**1. Introduction**

The concept of in vitro maturation of oocytes (IVM) was firstly mentioned in literature by Pincus and Enzmann, initially in 1935 [1]. They conducted experiments in which ova, taken from tubes at various intervals after fertile matting, were cultured in vitro*.* Thirty-four years later, Eppig and Schroeder [2] designed the possibility to use IVM rather than protocols of hormonal stimulation currently in use. These authors mentioned "it may be possible to recover immature oocytes from several antral follicles, excluding the dominant preovulatory follicle, and mature them in proper culture." At that time it was impossible to accomplish that assignment due to technical reasons. However, 2 years later, Cha et al. [3] mentioned, "this is an engineering problem with the ultrasound equipment that will be resolved in

the future." Recently, Cha et al. were the first to succeed with IVM in human using immature oocytes retrieved from antral follicles. Trounson et al. [4] were the first who put IVM firmly in the clinical realm, obtaining a live birth from oocytes, recovered from untreated polycystic ovarian patient who underwent in vitro maturation.

In vitro maturation of oocytes have potential advantages over conventional IVF: a simple protocol with decreased or no hormonal stimulation before oocyte retrieval, lower cost of the treatment cycle, and reduced psychological impact. Moreover, and not of less of importance, the risk of OHSS is entirely avoided. Despite these benefits, however, there are still many debatable problems surrounding this treatment. Until one can say that IVM could be an alternative to conventional IVF treatment, these advantages have to be weighed against the pregnancy and delivery rates, children outcome, and possible risks.

There are several basic differences between routine in vitro fertilization (IVF) and IVM. These differences might be related to the size of aspirated follicles, the nuclear and cytoplasmic maturity of the oocytes, and the laboratory procedures. In order to achieve proper fertilization and embryo development, Gougeon and Testart in [5] pointed out that the follicles at the time of the collection for IVM procedures should be in the early antral to antral stage (0.2–14 mm) vs. preovulatory stage (0.16–20 mm) for IVF. After retrieval, majority of the oocytes are immature (MI, GV). However, only few will proceed in vitro to metaphase II (MII) in culture media, some after 6 h, but most of them after 24–48 h and only then will be able to undergo successful fertilization after ICSI. This is in comparison with conventional IVF, where most of the retrieved oocytes are mature, and fertilization might occur immediately after ICSI.

There are many controversial areas of debate, especially regarding the process of oocyte maturation in vitro: (a) nuclear maturation—the process that reverses meiotic arrest at prophase I (GV), driving the progression to MII. This process is followed by the expansion of cumulus granulosa cells and loss of intercellular communications between the cumulus cells and also between cumulus and the surface of the oocyte (a visible laboratory course); (b) cytoplasmic maturation—metabolic and structural modifications within the oocyte that prepares the ovum for activation, fertilization, and embryonic development (an invisible laboratory process) [2, 4, 5]; it should, therefore, be kept in mind that IVM may not be free of possible future complications and counseling of the patients thoroughly before commencing the treatment is essential.

### **2. Guidelines for IVM treatment**

### **2.1 Ultrasound monitoring**

Follicle monitoring follow-up is mainly performed using ultrasound scans of the ovaries and endometrium. However, in contrast to IVF, in IVM cycles, there is no need for serum hormonal level (estradiol, progesterone) follow-up.

First ultrasound should be performed on days 2–3 of the menstrual (or induced) cycle in order to record the number and size of all follicles along with endometrial thickness and a second scan on days 6–8, to determine the presence and size of the largest follicle in each ovary. A third scan should be completed on day of hCG trigger, to measure the endometrial thickness (**Figure 1**).

### **2.2 FSH priming**

Fadini et al. [6] reported that 77.4% of retrieved immature oocytes underwent maturation in vitro followed by 29.8% pregnancy rate vs. 48.4 and 15.2% in primed and non-primed IVM cycles, respectively. Mikkelsen et al. [7] pointed that priming

**179**

*In Vitro Maturation and Fertilization of Oocytes: From Laboratory Bench to Clinical Practice*

with r-FSH for 2–3 days before the harvesting of immature oocytes from patients with polycystic ovarian syndrome (PCOS) may improve the maturational potential of the oocytes and the implantation rate. Similar results were described by other

**2.3 Human chorionic gonadotropin (hCG)/GnRh-agonist priming and retrieval**

The beneficial effect of hCG priming in IVM cycles and extension of the period of time from 35 to 36 h (routinely administrated in IVF cycles) to 38 h from hCG administration was demonstrated by Son et al. [12]. This hCG priming promotes GV oocytes to reach MI stage and increases the maturation rate of immature oocytes in vitro. Gonadotropin-releasing hormone agonist (GnRH-a) has been used recently in triggering oocyte maturation. In this approach, small follicles were stimulated with gonadotropins for 3–5 days. GnRH-a administration was performed, to trigger ovulation, when the largest follicles were 10–12 mm in diameter. Many immature oocytes, which underwent maturation in vitro, were then harvested, fertilized, and subsequently developed into blastocysts that resulted in live births [13]. This interesting observation might have a great importance in follicular cytoplasmic maturation due to the FSH surge obtained after GnRH triggering. The FSH surge might promote formation of LH receptors on the granulosa cells enhancing LH activity, induce plasminogen activator activity causing dissociation of oocytes from somatic cells of the follicle (therefore more immature oocytes could be obtained), and maintain the opening of gap junction between cumulus cells and oocyte which

Son et al. [18] conducted a study in which patients were triggered when the leading follicle was <10 mm, 10–14 mm, or >14 mm. In the group with a leading follicle >14 mm, only one pregnancy was obtained. The authors of the current paper administer, therefore, hCG trigger once a leading follicle of 10–12 mm is developed.

In IVM, estradiol levels are physiological, and oocyte collection is done before the endometrium is fully estrogenized. After aspiration, there is an insufficient support from the corpus luteum for endometrial receptivity. It was demonstrated

*DOI: http://dx.doi.org/10.5772/intechopen.91802*

investigators in PCOS patients [8–11].

*In vitro maturation oocytes: clinical protocol.*

contributes to oocyte maturation [14–17].

**2.4 Timing of collection: at what follicle size?**

**2.5 Endometrial preparation and luteal support**

**interval**

**Figure 1.**

*In Vitro Maturation and Fertilization of Oocytes: From Laboratory Bench to Clinical Practice DOI: http://dx.doi.org/10.5772/intechopen.91802*

**Figure 1.**

*Innovations in Assisted Reproduction Technology*

**2. Guidelines for IVM treatment**

**2.1 Ultrasound monitoring**

and delivery rates, children outcome, and possible risks.

the future." Recently, Cha et al. were the first to succeed with IVM in human using immature oocytes retrieved from antral follicles. Trounson et al. [4] were the first who put IVM firmly in the clinical realm, obtaining a live birth from oocytes, recovered from untreated polycystic ovarian patient who underwent in vitro maturation. In vitro maturation of oocytes have potential advantages over conventional IVF: a simple protocol with decreased or no hormonal stimulation before oocyte retrieval, lower cost of the treatment cycle, and reduced psychological impact. Moreover, and not of less of importance, the risk of OHSS is entirely avoided. Despite these benefits, however, there are still many debatable problems surrounding this treatment. Until one can say that IVM could be an alternative to conventional IVF treatment, these advantages have to be weighed against the pregnancy

There are several basic differences between routine in vitro fertilization (IVF) and IVM. These differences might be related to the size of aspirated follicles, the nuclear and cytoplasmic maturity of the oocytes, and the laboratory procedures. In order to achieve proper fertilization and embryo development, Gougeon and Testart in [5] pointed out that the follicles at the time of the collection for IVM procedures should be in the early antral to antral stage (0.2–14 mm) vs. preovulatory stage (0.16–20 mm) for IVF. After retrieval, majority of the oocytes are immature (MI, GV). However, only few will proceed in vitro to metaphase II (MII) in culture media, some after 6 h, but most of them after 24–48 h and only then will be able to undergo successful fertilization after ICSI. This is in comparison with conventional IVF, where most of the retrieved oocytes are mature, and fertilization might occur immediately after ICSI. There are many controversial areas of debate, especially regarding the process of oocyte maturation in vitro: (a) nuclear maturation—the process that reverses meiotic arrest at prophase I (GV), driving the progression to MII. This process is followed by the expansion of cumulus granulosa cells and loss of intercellular communications between the cumulus cells and also between cumulus and the surface of the oocyte (a visible laboratory course); (b) cytoplasmic maturation—metabolic and structural modifications within the oocyte that prepares the ovum for activation, fertilization, and embryonic development (an invisible laboratory process) [2, 4, 5]; it should, therefore, be kept in mind that IVM may not be free of possible future complications and counseling of the patients thoroughly before commencing the treatment is essential.

Follicle monitoring follow-up is mainly performed using ultrasound scans of the ovaries and endometrium. However, in contrast to IVF, in IVM cycles, there is no

First ultrasound should be performed on days 2–3 of the menstrual (or induced) cycle in order to record the number and size of all follicles along with endometrial thickness and a second scan on days 6–8, to determine the presence and size of the largest follicle in each ovary. A third scan should be completed on day of hCG trig-

Fadini et al. [6] reported that 77.4% of retrieved immature oocytes underwent maturation in vitro followed by 29.8% pregnancy rate vs. 48.4 and 15.2% in primed and non-primed IVM cycles, respectively. Mikkelsen et al. [7] pointed that priming

need for serum hormonal level (estradiol, progesterone) follow-up.

ger, to measure the endometrial thickness (**Figure 1**).

**178**

**2.2 FSH priming**

*In vitro maturation oocytes: clinical protocol.*

with r-FSH for 2–3 days before the harvesting of immature oocytes from patients with polycystic ovarian syndrome (PCOS) may improve the maturational potential of the oocytes and the implantation rate. Similar results were described by other investigators in PCOS patients [8–11].

### **2.3 Human chorionic gonadotropin (hCG)/GnRh-agonist priming and retrieval interval**

The beneficial effect of hCG priming in IVM cycles and extension of the period of time from 35 to 36 h (routinely administrated in IVF cycles) to 38 h from hCG administration was demonstrated by Son et al. [12]. This hCG priming promotes GV oocytes to reach MI stage and increases the maturation rate of immature oocytes in vitro. Gonadotropin-releasing hormone agonist (GnRH-a) has been used recently in triggering oocyte maturation. In this approach, small follicles were stimulated with gonadotropins for 3–5 days. GnRH-a administration was performed, to trigger ovulation, when the largest follicles were 10–12 mm in diameter. Many immature oocytes, which underwent maturation in vitro, were then harvested, fertilized, and subsequently developed into blastocysts that resulted in live births [13]. This interesting observation might have a great importance in follicular cytoplasmic maturation due to the FSH surge obtained after GnRH triggering. The FSH surge might promote formation of LH receptors on the granulosa cells enhancing LH activity, induce plasminogen activator activity causing dissociation of oocytes from somatic cells of the follicle (therefore more immature oocytes could be obtained), and maintain the opening of gap junction between cumulus cells and oocyte which contributes to oocyte maturation [14–17].

### **2.4 Timing of collection: at what follicle size?**

Son et al. [18] conducted a study in which patients were triggered when the leading follicle was <10 mm, 10–14 mm, or >14 mm. In the group with a leading follicle >14 mm, only one pregnancy was obtained. The authors of the current paper administer, therefore, hCG trigger once a leading follicle of 10–12 mm is developed.

### **2.5 Endometrial preparation and luteal support**

In IVM, estradiol levels are physiological, and oocyte collection is done before the endometrium is fully estrogenized. After aspiration, there is an insufficient support from the corpus luteum for endometrial receptivity. It was demonstrated

**Figure 2.** *Endometrial support.*

that endometrial thickness is an important predictor of IVM outcome [19]. Thus, endometrial preparation is an important factor for IVM success rates. The protocol for endometrial preparation, which the authors currently use, is similar to that originally described by Trounson et al. [19] and modified by Elizur et al. [20], depending upon the endometrial thickness at day of hCG administration: when endometrial thickness is less than 6 mm, 6–8 mm, or higher than 8 mm, supplementation with 10–12 mg/day, 8–10 mg/day, and 6 mg/day 17-beta estradiol is given, respectively, starting at the day of oocyte retrieval. This approach mimics the natural estrogen rise from the dominant follicle in a natural cycle. In terms of progesterone, supplementation usually begins on the day of oocyte aspiration (as this is the progesterone rise in a natural cycle) using vaginal micronized progesterone (Endometrin, Florish Ltd. Industrial Park Misgav, Israel, Ferring Pharmaceuticals Ltd.) 300 mg daily until pregnancy test is performed (**Figure 2**).

### **2.6 Oocyte retrieval by transvaginal ultrasound**

An high-resolution ultrasound device is obligatory. The oocyte retrieval is done with a single-channel needle 19 G (Swemed, Reduced Single Lumen, Vitrolife, Göteborg, Sweden AB), using a reduced aspiration pressure of 7.5 kPa. This is essential to minimize damage to the immature oocytes.

Usually general anesthesia/sedation is provided. However, local anesthesia with lidocaine 1%, 5 cc injected into the lateral fornixes, could be sufficient.

Oocytes from each ovary are aspirated in separate flask containing 15 ml of flushing medium (Origio, Denmark) and placed on a heated block. Collected cumulus oocyte complexes (OCCs) were classified immediately after OPU (Day 0).

Oocyte cumulus complexes could be classified in one of five groups: (1) expanded cumulus, slack and fluffy multilayer of granulosa cells; (2) full cumulus, multilayer of strictly compact and cubical granulosa cells; (3) full corona, thin layer of strictly compact and cubical granulosa cells; (4) partial cumulus, oocytes surrounded partially with cumulus cells; (5) nude, oocytes without cumulus cells (**Figure 3**). This classification may serve as a prognostic indication of the future maturation and fertilization rate of the immature collected oocytes.

Each complex was then separately cultured in IVM medium (Sage, Cooper Surgical Company, Trumbull, CT, USA) supplemented with FSH + LH (Menogon, Ferring GmbH, Kiel, Germany) with a final concentration of 75 mIU/ml (maturation medium). Oocyte maturation was assessed after 6 (a) and 24–30 h (b),

**181**

**Figure 4.**

*OPU (solid fill arrows); b*

*OPU (solid fill arrows); b*

*(A) In vitro maturation procedure. \*Day 0 = day of OPU; a*

*(B) in vitro maturation procedure. \*Day 0 = day of OPU; a*

*MII oocytes detected in maturation medium 6 h after*

*MII oocytes detected in maturation medium 6 h after*

*MII oocytes detected in maturation medium 24–30 h after OPU (open arrows) and*

*MII oocytes detected in maturation medium 24–48 h after OPU (open arrows).*

**Figure 3.**

*Classification of collected cumulus complexes.*

*In Vitro Maturation and Fertilization of Oocytes: From Laboratory Bench to Clinical Practice*

*DOI: http://dx.doi.org/10.5772/intechopen.91802*

*In Vitro Maturation and Fertilization of Oocytes: From Laboratory Bench to Clinical Practice DOI: http://dx.doi.org/10.5772/intechopen.91802*

**Figure 3.** *Classification of collected cumulus complexes.*

*Innovations in Assisted Reproduction Technology*

that endometrial thickness is an important predictor of IVM outcome [19]. Thus, endometrial preparation is an important factor for IVM success rates. The protocol for endometrial preparation, which the authors currently use, is similar to that originally described by Trounson et al. [19] and modified by Elizur et al. [20], depending upon the endometrial thickness at day of hCG administration: when endometrial thickness is less than 6 mm, 6–8 mm, or higher than 8 mm, supplementation with 10–12 mg/day, 8–10 mg/day, and 6 mg/day 17-beta estradiol is given, respectively, starting at the day of oocyte retrieval. This approach mimics the natural estrogen rise from the dominant follicle in a natural cycle. In terms of progesterone, supplementation usually begins on the day of oocyte aspiration (as this is the progesterone rise in a natural cycle) using vaginal micronized progesterone (Endometrin, Florish Ltd. Industrial Park Misgav, Israel, Ferring Pharmaceuticals

An high-resolution ultrasound device is obligatory. The oocyte retrieval is done

Usually general anesthesia/sedation is provided. However, local anesthesia with

Oocytes from each ovary are aspirated in separate flask containing 15 ml of flushing medium (Origio, Denmark) and placed on a heated block. Collected cumulus oocyte complexes (OCCs) were classified immediately after OPU (Day 0). Oocyte cumulus complexes could be classified in one of five groups: (1) expanded cumulus, slack and fluffy multilayer of granulosa cells; (2) full cumulus, multilayer of strictly compact and cubical granulosa cells; (3) full corona, thin layer of strictly compact and cubical granulosa cells; (4) partial cumulus, oocytes surrounded partially with cumulus cells; (5) nude, oocytes without cumulus cells (**Figure 3**). This classification may serve as a prognostic indication of the future

Each complex was then separately cultured in IVM medium (Sage, Cooper Surgical Company, Trumbull, CT, USA) supplemented with FSH + LH (Menogon, Ferring GmbH, Kiel, Germany) with a final concentration of 75 mIU/ml (maturation medium). Oocyte maturation was assessed after 6 (a) and 24–30 h (b),

with a single-channel needle 19 G (Swemed, Reduced Single Lumen, Vitrolife, Göteborg, Sweden AB), using a reduced aspiration pressure of 7.5 kPa. This is

lidocaine 1%, 5 cc injected into the lateral fornixes, could be sufficient.

maturation and fertilization rate of the immature collected oocytes.

Ltd.) 300 mg daily until pregnancy test is performed (**Figure 2**).

**2.6 Oocyte retrieval by transvaginal ultrasound**

essential to minimize damage to the immature oocytes.

**180**

**Figure 2.**

*Endometrial support.*

### **Figure 4.**

*(A) In vitro maturation procedure. \*Day 0 = day of OPU; a MII oocytes detected in maturation medium 6 h after OPU (solid fill arrows); b MII oocytes detected in maturation medium 24–30 h after OPU (open arrows) and (B) in vitro maturation procedure. \*Day 0 = day of OPU; a MII oocytes detected in maturation medium 6 h after OPU (solid fill arrows); b MII oocytes detected in maturation medium 24–48 h after OPU (open arrows).*

according to oocyte cumulus complex classification. This was followed by oocyte intracytoplasmic sperm injection (ICSI) for the injection of a single sperm cell into the MII oocytes on the day of OPU or 1–2 days afterward, according to oocyte maturation (**Figure 4A** (a and b) and **B** (a and b)). Two to three embryos were transferred 48–78 h post ICSI. Supplementary embryos were vitrified.


**3. Classification of CDH**

Congenital diaphragmatic defects (CDD) are classified as posterolateral (Bochdalek, 70-75%) and non-posterolateral according to the location. Nonposterolateral hernias can be retrosternal (Morgagni-Larrey, 23-28%) or central (2-7%), that involves the non-muscular or central tendinous portion of the diaphragm [15]. This anatomical classification has drawbacks as the actual site of lesion may not be easy to discern. Around 85% of the posterolateral CDH are on the left, while 10% are right sided and 5% bilateral. The diaphragmatic eventration is a rare anomaly in which a part or whole of the hemi diaphragm is abnormally elevated into the thoracic cavity, as the normal diaphragmatic musculature is partly or fully replaced by a thin fibro membranous membrane [15], allowing the abdominal viscera to protrude upwards. In order to elucidate the developmental pathways of diaphragmatic defects more accurately, Ackerman et al developed a phenotype worksheet to capture the precise morphological data on CDD by retrospectively analyzing autopsies of 181 cases [16]. They proposed a new classification system

**121**

the parents.

*Congenital Diaphragmatic Hernia: A Major Challenge for Neonatologists*

based on the location and other specifications. According to the fetal imaging CDH can also be classified as intrapleural and mediastinal; this might help in prenatal counselling [17]. Although inadequately studied, the patho-anatomy of the diaphragmatic defects is relevant for understanding the genetics of CDH, as well as for

Sixty percent of the CDH cases are isolated and not associated with any other major malformations. Pulmonary hypoplasia, gut malrotation, cardiac dextro position and left sided heart hypoplasia are considered to be parts of the sequence of CDH and are not regarded as separate entities. The remaining 40% of complex CDH cases are associated with major congenital anomalies in either syndromic or

Environmental factors contributing to CDH are not well investigated. Maternal vitamin A deficiency, nitrofen treatment and retinoid receptor knock-out in animal model are reported to result in CDH in their offsprings, suggesting a role of retinoid signaling pathway in the pathogenesis [18]. Similar effects are seen in WT1 and COUP-TFII mutant mouse models. Beurskens et al found a significantly higher risk of CDH in the infants born to mothers who had low vitamin A intake during pregnancy (Odds ratio, 7.2; 95% confidence interval, 1.5-34.4; p = 0.01)[19]. The cord blood in neonates with CDH is reported to have low retinol and retinol-binding protein levels. Thalidomide and quinine have also been implicated as possible causes

CDH is genetically heterogeneous although can be monogenic. A genetic cause is found in 30% of the cases, even though most cases are sporadic and about 1-2% familial [21]. The application of the next generation sequencing technologies, e.g. DNA array and whole genome sequencing has contributed to the identification of

Chromosomal anomalies account for about 10% of the CDH cases. The common chromosomal aneuploidies associated with CDH are trisomy 18, 13 and 21. Morgagni hernia is more frequently found in trisomy 21 than Bochdalek hernia. Pallister Killian syndrome (PKS), characterized by mosaic tetrasomy 12p is a common chromosomal anomaly associated with CDH and occurs sporadically. The karyotype in PKS is often found to be normal due to a tissue specific distribution of chromosome i12p and reduced yield with culture aging [22, 23]. Establishing diagnosis in such cases depends on the tissue examined and genetic test used [24]. Isochromosome 12p is rarely isolated from cord blood lymphocytes, whereas its yield from the skin fibroblast is close to 100% [25]. Also, the chorionic villus sampling may miss mosaicism, while detection rate of amniocentesis is nearly 90% [23, 26]. Chromosome 12p targeted-FISH, array comparative genomic hybridization (aCGH) or other newer genetic technologies should be used to prevent misdiagnosis in the condition [24, 27]. The availability of information regarding genetic etiology equips the clinicians with better understanding of the prognosis for discussion with

Some of the common monogenic syndromes associated with CDH are Cornelia de Lange, Donnai Barrow and Simpson-Golabi-Behmel, while, others, like Fryn

*DOI: http://dx.doi.org/10.5772/intechopen.94839*

comparing the outcomes.

non-syndromic form.

of CDH [20].

**4.2 Genetics of CDH**

cases with genetic etiology.

**4. Etiopathogenesis of CDH**

**4.1 Environmental factors**

*Congenital Diaphragmatic Hernia: A Major Challenge for Neonatologists DOI: http://dx.doi.org/10.5772/intechopen.94839*

based on the location and other specifications. According to the fetal imaging CDH can also be classified as intrapleural and mediastinal; this might help in prenatal counselling [17]. Although inadequately studied, the patho-anatomy of the diaphragmatic defects is relevant for understanding the genetics of CDH, as well as for comparing the outcomes.

Sixty percent of the CDH cases are isolated and not associated with any other major malformations. Pulmonary hypoplasia, gut malrotation, cardiac dextro position and left sided heart hypoplasia are considered to be parts of the sequence of CDH and are not regarded as separate entities. The remaining 40% of complex CDH cases are associated with major congenital anomalies in either syndromic or non-syndromic form.


**3. Constrained polycyclic derivatives**

Tricyclic and even larger constrained derivatives have been investigated for their melatoninergic potency. The synthesis of 6,7,8,9-tetrahydropyridino[1,2-*a*]indole (**36**) [47] is illustrated in **Figure 8**.

**Figure 8.** *6,7,8,9-Tetrahydropyridino[1,2-a]indole.*

**Figure 9.** *1,3,4,5-Tetrahydro[cd]indole.*

**9**

*Synthetic Melatonin Receptor Agonists and Antagonists DOI: http://dx.doi.org/10.5772/intechopen.91424*

*Isoindolo[2,1-a]indoles and benzo[*c*]azepeno[2,1-a]indoles.*

The 3-substituted 1,3,4,5-tetrahydro[*cd*]indoles exhibit higher melatonin receptor affinity than their more constrained congeners [30]. The key intermediate ketone **38** was obtained upon cyclization of the carboxylic acid **37** with polyphosphoric acid. As shown in **Figure 9**, the ketone **38** was converted to the corresponding cyanide **39**, in two steps. The latter gave then the respective acetamide **40**, and the final tricyclic adduct **41** was prepared by ester hydrolysis followed by decarboxylation of the cor-

Azaindoles have also been proven to exhibit melatoninergic potency. Some melatonin analogues based on 3*a*-aza-, 4-aza-, 6-aza-, and 7-azaindole cores are

In the synthetic route to the 3*a*-azamelatonin analogue **49**, El Kazzouli et al. [48] reported the treatment of 2-amino-5-bromopyridine (**42**) with 2-bromoacetone and the use of ethyl 2-azidoacetate for the formation of the key intermediate ester **45**. In the synthesis of 3-substituted-4-azaindole **49**, Mazeas et al. [49] have used 2-methoxy-5-nitropyridine (**50**), as starting material, and standard chemistry procedures. The 4-azaindole analogue **50** was proven to be a stronger agonist than melatonin at both melatonin receptors [50]. The preparation of 6-azamelatonin derivative **61** involves the Sonogashira reaction, as reported in the literature [49]. Finally, the 7-azamelatonin congener **67** presents promising melatoninergic potential [49].

The isoindolo[2,1-*a*]indoles and benzo[*c*]azepeno[2,1-*a*]indoles were prepared by Tsotinis et al. [51]. The appropriate *N*-acetyl tryptamine was coupled with the respective dibromide **68**, and the derived *N*-alkyl indole **70** was then cyclized in the

The pharmacological evaluation has shown that 6*H*-isoindolo[2,1-*a*]indoles (**71a**) are agonists, while the 5,6-dihydroindolo[2,1-*a*]isoquinolines (**71b**) are partial agonists/antagonists, and the 6,7-dihydro-5*H*-benzo[*c*]azepino[2,1-*a*]indoles (**71c**) are antagonists. Thus, the size of the linker between the phenyl ring and the pyrrole nitrogen atom serves as a switch pharmacological probe, spanning from agonist to

Some derivatives with constrained conformation also present chirality. Ramelteon is the most emblematic representative example of this class of

responding acid in boiling quinoline in the presence of copper powder.

presence of Pd(PPh3)4 to afford the desired products **71 (Figure 11)**.

described in **Figure 10**.

**Figure 11.**

antagonist melatoninergic action.

**4. Chiral melatonin analogues**

**Figure 10.** *Azaindoles.*

*Synthetic Melatonin Receptor Agonists and Antagonists DOI: http://dx.doi.org/10.5772/intechopen.91424*

#### **Figure 11.**

*Melatonin - The Hormone of Darkness and Its Therapeutic Potential and Perspectives*

**8**

**Figure 10.** *Azaindoles.*

**Figure 9.**

*1,3,4,5-Tetrahydro[cd]indole.*

*Isoindolo[2,1-a]indoles and benzo[*c*]azepeno[2,1-a]indoles.*

The 3-substituted 1,3,4,5-tetrahydro[*cd*]indoles exhibit higher melatonin receptor affinity than their more constrained congeners [30]. The key intermediate ketone **38** was obtained upon cyclization of the carboxylic acid **37** with polyphosphoric acid. As shown in **Figure 9**, the ketone **38** was converted to the corresponding cyanide **39**, in two steps. The latter gave then the respective acetamide **40**, and the final tricyclic adduct **41** was prepared by ester hydrolysis followed by decarboxylation of the corresponding acid in boiling quinoline in the presence of copper powder.

Azaindoles have also been proven to exhibit melatoninergic potency. Some melatonin analogues based on 3*a*-aza-, 4-aza-, 6-aza-, and 7-azaindole cores are described in **Figure 10**.

In the synthetic route to the 3*a*-azamelatonin analogue **49**, El Kazzouli et al. [48] reported the treatment of 2-amino-5-bromopyridine (**42**) with 2-bromoacetone and the use of ethyl 2-azidoacetate for the formation of the key intermediate ester **45**. In the synthesis of 3-substituted-4-azaindole **49**, Mazeas et al. [49] have used 2-methoxy-5-nitropyridine (**50**), as starting material, and standard chemistry procedures. The 4-azaindole analogue **50** was proven to be a stronger agonist than melatonin at both melatonin receptors [50]. The preparation of 6-azamelatonin derivative **61** involves the Sonogashira reaction, as reported in the literature [49]. Finally, the 7-azamelatonin congener **67** presents promising melatoninergic potential [49].

The isoindolo[2,1-*a*]indoles and benzo[*c*]azepeno[2,1-*a*]indoles were prepared by Tsotinis et al. [51]. The appropriate *N*-acetyl tryptamine was coupled with the respective dibromide **68**, and the derived *N*-alkyl indole **70** was then cyclized in the presence of Pd(PPh3)4 to afford the desired products **71 (Figure 11)**.

The pharmacological evaluation has shown that 6*H*-isoindolo[2,1-*a*]indoles (**71a**) are agonists, while the 5,6-dihydroindolo[2,1-*a*]isoquinolines (**71b**) are partial agonists/antagonists, and the 6,7-dihydro-5*H*-benzo[*c*]azepino[2,1-*a*]indoles (**71c**) are antagonists. Thus, the size of the linker between the phenyl ring and the pyrrole nitrogen atom serves as a switch pharmacological probe, spanning from agonist to antagonist melatoninergic action.

## **4. Chiral melatonin analogues**

Some derivatives with constrained conformation also present chirality. Ramelteon is the most emblematic representative example of this class of

**Figure 12.** *Ramelteon.*

**Figure 13.**

*Examples of chiral melatoninergic analogues and side chain conformationally constrained tricyclic derivatives 83 and 84.*

compounds. Ramelteon, *N*-{2-[(8*S*)-1,6,7,8-tetrahydro-2H-indeno[5,4-*b*]furan-8-yl]ethyl}propanamide (**76**) [52], is a melatonin analogue approved by the FDA as a sedative-hypnotic. The following synthetic route [53], illustrated in **Figure 12**, uses dibenzoyl-*L*-tartaric acid as an acid to form the salt at the end of hydrogenation and as the resolution agent as well.

Most of these chiral derivatives are prepared as racemates and, then, in some cases, resolved. The racemate mixture of enantiomers provides an initial estimation of the biology of these compounds, although asymmetric syntheses may then be required if one of the enantiomers exhibits a selective result. Substituents on the 3-side chain, particularly at the *β*-position, present a preference for the active conformation. This hypothesis has been investigated by assessing the melatoninergic potency of various compounds which bear in their side chain small to large substituents. An example of *α*- and *β*-methyl side chain functionalized molecules with enhanced activity is the *N*1-phenethyl-substituted indole derivatives **79** and **82** [54]. The characteristic steps of the synthesis of these probes are illustrated in **Figure 13**. Similar results, in terms of activities and related conformation, have been obtained for the analogues **83**, **84**, and **85** [55–57].

**11**

*Synthetic Melatonin Receptor Agonists and Antagonists DOI: http://dx.doi.org/10.5772/intechopen.91424*

The authors declare no conflict of interest.

Andrew Tsotinis\* and Ioannis P. Papanastasiou

provided the original work is properly cited.

\*Address all correspondence to<EMAIL_ADDRESS>
Division of Pharmaceutical Chemistry, Department of Pharmacy, School of Health

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Sciences, National and Kapodistrian University of Athens, Athens, Greece

28-fold selectivity for the MT2 receptor.

**5. Conclusions**

**Conflict of interest**

**Author details**

The *β*-methyl, *N*-methyl-substituted melatonin derivative **86** was prepared and resolved by chiral HPLC [58]. The (+) enantiomer has a tenfold higher potency in pigment aggregation in the *Xenopus laevis* protocol, while the (−) enantiomer has a

A selection of key melatoninergic derivatives was reported herein. We pointed out the synthetic routes towards these melatonin analogues, first of the aromatic nucleus, then of the functionalities that have been introduced to the nucleus, and finally of those analogues with restrained conformations and those that are optically active. Much more needs to be explored about the variant functions of melatonin and through which receptor type they exert their action. The range of small molecules having agonist or antagonist effects on the melatonin receptors is large, and new scaffolds keep appearing as drug candidates in different treatments. This work is hoped to assist those seeking to explore the melatonin and melatoninergic field.

The *β*-methyl, *N*-methyl-substituted melatonin derivative **86** was prepared and resolved by chiral HPLC [58]. The (+) enantiomer has a tenfold higher potency in pigment aggregation in the *Xenopus laevis* protocol, while the (−) enantiomer has a 28-fold selectivity for the MT2 receptor.


**Experimental Investigation on Premixed Combustion in a Diesel Engine with Ultra-Multihole Nozzle**

Xuelong Miao,Yusheng Ju, Xianyong Wang, Jianhai Hong and Jinbao Zheng *Wuxi Fuel Injection Equipment Research Institute China*


**3. Results**

The chemical composition of lemongrass oil used in process 1 is summarized in Table 1. This raw material reports high content of moisture, and organic compounds as myrcene, undecyne, nerol, geranial, among other [24]. These substances are the key compounds in the lemongrass extract and are used to guarantee the nanosize of the TiO2 nanoparticles. The oil content in the lemongrass is about 1.10% of the total mass while the moisture and solid contents are approximately 71.23% and 27.67%, respectively. The above implies that the oil extraction method has to be highly efficient to reach acceptable/required yields. Most of the solid contents in lemongrass is cellulosic biomass, therefore it was assumed (for simulation purpose) that all solid content is cellulose.

**Table 1.** Chemical composition of lemongrass dry basis.

#### *3.1. Processes Simulation Results*

3.1.1. Simulation of Large-Scale Production of Chitosan Microbeads Modified with TiO2 Nanoparticles via Green Chemistry

Process simulation flowsheet of large-scale production of chitosan microbeads modified with TiO2 nanoparticles is shown in Figure 4. And the pressure, temperature, composition and flows of main process streams are shown in Table 2. As is described in Section 2.2, the system is composed of three sections: (1) Lemongrass oil extraction; (2) TiO2 nanoparticles synthesis, and (3) CMTiO2 production. For subprocess (1), stream 1 represents the inlet flow of the main feedstock (lemongrass). This stream is sent to washing and drying stages for cellulosic material removal and moisture reduction, respectively. After feedstock cleaning, the material is sent to a crusher unit for size-reduction. The above is performed with the aim of increasing the surface contact between the lemongrass and the extraction media (water). The liquid-solid extraction (infusion stage) is developed by a heating-sedimentation stage where the oil extract is separated in the upper site of the unit while the residual solids remain at the bottom. The oil extract is obtained with high moisture content (99% w/w) so the stream passes

through an evaporation unit to reduce the water content close to 3% w/w. Finally, the oil extract is cooled until 28 ◦C, and it is stored. Subprocess (2) starts with the preparation of TTIP solution and the hydrolysis reaction where the TiO2 nanoparticles are synthesized (see stream 39). In the case of subprocess (3), this stage starts with the production of the chitosan gel considering inlet flow according to the composition of stream 43. Stream 39 is added to the mixing tank where the CMTiO2 are formed maintaining the required proportion of 1:1 for Chitosan and TiO2, respectively. Finally, the product obtained with a flow of 232.03 kg/h according to stream 56.

```
(a)
```
(**b**)

**Figure 4.** *Cont*.

**Figure 4.** Process flowsheet of large-scale production of chitosan microbeads modified with TiO2 nanoparticles via a green chemistry method: (**a**) Lemongrass oil extraction (**b**) TiO2 nanoparticles synthesis; (**c**) chitosan microbeads production.

**Table 2.** Main process streams for Chitosan microbeads modified with TiO2 nanoparticles.

3.1.2. Simulation of Large-Scale Production of Chitosan Microbeads Modified with TiO2 and Magnetite Nanoparticles

The simulation for the second processing route of bio-adsorbents production was performed based on general mass/energy balances, and operational conditions obtained from literature and lab experiments. Figure 5 shows the simulated process flowsheet of large-scale production of chitosan microbeads modified with TiO2 nanoparticles and magnetite. Table 3 summarizes the main streams and operational conditions of this process. As it was described, this designed system is constituted by two main subprocesses: (1) Magnetite nanoparticles synthesis, and (2) chitosan microbeads production (with nanoparticles modification). For magnetite nanoparticles synthesis, stream 1 and 2 represent the inlet flow of main feedstocks: FeCl2·4H2O and FeCL3·6H2O, respectively. The reaction between these iron chlorides and NaOH generates magnetite (desired product) along with hematite, water and sodium chloride. For simulation purpose, it was assumed that all produced hematite is magnetite. After reaction stage, the stream is sent to separations processes where all impurities are removed from

the product. The magnetic properties of these nanoparticles allow separating them using a magnetic field or a magnet [19]. Finally, the dried and purified magnetite nanoparticles are sent to the second subprocess, the chitosan microbeads modified with the nanoparticles (TiO2 and magnetite) are sent to purification stage where the desired product is obtained dried and with high purity with a mass flow rate of 307.86 kg/h.

**Figure 5.** Process flowsheet of Large-scale production of chitosan microbeads modified with TiO2 and magnetite nanoparticles: (**a**) Magnetite nanoparticles synthesis; (**b**) chitosan microbeads with modifications subprocess.

**Table 3.** Main process streams for chitosan microbeads modified with TiO2 and magnetite nanoparticles.

3.1.3. Simulation of Large-Scale Production of Chitosan Microbeads Modified with Thiourea

The simulation of the third processing route was developed according to the subprocess stage for chitosan microbeads production described for CMTiO2 and CMTiO2-Mag processes. In this case, the modification of the microbeads does not require the synthesis of any nanoparticles because the thiourea is introduced to the process as an available raw material. After this stage, the stream is sent to microbeads production and purification stages. Table 4 reports the main mass flows and operational conditions of this process. Figure 6 shows the simulated process flowsheet of large-scale production of chitosan microbeads modified thiourea. Finally, in this process alternative are produced 155.23 kg/h of chitosan microbeads modified with thiourea according to stream 13.

**Table 4.** Main process streams for chitosan microbeads modified with thiourea.

#### *3.2. Exergy Analysis of the Routes Simulated*

These large-scale production processes can be generally divided into two main stages: nanoparticles preparation and chitosan microbeads formation. Considering the results of simulations and composition of each stream, chemical and physical exergies were estimated. Table 5 shows the estimated and reported chemical exergies of main components for bio-adsorbent processing routes.

**Table 5.** Chemical exergy of main components for bio-adsorbent production processes.

The higher chemical exergies correspond to those compounds with the longest carbon chains in its molecular structures (undecyne, myrcene and chitosan). Chemical exergy for common components as water, NaOH, or acetic acid was found in literature [25]. Table 6 reports the results obtained for exergy performance indicators for each processing route.

**Table 6.** Results for exergy performance of each processing route.

CMTiO2 route presents the lowest exergy efficiency performance with a 0.04%. For CMTiO2-Mag and CMThi processes were obtained a corresponding exergy efficiency of 2.83% and 2.50%, respectively. From a general viewpoint, the performance of the exergy efficiency was significantly low for all bio-adsorbent processes. This result implies that these designs might require technological improvements to reach better exergy and energy performances. In this sense, CMTiO2 requires special attention due to its exergy efficiency shows that almost 100% of the inlet exergy is lost through the operation.

Figure 7 shows the exergy destruction of each bio-adsorbent processing route. The process with higher irreversibilities is CMTiO2-Mag with an exergy flow of 182,698.34 MJ/h, followed by CMTiO2 route with destroyed exergy of 144,445.08 MJ/h. The performance of this parameter is congruent respect to exergy efficiencies obtained for all processes. For CMTiO2 route, it was found that the stage with the highest irreversibilities was the separation train. This stage is composed of three consecutive centrifuges representing approximately 53.00% of total irreversibilities for this process. The use of other separation technologies may contribute to reduce exergy losses. For the case of CMTiO2-Mag route, it was obtained that microbeads-drying unit was the stage with the highest irreversibilities with a contribution of 41.05%, followed by washing unit in the separation stage (see "Lav4" in Figure 5b) with a 24.20%. In the case of CMThi route, drying unit was the stage with the highest exergy destruction representing 92.48% of total irreversibilities. The above results (for all cases) imply that these designs require better/improved separation technologies/stages to avoid several irreversibilities. This also could contribute to obtaining higher exergy efficiencies for each process.

**Figure 7.** Comparison of exergy destruction for each processing route.

On the other hand, the exergy of residues was higher for CMTiO2-Mag route, which was expected due to this process presents higher mass inventory respect to the other processing routes. Figure 8 shows the exergy of residues for each assessed process. CMThi process destroys less exergy due to residues with a flow of 33,654.48 MJ/h, while for CMTiO2 and CMTiO2-Mag were obtained exergy flows of 144,405.08 MJ/h and 182,698.34 MJ/h, respectively.

**Figure 8.** Comparison of exergy of residues for each processing route.

Finally, the exergy of utilities was estimated for each processing route. Figure 9 shows the comparison of this parameter for each bio-adsorbent processing route. The results for exergy of utilities present a similar performance for the three alternatives, obtaining an exergy flow of 91,033.74 MJ/h for CMTiO2, 88,030.46 MJ/h for CMTiO2-Mag, and 105,964.41 MJ/h for CMThio. For the case of CMTiO2-Mag route, drying unit was the most significant stage representing an around 99% of the total exergy by utilities for this process. The above result indicates that this unit probably has important energy requirements that implies a high demand of industrial utilities. In this sense, the application of process optimization techniques could contribute to decrease the energy requirements, or obtain a better energy distribution. For CMTiO2 process was found that hydrolysis reactor is a critical stage due to the most of exergy utility is consumed in this unit with a contribution of 47.22% of the total. It is

explained by studying the thermodynamics of this reaction (hydrolysis of TTIP) because it is highly exothermic, thus, a cooling system is needed to maintain the reaction temperature constant.

**Figure 9.** Comparison of exergy utilities for each processing route.

As described by CMTiO2-Mag route, for CMThio alternative, the drying stage was also the most critical unit for exergy of utilities parameter with an exergy flow of 105,942.41 MJ/h which represents almost a 100% of the total. This result confirms the described behavior for exergy destruction performance which was previously explained for this bio-absorbent route.


**4.2 Radar hardware overview**

In the transmitter unit of the radar system, a 'saw-tooth' voltage sequence is generated by the 'saw-tooth' generator. It is used to control a VCO to generate a frequency modulated radio frequency (RF) signal. The receiver channel, with the help of the beam forming array, receives the signal reflected by obstructions. The fundamental frequency output from VCO is connected to the transmit antenna array and halved frequency output. Signals received from the antenna array are mixed with the transmitted signal in the sub-harmonic mixer. The intermediate frequency (IF) signal obtained from the mixer is then amplified by the variable gain amplifier (VGA) and filtered by a band-pass filter (BPF). These two components are controlled by the radars electronic control unit (ECU) unit, which also communicates with the PC host via the USB interface [27–30]. The photograph of the manufactured FMCW radar unit is shown in **Figure 3**.