42 95 gestational diabetes is the third main form and occurs Federation 2015). Considering global costs of DM, it 43 96 when pregnant women without a previous history of has been reported that at least $612 billion were spent 44 97 diabetes develop high blood sugar levels. globally on diabetes in 2014, representing 11% of all 45 98 DM is one of the most common chronic conditions global health expenditures. This represents an increase 46 99 in nearly all countries. In 2014, the International of 12% compared to the data published in 2013 ($548 47 100 Diabetes Federation (IDF) assessed that 8.2% of adults billion), due to increases in the total number of people 48 101 between 20 and 79 (around 387 million people) were with diabetes (Fernandes et al. 2016). In 2015, 12% 49 102 living with diabetes, an increase compared to 382 mil- of global health expenditure (around $673 billion) 50 103 lion people in 2013 (Fernandes et al. 2016). was dedicated to diabetes treatment and related 51 104

52 CONTACT Andres R. Alcantara [email protected] Biotransformations Group, Organic & Pharmaceutical Chemistry Department, Faculty of Pharmacy, 105 53 Q2 Complutense University of Madrid, Campus de Moncloa, E-28040 Madrid, Spain 106 ß 2017 Informa UK Limited, trading as Taylor & Francis Group 2 C. M. ALCANTARA AND A. R. ALCANTARA

107 complications, and the majority of countries spent To focus this article, we will comment some exam- 160 108 between 5% and 20% of their total health expenditure ples illustrating the use of chemoenzymatic protocols 161 109 on DM (International Diabetes Federation 2015; for the sustainable synthesis of antidiabetic drugs. For 162 110 Williams 2016). this purpose, first we will mention some cases in 163 111 Therefore, the world market for diabetes drugs is which biocatalysis has been useful for the preparation 164 112 really huge, $35.6 billion in 2012 (Visiongain 2013), of insulin analogues (treatment of DM Type 1 and 2), 165 113 growing up to 51.1 billion in 2015 (Global Market and subsequently we will focus in the chemoenzy- 166 114 Insight, Inc. 2016), and it is estimated to reach $55.3 matic synthesis of drugs specially designed for the 167 115 billion in 2017 (Visiongain 2013) and more than treatment of Type 2 DM. 168 116 double, $116.1 billion, by 2023 (Global Market Insight, 169 Inc. 2016). While Type 1 DM can only be treated with 117 Insulin and insulin analogues 170 118 insulin or synthetic insulin analogues (Freeland and 171 119 Farber 2016; Zaykov et al. 2016), for the treatment of Semisynthetic human insulin was commercially devel- 172 120 Type 2 DM either insulin or other types of mostly oral oped in the 1970s by Novo Nordisk A/S, starting from 173 121 drugs, either as a single API or a combination of them, porcine insulin, by substituting the B-30 alanine resi- 174 122 can be used (Freeland and Farber 2015; Kokil et al. due of porcine insulin with a threonine residue 175 123 2015; Gaitonde et al. 2016) (Markussen 1981; Andresen and Balschmidt 1982), as 176 124 Taking into consideration all of these data, it is cer- shown in Figure 1. 177 125 tainly understandable that the development of effi- For these biotransformations, five steps were 178 126 cient and sustainable methodologies for synthesizing required (Barfoed 1987): insulin was first extracted 179 127 antidiabetic drugs is highly desirable. For this purpose, from frozen porcine pancreas glands. In a second step, 180 128 the use of biocatalyzed protocols is increasingly of the purified porcine insulin was converted into 181 129 becoming recognized as a very important part inside human insulin in a medium that contains only a small 182 130 Green Chemistry (Malhotra et al. 2015; Sheldon 2016), amount of water and trypsin and a large quantity of 183 131 because those synthetic routes mediated by enzymes organic solvent and threonine ester. Subsequently, 184

132 or cells are generally conducted under mild reaction trypsin hydrolyzed insulin at LysB29-AlaB30, while at the 185 133 conditions, at ambient temperature and can use water same time catalyzed the reverse reaction in which the 186 134 as reaction medium in many cases (Hoyos et al. 2013); threonine ester displaced alanine from position B30 in 187 135 moreover, their high selectivity avoids the need of the insulin molecule. This transpeptidation of porcine 188 136 functional group activation and protection/deprotec- insulin to human insulin was optimized to 97% yield 189 137 tion steps usually required in traditional organic using soluble trypsin (Morihara et al. 1979). This was 190 138 synthesis. Thus, biocatalysis provides processes which followed by chromatographic purification to reduce 191 139 are shorter, produce less waste (generally measured measurable levels of proinsulin and remove the other 192 140 using E-factor value, ratio between the kilograms of reagents, pharmaceutical formulation and distribution 193 141 waste to the kilograms of desired product (Sheldon into the market. Finally, the product was formulated 194 142 2017)) and reduce manufacturing costs and environ- and then filled under sterile conditions, packaged, and 195 143 mental impact. These features are even more signifi- distributed. Transpeptidation was also catalyzed by 196 144 cant in drug synthesis, because it is well known that immobilized trypsin, although the yield was lower 197 145 Pharma Industry produces a great amount of waste, so (80%) (Ueno and Morihara 1989). Another protease, 198 146 that implementing biocatalyzed protocols is increas- from Achromobacter lyticus, was also found to be com- 199 147 200 ingly being employed (Hoyos et al. 2014; Patel 2016a, pletely specific in the hydrolysis of LysB29-AlaB30 and 148 2016b, 2016c). the subsequent condensation con H-Thr-OBut 201 149 202 150 203 151 204 152 205 153 206 154 207 155 208 156 209 157 210 158 211 159 Figure 1. Semisynthesis of human insulin from porcine insulin by trypsin-catalyzed transpeptidation. 212 BIOCATALYSIS AND BIOTRANSFORMATION 3

213 (Morihara et al. 1980; Morihara and Ueno 1991). Also, caboxypeptidase to delete two Arg residues from 266 214 the use of carboxypeptidase A for the same procedure BThr30, yield the human insulin (Thim et al. 1986; 267 215 was described (Andresen et al. 1983). Ladisch and Kohlmann 1992). 268 216 Human insulin was the first animal protein to be Since those innovative methods, many other proto- 269 217 made in bacteria in a sequence identical to that of the cols based on genetic engineering have been devel- 270 218 human pancreatic peptide, as early as 1978 by oped, and nowadays recombinant human insulin is 271 219 Genentech (lab scale) and Eli Lilly and Co. (scale-up) mainly produced either in E. coli or S. cerevisiae, 272 220 (Johnson 1983), working together to achieve the although several other alternate yeast strains have 273 221 expression of recombinant human insulin in been explored for insulin production, as well as mam- 274 222 Escherichia coli K-12 using genes for the insulin A and malian cells, transgenic animals or plant expression 275 223 B chains; thus, each insulin chain was produced as a systems have been also employed as a host for large- 276 224 b-galactosidase fusion protein in separate fermenta- scale production of recombinant insulin (Walsh 2005; 277 225 tions using E. coli cells transformed with plasmids con- Baeshen et al. 2014). 278 226 taining either the A or B insulin peptide sequence. The On the other hand, by using recombinant DNA 279 227 intracellular products, once removed from the inclu- technology different insulin analogues have been syn- 280 228 sion bodies, were chemically cleavage by CNBr at the thesized. This term refers to an altered form of insulin, 281 b 229 Met residue between the -gal and the A or B chains, different from any occurring in nature, still available to 282 230 purified, suffered an oxidative sulfitolysis and chem- the human body for performing the same action as 283 231 ically linked to afford crude insulin. A final purification human insulin in terms of glycaemic control, but dis- 284 232 process led to the first production of recombinant 285 playing improved ADME (absorption, distribution, 233 human insulin, approved by drug regulatory agencies 286 metabolism, and excretion) characteristics (Zaykov 234 in 1982 (Ladisch and Kohlmann 1992). 287 et al. 2016). Officially, the U.S. Food and Drug 235 After this pioneering work, some other strategies 288 Administration (FDA) refers to these as “insulin recep- 236 were developed using recombinant microorganisms 289 tor ligands”, although they are more commonly named 237 that produce intact proinsulin instead of the A or B 290 as insulin analogues, which can be classified into two 238 chains separately. For instance, Novo used 291 main classes: 239 Saccharomyces cerevisiae to secrete insulin as a single- 292 240 chain insulin precursor, in which amino acid 30 of the 293 a. those that are more readily absorbed from the 241 B chain of insulin was connected to amino acid 1 of 294 injection site and therefore act faster than natural 242 the A chain by a peptide (Chain C), as shown in 295 insulin injected subcutaneously, intended to sup- 243 Figure 2. Two enzymatic cleavages, the first one cata- 296 244 lyzed by trypsin leading to the removal of most of ply the bolus level of insulin needed at mealtime 297 245 the C chain, and the second one catalyzed by (prandial insulin) 298 246 299 247 300 248 301 249 302 250 303 251 304 252 305 253 306 254 307 255 308 256 309 257 310 258 311 259 312 260 313 261 314 262 315 263 316 264 317 265 Figure 2. Preparation of human insulin from a precursor. 318 4 C. M. ALCANTARA AND A. R. ALCANTARA

319 b. those that are released slowly over a period of (Howey et al. 1994; Torlone et al. 1994), therefore 372 320 between 8 and 24 h, intended to supply the basal allowing larger amounts of active monomeric insulin 373 321 level of insulin during the day and particularly at to be available for postprandial (after meal) injections 374 322 night time (basal insulin). (Anderson et al. 1997). The second entry in this class, 375 323 insulin aspart (Novolog, Novo Nordisk), was first mar- 376 324 Among fast-action analogues, Eli Lilly and Co. devel- keted in 2000 and utilizes an Asp at position B28 377 325 oped and marketed in 1996 Humalog, the first rapid- (Brange et al. 1988; Home et al. 1998, 2000). The most 378 326 acting insulin analogue (insulin lispro rDNA). This recent rapid-acting analogue, insulin glulisine (Apidra, 379 327 analogue was engineered through recombinant DNA Sanofi), was marketed in 2006 and is based on 380 328 technology, so that the penultimate lysine and proline replacements of LysB29 with Glu and of AsnB3 with Lys 381 329 residues on the C-terminal end of the B-chain (Becker et al. 2005; Dreyer et al. 2005; Becker and Frick 382 330 (ProB28LysB29) were reversed (Figure 3). This modifica- 2008). 383 331 tion did not alter the insulin receptor binding, but Considering those analogues intended to control 384 332 blocked the formation of insulin dimers and hexamers basal insulin levels, insulin glargine (Lantus, Sanofi), 385 333 386 334 387 335 388 336 389 337 390 338 391 339 392 340 393 341 394 342 395 343 396 344 397 345 398 346 399 347 400 348 401 349 402 350 403 351 404 352 405 353 406 354 407 355 408 356 409 357 410 358 411 359 412 360 413 361 414 362 415 363 416 364 417 365 418 366 419 367 420 368 421 369 422 370 423 371 Figure 3. Some insulin analogues. 424 BIOCATALYSIS AND BIOTRANSFORMATION 5

425 launched in 2001, uses a shift in isoelectric point insulin (Freeland and Farber 2016; Pettus et al. 2016; 478 426 (achieved through two additional Arg residues at posi- Zaykov et al. 2016; Sherr et al. 2017) and new and 479 427 tions B31 and B32) in order to dramatically lower solu- faster formulations (Sherr et al. 2017), because there is 480 428 bility at physiological pH, rendering the insulin far less little to no alternatives to brand-named analogue insu- 481 429 soluble at the injection site. This results in an lin and non-analogue human alternatives for treating 482 430 extended time–action profile as the analogue slowly both types of DM in low- and middle-income countries 483 431 re-solubilizes (Rosenstock et al. 2001). Additionally, a (Kaplan and Beall 2017). 484 432 Gly residue is introduced at position A21 to maintain 485 433 chemical stability in the aqueous, acidic formulation. Other antidiabetic drugs 486 434 Another different tactic consists in attaching a long- 487 435 chain fatty acid with the purpose of slowing adsorp- Inside this category we will include: 488 436 tion and facilitating extended plasma circulation 489 437 through non-covalent albumin binding. This strategy a. drugs that increase the sensitivity of target organs 490 438 was reported by Eli Lilly with insulin lipidated at to insulin, called sensitizers 491 439 LysB29 with palmitic acid (W99-S-32) (Myers et al. b. agents that increase the amount of insulin 492 440 1995) and by Novo Nordisk with LysB29-myristyl secreted by the pancreas, known as 493 441 desB30-insulin, launched in 2006 under the name of secretagogues 494 442 insulin detemir (Levemir) (Havelund et al. 2004; c. agents that decrease the rate at which glucose is 495 443 Hermansen et al. 2006). Very recently, two new basal absorbed from the gastrointestinal tract. 496 444 insulin analogues completed Phase III trials (Pettus d. enzyme inhibitors 497 445 et al. 2016): insulin degludec (Tresiba, Novo Nordisk), a 498 446 des-B30 human insulin that is uniquely fatty-acylated We will show now different examples of biocata- 499 447 at LysB29 with hexadecanedioic acid via gamma-L-glu- lyzed synthesis of some of these compounds. The use 500 448 tamyl spacer (Wang et al. 2012; Gough et al. 2013), of biocatalysts is especially adequate for the stereo- 501 449 which have been recently approved by FDA in the selective synthesis of those drugs possessing at least 502 450 USA, and insulin peglispro (Ly2605541), derived by one stereogenic centre. 503 451 covalent attachment of a linear 20 kD polyethylene 504 452 glycol (PEG) polymer to the LysB28 side-chain in insu- Sensitizers 505 453 lin lispro (Henry et al. 2014). 506 Drugs belonging to this category are biguanides (such 454 Generally speaking, the use of synthetic biology for 507 as metformin, 1), thiazolidinediones (TZDs, also named 455 creating cell factories is the methodology used at 508 glitazones, 2), and glitazares 3), shown in Figure 4. The 456 industrial scale in the preparation of these insulin ana- 509 first class is represented by metformin, the first-line 457 logues (Baeshen, Baeshen et al. 2014; Sanchez-Garcia 510 medication for the treatment of Type 2 DM (Maruthur 458 et al. 2016). For sure, the improvement of metabolic 511 et al. 2016; Wise 2016), which is synthetized only by 459 pathways for increasing their production can be classi- 512 classical chemical methods, so that we will mention 460 fied as sequential (or cascade) biocatalytical processes, 513 the other two classes, glitazones and glitazars. 461 and not many examples of protease modifications of 514 462 precursors leading to insulin analogues, which can be 515 Peroxisome proliferator-activated receptors (PPAR) 463 sensu stricto considered biotransformations, can be 516 agonists 464 found in literature (Bogsnes et al. 2003; Habermann 517 465 518 and Zocher 2008). Anyway, ample research is being The PPARs are ligand-dependent transcription factors. 466 519 carried out in the development of new analogues of The three mammalian PPARs (a, b/d and c) 467 520 468 521 469 522 470 523 471 524 472 525 473 526 474 527 475 528 476 529 477 Figure 4. General structure of insulin sensitizers. 530 6 C. M. ALCANTARA AND A. R. ALCANTARA

531 (Nevin et al. 2011; Wright et al. 2014) are crucial regu- Jamali et al. 2008), as also shown in Figure 5. 584 532 lators of fatty acid and lipoprotein metabolism, glu- Consequently, in animal models, the individual enan- 585 533 cose homeostasis, cellular proliferation/differentiation tiomers and racemates of glitazones appear to show 586 534 and the immune response. Therefore, PPARs are key equivalent activity as antidiabetic agents, so that most 587 535 targets in the treatment of metabolic disorders such as synthetic methodologies are conducted following con- 588 536 insulin resistance and Type 2 DM; furthermore, PPARs ventional chemical steps (Ortiz and Sansinenea 2011). 589 537 are also involved in chronic inflammatory diseases However, Parks et al. (1998), through the in vitro 590 538 such as atherosclerosis, arthritis, chronic pulmonary analysis of rosiglitazone enantiomers in a PPARc- 591 539 inflammation, pancreatitis, inflammatory bowel dis- binding assay, suggested that the (S)-isomer is the 592 540 ease, psoriasis, blood pressure regulation, neuroinflam- main responsible for the antidiabetic activity, and a 593 541 mation, nerve-cell protection, inflammatory pain similar behaviour of the (S)-eutomer was described for 594 542 reduction, and hypothalamic control of metabolism rivoglitazone, another glitazone which failed to reach 595 543 (Menendez-Gutierrez et al. 2012). However, PPAR-c, the drug market (Izumi et al. 2013). On the other 596 544 which is expressed in adipose tissue, lower intestine, hand, pioglitazone is currently undergoing clinical tri- 597 545 and cells involved in immunity, is the most extensively als for treatment of Alzheimer’s disease (AD): when 598 546 investigated PPAR, while PPAR-d, which regulates mice were dosed with racaemic pioglitazone, the con- 599 547 several metabolic processes, has also been investi- centration of (R)-(þ)-pioglitazone was 46.6% higher 600 548 gated for the development of new drugs for treating than that of (S)-()-pioglitazone in brain tissue and 601 549 DM (Wright et al. 2014). 67.7% lower than that of (S)-pioglitazone in plasma, 602 550 There are three marketed TZDs, acting as PPAR-c and dosing mice with pure (R)-pioglitazone led to a 603 551 agonist: pioglitazone (2a, ActosTM or GlustinTM, Takeda 76% increase in brain exposure levels compared to 604 552 Pharmas USA and Eli Lilly), rosiglitazone (2b, those from an equivalent dose of racaemic 605 553 AvandiaTM, GlaxoSmithKline), and lobeglitazone (2c, pioglitazone (Chang et al. 2015). Furthermore, pure 606 554 DuvieTM, Chong Kun Dang), whose chemical structures (R)-pioglitazone was also shown to have comparable 607 555 are shown in Figure 5. Common for the chemical amyloid-lowering capabilities to the racaemic pioglita- 608 556 structure of TZDs compounds is the chiral centre at zone in an in vitro AD model, so that dosing with (R)- 609 557 610 the C-5 of the ring, prone to racemization at physio- pioglitazone instead of the racaemic mixture may 558 611 logical pH (Welch et al. 2003; Rippley et al. 2007; result in higher levels of brain exposure to 559 612 560 613 561 614 562 615 563 616 564 617 565 618 566 619 567 620 568 621 569 622 570 623 571 624 572 625 573 626 574 627 575 628 576 629 577 630 578 631 579 632 580 633 581 634 582 635 583 Figure 5. Chemical structure of glitazones. 636 BIOCATALYSIS AND BIOTRANSFORMATION 7

637 pioglitazone, thus potentially improving the develop- possess a common chemical structure (2-methyl-2-ary- 690 638 ment of pioglitazone treatment of AD. loxypropionic acids or esters), not displaying any chiral 691 639 Thus, it is possible to find in literature some exam- centre. In an attempt to obtain an enantiopure PPAR-a 692 640 ples of the resolution of both enantiomer of agonist, Astra Zeneca synthesized AZD 4619 (Figure 7, 693 641 glitazones, either through their derivatization to dia- (S)-6), an a agonist, by means of an enzymatic 694 642 stereoisomers (Sohda et al. 1984; Gahafu et al. 2010; dynamic kinetic resolution (DKR) of the corresponding 695 643 van Niel et al. 2011a, 2011b) or by preparative chiral racaemic thioester, using an organic base to promote 696 644 HPLC (Calixto and Bonato 2013). On the other hand, the racemization (Brown et al. 2006), as shown 697 645 the first example of stereoselective biocatalytic synthe- in Figure 7. 698 646 sis was described by researchers at SmithKline The thioester rac-5 was resolved with Pseudomonas 699 647 Beecham by means of the bioreduction of the precur- cepacia lipase in the presence of a tert-amine base, tri- 700 648 sor benzylidine compound 4 (Figure 6), catalyzed by octylamine. The desired acid (S)-6 is stable, and 701 649 whole cells from red yeast Rhodotorula rubra CBS 6469 residual (R)-thioester was racemized by deprotonation 702 650 (Cantello et al. 1994). and reprotonation catalyzed by the organic base, 703 651 These authors observed that the bioreduction pro- which cannot make a similar undesired racemization 704 652 ceeded at basic pH values with a high degree of step of (S)-6, because the a protons of the carboxylate 705 653 stereoselectivity, but that the product was undergoing product are not acidic enough to be deprotonated by 706 654 racemization, the rate of racemization being slower tert-amine bases. Although this process was scaled to 707 655 than the rate of product formation under these condi- grams, AZD 4619 was discontinued because of hepato- 708 656 tions. Thus, the biotransformation was carried out toxicity problems detected in Phase I (Thulin et al. 709 657 under acidic pH conditions. Over a 4 h reaction at pH 2008). Similarly, the resolution of the racaemic alpha- 710 658 3.75, the product was found to be of >98% enantio- chloro thioester intermediate 7 has been described 711 659 meric purity. These authors also described the use of using the same strategy, shown in Figure 8 (Dow et al. 712 660 713 alginate-entrapped immobilized cells for performing 2012). Remarkably, the use of a protease instead of a 661 714 the bioreduction (Cantello et al. 1994), which was fur- lipase allowed the synthesis of the antipode (R)-8, 662 715 ther scaled-up by some other scientists of the same although with a lower ee (90% with Savinase versus 663 716 company (Heath et al. 1997). 98% with lipase). 664 717 On the other hand, PPAR-a agonists serve as cellu- Glitazars (Figure 9) are dual PPAR a/c agonists 665 718 lar receptor for fibrates, a class of drugs used in the that improve the lipid profile and exert an antidia- 666 719 treatment of dyslipidaemia, and also used for the betic action, similar to a combination of a fibrate 667 720 treatment of vascular complications associated with and a thiazolidinedione, so that they are 668 721 Type 2 DM (Verges 2004; Steiner 2007). These fibrates considered as “two drugs in one” (Wilding 2012). 669 722 670 723 671 724 672 725 673 726 674 727 675 728 676 729 677 730 Figure 6. Biocatalyzed synthesis of (R)-(þ)-rosiglitazone. 678 731 679 732 680 733 681 734 682 735 683 736 684 737 685 738 686 739 687 740 688 741 689 Figure 7. DKR process to synthesize AZD 4619. 742 8 C. M. ALCANTARA AND A. R. ALCANTARA

743 796 744 797 745 798 746 799 747 800 748 801 749 802 750 803 751 804 752 805 753 806 754 807 755 808 756 809 Figure 8. DKR process to synthesize a precursor of AZD 4619. 757 810 758 811 759 812 760 813 761 814 762 815 763 816 764 817 765 818 766 819 767 820 768 821 769 822 770 823 771 824 772 825 773 826 774 827 775 828 776 829 777 830 778 831 779 832 780 833 781 834 782 835 783 836 784 837 785 838 786 839 787 840 788 841 789 842 790 843 791 844 792 845 793 846 794 847 795 Figure 9. Some glitazars. 848 BIOCATALYSIS AND BIOTRANSFORMATION 9

849 902 850 903 851 904 852 905 853 906 854 907 855 908 856 909 857 910 858 911 859 912 860 913 Figure 10. Chemical synthesis of (S)-2-ethoxy-3-(4-hydroxyphenyl)propanoic acid ((S)-9) starting form L-tyrosine. 861 914 862 915 863 916 864 917 865 918 866 919 867 920 868 921 869 922 870 923 871 924 872 925 873 926 874 927 875 928 876 929 877 930 878 931 879 Figure 11. Enzymatic kinetic resolution of rac-17 by an enantioselective hydrolysis. 932 880 933 881 934 882 Ragaglitazar (Figures 9 and 10) was discontinued by accepted for launch in India by the Drug Controller 935 883 Novo Nordisk and Dr. Reddy’s Laboratories because General of India (DCGI) for the treatment of diabetic 936 884 of its adverse effects after detecting urinary bladder dyslipidaemia or hypertriglyceridaemia in patients 937 885 938 tumour in mice. After different clinical trials, in May with Type II DM not controlled by statins alone 886 939 2006 the two glitazars most advanced in develop- (Agrawal 2014; Sharma et al. 2015; Dwivedi et al. 887 940 ment at that time, muraglitazar (16, PargluvaTM, 2015b). 888 941 developed by Brystol Myers Squibb) and tesaglitazar As can be seen, the structure of (S)-2-ethoxy-3-(4- 889 942 (11, GalidaTM, Astra Zeneca) were discontinued. In hydroxyphenyl)propanoic acid ((S)-9, shown in bold 890 943 fact, 16 was associated with an increased incidence font in Figure 9) is common feature for many glitazars; 891 944 of heart failure, while ragaglitazar 10 was associated its synthesis has been described starting from L-tyro- 892 945 with decreased glomerular filtration (Conlon 2006). sine (Dwivedi et al. 2014), in a rather dull 4-step meth- 893 946 894 Some other newer glitazars are aleglitazar 13, from odology depicted in Figure 10. 947 895 Hoffmann-La Roche (Wilding 2012), which has been Therefore, establishing an alternative greener meth- 948 896 discontinued in July 2013 after Phase III trials, and odology using biocatalyzed protocols would be highly 949 897 cevoglitazar 15 (Chen et al. 2010; LBM-642, from desirable. In fact, in the synthesis of ragaglitazar 950 898 Novartis AG), which failed to pass Phase I. Very (Figure 11), the key intermediate (S)-9 was obtained 951 899 recently, in June 2013, the Indian company Zydus through a very mild enantioselective hydrolysis of the 952 TM 900 Cadila has presented Lipaglyn (Saroglitazar 14), racaemic ethyl ester 17, catalyzed by an esterase from 953 901 the first glitazar to be approved in the world, Aspergillus oryzae; this process was run on a 44 kg pilot 954 10 C. M. ALCANTARA AND A. R. ALCANTARA

955 1008 956 1009 957 1010 958 1011 959 1012 960 1013 961 1014 962 1015 963 1016 964 1017 965 1018 966 1019 967 1020 968 1021 969 1022 970 1023 971 1024 972 1025 973 1026 974 1027 975 1028 976 Figure 12. Enzymatic kinetic resolution of rac-18a and 18b by an enantioselective hydrolysis. 1029 977 1030 978 1031 979 1032 980 1033 981 1034 982 1035 983 1036 984 1037 985 1038 986 1039 987 1040 988 1041 989 1042 990 1043 991 1044 992 1045 993 1046 994 1047 995 1048 996 1049 997 1050 S S 998 Figure 13. Preparation of enantiopure ethyl-( )-2-ethoxy-3-(p-methoxyphenyl)propanoate (EEHP) ( )-19a by bioreduction. 1051 999 1052 1000 scale, to produce enantiopure ragaglitazar (S)-10 in 43- a-chymotrypsin-catalyzed hydrolysis of racaemic 18a 1053 1001 48% yields with ee values between 98.8% and 99.6% or 18b, and subsequent work up to finally obtain 1054 1002 (Deussen et al. 2003). (S)-20a or (S)-20b with moderate overall yields 1055 1003 On the other hand, Brenna and coworkers have (Brenna et al. 2009a)(Figure 12). 1056 1004 reported two different biocatalytic approaches Due to the lower yield obtained, this research 1057 1005 to obtain another enantiopure precursor for the group decided to change to a reductase-catalyzed 1058 1006 preparation of glitazars. In a first strategy, the prep- strategy, depicted in Figure 13(a), finally leading to the 1059 1007 aration of (S)-20a or (S)-20b was initiated by an corresponding compounds (S)-19 (after chemical 1060 BIOCATALYSIS AND BIOTRANSFORMATION 11

1061 1114 1062 1115 1063 1116 1064 1117 1065 1118 1066 1119 1067 1120 1068 1121 1069 1122 1070 1123 1071 1124 1072 1125 1073 1126 1074 1127 1075 1128 1076 1129 1077 1130 1078 1131 1079 1132 1080 1133 1081 1134 1082 1135 1083 1136 1084 1137 Figure 14. Preparation of enantiopure methyl (S)-2-bromobutyrate (S)-26 and (S)-2-bromobutyric acid (S)-24 by bioreduction. 1085 1138 1086 1139 oxidation of (S)-22) in good yield of 78% and an excel- ester 25. By using baker’s yeast, it was possible to pre- 1087 1140 lent ee of 99%, using baker’s yeast and an in situ sub- pare the corresponding (S)-2-bromobutyric acid (S)-24 1088 1141 strate feeding product removal (SFPR) technique (78% conversion, 97% ee) starting from the same sub- 1089 1142 (Brenna et al. 2009b). Nevertheless, it suffers from (i) strate. These enantiopure molecules can be useful as 1090 1143 an extremely low productivity (0.39 g L 1 d 1), (ii) a chiral building blocks in the preparation of compounds 1091 1144 nonquantitative conversion, (iii) a complex purification such as 27 (Liu et al. 2009)or28 (Acton et al. 2009), 1092 1145 process based on the chemoselective oxidation of the 3-acyl-1-(phenyl or benzyl)indolecarboxylic acids which 1093 1146 byproduct, the undesired allylic alcohol, and (iv) a use- were also tested as PPAR-c modulators (Pirat et al. 1094 1147 less and counterproductive reduction of the carbonyl 2012). 1095 1148 group (by alcohol dehydrogenases present in baker’s 1096 1149 yeast) since the final target is an ester. Therefore, 1097 Secretagogues 1150 1098 Brenna and coworkers improved the methodology 1151 These are drugs that increase insulin output from the 1099 (Figure 13(b)) by using a genetically engineered ene- 1152 pancreas, and they can be divided in two main types: 1100 reductase (Old Yellow enzyme, OYE) from S. cerevisiae 1153 sulfonylureas and non-sulfonylureas secretagogues. As 1101 expressed in E. coli, and glucose dehydrogenase for 1154 1102 cofactor regeneration; thus, it was possible to improve long as most of sulfonylureas do not possess any 1155 1 1 1103 the productivity (up to 55.6 g L d , 74% yield, 98% chiral centre in their structures, their syntheses are car- 1156 1104 ee), at gram scale (Bechtold et al. 2012) in the prepar- ried out following classical chemical methodologies. 1157 1105 ation of the desired ethyl-(S)-2-ethoxy-3-(p-methoxy- There are different types of non-sulfonylureas secre- 1158 phenyl)propanoate (EEHP) (S)-19a, ultimately obtained tagogues. For instance, meglitinides, which bind to an 1106 þ 1159 1107 through chemical oxidation of intermediate aldehyde ATP-dependent K (KATP) channel on the cell mem- 1160 1108 (S)-23. brane of pancreatic beta cells in a similar manner to 1161 1109 Using this same cloned OYE, a similar strategy sulfonylureas (Proks et al. 2002), although with weaker 1162 1110 (Figure 14) has been developed by the same group binding affinity and faster dissociation (Dornhorst 1163 1111 (Brenna et al. 2012) for the preparation of enantiopure 2001). The only described example in which a biocata- 1164 1112 methyl 2-bromobutyrate (S)-26 (100% conversion, 97% lyzed protocol is employed in the synthesis of this 1165 1113 ee) starting from the corresponding Z-a,b-insaturated type of compounds is the preparation of (S)-(þ)-3- 1166 12 C. M. ALCANTARA AND A. R. ALCANTARA

1167 1220 1168 1221 1169 1222 1170 1223 1171 1224 1172 1225 1173 1226 1174 1227 Figure 15. Preparation of enantiopure (S)-(þ)-3-methyl-1-(2-(1-piperidinyl)phenyl)butylamine (S)-29. 1175 1228 1176 1229 1177 methyl-1-(2-(1-piperidinyl)phenyl)butylamine (S)-29 meglitinides, due to their lower risk of causing hypo- 1230 1178 through enzymatic catalysis, as described in Figure 15 glycaemia (Garber 2012). 1231 1179 (Zhao et al. 2010). Like so, (S)-2-amino-3-(6-o-tolylpyridin-3-yl)propa- 1232 1180 This reaction was carried out in AcOEt, acting as noic acid (S)-32, Figure 16, is a key intermediate 1233 1181 solvent and acyl donor, and the enzyme used was needed for synthesis of GLP-1 mimics or GLP-1 recep- 1234 1182 Novozyme 435, a commercial preparation of immobi- tor modulators: its synthesis has been described using 1235 1183 lized lipase from Candida antarctica. The yields were three different enzymatic procedures (Chen et al. 1236 1184 not very high, although the use of an immobilized bio- 2011); in the first one, depicted in Figure 16,(S)-32 1237 1185 catalyst allows its recycling to afford pure (S)-29 and was prepared (gram scale) in 68% solution yield and 1238 1186 the subsequent synthesis of repaglinide 31 (PrandinTM, 54% isolated yield (100% ee), starting from racaemic 1239 1187 Novo Nordisk) (Scott 2012). 32 using a recombinant (R)-amino acid oxidase from 1240 1188 Trigonopsis variabilis, cloned and overexpressed in E. 1241 1189 GLP-1 analogues coli and then immobilized on Celite, and an (S)-amino 1242 1190 1243 The main role of pancreatic b cells, to synthesize and acid dehydrogenase from Sporosarcina ureae. The 1191 1244 secrete insulin, is somewhat modulated by a group of cofactor NADH required for the reductive amination 1192 1245 heterotrimeric G proteins, which are the immediate reaction was regenerated using formate and formate 1193 1246 downstream targets of diverse G protein-coupled dehydrogenase (FDH). 1194 1247 receptors (GPCRs). Hence, different GPCRs expressed In a second strategy (Figure 17), (S)-32 could be 1195 1248 by pancreatic b cells regulate insulin secretion, and prepared in 73% isolated yield with 99.9% ee from 1196 1249 therefore these compounds, acting as insulin secreta- racaemic amino acid using the same initial enzyme, 1197 1250 gogues, are potential therapeutic targets for treating (R)-amino acid oxidase from T. variabilis expressed in 1198 1251 Type 2 DM (Ahren 2009; Lovshin and Drucker 2009). E. coli, but now in combination with an (S)-amino- 1199 1252 One of them is the receptor for the glucagon-like transferase (purified from a soil organism identified 1200 1253 peptide-1 (GLP-1R), which binds to and is activated by as Burkholderia sp., also cloned and expressed in E. 1201 1254 glucagon-like peptide-1 (GLP-1), a 30 amino acid resi- coli), and using (S)-aspartate as amino donor. This 1202 1255 procedure had the advantage that both enzymes 1203 due peptide, originated from preproglucagon, synthe- 1256 could be added at the start of reaction in a one-pot 1204 sized in the L-cells in the distal ileum, in the pancreas 1257 system, and several batches containing 9.11 g (15 g 1205 and in the brain. GLP-1 is member of the incretin hor- 1258 of the monosulfate monohydrate) of rac-32 were run 1206 mone family, a term that refers to the observation that 1259 in a 2-L reactor at 30 C 22 h, to produce 85% yield 1207 orally administered glucose results in a larger increase 1260 (73% after crystallization, ee 99.9%). Hence, the reac- 1208 in plasma insulin levels and insulin-dependent 1261 tion was scaled up with 607 g (1 kg of the monosul- 1209 decrease in blood glucose concentration when com- 1262 1210 pared to the same amount of glucose given intraven- fate monohydrate) of rac-32 to give a 66% isolated 1263 1211 ously (Rondas et al. 2013). Then, injectable GLP-1 yield of (S)-32 as the monosulfate monohydrate 1264 1212 mimetics (synthetic polypeptides such as exenatide (ee 99.9%). 1265 TM TM 1213 (Amylin Pharmaceuticals, Byetta /Bydureon ), lira- Finally, a cyclic deracemization of rac-32 by 1266 TM TM 1214 glutide (Novo Nordisk, Victoza , Saxenda ), lixisena- (R)-selective oxidation was developed using Celite- 1267 TM TM 1215 tide (Sanofi, Lyxumia ), albiglutide (GSK, Tanzeum ), immobilized (R)-amino acid oxidase, in combination 1268 TM 1216 dulaglutide (Eli Lilly, Trulicity ), taspoglutide (phase III with chemical imine reduction using the borane- 1269 1217 halted Sept 2010) or semaglutide are used in the treat- ammonia complex (Figure 18). Before the imine 34 1270 1218 ment Type 2 DM, displaying an advantage over older (bounded to the enzyme as initial product of the oxi- 1271 1219 insulin secretagogues, such as sulfonylureas or dase reaction) gets hydrolyzed to the keto acid 31, the 1272 BIOCATALYSIS AND BIOTRANSFORMATION 13

1273 1326 1274 1327 1275 1328 1276 1329 1277 1330 1278 1331 1279 1332 1280 1333 1281 1334 1282 1335 1283 1336 1284 1337 1285 1338 1286 1339 1287 1340 1288 1341 1289 1342 1290 1343 1291 1344 1292 1345 1293 1346 1294 1347 1295 Figure 16. Synthesis of (S)-32 using a (R)-amino acid oxidase and an (S)-amino acid dehydrogenase. 1348 1296 1349 1297 1350 1298 1351 1299 1352 1300 1353 1301 1354 1302 1355 1303 1356 1304 1357 1305 1358 1306 1359 1307 1360 1308 1361 1309 1362 1310 1363 1311 1364 1312 1365 1313 1366 1314 1367 1315 1368 1316 1369 1317 1370 1318 Figure 17. Synthesis of (S)-32 using a (R)-amino acid oxidase and an (S)-aminotransferase. 1371 1319 1372 1320 borane-ammonia reduces it to regenerate rac-32 in a GPR119 agonists 1373 1321 dynamic process. Through this strategy, a maximum 1374 1322 The GPCR 119 (GPR119, also named “glucose-depend- 1375 yield of 76–79%, was obtained at pH 6.0–7.0, with ee 1323 ent insulinotropic receptor”) is a recent potential tar- 1376 values reaching >99.9% at pH 6–8 using 10 equiv. of 1324 get for the development of oral antidiabetic drugs 1377 the borane–ammonia complex. 1325 (Overton et al. 2008; Ritter et al. 2016). In fact, more 1378 14 C. M. ALCANTARA AND A. R. ALCANTARA

1379 than 20 pharmaceutical companies have been devel- pyrimidinylpiperidinyloxypyridone analogues 39 1432 1380 oping GPR119 agonists, but many clinical candidates (Figure 19) possessing chirality (Broekema et al. 2013), 1433 1381 have been discontinued for different reasons not and for this purpose, an scalable synthesis of enan- 1434 1382 always explained (Buzard et al. 2012; Ritter et al. 2016). tiomers of N-substituted 3-hydroxypyrrolidin-2-ones 1435 1383 For instance, Bristol-Myers Squibb, after disclosing have been recently reported (Singh et al. 2015), as 1436 1384 some pyridone, pyridazone, benzothiazole, dihydro- shown in Figure 19. 1437 1385 benzofuran, bicyclic pyrimidines and piperidinyl Thus, lipase PS 30 from P. cepacia immobilized on 1438 1386 sulfone GPR119 agonists (Wacker et al. 2014; Ye et al. polypropylene catalyzed the enantioselective esterifica- 1439 1387 2014), is working now on some new tion of racaemic-1-(2-fluoro-4-iodophenyl)-3-hydroxy- 1440 1388 pyrrolidin-2-one 35 with succinic anhydride and 1441 1389 2-methyltetrahydrofuran at 4 C, leading to (S)-35 in 1442 1390 high enantiomeric excess >99% and yield 40%, after 1443 1391 an easy separation from (R)-36 (Singh et al. 2015). 1444 1392 Following the initial experiments, it was possible to 1445 1393 scale-up to 50 g/L of substrate; in this process, after 1446 1394 8.5 h, immobilized enzyme was removed by simple fil- 1447 1395 tration, and (S)-35 was isolated in 40% yield and 1448 1396 ee >99%. 2-Methyltetrahydrofuran, a green biosolvent 1449 1397 (Pace et al. 2012, 2014) served as a reaction medium 1450 1398 and a solvent to extract the desired compound from 1451 1399 the reaction mixture, eliminating the use of chroma- 1452 S R 1400 Figure 18. Synthesis of ( )-32 using an ( )-amino acid oxidase tography. On the other hand, Novozyme 435 (C. ant- 1453 combined with a chemical racemization protocol. 1401 arctica lipase B) was employed for the resolution of 1454 1402 1455 1403 1456 1404 1457 1405 1458 1406 1459 1407 1460 1408 1461 1409 1462 1410 1463 1411 1464 1412 1465 1413 1466 1414 1467 1415 1468 1416 1469 1417 1470 1418 1471 1419 1472 1420 1473 1421 1474 1422 1475 1423 1476 1424 1477 1425 1478 1426 1479 1427 1480 1428 1481 1429 1482 1430 1483 1431 Figure 19. Enzymatic resolution of N-substituted 3-hydroxypyrrolidin-2-ones. 1484 BIOCATALYSIS AND BIOTRANSFORMATION 15

1485 racaemic acetate 37 for obtaining the desired alcohol Figure 20, 40), precursor of the commercialized 1538 TM 1486 (R)-38 in 37% isolated yield and high enantiomeric Miglitol 41 (Glyset , Pfizer) or Miglustat 42 1539 TM 1487 excess (ee >99.4%). This process was scaled up to kg (Zavesca , Actelion). Other type of iminocyclitols 1540 1488 scale (two successive campaigns (4.1 kg, ee >99.4% described in that review by Alcantara and coworkers 1541 1489 and 5.5 kg, ee >99.5%), and the major disadvantage of are polyhydroxylated pyrrolidines, such as 2,5-dideoxy- 1542 1490 a kinetic resolution (using only 50% of starting mater- 2,5-imino-D-mannitol 43, commonly known as DMDP, 1543 1491 ial) was overcome by recycling the undesired enantio- found in many plants and microorganisms, have been 1544 1492 mer into the desired enantiomer via Mitsunobu studied for their antihyperglycaemic properties (Horne 1545 1493 inversion (probed at gram scale) (Singh et al. 2015). et al. 2011). A representative member of polyhydroxy- 1546 1494 lates indolizidine 44 is the toxic alkaloid castanosper- 1547 1495 mine, a potent inhibitor of lysosomal a-glucosidase 1548 Enzyme inhibitors 1496 (Lahiri et al. 2013). DNJ, DMDP, and castanospermine 1549 1497 The inhibition of enzymes involved in metabolic path- also inhibit glycoprotein-processing enzymes to vary- 1550 1498 ways is undoubtedly one of the most active areas ing degrees (Asano et al. 2000). Casuarine 45 is an 1551 1499 inside Medicinal Chemistry (Harriman et al. 2010; example of a pyrrolizidines, which are also isolated 1552 1500 Copeland 2013). There are many enzymatic processes from plants and have been used in the treatment of 1553 1501 which inhibition could lead to a beneficial effect on breast cancer, diabetes, and bacterial infections 1554 1502 patients suffering from diabetes; in fact, two main (Wardrop and Waidyarachchi 2010). Finally, Calystegine 1555 a 1503 types of enzyme inhibitors, such as -glucosidase A3 46 belongs to nortropane-type alkaloids possessing 1556 1504 inhibitors and dipeptidyl peptidase-4 (DPP-4) inhibitors glycosidase inhibitory activity (Asano et al. 2000). 1557 1505 have already been commercialized and frequently pre- The chemoenzymatic preparation of another type 1558 1506 scribed, while some others are still under evaluation at of a-glucosidase inhibitors, aminocyclitols, are also 1559 1507 different (pre)clinical stages. In the following sections, described in the above-mentioned review (Alcantara 1560 1508 we will describe both types, showing how biocatalysis et al. 2014), illustrating biocatalyzed protocols for syn- 1561 1509 can help in the synthesis of their chemical structures. thesizing Voglibose 47 (VoglibTM, marketed by Mascot 1562 1510 Health Series), or Acarbose 48, generic sold in Europe 1563 1511 a-Glucosidase inhibitors and China as GlucobayTM (Bayer AG), in North America 1564 1512 as PrecoseTM (Bayer Pharmaceuticals), and in Canada 1565 It is well known that inhibitors of intestinal 1513 as PrandaseTM (Bayer AG). 1566 a-glucosidase enzymes promote a delay in the absorp- 1514 1567 tion of sugars because of the retard in the final steps 1515 DPP-4 inhibitors 1568 of carbohydrate digestion, so that they are useful for 1516 1569 reducing postprandial hyperglycaemia in DM (Derosa 1517 DPP-4, also known as adenosine deaminase complex- 1570 and Maffioli 2012; Campo et al. 2013). These a-glucosi- 1518 ing protein 2 or CD26 (cluster of differentiation 26) is 1571 dase inhibitors act as glycomimetics, because they 1519 a homodimer protein consisting of 766 amino acids 1572 1520 bear a certain grade of resemblance to the natural car- with cytoplasmic, transmembrane, and extracellular 1573 1521 bohydrates, but the differential part of their structure regions, playing a pivotal role in glucose metabolism, 1574 1522 promotes a blockade of enzymatic action (Ernst and because it is responsible for the degradation of the 1575 1523 Magnani 2009). The use of iminosugars (N atom GLP-1 incretins previously mentioned. In fact, DPP-4 is 1576 1524 replacing O) (Winchester 2009; Horne et al. 2011), thio- a serine exodipeptidase highly specific in recognizing 1577 1525 sugars (S instead of O) (Witczak and Culhane 2005)or peptide substrates with proline or alanine in the last 1578 1526 carbasugars (ethereal bridge substituted by a methy- position (P1) prior to the scissile amide bond of the 1579 1527 lene) (Mayato et al. 2012) as glycomimetics is a well- N-terminal of incretins (Deacon and Holst 2013). 1580 1528 developed strategy. More specifically, iminosugars Thus, DPP4 inhibitors are a pharmacological class 1581 1529 mimics transition state (oxocarbenium) of glycosidases of glucose-lowering agents that open up new per- 1582 1530 mechanism, due to the nitrogen protonation at spectives for Type 2 DM treatment because of their 1583 1531 physiological pH values (Caines et al. 2007; Winchester unique mechanism of action (Scheen 2012; Deacon 1584 1532 2009). Recently, Alcantara and coworkers published a and Holst 2013; Mize and Salehi 2013). Furthermore, 1585 1533 review covering chemo-enzymatic protocols for syn- recently the cardioprotective effects of these com- 1586 1534 thesizing this type of glycomimetics (Alcantara et al. pounds have been described (Dai et al. 2013; Wang 1587 1535 2014). These authors described the biocatalyzed syn- et al. 2013; Juillerat-Jeanneret 2014), so that this 1588 1536 thesis of different iminocyclitols, such as the polyhy- type of drugs, called generically gliptins and shown 1589 1537 droxylated piperidine 1-deoxynojirimycin (DNJ, in Figure 21, are becoming increasingly more studied 1590 16 C. M. ALCANTARA AND A. R. ALCANTARA

1591 1644 1592 1645 1593 1646 1594 1647 1595 1648 1596 1649 1597 1650 1598 1651 1599 1652 1600 1653 1601 1654 1602 1655 1603 1656 1604 1657 1605 1658 1606 1659 1607 1660 1608 1661 1609 1662 1610 1663 1611 1664 1612 1665 1613 1666 1614 1667 1615 1668 Figure 20. Some inhibitors of a-glucosidases. 1616 1669 1617 (Mittermayer et al. 2015; Cahn et al. 2016; Doggrell Sitagliptin is the most widely sold DPP-4 inhibitors in 1670 1618 and Dimmitt 2016; Thomas et al. 2016), as they are the USA and worldwide, reaching sales of US$6358 1671 1619 commonly used as second-line therapy for diabetes million in 2014 with an expected rise to 7525 in 2020. 1672 1620 1673 in high-income regions (Cahn et al. 2016). Sitagliptin was the second leading antidiabetic product 1621 1674 Although in many cases, purely chemical syntheses in 2014, after insulin glargine, and is predicted to be 1622 1675 have been described for the preparation of gliptins, the leading product by 2020 (Cahn et al. 2016; 1623 1676 there are some very attractive examples of biocata- Fernandes et al. 2016). 1624 1677 lyzed protocols for preparing the homochiral building The first chemical synthesis of sitagliptin (Hansen 1625 1678 blocks required for their synthesis; in some cases, the et al. 2009) involved in asymmetric hydrogenation of 1626 1679 chirality comes directly from commercially available an enamine 65 using a rhodium-based chiral catalyst 1627 1680 proline, which is transformed either into (S)-pyrrolidine (Rh[Josiphos]) at high pressure (Figure 22); neverthe- 1628 1681 2-carbonitrile, as for the preparation of anagliptin 56 less, this process is not stereoselective enough (97% 1629 1682 (Kato et al. 2011), or into the corresponding proline ee), and the final product is contaminated with rho- 1630 1683 amide, as required for the synthesis of Vildagliptin 50 dium, so that different additional purification steps are 1631 1684 required. Some other chemical syntheses have been 1632 (Pellegatti and Sedelmeier 2015). In other cases, chiral- 1685 1633 ity comes from other compounds, and these examples recently reviewed by Davies et al. (2015). 1686 1634 will be commented in the following paragraphs. Nevertheless, an enzymatic process has substantially 1687 1635 improved the efficiency of sitagliptin manufacturing 1688 1636 Sitagliptin. Sitagliptin (Figure 21, 49) (sold under the (Savile et al. 2010a, 2010b); in fact, using an engi- 1689 TM 1637 trade name Januvia by Merck Sharp & Dhome) was neered transaminase, developed at Codexis by rational 1690 1638 the first marketed oral antihyperglycaemic drug design, a biocatalyst with broad applicability towards 1691 1639 belonging to the gliptin family (Aroda et al. 2012). the synthesis of chiral amines was obtained. Under 1692 1640 Sitagliptin can be used either alone or combined with optimal conditions, the best variant converted 200 g/L 1693 1641 metformin or thiazolidinedione, another oral antihy- prositagliptin ketone 64 (Figure 22) to sitagliptin 49 1694 1642 perglycaemic agents in the treatment of Type 2 DM, with a 92% yield and an enantiomeric excess higher 1695 1643 already commented before (Kim et al. 2005). that 99%, by using 6 g/L enzyme in 50% dimethyl 1696 BIOCATALYSIS AND BIOTRANSFORMATION 17

1697 1750 1698 1751 1699 1752 1700 1753 1701 1754 1702 1755 1703 1756 1704 1757 1705 1758 1706 1759 1707 1760 1708 1761 1709 1762 1710 1763 1711 1764 1712 1765 1713 1766 1714 1767 1715 1768 1716 1769 1717 1770 1718 1771 1719 1772 1720 1773 1721 1774 1722 1775 1723 1776 1724 1777 1725 1778 1726 1779 1727 1780 1728 1781 1729 1782 1730 1783 1731 1784 1732 1785 1733 1786 1734 1787 1735 1788 1736 1789 1737 1790 1738 1791 1739 1792 1740 1793 1741 Figure 21. Chemical structure of several DPP4 inhibitors registered for clinical uses (49–60) and some other discontinued ones 1794 1742 (61–63). 1795 1743 1796 1744 1797 1745 sulfoxide. The biocatalytic process provides sitagliptin total manufacturing cost. Furthermore, the enzymatic 1798 1746 with a 10–13% increase in overall yield compared to reaction is run in multipurpose vessels, so that special- 1799 1747 the chemical process, a 53% increase in productivity ized high pressure hydrogenation equipment is no 1800 1748 (kg/L per day), a 19% reduction in total waste, the longer needed. Full details of this process, which 1801 1749 elimination of all heavy metals, and a reduction in obtained the Presidential Green Chemistry Challenge 1802 18 C. M. ALCANTARA AND A. R. ALCANTARA

1803 1856 1804 1857 1805 1858 1806 1859 1807 1860 1808 1861 1809 1862 1810 1863 1811 1864 1812 1865 1813 1866 1814 1867 1815 1868 1816 1869 1817 1870 1818 1871 1819 1872 1820 1873 1821 1874 1822 1875 1823 1876 1824 1877 1825 1878 1826 1879 1827 1880 1828 1881 1829 1882 1830 1883 1831 1884 1832 1885 1833 1886 1834 1887 Figure 22. Chemical versus biocatalyzed synthesis of JanuviaTM, sitagliptin phosphate 66. 1835 1888 1836 Award (Greener Reaction Conditions Award) from the as depicted in Figure 23. A modified form of a recom- 1889 1837 1890 U.S. Environmental Protection Agency (EPA) in 2010 binant phenylalanine dehydrogenase cloned from 1838 1891 (http://www.epa.gov/greenchemistry/pubs/pgcc/past. Thermoactinomyces intermedius and expressed in Pichia 1839 1892 html), can be found in literature (Moore et al. 2012; pastoris as well as in E. coli was used for this process 1840 þ 1893 Willies et al. 2012; Busto et al. 2016). Since this innova- development and scale-up. NAD produced during 1841 1894 tive approach of biocatalyzed synthesis of sitagliptin the reaction was recycled to NADH using FDH cloned 1842 1895 using transaminases, other similar examples have been and overexpressed in E. coli. The modified phenylalan- 1843 1896 described (Hou et al. 2016; Wei et al. 2016). ine dehydrogenase contains two amino acid changes 1844 1897 at the C-terminus and a 12 amino acid extension of 1845 1898 Saxagliptin. Saxagliptin (OnglyzaTM, 51) is another the C-terminus (Hanson et al. 2007). The production of 1846 1899 inhibitor of DPP-4 developed by Bristol-Myers Squibb 1847 multikilogram batches was originally carried out with 1900 1848 (Kania et al. 2011). This compound inhibits DDP-4 by extracts of P. pastoris expressing the modified phenyl- 1901 1849 covalent bonding to the catalytic serine presented in alanine dehydrogenase from T. intermedius and 1902 1850 DDP-4 active site (Aroda et al. 2012). Its synthesis endogenous FDH. The reductive amination process 1903 1851 (Savage et al. 2009) requires (S)-N-Boc-3-hydroxyada- was further scaled up using a preparation of the two 1904 1852 mantylglycine 69 as a key chiral intermediate. For its enzymes, FDH and phenylalanine dehydrogenase, 1905 1853 preparation, a process for conversion of the keto acid expressed in a single recombinant E. coli. The amino 1906 1854 67 to the corresponding amino acid 68 using acid 68 was directly protected as its Boc derivative 69 1907 1855 (S)-amino acid dehydrogenases was developed, without isolation to afford the intermediate. 1908 BIOCATALYSIS AND BIOTRANSFORMATION 19

1909 1962 1910 1963 1911 1964 1912 1965 1913 1966 1914 1967 1915 1968 1916 1969 1917 1970 1918 1971 1919 1972 1920 1973 1921 1974 1922 1975 1923 1976 1924 1977 1925 1978 1926 1979 1927 1980 1928 1981 1929 Figure 23. Enzymatic preparation of two intermediates in the synthesis of saxagliptin 51. 1982 1930 1983 Yields before isolation were close to 98% with 100% improvement (79% amide and 13% side-products), as 1931 1984 ee. This process has now been used to prepare several well as the use of sodalime and ascarite, respectively, 1932 1985 hundred kilograms of 69 to support the development at 200 g/L in the reaction headspace (increase in 1933 1986 and manufacturing of saxagliptin. amide yield to 84 and 95%), this presumably by way 1934 1987 Also (S)-5-aminocarbonyl-4,5-dihydro-1H-pyrrole-1- of adsorption of carbon dioxide liberated from the 1935 1988 carboxylic acid,1-(1,1-dimethylethyl)-ester 70 is decomposition of ammonium carbamate. A further 1936 1989 required in the synthetic scheme for obtaining saxa- increase in yield to 98% was attained via the com- 1937 1990 gliptin. Direct chemical ammonolysis was hindered by bined use of 100 g/L of calcium chloride and 200 g/L 1938 1991 reaction conditions, which resulted in unacceptable 1939 of ascarite. A prep-scale reaction with the process 1992 levels of amide racemization and side-product forma- 1940 ester feed was used. So, in the optimized process, 70 1993 1941 tion, whereas milder two-step hydrolysis condensation (220 g/L) was reacted with 90 g/L (1.25 mol equiv.) of 1994 1942 protocols using coupling agents such as 4-(4,6-dime- ammonium carbamate, 33 g/L (15% w/w of ester 1995 thoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride input) of CAL-B, 110 g/L calcium chloride, and 216 g/L 1943 1996 1944 (DMT-MM), were compromised by reduced overall of ascarite (in the headspace) and run at 50 C for 3 1997 1945 yields (Kunishima et al. 2001). To address this issue, a days. Complete conversion of ester was achieved, with 1998 1946 biocatalytic procedure was developed, based upon the the formation of 96% (182 g/L) of 71 and 4% of side- 1999 1947 CAL-B-mediated ammonolysis of 68 with ammonium products; finally, after workup, 98% potency amide in 2000 > 1948 carbamate to furnish amide 71 without racemization 99.9% ee was isolated in 81% yield (Gill and Patel 2001 1949 and with low levels of side-product formation (Gill and 2006). 2002 1950 Patel 2006). Experiments utilized process stream ester 2003 1951 feed, which consisted of 22% w/v (0.91 M) of the Aloglitin, linagliptin and trelagliptin. As can be seen 2004 1952 ester in toluene. Since the latter precluded the use of in Figure 24, three gliptins, alogliptin 52, linagiptin 53 2005 1953 free ammonia due to its low solubility in toluene, solid and trelagliptin 58 shared a common moiety, (R)-piper- 2006 1954 ammonium carbamate was employed. Reactions were idin-3-amine (R)-74, in their chemical structures, which 2007 1955 performed using a mixture of neat process feed, apropos is the only chiral centre present in these 2008 1956 ammonium carbamate (71 g/L, 2 mol equiv. of ammo- drugs. Therefore, this enantiopure amine is required in 2009 1957 nia), and biocatalyst (25 g/L) and shaken at 400 rpm, the chemical synthesis of 52 (Feng et al. 2007a, 2007b; 2010 1958 50 C. Under these conditions, CAL-B provided optically Ludescher et al. 2010), 53 (Eckhardt et al. 2007) and 2011 1959 pure amide 71 with yields of 69%, together with 21% 58 (Zhang et al. 2011). Although there are different 2012 1960 of side-products (by HPLC). The inclusion of drying chemical methods to produce optically pure (R)-74, 2013 1961 agents such as calcium chloride gave significant the use of transaminases allows very clean 2014 20 C. M. ALCANTARA AND A. R. ALCANTARA

2015 2068 2016 2069 2017 2070 2018 2071 2019 2072 2020 2073 2021 2074 2022 2075 2023 2076 2024 2077 2025 2078 2026 2079 2027 2080 2028 2081 2029 2082 2030 2083 2031 2084 2032 2085 2033 2086 2034 2087 2035 2088 Figure 24. Enzymatic transaminase-catalyzed kinetic resolution of 72 to produce enantiopure (R)-74. 2036 2089 2037 2090 2038 2091 2039 2092 2040 2093 2041 2094 2042 2095 2043 2096 2044 2097 2045 2098 2046 2099 2047 2100 Figure 25. Enzymatic preparation of (R)-74, through a transaminase-catalyzed asymmetric synthesis. 2048 2101 2049 2102 2050 methodologies. Thus, Hoehne et al. (2008) described production of (R)-74 starting from Cbz-protected pyr- 2103 2051 the kinetic resolution of racaemic N-protected 3-ami- imidine-3-one 73, and a subsequent deprotection of 2104 2052 nopiperidine 72, as shown in Figure 24. the correspondent amine (R)-72 allowed a good yield 2105 2053 In this process, the x-transaminase of Alcaligenes (up to 92%) of (R)-74 (Yang et al. 2014). Furthermore, 2106 2054 denitrificans (AdeTA) was the selected catalyst for the similar results regarding yield and optical purity could 2107 2055 kinetic resolution of 72, which was coupled to the sim- be obtained starting from Boc- or Bn-protected pyrimi- 2108 2056 ultaneous transformation of pyruvic acid into L-Ala, dine-3-one. 2109 2057 and the desired amine (R)-72 was obtained with 41% Recently, a similar approach has been proposed 2110 2058 isolated yield and 97% ee. This transaminase was also employing a recombinant transaminase from 2111 2059 tested in the synthesis of homologous N-protected Mycobacterium vanbaalenii, both as isolated enzyme 2112 2060 3-aminopyrrolidine, through an analogous kinetic reso- and whole cells (and also in their immobilized forms), 2113 2061 lution. Anyhow, the drawback of these resolutions is leading to good yield and optical purity (Luo et al. 2114 2062 their inherent limited yield (max. 50%). 2016). 2115 2063 On the other hand, enantiopure (R)-74 can be syn- 2116 2064 thesized using a transaminase-catalyzed asymmetric Teneligliptin and gosogliptin. Teneligliptin 55 is a 2117 2065 synthesis (not limited to 50% yield), as depicted in DPP4-inhibitor initially developed by Mitsubishi 2118 TM 2066 Figure 25. In this process, the use of a transaminase Tanabe Pharma under the name of Tenelia (recently 2119 TM 2067 and isopropilamine as sacrificial substrate allowed the available also in Argentina (Teneglucon ) and India 2120 BIOCATALYSIS AND BIOTRANSFORMATION 21

2121 2174 2122 2175 2123 2176 2124 2177 2125 2178 2126 2179 2127 2180 2128 2181 2129 2182 2130 2183 2131 2184 2132 2185 2133 2186 2134 2187 2135 2188 2136 Figure 26. Schematic synthesis of teneligliptin 55 and gosogliptin 60. 2189 2137 2190 2138 2191 (TenepureTM; TenezaTM) at relatively affordable price, 2139 2192 with an unique structure characterized by five con- 2140 2193 secutive rings, explaining its powerful activity and its 2141 2194 extremely long half-life (24.2 h), with resulting DPP-4 2142 2195 inhibition throughout the day (Scott 2015; Pujadas 2143 2196 et al. 2016; Sharma et al. 2016). Gosogliptin 60 was 2144 2197 developed by Pfizer, but it was discontinued in 2012 2145 2198 in Phase II trials; on June 2012 exclusive rights were 2146 Figure 27. Synthesis of Hyp 75 catalyzed by P4H. 2199 granted to SatRx LLC, a Russian company, for further 2147 2200 development, and it was launched in Russian market 2148 are usually difficult to process (Huttel 2013; Wu et al. 2201 in 2016 under the trade name SatRxTM. 2149 2016). 2202 As can be seen in Figure 26, teneligliptin 55 and 2150 Another chemoenzymatic methodology for produc- 2203 gosogliptin 60 share a somehow similar structure, in 2151 ing Hyp starts from racaemic ethyl 2-Boc-amino-4-pen- 2204 which the chirality in the molecule is introduced by 2152 tenoate 78, obtained from the corresponding 2205 using enantiopure N-Boc-trans-4-hydroxy-L-proline (76, 2153 malonate derivative 77, as shown in Figure 28 2206 Figure 26), as described for teneligliptin (Yoshida et al. 2154 (Krishnamurthy et al. 2014). 2207 2012; Dwivedi et al. 2015a) and gosogliptin (Lafrance 2155 Racaemic 78 is resolved by enantioselective 2208 2156 and Caron 2012). hydrolysis catalyzed by subtilisin, leading to acid 2209 2157 4-(R)-Hydroxyproline (Hyp, 75) is a non-proteino- (S)-79, which is subsequently converted into the ben- 2210 2158 genic amino acid present in collagen, and which cylic ester and epoxidized, to furnish diastereomers 80 2211 2159 abundance among the residues in animal proteins is (69% yield, 57:43 (2S,4R):(2S,4S), as detected by NMR). 2212 2160 very high, around 4%, a value calculated from the Amine deprotection with HCl (4M in dioxane) for 2.5 h 2213 2161 abundance of collagen amongst animal proteins (1/3) quantitatively produced the hydrochloride 81 (not iso- 2214 2162 and that of Hyp within collagen ( 38% 1/3). There lated) as a white solid upon solvent evaporation. 2215 2163 are different biocatalyzed methodologies for the syn- Subsequently, 81 was dissolved in DMF (4.6 mmol in 2216 2164 thesis of 75; the most obvious one requires the 30 mL DMF), and two equivalents of Et3N were added 2217 2165 employ of prolyl 4-hydroxylase (P4H, E.C. 1.14.11.2, to neutralize HCl. After 72 h, TLC results showed the 2218 H 2166 also named procollagen-proline 4-dioxygenase), an disappearance of 81, and 1 NMR analysis of the 2219 2167 2-oxoacid dioxygenase requiring 2-oxoglutaric acid evaporated reaction mixture showed the absence of 2220 2168 and molecular oxygen as cosubstrates (Gorres and characteristic methylene epoxide signals, strongly sug- 2221 2169 Raines 2010), as depicted in Figure 27. Nevertheless, gest that the nucleophilic ring opening of the 81 2222 2170 although there are many references for this proced- occurred intramolecularly, possibly to produce both 2223 2171 ure at lab scale (Hara et al. 2014; Yi et al. 2014; Chen cis- and trans-diastereomers of L-hydroxyproline benzyl 2224 2172 et al. 2015; Pozzolini et al. 2015), its technical applica- ester 82. Nonetheless, these products were not iso- 2225 2173 tion is limited, because these 2-oxoacid dioxygenases lated, and this reaction mixture was re-dissolved in 2226 22 C. M. ALCANTARA AND A. R. ALCANTARA

2227 2280 2228 2281 2229 2282 2230 2283 2231 2284 2232 2285 2233 2286 2234 2287 2235 2288 2236 2289 2237 2290 2238 2291 2239 2292 2240 2293 2241 2294 2242 2295 2243 2296 2244 2297 2245 2298 2246 2299 2247 2300 2248 2301 2249 2302 2250 2303 2251 2304 2252 2305 2253 2306 2254 2307 Figure 28. Chemoenzymatic synthesis of Hyp ester 83. 2255 2308 2256 2309 2257 2310 2258 2311 2259 2312 2260 2313 2261 2314 2262 2315 2263 2316 2264 2317 2265 2318 2266 2319 2267 2320 2268 2321 2269 2322 2270 2323 2271 2324 2272 2325 2273 2326 2274 2327 2275 2328 2276 2329 2277 2330 Figure 29. Chemoenzymatic synthesis of (R)-ProP 91. 2278 2331 2279 2332 BIOCATALYSIS AND BIOTRANSFORMATION 23

2333 2386 2334 2387 2335 2388 2336 2389 2337 2390 2338 2391 2339 2392 2340 2393 2341 2394 2342 2395 2343 2396 2344 2397 2345 2398 2346 2399 2347 2400 2348 2401 2349 2402 2350 2403 2351 2404 2352 2405 2353 Figure 30. Enantioselective preparation of both enantiomers of benzyl-2-(dimethoxyphosphoryl)pyrrolidine-1-carboxylate 94. 2406 2354 2407 2355 2408 2356 2409 2357 dioxane and reacted with a small excess of Boc2O 86 mediated by a lipase from Aspergillus niger,as 2410 2358 using Et3N to re-protect the secondary amino groups, depicted in Figure 29 (Wuggenig et al. 2011). 2411 2359 and to furnish after 24 h both diastereomers N-Boc-cis- Subsequently, the a-hydroxyphosphonate (S)-87 2412 2360 L-hydroxyproline benzyl ester (cis-(S,S)-83) and (obtained in 40% yield and 86% ee) was mesylated in 2413 2361 N-Boc-trans-L-hydroxyproline benzyl ester (trans-(S,R)- 93% yield and then selectively converted (NaN3/18- 2414 2362 83), as a yellow oil. Column chromatography (silica crown-6/MeCN/24 h/reflux) into the monoazide (S)-88 2415 2363 gel, hexane–50% EtOAc (v/v) as eluent) allowed the in 91% yield; subsequently, a Staudinger reaction with 2416 2364 separation of L-cis-hydroxyproline lactone (S,S)-84 Ph3P in DMF furnished the intermediate iminophos- 2417 2365 (obtained via intramolecular cyclization, white solid, phorane (S)-89, which cyclized to the protected 2418 2366 29–42%) and the desired Hyp ester cis-(S,S)-83 L-phosphaproline (R)-90, and after deprotection and 2419 P 2367 (yellowish oil, 18–38%). purification yielded (R)-Pro 91. 2420 P 2368 Recently, a kinetic resolution of racaemic Pro has 2421 2369 Other DPP4 inhibitors: phosphoproline containing been described (Arizpe et al. 2015), using lipases for 2422 2370 dipeptides. Dipeptides containing phosphoproline the enantioselective synthesis of carbamates, as 2423 P 2371 (Pro , the phosphonic counterpart of proline) are depicted in Figure 30. 2424 2372 known to inhibit several serine DPP4 proteases, as well The starting racaemic dimethyl pyrrolidin- 2425 2373 as other serine proteases (Boduszek et al. 1994; 2-ylphosphonate 92 was chemically derived from pyr- 2426 2374 Moonen et al. 2004; Mucha et al. 2011). For preparing rolidin-2-one. Different lipases were tested to check 2427 2375 these enantiopure dipeptides, it is thus mandatory to the best option for enantioselective alkoxycarbonyla- 2428 P 2376 prepare homochiral Pro , which chemical asymmetric tion with several carbonates; best results were 2429 2377 synthesis (Katritzky et al. 1999; Davis et al. 2004;Ma obtained using allyl 3-methoxyphenylcarbonate and 2430 2378 et al. 2011; Ordonez~ et al. 2015) or chemical resolution C. antarctica lipase type A (CAL-A), which catalyzed the 2431 2379 by diastereomers preparation (Kaboudin et al. 2013) allyloxycarbonylation of rac-92 to yield the unreacted 2432 2380 have been described in literature. substrate (S)-92 with 90% ee and the allyl carbamate 2433 2381 The development of biocatalyzed protocols for the (R)-93 with 20% ee (Figure 30) after 92 h of reaction. 2434 P 2382 preparation of enantiopure Pro is a very recent Nevertheless, separation of these compounds by silica- 2435 2383 research area, and not many cases have been gel column chromatography was not possible, because 2436 P 2384 described; in fact, (R)-Pro was obtained by kinetic reso- of the lability of 92, so that the crude obtained from 2437 2385 lution of d-bromo-a-(chloroacetoxy)butylphosphonate the enzymatic reaction was treated with benzyl 2438 24 C. M. ALCANTARA AND A. R. ALCANTARA

2439 2492 2440 2493 2441 2494 2442 2495 2443 2496 2444 2497 2445 2498 2446 2499 2447 2500 2448 2501 2449 2502 2450 2503 2451 2504 2452 2505 P 2453 Figure 31. Enantioselective preparation of carbamates of Pro dimethyl esters. 2506 2454 2507 2455 chloroformate to give a mixture of optically active car- C-glycosidic bond, but now between the aglycon and 2508 2456 bamates (S)-94 and (R)-93, which could be separated, the corresponding 5-thio-D-glucopiranose. 2509 2457 and finally (R)-93 was converted into (R)-94. In all Remogliflozin etabonate 102 is the only member of 2510 2458 cases, the enantiomers could be separated by chiral the family possessing the classical O-linkage, 2511 2459 chromatography (Arizpe et al. 2015). These same while tofogliflozin 100 is a spiranic compound, so that 2512 2460 authors described a somehow simpler protocol by C- and O-linkages are simultaneously presented. 2513 2461 using benzyl 3-methoxyphenyl carbonate for catalyz- Any biocatalytic method for creating the 2514 2462 ing the carbamoylation (after 84 h, 82% of (R)-94 and C-glycoside would demand the use of Leloir C-glycosil- 2515 2463 94% ee of (S)-92), and then an easier separation by a transferases (C-GTs) (Gutmann and Nidetzky 2013), but 2516 2464 previous tosilation of non-converted (S)-92 (Figure 31). this possibility has not been developed for gliflozins, 2517 2465 as far as we know. Nevertheless, empagliflozin 98 2518 2466 Sodium–glucose co-transporter 2 (SGLT2) inhibitors: does contain a chiral fragment in its structure, (S)- 2519 2467 gliflozins tetrahydrofuran-3-ol (S)-103, required for the synthesis 2520 2468 2521 – – of the drug (Wang et al. 2014), and different biotrans- 2469 Sodium glucose co-transporters or sodium glucose- 2522 formations can be found in literature for producing 2470 linked transporter (SGLTs) play an important role in 2523 both enantiomers of 103, as shown in Figure 33. The 2471 the intake and elimination of glucose. SGLTs are 2524 first strategy is based on a kinetic resolution of the 2472 located in the intestinal mucosa (enterocytes) of the 2525 corresponding racaemic alcohol, but this is not that 2473 small intestine (SGLT1), and the proximal tubule of the 2526 trivial because of the small differences in the size of 2474 nephron (SGLT2 in proximal convoluted tubule, SGLT1 2527 both groups attached to the carbinol moiety: in fact, 2475 in proximal straight tubule). SGLT2 is the main respon- 2528 Baumann and coworkers did not find any measurable 2476 sible for reabsorption of glucose in kidney; thus, inhib- 2529 enantioselectivity in the hydrolysis of different racae- 2477 ition of SGLT2 would lead to a very low or even null 2530 mic tetrahydrofuran-3-yl esters after testing more than 2478 glucose reabsorption and an increased glycosuria, 2531 100 commercial hydrolases (Baumann et al. 2000). 2479 highly desirable for patients suffering Type 2 DM 2532 Using enzymes modified by mutations of amino acids 2480 (Madaan et al. 2016; Solini 2016). 2533 2481 There are several SGLT2 inhibitors, called generically it became possible to increase the enantioselectivity in 2534 2482 gliflozins, already marketed (Figure 32): dapagliflozin the hydrolysis, but only up to a moderate value 2535 ¼ ¼ 2483 96, canagliflozin 97, empagliflozin 98, ipragliflozin 99, (enantiomeric ratio E 10, compared to E 4.3 with 2536 2484 tofogliflozin 100, and luseogliflozin 101 are approved the wild-type enzyme) using the D31T/L93F double 2537 2485 in different countries, and the prodrug remogliflozin mutant of an esterase from Bacillus stearothermophilus 2538 2486 etabonate 102 is under study for commercialization (Nobili et al. 2013). 2539 2487 (Madaan et al. 2016). Similarly, the bioreduction of the corresponding 2540 2488 The chemical structures of dapagliflozin 96, canagli- ketone dihydrofuran-3(2H)-one 105 did not lead to 2541 2489 flozin 97, empagliflozin 98 and ipragliflozin 99 present high enantioselectivity values, once again because of 2542 2490 a C-glycosidic linkage between the glucose moiety the similar size of both methylene groups around the 2543 2491 and the aglycon; luseogliflozin 101 also possess the carbonyl moiety; in fact, Sun et al. (2016) tested this 2544 BIOCATALYSIS AND BIOTRANSFORMATION 25

2545 2598 2546 2599 2547 2600 2548 2601 2549 2602 2550 2603 2551 2604 2552 2605 2553 2606 2554 2607 2555 2608 2556 2609 2557 2610 2558 2611 2559 2612 2560 2613 2561 2614 2562 2615 2563 2616 2564 2617 2565 2618 2566 2619 2567 2620 2568 2621 2569 2622 2570 2623 2571 2624 2572 2625 2573 2626 2574 2627 2575 2628 2576 2629 2577 2630 2578 2631 2579 2632 2580 2633 2581 2634 2582 2635 Figure 32. Some marketed gliflozins. 2583 2636 2584 2637 2585 2638 2586 bioreduction using two commercial ADH kits An indirect biocatalyzed methodology for produc- 2639 2587 (47 enzymes), describing only a 22% ee for the best ing (S)-103 was described by Pienaar et al. (2008), 2640 2588 (R)-selective enzyme and 91% ee for the best (S)- using a chemoenzymatic approach also presented in 2641 2589 selective one, but under suboptimal conversions. So, Figure 33. Hence, the racaemic epoxide 106 2642 2590 this group decided to modify the ADH from was opened using whole cells of Yarrowia lipolytica 2643 2591 Thermoethanolicus brockii (TbSADH, slightly (R)-select- containing epoxide hydrolase activity, and the non- 2644 2592 ive (23% ee) at full conversion) by genetic engineering, converted substrate (S)-106 (20% yield, 97.8% ee) was 2645 2593 using triple-code saturation mutagenesis (TCSM), chemically opened leading to halohydrin (S)-108, 2646 2594 obtaining highly (R)- and (S)-selective variants (62–94% which upon a lipase-catalyzed hydrolysis and acid 2647 2595 ee for (S)-selective, 95%99% ee for (R)-slective) with catalysis furnished (S)-103 with moderate yields (79%), 2648 2596 minimal screening at semipreparative scale (Sun et al. not altering the good enantioselectivity obtained in 2649 2597 2016). the preparation of starting (S)-106. 2650 26 C. M. ALCANTARA AND A. R. ALCANTARA

2651 2704 2652 2705 2653 2706 2654 2707 2655 2708 2656 2709 2657 2710 2658 2711 2659 2712 2660 2713 2661 2714 2662 2715 2663 2716 2664 2717 2665 2718 2666 2719 2667 2720 2668 2721 2669 2722 2670 2723 2671 2724 2672 2725 2673 2726 2674 2727 2675 2728 2676 2729 2677 2730 2678 2731 2679 Figure 33. Some biocatalyzed methods for obtaining (S)-tetrahydrofuran-3-ol (S)-103. 2732 2680 2733 2681 11b-hydroxysteroid dehydrogenase Type 1 (11b- in this area, developing many different chemical struc- 2734 2682 HSD1) inhibitors tures displaying inhibition of 11b-HSD1 (Scott et al. 2735 2683 2014); many of these chemical structures present ster- 2736 An increased abnormal concentration of glucocorti- 2684 eogenic centres, and thus they could be synthesized 2737 coids (GCs) may lead to its precipitation, with the con- 2685 with the help of biocatalysis. We will show only some 2738 comitant aggravation of truncal obesity, insulin 2686 examples covering this field, being the first one oxazo- 2739 resistance, hepatic triacylglycerol accumulation, hyper- 2687 lone 112 (Figure 34) reported by Biovitrum, having 2740 2688 glycaemia, hypertension and dyslipidaemia. This is 2741 reasonable potency tested in vitro (Sutin et al. 2007). 2689 known as metabolic syndrome (Grundy et al. 2004), 2742 Preparation of the chiral amine (S)-111, required for 2690 and it represents a major risk factor for Type 2 DM and 2743 synthesizing 112, has been recently described by 2691 cardiovascular disease. Thus, interventions to reduce 2744 Martinez-Montero and coworkers, by means of a trans- 2692 GC action can prevent and reverse these effects. One 2745 amination of the corresponding ketone 110, in high 2693 of the most important enzymes involved in GCs activ- 2746 b b yield (95% conversion) and stereoselectivity (>99% ee) 2694 ity is 11 -hydroxysteroid dehydrogenase 1 (11 -HSD1), 2747 (Martinez-Montero et al. 2017). 2695 which converts cortisone to cortisol, the primary GC in 2748 In another example, monoester 114, required for 2696 humans, mostly in liver and adipose tissue, so that the 2749 b 2697 inhibition of this enzyme could reduce cortisol produc- preparing the 11 -HSD1 inhibitor 115 (Peddi et al. 2750 2698 tion within these tissues without substantially affecting 2010), has been prepared starting from diester 113,by 2751 2699 circulating cortisol (Anderson and Walker 2013). This is using a lipase catalyzed mono-hydrolysis, not continu- 2752 2700 the reason why 11b-HSD1 has been proposed as an ing to the diacid (Guo et al. 2014). Thus, about 100 kg 2753 2701 innovative therapeutic target for the treatment of of 114 were prepared in 78% yield by hydrolysis of 2754 2702 Type 2 DM (Bailey et al. 2016; Bailey 2017). There are 113 with a commercially available lipase from 2755 2703 many pharmaceutical companies working very actively Burkholderia cepacia: a more efficient enzymatic 2756 BIOCATALYSIS AND BIOTRANSFORMATION 27

2757 2810 2758 2811 2759 2812 2760 2813 2761 2814 2762 2815 2763 2816 2764 2817 2765 2818 2766 2819 2767 2820 2768 2821 2769 2822 2770 2823 2771 2824 2772 Figure 34. Biocatalytic procedures for preparing some 11b-HSD1 inhibitors. 2825 2773 2826 2774 2827 2775 2828 2776 2829 2777 2830 2778 2831 2779 2832 2780 2833 2781 2834 2782 2835 2783 2836 2784 2837 2785 2838 2786 2839 2787 2840 2788 2841 2789 2842 2790 2843 2791 2844 2792 2845 2793 2846 2794 Figure 35. Some 11b-HSD1 inhibitors possessing an adamantyl moiety in their structures. 2847 2795 2848 2796 2849 2797 2850 2798 process was developed for the hydrolysis of 113 that 120 (Venier et al. 2011), developed by Sanofi, are at 2851 2799 gave the monoester 114 in 82% yield using signifi- different levels of clinic assays. 2852 2800 cantly lower amounts of the commercially available Microbial oxidation of adamantane 121 (Figure 36) 2853 2801 immobilized lipase B from C. antarctica. is a good alternative for obtaining hydroxylated deriv- 2854 2802 As we commented before, there are many pharma- atives, because chemical methods require harsh 2855 2803 ceutical companies working very actively in the prep- oxidants, are poorly selective and are prone to overox- 2856 b 2804 aration of 11 -HSD1 inhibitors; many of the candidates idation; subsequently, these hydroxylated derivatives 2857 2805 present an adamantyl moiety in its structure, as shown can be chemically converted in other structures 2858 2806 in Figure 35. In fact, compounds such as 116, from required for the preparation of those drug candidates 2859 2807 Pfizer (Cheng et al. 2010); 117 (Scott et al. 2012a) and presented before. 2860 2808 118 (Scott et al. 2012b), from AstraZeneca; 119 Biohidroxylation of 121 catalyzed by Streptomyces 2861 2809 (Becker et al. 2008; An et al. 2013) from Abbott, or griseoplanus was described by Mitsukura and 2862 28 C. M. ALCANTARA AND A. R. ALCANTARA

2863 2916 2864 2917 2865 2918 2866 2919 2867 2920 2868 2921 2869 2922 2870 2923 2871 2924 2872 2925 2873 2926 2874 2927 2875 2928 2876 2929 2877 2930 2878 2931 Figure 36. Microbial hydroxylation of adamantane and derivatives. 2879 2932 2880 2933 coworkers in 2006: among 470 strains tested, S. griseo- The focus has been centred in the different types of 2881 2934 planus was highly regioselective to give 1-adamanta- drugs actually being used, and we have not considered 2882 2935 nol 122 in 32% molar conversion yield after 72-h the effect of combined drugs (a very common thera- 2883 2936 cultivation in the presence of 3% (v/v) Tween 60. peutic strategy), because this is out of the aim of this 2884 2937 This same group also described the production of revision. According to the market volume of these 2885 2938 1,3-adamantanediol 123 by a regioselective monohy- types of drugs, we can foresee a clear increase in the 2886 2939 droxylation of 122 using Streptomyces sp. SA8, produc- 2887 use of versatile biocatalyzed protocols for the produc- 2940 ing 5.9 g L1 of 123 starting from 6.2 g L1 of 122 in 2888 tion of antidiabetic drugs. 2941 culture broth after 120 h at 25 C (Mitsukura et al. 2889 2942 2010). Using resting cells, 2.3 g L1 of 122 was pro- 2890 2943 duced after 96 h of incubation at a 69% conversion Acknowledgements 2891 2944 rate. In both cases, 1,4-adamantanediol 124 was 2892 The author would like to give special thanks to the library of 2945 formed as a byproduct at a rate of about 15%; this 2893 the Faculty of Pharmacy for the technical help in finding and 2946 strain SA8 was also able to hydroxylate 2-adamantanol 2894 management of scientific literature used for this review. 2947 2895 and 2-methyl-2-adamantanol. Similarly, washed cells 2948 2896 (62 mg) of Kitasatospora sp. GF12 in 4 mL buffer (pH 7) 2949 Disclosure statement 2897 were used for catalyzing the regioselective hydroxyl- 2950 ation of 60 mM 123 to 30.9 mM 1,3,5-adamantanetriol The author reports no declarations of interest. 2898 2951 2899 125 over 120 h at 24 C (Mitsukura et al. 2012), adding 2952 glycerol (400 mM) to the reaction mixture to recycle 2900 References 2953 2901 the intracellular NADH/NADPH. The same cells also cat- 2954 2902 alyzed the hydroxylation of 10 mM of 122 directly to Acton JJ, Akiyama TE, Chang CH, Colwell L, Debenham S, 2955 Doebber T, Einstein M, Liu K, McCann ME, Moller DE, et al. 2903 125 (3.6 mM). 2956 2009. Discovery of (2R)-2-(3-{3-(4-methoxyphenyl)carbonyl- 2904 2-methyl-6-(trifluoromethoxy)-1H-indol-1-yl}phenoxy)buta- 2957 2905 Conclusions noic acid (MK-0533): a novel selective peroxisome prolifer- 2958 2906 ator-activated receptor gamma modulator for the 2959 2907 We have presented in this review some examples of treatment of type 2 diabetes mellitus with a reduced 2960 2908 how biocatalysis can help in the development of drugs potential to increase plasma and extracellular fluid vol- 2961 2909 possessing antidiabetic activity. Our objective has been ume. J Med Chem 52:3846–3854. 2962 to illustrate how the employ of biocatalysts, enzymes Agrawal R. 2014. The first approved agent in the Glitazar’s 2910 – 2963 (wild-type or genetically modified, soluble or immobi- class: Saroglitazar. Curr Drug Targets 15:151 155. 2911 Ahren B. 2009. Islet G protein-coupled receptors as potential 2964 lized) or whole cells (in any state) points towards a 2912 targets for treatment of type 2 diabetes. Nat Rev Drug 2965 2913 definitive improve in the sustainability of the synthetic Discov 8:369–385. 2966 2914 process, because of the excellent biocatalytic precision Alcantara AR, Pace V, Hoyos P, Sandoval M, Holzer W, 2967 2915 (chemoselectivity, regioselectivity, stereoselectively). Hernaiz MJ. 2014. Chemoenzymatic synthesis of 2968 BIOCATALYSIS AND BIOTRANSFORMATION 29

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